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Sample records for fluorescence spectroscopy trlfs

  1. Speciation of Eu3+ bound to humic substances by time-resolved laser fluorescence spectroscopy (TRLFS) and parallel factor analysis (PARAFAC)

    NASA Astrophysics Data System (ADS)

    Lukman, Steven; Saito, Takumi; Aoyagi, Noboru; Kimura, Takaumi; Nagasaki, Shinya

    2012-07-01

    The bioavailability and toxicity of metal ions including radionuclides in the biosphere are greatly influenced by their speciation. Humic substances (HSs) are important constituents of various soil and water systems and have significant impact on the speciation and mobility of metal ions because of their high affinity to metal ions. In this study, the speciation of europium (Eu3+), a chemical homologue of trivalent actinides, with HSs collected from various origins was investigated by time-resolved laser fluorescence spectroscopy (TRLFS). The difficulties associated with the separation of the contribution of different Eu3+ species due to overlapping spectra or similar fluorescence lifetimes were addressed and mitigated by applying a multi-mode factor analysis, parallel factor analysis (PARAFAC), which resulted in the number, spectra, decay curves and relative fluorescence intensity profiles of different Eu3+ species. Subsequently, the interpretation of the Eu3+ species, was tackled by principal component analysis (PCA) and partial linear square (PLS) regression to deduce the nature of the Eu3+ species by taking into account the physicochemical properties of the HSs. Three factors corresponding to different Eu3+ species were obtained at 70 μM Eu3+ for all HSs investigated except for one humic acid. One of the factors corresponded to free Eu3+ ion interacting with HSs via diffusion. The remaining two factors were thought to be Eu3+ bound to HSs: one bound to acidic functional groups of HSs and the other to the sites of HSs influenced by the carbon backbone structures. It was also found that the latter factor exhibited strong energy transfer from the excited Eu3+ center to HSs. At lower Eu3+ concentration (10 μM), two factors having similar fluorescent characteristics to those of the second and third factors were obtained.

  2. Nanosecond fluorescence spectroscopy

    SciTech Connect

    Leskovar, B.

    1985-03-01

    This article is a summary of a short course lecture given in conjunction with the 1984 Nuclear Science Symposium. Measuring systems for nanosecond fluorescence spectroscopy using single-photon counting techniques are presented. These involve systems based on relaxation-type spark gap light pulser and synchronously pumped mode-locked dye lasers. Furthermore, typical characteristics and optimization of operating conditions of the critical components responsible for the system time resolution are discussed. A short comparison of the most important deconvolution methods for numerical analysis of experimental data is given particularly with respect to the signal-to-noise ratio of the fluorescence signal. 22 refs., 8 figs.

  3. Smartphone fluorescence spectroscopy.

    PubMed

    Yu, Hojeong; Tan, Yafang; Cunningham, Brian T

    2014-09-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of specific nucleic acid sequences in a liquid test sample and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route toward portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins. PMID:25098859

  4. Smartphone fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Yu, Hojoeng; Tan, Yafang; Cunningham, Brian T.

    2014-03-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

  5. High-Pressure Fluorescence Spectroscopy.

    PubMed

    Maeno, Akihiro; Akasaka, Kazuyuki

    2015-01-01

    The combination of fluorescence and pressure perturbation is a widely used technique to study the effect of pressure on a protein system to obtain thermodynamic, structural and kinetic information on proteins. However, we often encounter the situation where the available pressure range up to 400 MPa of most commercial high-pressure fluorescence spectrometers is insufficient for studying highly pressure-stable proteins like inhibitors and allergenic proteins. To overcome the difficulty, we have recently developed a new high-pressure fluorescence system that allows fluorescence measurements up to 700 MPa. Here we describe the basic design of the apparatus and its application to study structural and thermodynamic properties of a couple of highly stable allergenic proteins, hen lysozyme and ovomucoid, using Tryptophan and Tyrosine/Tyrosinate fluorescence, respectively. Finally, we discuss the utility and the limitation of Trp and Tyr fluorescence. We discuss pitfalls of fluorescence technique and importance of simultaneous use of other high-pressure spectroscopy, particularly high-pressure NMR spectroscopy. PMID:26174405

  6. Fluorescence Spectroscopy in a Shoebox

    NASA Astrophysics Data System (ADS)

    Farooq Wahab, M.

    2007-08-01

    This article describes construction of a simple, inexpensive fluorometer. It utilizes a flashlight or sunlight source, highlighter marker ink, bowl of water with mirror as dispersing element, and colored cellophane sheets as filters. The human eye is used as a detector. This apparatus is used to demonstrate important concepts related to fluorescence spectroscopy. Using ink from a highlighter marker, one can demonstrate the difference between light scattering and fluorescence emission, the need for an intense light source, phenomenon of the Stokes shift, the choice of filters, the preferred geometry of excitation source and emission detector, and the low detection limits that can be achieved by fluorescence measurements. By reflecting the fluorescence emission from a compact disk, it can be seen that the light emitted by molecules is not monochromatic. Furthermore, a spectrofluorometer is constructed using gratings made from a DVD or a CD. The shoebox fluorometer and spectrofluorometer can serve as useful teaching aids in places where commercial instruments are not available, and it avoids the black box problem of modern instruments.

  7. Supercritical Angle Fluorescence Correlation Spectroscopy

    PubMed Central

    Ries, Jonas; Ruckstuhl, Thomas; Verdes, Dorinel; Schwille, Petra

    2008-01-01

    We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves. PMID:17827221

  8. Modified diglycol-amides for actinide separation: solvent extraction and time-resolved laser fluorescence spectroscopy complexation studies

    SciTech Connect

    Wilden, A.; Modolo, G.; Lange, S.; Sadowski, F.; Bosbach, D.; Beele, B.B.; Panak, P.J.; Skerencak-Frech, A.; Geist, A.; Iqbal, M.; Verboom, W.

    2013-07-01

    In this work, the back-bone of the diglycolamide-structure of the TODGA extractant was modified by adding one or two methyl groups to the central methylene carbon-atoms. The influence of these structural modifications on the extraction behavior of trivalent actinides and lanthanides and other fission products was studied in solvent extraction experiments. The addition of methyl groups to the central methylene carbon atoms leads to reduced distribution ratios, also for Sr(II). This reduced extraction efficiency for Sr(II) is beneficial for process applications, as the co-extraction of Sr(II) can be avoided, resulting in an easier process design. The use of these modified diglycol-amides in solvent extraction processes is discussed. Furthermore, the complexation of Cm(III) and Eu(III) to the ligands was studied using Time-Resolved-Laser-Fluorescence-Spectroscopy (TRLFS). The complexes were characterized by slope analysis and conditional stability constants were determined.

  9. Fluorescence spectroscopy applied to orange trees

    NASA Astrophysics Data System (ADS)

    Marcassa, L. G.; Gasparoto, M. C. G.; Belasque, J., Jr.; Lins, E. C.; Dias Nunes, F.; Bagnato, V. S.

    2006-05-01

    In this work, we have applied laser-induced fluorescence spectroscopy to investigate biological processes in orange trees (Citrus aurantium L.). We have chosen to investigate water stress and Citrus Canker, which is a disease caused by the Xanthomonas axonopodis pv. citri bacteria. The fluorescence spectroscopy was investigated by using as an excitation source a 442-nm 15-mW HeCd gas multimode discharge laser and a 532-nm 10-mW Nd3+:YAG laser. The stress manifestation was detected by the variation of fluorescence ratios of the leaves at different wavelengths. The fluorescence ratios present a significant variation, showing the possibility to observe water stress by fluorescence spectrum. The Citrus Canker’s contaminated leaves were discriminated from the healthy leaves using a more complex analysis of the fluorescence spectra. However, we were unable to discriminate it from another disease, and new fluorescence experiments are planned for the future.

  10. Fluorescence spectroscopy of rhodopsins: insights and approaches.

    PubMed

    Alexiev, Ulrike; Farrens, David L

    2014-05-01

    Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks. PMID:24183695

  11. Fluorescence spectroscopy of rhodopsins: Insights and approaches

    PubMed Central

    Alexiev, Ulrike; Farrens, David L.

    2014-01-01

    Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. PMID:24183695

  12. Fluorescence spectroscopy for neoplasms control

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    Investigation of malignant skin tumors diagnosis was performed involving two setups for native tissues fluorescence control in visible and near infrared regions. Combined fluorescence analysis for skin malignant melanomas and basal cell carcinomas was performed. Autofluorescence spectra of normal skin and oncological pathologies stimulated by 457 nm and 785 nm lasers were registered for 74 skin tissue samples. Spectra of 10 melanomas and 27 basal cell carcinomas were registered ex vivo. Skin tumors analysis was made on the basis of autofluorescence spectra intensity and curvature for analysis of porphyrins, lipo-pigments, flavins and melanin. Separation of melanomas and basal cell carcinomas was performed on the basis of discriminant analysis. Overall accuracy of basal cell carcinomas and malignant melanomas separation in current study reached 86.5% with 70% sensitivity and 92.6% specificity.

  13. Fluorescence lifetime spectroscopy of glioblastoma multiforme.

    PubMed

    Marcu, Laura; Jo, Javier A; Butte, Pramod V; Yong, William H; Pikul, Brian K; Black, Keith L; Thompson, Reid C

    2004-01-01

    Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time-resolved, laser-induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time-resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time-resolved spectra were detected in a 360-550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral- (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high-grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectral- and the time domain can enhance the ability of fluorescence-based techniques to diagnose and detect brain tumor margins intraoperatively. PMID:15339216

  14. Online fluorescence suppression in modulated Raman spectroscopy.

    PubMed

    De Luca, Anna Chiara; Mazilu, Michael; Riches, Andrew; Herrington, C Simon; Dholakia, Kishan

    2010-01-15

    Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and

  15. Multipoint fluorescence correlation spectroscopy with total internal reflection fluorescence microscope.

    PubMed

    Ohsugi, Yu; Kinjo, Masataka

    2009-01-01

    We report simultaneous determination of diffusion coefficients at different points of a cell membrane using a multipoint fluorescence correlation spectroscopy (FCS) system. A system carrying seven detection areas in the evanescent field is achieved by using seven optical fibers on the image plane in the detection port of an objective-type total internal reflection FCS (TIR-FCS) system. Fluctuation of fluorescence intensity is monitored and evaluated using seven photomultiplier tubes (PMTs) and a newly constructed multichannel correlator. We demonstrate simultaneous-multipoint FCS, with a 3-mus time resolution, to investigate heterogeneous structures such as cell membranes and membrane-binding molecular dynamics near glass surfaces in live cells. PMID:19256718

  16. Ultraviolet, Visible, and Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Penner, Michael H.

    Spectroscopy in the ultraviolet-visible (UV-Vis) range is one of the most commonly encountered laboratory techniques in food analysis. Diverse examples, such as the quantification of macrocomponents (total carbohydrate by the phenol-sulfuric acid method), quantification of microcomponents, (thiamin by the thiochrome fluorometric procedure), estimates of rancidity (lipid oxidation status by the thiobarbituric acid test), and surveillance testing (enzyme-linked immunoassays), are presented in this text. In each of these cases, the analytical signal for which the assay is based is either the emission or absorption of radiation in the UV-Vis range. This signal may be inherent in the analyte, such as the absorbance of radiation in the visible range by pigments, or a result of a chemical reaction involving the analyte, such as the colorimetric copper-based Lowry method for the analysis of soluble protein.

  17. Differentiating tissue by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Woessner, Stefan; Huen, Julien; Malthan, Dirk

    2004-03-01

    A common problem in several surgical applications is the lack of navigational information. Most often, the only source of information about the location of crucial structures, in relation to the surgical instrument, is the visible and tactile sensory input of the surgeon. In some cases, this leads to time-consuming procedures and a high risk for the patient. Therefore, we developed a spectroscopic sensor system for automatic differentiation between several tissue types. For example in milling processes, a sensor that is able to detect bone in contrast to nerve or vein tissue can be used to control the milling process. We showed exemplarily for the cochlea implant, a typical ENT-surgery, that with the help of our sensor system, the milling of bone can be accelerated without increasing the risk for the patient. It is also possible to use this type of sensor system in the area of medical robotics in soft-tissue applications. With real-time information, a continuous registration can take place, in contrast to a registration that is done using static preoperatively acquired images. We showed that our sensor system can be used to dynamically update the location of the patient in relation to CT or MR-images. In conclusion, we have been able to show that well-known spectroscopy sensors can be used to open new possibilities in medical treatment with and without the use of robotics.

  18. A TRLFS study on the complexation of novel BTP type ligands with Cm(III).

    PubMed

    Beele, Björn B; Rüdiger, Elias; Schwörer, Felicitas; Müllich, Udo; Geist, Andreas; Panak, Petra J

    2013-09-14

    Two BTP-type N-donor ligands with different numbers of aromatic nitrogen atoms (2,6-bis(4-ethyl-pyridazin-1-yl)pyridine, Et-BDP and 2,6-bis(4-(n)propyl-2,3,5,6-tetrazine-1-yl)pyridine, (n)Pr-Tetrazine) have been synthesized and characterized by NMR and MS techniques. The complexation with Cm(III) in 2-propanol-water (1 : 1, vol.) is studied for both ligands using time resolved laser-induced fluorescence spectroscopy (TRLFS) and the complexation properties are compared to (n)Pr-BTP. With increasing the ligand concentration three different species, the 1 : 1-, 1 : 2- and 1 : 3-complex, were found. Log β3 values of 7.6 for the formation of Cm(Et-BDP)3 and 9.2 for the formation of Cm((n)Pr-Tetrazine)3 are determined. The complexation with (n)Pr-Tetrazine shows slow kinetics. Thermodynamic data of the complexation reactions are determined in a temperature range of 25 °C-60 °C. The complexation with Et-BDP is exothermic (ΔH = -16.3 ± 1.2 kJ mol(-1)) and exergonic (ΔG = -43.8 ± 2.6 kJ mol(-1)) whereas the complexation with (n)Pr-Tetrazine is endothermic (ΔH = 43.9 ± 3.1 kJ mol(-1)) and exergonic (ΔG = -51.7 ± 2.2 kJ mol(-1)). In the case of the latter the complexation is driven by a highly positive reaction entropy change (ΔS = 320.6 ± 15.4 J mol(-1) K(-1)). In comparison to (n)Pr-BTP, less negative ΔG values were found for the complexation of Cm(III) with both ligands. PMID:23552476

  19. Fluorescence spectroscopy for wastewater monitoring: A review.

    PubMed

    Carstea, Elfrida M; Bridgeman, John; Baker, Andy; Reynolds, Darren M

    2016-05-15

    Wastewater quality is usually assessed using physical, chemical and microbiological tests, which are not suitable for online monitoring, provide unreliable results, or use hazardous chemicals. Hence, there is an urgent need to find a rapid and effective method for the evaluation of water quality in natural and engineered systems and for providing an early warning of pollution events. Fluorescence spectroscopy has been shown to be a valuable technique to characterize and monitor wastewater in surface waters for tracking sources of pollution, and in treatment works for process control and optimization. This paper reviews the current progress in applying fluorescence to assess wastewater quality. Studies have shown that, in general, wastewater presents higher fluorescence intensity compared to natural waters for the components associated with peak T (living and dead cellular material and their exudates) and peak C (microbially reprocessed organic matter). Furthermore, peak T fluorescence is significantly reduced after the biological treatment process and peak C is almost completely removed after the chlorination and reverse osmosis stages. Thus, simple fluorometers with appropriate wavelength selectivity, particularly for peaks T and C could be used for online monitoring in wastewater treatment works. This review also shows that care should be taken in any attempt to identify wastewater pollution sources due to potential overlapping fluorophores. Correlations between fluorescence intensity and water quality parameters such as biochemical oxygen demand (BOD) and total organic carbon (TOC) have been developed and dilution of samples, typically up to ×10, has been shown to be useful to limit inner filter effect. It has been concluded that the following research gaps need to be filled: lack of studies on the on-line application of fluorescence spectroscopy in wastewater treatment works and lack of data processing tools suitable for rapid correction and extraction of

  20. Interaction of europium and nickel with calcite studied by Rutherford Backscattering Spectrometry and Time-Resolved Laser Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Sabau, A.; Pipon, Y.; Toulhoat, N.; Lomenech, C.; Jordan, N.; Moncoffre, N.; Barkleit, A.; Marmier, N.; Brendler, V.; Surblé, S.; Giffaut, E.

    2014-08-01

    This study aims at elucidating the mechanisms regulating the interaction of Eu and Ni with calcite (CaCO3). Calcite powders or single crystals (some mm sized) were put into contact with Eu or Ni solutions at concentrations ranging from 10-3 to 10-5 mol L-1 for Eu and 10-3 mol L-1 for Ni. The sorption durations ranged from 1 week to 1 month. Rutherford Backscattering Spectrometry (RBS) well adapted to discriminate incorporation processes such as: (i) adsorption or co precipitation at the mineral surfaces or, (ii) incorporation into the mineral structure (through diffusion for instance), has been carried out. Moreover, using the fluorescence properties of europium, the results have been compared to those obtained by Time-Resolved Laser Fluorescence Spectroscopy (TRLFS) on calcite powders. For the single crystals, complementary SEM observations of the mineral surfaces at low voltage were also performed. Results showed that Ni accumulates at the calcite surface whereas Eu is also incorporated at a greater depth. Eu seems therefore to be incorporated into two different states in calcite: (i) heterogeneous surface accumulation and (ii) incorporation at depth greater than 160 nm after 1 month of sorption. Ni was found to accumulate at the surface of calcite without incorporation.

  1. Fluorescence Correlation Spectroscopy: The Case of Subdiffusion

    PubMed Central

    Lubelski, Ariel; Klafter, Joseph

    2009-01-01

    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. PMID:19289033

  2. Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy

    PubMed Central

    Lakowicz, Joseph R.; Ray, Krishanu; Chowdhury, Mustafa; Szmacinski, Henryk; Fu, Yi; Zhang, Jian; Nowaczyk, Kazimierz

    2009-01-01

    Fluorescence spectroscopy is widely used in biological research. Until recently, essentially all fluorescence experiments were performed using optical energy which has radiated to the far-field. By far-field we mean at least several wavelengths from the fluorophore, but propagating far-field radiation is usually detected at larger macroscopic distances from the sample. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. Near-field interactions are those occurring within a wavelength distance of an excited fluorophore. The spectral properties of fluorophores can be dramatically altered by near-field interactions with the electron clouds present in metals. These interactions modify the emission in ways not seen in classical fluorescence experiments. In this review we provide an intuitive description of the complex physics of plasmons and near-field interactions. Additionally, we summarize the recent work on metal–fluorophore interactions and suggest how these effects will result in new classes of experimental procedures, novel probes, bioassays and devices. PMID:18810279

  3. Position-Sensitive Scanning Fluorescence Correlation Spectroscopy

    PubMed Central

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

    2005-01-01

    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. PMID:15894645

  4. Ultrafast Nonlinear Spectroscopy of Red Fluorescent Proteins

    NASA Astrophysics Data System (ADS)

    Konold, Patrick Eugene

    Red-emitting homologues (RFPs) of the native Green Fluorescent Protein (GFP) with emission wavelengths beyond 650 nm are desirable probes for in vivo imaging experiments. They offer the potential for deeper tissue penetration and lower background scatter given a cleaner spectral window. However, bioimaging applications are hindered by poor photophysics ( e.g. low fluorescence quantum yield, high photobleaching), which limits experimental resolution and represents a significant obstacle towards utilization for low copy-number, long-duration imaging applications. In this thesis, a variety of femtosecond nonlinear electronic spectroscopies were employed jointly with site-directed mutagenesis to investigate the photophysical properties of RFPs. In one study, the molecular mechanism of red emission was pursued in two notable RFPs, mPlum and TagRFP675. Solvation dynamics observed with time-resolved transient grating spectroscopy were interpreted with the aid of molecular dynamics simulations to indicate that their red-emission is correlated with the ability of specific chromophore-sidechain hydrogen-bonding interactions to interconvert between direct and water-mediated states. In a second set of studies, two-dimensional double quantum coherence spectroscopy was used to probe the electronic transitions of mPlum. It was discovered that it displayed a response distinctly different from an organic dye in bulk solvent. Modeling indicate of these spectra indicate the spectral features may be attributed to the existence of multiple high-lying (n>1) excited states. The results provide new insight into the electronic structure of these widely used fluorescent probes.

  5. 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.

  6. APD detectors for biological fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Mazères, S.; Borrel, V.; Magenc, C.; Courrech, J. L.; Bazer-Bachi, R.

    2006-11-01

    Fluorescence spectroscopy is a very convenient and widely used method for studying the molecular background of biological processes [L. Salomé, J.L. Cazeil, A. Lopez, J.F. Tocanne, Eur. Biophys. J. 27 (1998) 391-402]. Chromophores are included in the structure under study and a flash of laser light induces fluorescence (Fluorescence Recovery After Photo-bleaching), the decay of which yields information on the polarity, the speed of rotation, and the speed of diffusion as well as on the temporal and spatial evolution of interactions between molecular species. The method can even be used to study living cells [J.F. Tocanne, L. Cézanne, A. Lopez, Prog. Lipid Res. 33 (1994) 203-237, L. Cezanne, A. Lopez, F. Loste, G. Parnaud, O. Saurel, P. Demange, J.F. Tocanne, Biochemistry 38 (1999) 2779-2786]. This is classically performed with a PM-based system. For biological reasons a decrease of the excitation of the cells is highly desirable. Because the fluorescence response then becomes fainter a significant improvement in detector capability would be welcome. We present here results obtained with an Avalanche Photo Diode (APD)-based system. The small sensitive area of detection allows a very significant improvement in signal/noise ratio, improvement in gain, and the opening-up of a new parameter space. With these new detectors we can begin the study of information transmission between cells through morphine receptors. This work involves both electronics engineers and biophysicists, so results and techniques in both fields will be presented here.

  7. Fluorescence Spectroscopy Investigations of Cutaneous Tissues

    NASA Astrophysics Data System (ADS)

    Borisova, E.; Bliznakova, I.; Momchilov, N.; Troyanova, P.; Avramov, L.

    2007-04-01

    Fluorescence Spectroscopy of the human skin is very prominent for early diagnosis and differentiation of cutaneous diseases. Selection of proper excitation sources and sensitive detectors gives wide range of possibilities related to real-time determination of existing pathological conditions. A problem with using laser as an excitation source is the high expenses associated with the operation of these types of installations. This is why we test capability of a cheaper excitation sources - ultraviolet and blue light-emitting diodes. Initially, we investigate the spectral response of normal skin from different anatomic areas, as well as from different phototypes volunteers. Our first results obtained demonstrated promising possibility to implement an inexpensive system for detection of cutaneous lesions with wide clinical applications. The results achieved will be introduced in development of diagnostic algorithms for improvement of diagnostic sensitivity of benign and malignant tumor lesions determination.

  8. Glucose Recognition in Vitro Using Fluorescent Spectroscopy

    SciTech Connect

    Noronha, G; Heiss, A M; Reilly, J R; Vachon, Jr, D J; Cary, D R; Zaitseva, N P; Reibold, R A; Lane, S M; Peyser, T A; Satcher, J H

    2001-04-25

    Diabetes is a disease that affects over 16 million people in the USA at a cost of 100 billion dollars annually. The ability to regulate insulin delivery in people with Type 1 diabetes is imperative as is the need to manage glucose levels in all people with this disease. Our current method for monitoring glucose is a (FDA approved) minimally invasive enzymatic sensor that can measure glucose levels in vivo for three days. We are focused on developing a noninvasive implantable glucose sensor that will be interrogated by an external device. The material must be robust, easy to process, biocompatible and resistant to biofouling. In this Presentation we will discuss the development of a new polymeric matrix that can recognize physiological levels of glucose in vitro using fluorescent spectroscopy.

  9. Fluorescence Spectroscopy of Neoplastic and Non-Neoplastic Tissues

    PubMed Central

    Ramanujam, Nirmala

    2000-01-01

    Abstract Fast and non-invasive, diagnostic techniques based on fluorescence spectroscopy have the potential to link the biochemical and morphologic properties of tissues to individual patient care. One of the most widely explored applications of fluorescence spectroscopy is the detection of endoscopically invisible, early neoplastic growth in epithelial tissue sites. Currently, there are no effective diagnostic techniques for these early tissue transformations. If fluorescence spectroscopy can be applied successfully as a diagnostic technique in this clinical context, it may increase the potential for curative treatment, and thus, reduce complications and health care costs. Steady-state, fluorescence measurements from small tissue regions as well as relatively large tissue fields have been performed. To a much lesser extent, time-resolved, fluorescence measurements have also been explored for tissue characterization. Furthermore, sources of both intrinsic (endogenous fluorophores) and extrinsic fluorescence (exogenous fluorophores) have been considered. The goal of the current report is to provide a comprehensive review on steady-state and time-resolved, fluorescence measurements of neoplastic and non-neoplastic, biologic systems of varying degrees of complexity. First, the principles and methodology of fluorescence spectroscopy are discussed. Next, the endogenous fluorescence properties of cells, frozen tissue sections and excised and intact bulk tissues are presented; fluorescence measurements from both animal and human tissue models are discussed. This is concluded with future perspectives. PMID:10933071

  10. Multiphoton excitation fluorescence correlation spectroscopy of fluorescent DNA base analogs

    NASA Astrophysics Data System (ADS)

    Katilius, Evaldas; Woodbury, Neal W.

    2004-06-01

    Two- and three-photon excitation was used to investigate the properties of two fluorescent DNA base analogs: 2-aminopurine and 6-methylisoxanthopterin. 2-aminopurine is a widely used fluorescent analog of the DNA base adenine. Three-photon excitation of 2-aminopurine is achievable by using intense femtosecond laser pulses in 850-950 nm spectral region. Interestingly, the three-photon excitation spectrum is blue-shifted relative to the three-times-wavelength single-photon excitation spectrum. The maximum of the absorbance band in the UV is at 305 nm, while the three-photon excitation spectrum has a maximum at around 880 nm. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2-aminopurine for DNA-protein interaction studies. However, due to relatively small three-photon absorption cross-section, a good signal-to-noise fluorescence correlation curves take very long time to obtain. Fluorescence properties of 6-methylisoxanthopterin, the fluorescent analog of guanine, were investigated using two-photon excitation. This molecule has the lowest energy absorption band centered around 350 nm, thus, two-photon excitation is attainable using 700 to 760 nm output of Ti-sapphire laser. The excitation spectrum of this molecule in the infrared well matches the doubled-wavelength single-photon excitation spectrum in the UV. The high fluorescence quantum yield of 6-methylisoxanthopterin allows efficient fluorescence correlation measurements and makes this molecule a very good candidate for using in in vitro DNA-protein interaction studies.

  11. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  12. Native fluorescence spectroscopy of thymus and fat tissues

    NASA Astrophysics Data System (ADS)

    Tang, Gui C.; Oz, Mehmet C.; Reid, V.; Steinglass, K.; Ginsberg, Mark D.; Jacobowitz, Larry; Alfano, Robert R.

    1993-08-01

    Fluorescence spectroscopy of the human thymus gland and surrounding mediastinal fat were measured to evaluate this approach in distinguishing between thymus and fat tissues during therapeutic surgery for myasthenia gravis disease.

  13. Quantum dots fluorescence quantum yield measured by Thermal Lens Spectroscopy.

    PubMed

    Estupiñán-López, Carlos; Dominguez, Christian Tolentino; Cabral Filho, Paulo E; Fontes, Adriana; de Araujo, Renato E

    2014-01-01

    An essential parameter to evaluate the light emission properties of fluorophores is the fluorescence quantum yield, which quantify the conversion efficiency of absorbed photons to emitted photons. We detail here an alternative nonfluorescent method to determine the absolute fluorescence quantum yield of quantum dots (QDs). The method is based in the so-called Thermal Lens Spectroscopy (TLS) technique, which consists on the evaluation of refractive index gradient thermally induced in the fluorescent material by the absorption of light. Aqueous dispersion carboxyl-coated cadmium telluride (CdTe) QDs samples were used to demonstrate the Thermal Lens Spectroscopy technical procedure. PMID:25103802

  14. Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy.

    PubMed

    Boens, Noël; Qin, Wenwu; Basarić, Nikola; Hofkens, Johan; Ameloot, Marcel; Pouget, Jacques; Lefèvre, Jean-Pierre; Valeur, Bernard; Gratton, Enrico; vandeVen, Martin; Silva, Norberto D; Engelborghs, Yves; Willaert, Katrien; Sillen, Alain; Rumbles, Garry; Phillips, David; Visser, Antonie J W G; van Hoek, Arie; Lakowicz, Joseph R; Malak, Henryk; Gryczynski, Ignacy; Szabo, Arthur G; Krajcarski, Don T; Tamai, Naoto; Miura, Atsushi

    2007-03-01

    A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-l-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 degrees C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and the solvent, the excitation wavelengths used range from 284 to 575 nm, the emission from 330 to 630 nm. These lifetime standards may be used to either calibrate or test the resolution of time- and frequency-domain instrumentation or as reference compounds to eliminate the color effect in photomultiplier tubes. Statistical analyses by means of two-sample charts indicate that there is no laboratory bias in the lifetime determinations. Moreover, statistical tests show that there is an excellent correlation between the lifetimes estimated by the time-domain and frequency-domain fluorometries. Comprehensive tables compiling the results for 20 (fluorescence lifetime standard/solvent) combinations are given. PMID:17269654

  15. Absorption and fluorescence spectroscopy on a smartphone

    NASA Astrophysics Data System (ADS)

    Hossain, Md. Arafat; Canning, John; Cook, Kevin; Ast, Sandra; Rutledge, Peter J.; Jamalipour, Abbas

    2015-07-01

    A self-powered smartphone-based field-portable "dual" spectrometer has been developed for both absorption and fluorescence measurements. The smartphone's existing flash LED has sufficient optical irradiance to undertake absorption measurements within a 3D-printed case containing a low cost nano-imprinted polymer diffraction grating. A UV (λex ~ 370 nm) and VIS (λex ~ 450 nm) LED are wired into the circuit of the flash LED to provide an excitation source for fluorescence measurements. Using a customized app on the smartphone, measurements of absorption and fluorescence spectra are demonstrated using pH-sensitive and Zn2+-responsive probes. Detection over a 300 nm span with 0.42 nm/pixel spectral resolution is demonstrated. Despite the low cost and small size of the portable spectrometer, the results compare well with bench top instruments.

  16. Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs.

    PubMed

    Visser, A J W G; Laptenok, S P; Visser, N V; van Hoek, A; Birch, D J S; Brochon, J-C; Borst, J W

    2010-01-01

    Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET-FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive. PMID:19693494

  17. Laser-induced fluorescence spectroscopy in tissue local necrosis detection

    NASA Astrophysics Data System (ADS)

    Cip, Ondrej; Buchta, Zdenek; Lesundak, Adam; Randula, Antonin; Mikel, Bretislav; Lazar, Josef; Veverkova, Lenka

    2014-03-01

    The recent effort leads to reliable imaging techniques which can help to a surgeon during operations. The fluorescence spectroscopy was selected as very useful online in vivo imaging method to organics and biological materials analysis. The presented work scopes to a laser induced fluorescence spectroscopy technique to detect tissue local necrosis in small intestine surgery. In first experiments, we tested tissue auto-fluorescence technique but a signal-to-noise ratio didn't express significant results. Then we applied a contrast dye - IndoCyanine Green (ICG) which absorbs and emits wavelengths in the near IR. We arranged the pilot experimental setup based on highly coherent extended cavity diode laser (ECDL) used for stimulating of some critical areas of the small intestine tissue with injected ICG dye. We demonstrated the distribution of the ICG exciter with the first file of shots of small intestine tissue of a rabbit that was captured by high sensitivity fluorescent cam.

  18. Fluorescence correlation spectroscopy: inception, biophysical experimentations, and prospectus.

    PubMed

    Webb, W W

    2001-08-20

    Fluorescence correlation spectroscopy examines the chemical and the photophysical dynamics of dilute molecular solutions by measurement of the dynamic optical fluctuations of the fluorescence of a few molecules, even averaging less than one molecule at a time, in open focal volumes that are usually less than a femtoliter (<10(-18) m(3)). It applies the same principles of statistical thermodynamics as does quasi-elastic light scattering. Molecular interactions, conformational changes, chemical reactions, and photophysical dynamics that are not ordinarily detectable by quasi-elastic light scattering can be analyzed by fluorescence correlation spectroscopy in cases in which molecular fluorescence changes in the dynamic range 10(-7)-10(2) s. PMID:18360431

  19. [Study on interaction of caffeine with myoglobin by fluorescence spectroscopy].

    PubMed

    Huang, He-Yong; Gu, Xiao-Tian; Ding, Yan; Zhou, Jia-Hong; Feng, Yu-Ying

    2009-10-01

    The interaction of caffein and myoglobin was investigated by fluorescence spectroscopy and synchronous fluorescence spectroscopy. The intrinsic fluorescence of myoglobin was significantly quenched by caffein under the physiological condition (pH 7.4). The results indicated that caffeine was capable of binding with myoglobin to form a 1:1 complex and the quenching mechanism of myoglobin affected by caffeine was shown to be a static quenching procedure by calculating quenching constant, binding sites and binding constant. According to the thermodynamic parameters, the main binding force of the interaction is electrostatic force and hydrophobic force. The change in the micro-circumstance of aminos of myoglobin was analyzed by synchronous fluorescence spectrometry. The result indicated that caffeine can change the conformation of the protein, leading to the change in the micro-environment of tryptophane and tyrosine residues from hydrophobic environment to hydrophilic environment to different extent. PMID:20038063

  20. Chromophore detection by fluorescence spectroscopy in tissue-like phantoms

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert E.; Fantini, Sergio; Maier, John S.; Mantulin, William W.; Gratton, Enrico

    1997-08-01

    In the near-infrared spectral region (700 - 900 nm) light penetrates a few centimeters into tissues and hemoglobin dominates the absorption. Consequently, in vivo near-infrared tissue absorption spectroscopy becomes difficult for endogenous compounds of biological interest other than hemoglobin. Exogenous chromophore detection by fluorescence spectroscopy has the potential to provide enhanced sensitivity and specificity for in vivo optical tissue spectroscopy, facilitating the study of many important metabolites in tissues other than hemoglobin. We have performed measurements of the dc fluorescence intensity generated by a fluorophore (rhodamine B) homogeneously dissolved inside a highly scattering tissue-simulating phantom (aqueous suspension of titanium-dioxide particles). The phantom was prepared with optical coefficients (absorption and reduced scattering) similar to those of tissue in the near-infrared; these coefficients were measured with a frequency-domain spectrometer. Measurable dc fluorescence intensity signals from 1 nM rhodamine concentrations inside the phantom are reported. Furthermore, we were able to resolve changes in rhodamine concentration on the order of 1% using the dc fluorescence intensity. This dc fluorescence sensitivity is characterized experimentally at two concentrations (55 and 360 nM) and over a range of source-detector separations. Other aspects of the sensitivity are discussed over a large range of concentrations using a fluorescence photon migration model.

  1. Pancreatic tissue assessment using fluorescence and reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    Chandra, Malavika; Heidt, David; Simeone, Diane; McKenna, Barbara; Scheiman, James; Mycek, Mary-Ann

    2007-07-01

    The ability of multi-modal optical spectroscopy to detect signals from pancreatic tissue was demonstrated by studying human pancreatic cancer xenografts in mice and freshly excised human pancreatic tumor tissue. Measured optical spectra and fluorescence decays were correlated with tissue morphological and biochemical properties. The measured spectral features and decay times correlated well with expected pathological differences in normal, pancreatitis and adenocarcinoma tissue states. The observed differences between the fluorescence and reflectance properties of normal, pancreatitis and adenocarcinoma tissue indicate a possible application of multi-modal optical spectroscopy to differentiating between the three tissue classifications.

  2. Combined fiber probe for fluorescence lifetime and Raman spectroscopy

    PubMed Central

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Marple, Eric; Urmey, Kirk; Wachsmann-Hogiu, Sebastian; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-01-01

    In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. PMID:26093843

  3. Intramolecular Fluorescence Correlation Spectroscopy in a Feedback Tracking Microscope

    NASA Astrophysics Data System (ADS)

    McHale, Kevin; Mabuchi, Hideo

    2010-07-01

    We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system, and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity and long time-scale decays in comparison to traditional fluorescence correlation spectroscopy. We demonstrate our approach by using a three-dimensional tracking microscope to study genomic $\\lambda$-phage DNA molecules with various fluorescence label configurations.

  4. The development of attenuation compensation models of fluorescence spectroscopy signals

    NASA Astrophysics Data System (ADS)

    Dremin, Victor V.; Zherebtsov, Evgeny A.; Rafailov, Ilya E.; Vinokurov, Andrey Y.; Novikova, Irina N.; Zherebtsova, Angelina I.; Litvinova, Karina S.; Dunaev, Andrey V.

    2016-04-01

    This study examines the effect of blood absorption on the endogenous fluorescence signal intensity of biological tissues. Experimental studies were conducted to identify these effects. To register the fluorescence intensity, the fluorescence spectroscopy method was employed. The intensity of the blood flow was measured by laser Doppler flowmetry. We proposed one possible implementation of the Monte Carlo method for the theoretical analysis of the effect of blood on the fluorescence signals. The simulation is constructed as a four-layer skin optical model based on the known optical parameters of the skin with different levels of blood supply. With the help of the simulation, we demonstrate how the level of blood supply can affect the appearance of the fluorescence spectra. In addition, to describe the properties of biological tissue, which may affect the fluorescence spectra, we turned to the method of diffuse reflectance spectroscopy (DRS). Using the spectral data provided by the DRS, the tissue attenuation effect can be extracted and used to correct the fluorescence spectra.

  5. Tracking-FCS: Fluorescence correlation spectroscopy of individual particles

    NASA Astrophysics Data System (ADS)

    Berglund, Andrew J.; Mabuchi, Hideo

    2005-10-01

    We exploit recent advances in single-particle tracking to perform fluorescence correlation spectroscopy on individual fluorescent particles, in contrast to traditional methods that build up statistics over a sequence of many measurements. By rapidly scanning the focus of an excitation laser in a circular pattern, demodulating the measured fluorescence, and feeding these results back to a piezoelectric translation stage, we track the Brownian motion of fluorescent polymer microspheres in aqueous solution in the plane transverse to the laser axis. We discuss the estimation of particle diffusion statistics from closed-loop position measurements, and we present a generalized theory of fluorescence correlation spectroscopy for the case that the motion of a single fluorescent particle is actively tracked by a time-dependent laser intensity. We model the motion of a tracked particle using Ornstein-Uhlenbeck statistics, using a general theory that contains a umber of existing results as specific cases. We find good agreement between our theory and experimental results, and discuss possible future applications of these techniques to passive, single-shot, single-molecule fluorescence measurements with many orders of magnitude in time resolution.

  6. "FluSpec": A Simulated Experiment in Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Bigger, Stephen W.; Bigger, Andrew S.; Ghiggino, Kenneth P.

    2014-01-01

    The "FluSpec" educational software package is a fully contained tutorial on the technique of fluorescence spectroscopy as well as a simulator on which experiments can be performed. The procedure for each of the experiments is also contained within the package along with example analyses of results that are obtained using the software.

  7. Fibre optic fluorescence spectroscopy for monitoring fish freshness

    NASA Astrophysics Data System (ADS)

    Wu, Chi-Wu; Hsiao, Tzu-Chien; Chu, Shou-Chia; Hu, Hung-Hsi; Chen, Jyh-Cheng

    2012-01-01

    In this study, a portable Y-type fibreoptic fluorescence spectroscopy measurement system was used to evaluate the freshness of eight cobias (Rachycentron canadum). The results showed that the ratio of fluorescent intensity, which F480 nm/Fexci+50 nm was belong with the range of collagen type I and type V characteristic spectra, was positive correlated to the frozen time by hours. It was a strong approach to be a potential index for differentiating the fish freshness during delivery process. Besides, the different pattern results of dorsum and abdomen were shown in this study. In further, fibreoptic fluorescence spectroscopy could be a way not only to measure and quantify the freshness of different fish body but also to verify the level of taste.

  8. Solid-phase fluorescence spectroscopy to characterize organic wastes.

    PubMed

    Muller, Mathieu; Milori, Débora Marcondes Bastos Pereira; Déléris, Stéphane; Steyer, Jean-Philippe; Dudal, Yves

    2011-01-01

    The production of solid organic waste (SOW) such as sewage sludge (SS) or municipal solid waste (MSW) has been continuously increasing in Europe since the beginning of the 1990'. Today, the European Union encourages the stabilization of these wastes using biologic processes such as anaerobic digestion and/or composting to produce bio-energy and organic fertilizers. However, the design and management of such biologic processes require knowledge about the quantity and quality of the organic matter (OM) contained in the SOW. The current methods to characterize SOW are tedious, time-consuming and often insufficiently informative. In this paper, we assess the potential of solid-phase fluorescence (SPF) spectroscopy to quickly provide a relevant characterization of SOW. First, we tested well known model compounds (tryptophan, bovine serum albumin, lignin and humic acid) and biologic matrix (Escherichia coli) in three dimensional solid-phase fluorescence (3D-SPF) spectroscopy. We recorded fluorescence spectra from proteinaceous samples but we could not record the fluorescence emitted by lignin and humic acid powders. For SOW samples, fluorescence spectra were successfully recorded for MSW and most of its sub-components (foods, cardboard) but impossible for SS, sludge compost (SC) and ligno-cellulosic wastes. Based on visual observations and additional assays, we concluded that the presence of highly light-absorptive chemical structures in such dark-colored samples was responsible for this limitation. For such samples, i.e. lignin, humic acid, SS, SC and ligno-cellulosic wastes, we show that laser induced fluorescence (LIF) spectroscopy enables the acquisition of 2D fluorescence spectra. PMID:21696938

  9. Cryogenic Laser Induced U(VI) Fluorescence Studies of a U(VI) Substituted Natural Calcite: Implications to U(VI) Speciation in Contaminated Hanford Sediments

    SciTech Connect

    Wang, Zheming; Zachara, John M.; McKinley, James P.; Smith, Steven C.

    2005-04-14

    Time-resolved laser-induced fluorescence spectroscopy (TRLFS) and imaging spectromicroscopy (TRLFISM) were used to examine the chemical speciation of uranyl in contaminated subsurface sediments from the Hanford Site, Washington. Spectroscopic measurements for contaminant U(VI) were compared to those from a natural, uranyl-bearing calcite (NUC) that had been found via X-ray absorption spectroscopy (XAS) to include uranyl in the same coordination environment as calcium (1). Spectral deconvolution of TRLFS measurements on the NUC revealed the unexpected presence of two distinct chemical environments consistent with published spectra of U(VI)-substituted synthetic calcite and aragonite. Apparently, some U(VI) substitution sites in calcite distorted to exhibit a local, more energetically favorable aragonite structure. TRLFS measurements of the Hanford sediments were similar to the NUC in terms of peak positions and intensity, despite a small CaCO3 content (<0.1 to 3.2 mass%). Spectral deconvolution of the sediment measurements also revealed the presence of U(VI) in calcite and aragonite structural environments. TRLFISM measurements at multiple locations in the different sediments displayed only minor variation indicating a uniform speciation pattern. Collectively, the measurements implied that waste U(VI), long-resident beneath the sampled disposal pond (32 y), had co-precipitated within newly formed carbonates. These results have major implications for the solubility and fate of the contaminated U(VI).

  10. Fluorescence spectroscopy to assess apoptosis in myocardium

    NASA Astrophysics Data System (ADS)

    Ranji, Mahsa; Matsubara, Muneaki; Grosso, Michael A.; Jaggard, Dwight L.; Chance, Britton; Gorman, Robert C.; Gorman, Joseph H., III

    2007-02-01

    Apoptosis induced mitochondrial destruction and dysfunction has been shown to play an important role in the pathogenesis of both acute cardiac ischemia-reperfusion injury and chronic myocardial infarction-induced ventricular remodeling. Unfortunately this understanding has not translated into effective therapeutic strategies for either condition-mostly due to an inability to assess mitochondrial dysfunction/apoptosis effectively in humans. All current measures of apoptosis are pseudo-quantitative and require invasive tissue biopsy. Our group has developed an optical, non-tissue destructive catheter based device that allows the quantitative regional assessment of this pathological process in vivo. This instrument has been designed to acquire fluorescence signals of intrinsic mitochondrial fluorophores, Nicotinamide Adenine Dinucleotide (NAD) and Flavoprotein (FP). The normalized ratio of these fluorophores (FP/FP+NADH) called the redox ratio, is an indicator of the in vivo mitochondrial dysfunction. 1-3 We have demonstrated in a rabbit reperfusion model of apoptotic myocyte injury that this redox ratio is drastically increased which is consistent with profound apoptosis-induced "unhinging" of the mitochondrial respiratory function.

  11. Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook

    PubMed Central

    Kapusta, Peter; Macháň, Radek; Benda, Aleš; Hof, Martin

    2012-01-01

    Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector after pulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined. PMID:23202928

  12. Fluorescence correlation spectroscopy: Diagnostics for sparse molecules

    PubMed Central

    Maiti, Sudipta; Haupts, Ulrich; Webb, Watt W.

    1997-01-01

    The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions. PMID:9342306

  13. Fluorescence suppression using micro-scale spatially offset Raman spectroscopy.

    PubMed

    Conti, Claudia; Botteon, Alessandra; Colombo, Chiara; Realini, Marco; Matousek, Pavel

    2016-09-21

    We present a new concept of fluorescence suppression in Raman microscopy based on micro-spatially offset Raman spectroscopy which is applicable to thin stratified turbid (diffusely scattering) matrices permitting the retrieval of the Raman signals of sublayers below intensely fluorescing turbid over-layers. The method is demonstrated to yield good quality Raman spectra with dramatically suppressed fluorescence backgrounds enabling the retrieval of Raman sublayer signals even in situations where conventional Raman microscopy spectra are fully overwhelmed by intense fluorescence. The concept performance was studied theoretically using Monte Carlo simulations indicating the potential of up to an order or two of magnitude suppression of overlayer fluorescence backgrounds relative to the Raman sublayer signals. The technique applicability was conceptually demonstrated on layered samples involving paints, polymers and stones yielding fluorescence suppression factors between 12 to above 430. The technique has potential applications in a number of analytical areas including cultural heritage, archaeology, polymers, food, pharmaceutical, biological, biomedical, forensics and catalytic sciences and quality control in manufacture. PMID:27338230

  14. Fluorescence spectroscopy of excitation transfer in Photosystem 1

    SciTech Connect

    Mukerji, I.

    1990-12-01

    This thesis centers on the study of excitation transfer in a photosynthetic antenna array. The spectroscopic properties of two pigment-protein complexes were investigated. These complexes, isolated from higher plants, display an unusual temperature dependent fluorescence behavior. The author have chosen to study this fluorescence behavior with respect to energy transfer to the reaction center and in an isolated intact antenna preparation. A Photosystem 1 complex, PSI-200, was isolated from spinach. We have characterized this system by both steady state and time-resolved fluorescence spectroscopy. Fluorescence polarization measurements indicate that this emission arises from pigments which absorb in the long wavelength region of the spectrum and comprise a relatively small portion of the antenna population. Comparison of spectral characteristics were made with a PSI complex isolated from the thermophilic cyanobacterium, Synechococcus, sp. To address the role of Chl b in stimulating long wavelength fluorescence and the temperature dependence of the system, we have studied the energy transfer dynamics in an antenna complex, LHC-I isolated from PSI-200. Kinetic measurements indicate that initially absorbed excitation is rapidly redistributed to longer wavelength emitting pigments within 40 ps. The temperature dependence of F685 results from increased back transfer from long wavelength emitters to F685. We suggest that changes in excitation transfer between the various emitting species and a non-radiative fluorescence quenching mechanism account for the temperature dependence of the system. 144 refs., 50 figs., 3 tabs.

  15. Investigation on the interaction of cefpirome sulfate with lysozyme by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy.

    PubMed

    Han, Rong; Liu, Baosheng; Li, Gaixia; Zhang, Qiuju

    2016-03-01

    The reaction mechanism of cefpirome sulfate with lysozyme at different temperatures (298, 310 and 318 K) was investigated using fluorescence quenching and synchronous fluorescence spectroscopy under simulated physiological conditions. The results clearly demonstrated that cefpirome sulfate caused strong quenching of the fluorescence of lysozyme by a static quenching mechanism. The binding constants obtained using the above methods were of the same order of magnitude and very similar. Static electric forces played a key role in the interaction between cefpirome sulfate and lysozyme, and the number of binding sites in the interaction was close to 1. The values of Hill's coefficients were > 1, indicating that drugs or proteins showed a very weakly positive cooperativity in the system. In addition, the conclusions obtained from the two methods using the same equation were consistent. The results indicated that synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the fluorescence quenching method. In addition, the effect of cefpirome sulfate on the secondary structure of lysozyme was analyzed using circular dichroism spectroscopy. PMID:26304690

  16. Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy

    PubMed Central

    Blom, Hans; Hassler, Kai; Chmyrov, Andriy; Widengren, Jerker

    2010-01-01

    Electrostatic interactions between dielectric surfaces and different fluorophores used in ultrasensitive fluorescence microscopy are investigated using objective-based Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacial dynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein, zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengths at physiological pH. As analyzed by means of the amplitude and time-evolution of the autocorrelation function, the fluorescent molecules experience electrostatic attraction or repulsion at the glass surface depending on their charges. Influences of the electrostatic interactions are also monitored through the triplet-state population and triplet relaxation time, including the amount of detected fluorescence or the count-rate-per-molecule parameter. These TIR-FCS results provide an increased understanding of how fluorophores are influenced by the microenvironment of a glass surface, and show a promising approach for characterizing electrostatic interactions at interfaces. PMID:20386645

  17. Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy.

    PubMed

    Winterflood, Christian M; Seeger, Stefan

    2016-05-01

    We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time. PMID:27001472

  18. Fluorescence spectroscopy using indocyanine green for lymph node mapping

    NASA Astrophysics Data System (ADS)

    Haj-Hosseini, Neda; Behm, Pascal; Shabo, Ivan; Wârdell, Karin

    2014-02-01

    The principles of cancer treatment has for years been radical resection of the primary tumor. In the oncologic surgeries where the affected cancer site is close to the lymphatic system, it is as important to detect the draining lymph nodes for metastasis (lymph node mapping). As a replacement for conventional radioactive labeling, indocyanine green (ICG) has shown successful results in lymph node mapping; however, most of the ICG fluorescence detection techniques developed are based on camera imaging. In this work, fluorescence spectroscopy using a fiber-optical probe was evaluated on a tissue-like ICG phantom with ICG concentrations of 6-64 μM and on breast tissue from five patients. Fiber-optical based spectroscopy was able to detect ICG fluorescence at low intensities; therefore, it is expected to increase the detection threshold of the conventional imaging systems when used intraoperatively. The probe allows spectral characterization of the fluorescence and navigation in the tissue as opposed to camera imaging which is limited to the view on the surface of the tissue.

  19. In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Mütze, Jörg; Ohrt, Thomas; Petrášek, Zdeněk; Schwille, Petra

    In this manuscript, we describe the application of Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and scanning FCS (sFCS) to two in vivo systems. In the first part, we describe the application of two-photon standard and scanning FCS in Caenorhabditis elegans embryos. The differentiation of a single fertilized egg into a complex organism in C. elegans is regulated by a number of protein-dependent processes. The oocyte divides asymmetrically into two daughter cells of different developmental fate. Two of the involved proteins, PAR-2 and NMY-2, are studied. The second investigated system is the mechanism of RNA interference in human cells. An EGFP based cell line that allows to study the dynamics and localization of the RNA-induced silencing complex (RISC) with FCS in vivo is created, which has so far been inaccessible with other experimental methods. Furthermore, Fluorescence Cross-Correlation Spectroscopy is employed to highlight the asymmetric incorporation of labeled siRNAs into RISC.

  20. Principles and applications of fluorescence lifetime correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Beranová, Lenka; Humpolícková, Jana; Hof, Martin

    2009-05-01

    Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photon counting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new technique with several experimental benefits. In FLCS experiments, pulsed excitation is used and data are stored in a special time-tagged time-resolved mode. Mathematical treatment of TCSPC decay patterns of distinct fluorophores and their mixture enables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties and concentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed and information on interaction of the species can be obtained. This technique is particularly helpful for distinguishing different states of the same fluorophore in different microenvironments. The first application of that concept represents the simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dye is considerably quenched in the layer near the light-absorbing surface. This concept was also used in other applications: a) investigation of a conformational change of a labeled protein, b) detection of small amounts of labeled oligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNA molecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.

  1. Stark Spectroscopy of Rubrene. II. Stark Fluorescence Spectroscopy and Fluorescence Quenching Induced by an External Electric Field.

    PubMed

    Iimori, Toshifumi; Ito, Ryuichi; Ohta, Nobuhiro

    2016-07-21

    We report Stark fluorescence spectroscopy investigation of rubrene dispersed in a poly(methyl methacrylate) film. The features of the fluorescence spectrum are analogous to those in solutions. In the Stark fluorescence spectrum, the decrease of the fluorescence quantum yield in the presence of an external electric field is observed. This result shows that the yield of nonradiative decay processes is increased by the application of an external electric field. It is known that the fluorescence quantum yield for rubrene, which is nearly unity at room temperature, depends on temperature, and a major nonradiative decay process in photoexcited rubrene is ascribed to a thermally activated intersystem crossing (ISC). Equations that express the field-induced fluorescence quenching in terms of the molecular parameters are derived from the ensemble average of electric field effects on the activation energy of the reaction rate constant in random orientation systems. The molecular parameters are then extracted from the observed data. It is inferred that the field-induced increase in the yield of other intramolecular and intermolecular photophysical processes in addition to the ISC should be taken into account. PMID:27341859

  2. Synchronous fluorescence spectroscopy for analysis of wine and wine distillates

    NASA Astrophysics Data System (ADS)

    Andreeva, Ya.; Borisova, E.; Genova, Ts.; Zhelyazkova, Al.; Avramov, L.

    2015-01-01

    Wine and brandies are multicomponent systems and conventional fluorescence techniques, relying on recording of single emission or excitation spectra, are often insufficient. In such cases synchronous fluorescence spectra can be used for revealing the potential of the fluorescence techniques. The technique is based on simultaneously scanning of the excitation and emission wavelength with constant difference (Δλ) maintained between them. In this study the measurements were made using FluoroLog3 spectrofluorimeter (HORIBA Jobin Yvon, France) and collected for excitation and emission in the wavelength region 220 - 700 nm using wavelength interval Δλ from 10 to 100 nm in 10 nm steps. This research includes the results obtained for brandy and red wine samples. Fluorescence analysis takes advantage in the presence of natural fluorophores in wines and brandies, such as gallic, vanillic, p-coumaric, syringic, ferulic acid, umbelliferone, scopoletin and etc. Applying of synchronous fluorescence spectroscopy for analysis of these types of alcohols allows us to estimate the quality of wines and also to detect adulteration of brandies like adding of a caramel to wine distillates for imitating the quality of the original product aged in oak casks.

  3. 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.

  4. Fluorescence Spectroscopy of Gas-phase Polycyclic Aromatic Hydrocarbons

    NASA Technical Reports Server (NTRS)

    Thomas, J. D.; Witt, A. N.

    2006-01-01

    The purpose of this investigation was to produce fluorescence spectra of polycyclic aromatic hydrocarbon (PAH) molecules in the gas-phase for comparison with blue luminescence (BL) emission observed in astrophysical sources Vijh et al. (2004, 2005a,b). The BL occurs roughly from 350 to 450 nm, with a sharp peak near 380 nm. PAHs with three to four rings, e.g. anthracene and pyrene, were found to produce luminescence in the appropriate spectral region, based on existing studies. Relatively few studies of the gas-phase fluorescence of PAHs exist; those that do exist have dealt primarily with the same samples commonly available for purchase such as pyrene and anthracene. In an attempt to understand the chemistry of the nebular environment we also obtained several nitrogen substituted PAHs from our colleagues at NASA Ames. In order to simulate the astrophysical environment we also took spectra by heating the PAHs in a flame. The flame environment counteracts the formation of eximers and permits the spectroscopy of free-flying neutral molecules. Experiments with coal tar demonstrate that fluorescence spectroscopy reveals primarily the presence of the smallest molecules, which are most abundant and which possess the highest fluorescence efficiencies. One gas-phase PAH that seems to fit the BL spectrum most closely is phenanthridine. In view of the results from the spectroscopy of coal tar, a compound containing a mixture of PAHs ranging from small to very large PAH molecules, we can not preclude the presence of larger PAHs in interstellar sources exhibiting BL.

  5. 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.

  6. The complexation of Cm(III) with oxalate in aqueous solution at T = 20-90 °C: a combined TRLFS and quantum chemical study.

    PubMed

    Skerencak-Frech, Andrej; Maiwald, Martin; Trumm, Michael; Froehlich, Daniel R; Panak, Petra J

    2015-02-16

    The complexation of Cm(III) with oxalate is studied in aqueous solution as a function of the ligand concentration, the ionic strength (NaCl), and the temperature (T = 20–90 °C) by time-resolved laser fluorescence spectroscopy (TRLFS) and quantum chemical calculations. Four complex species ([Cm(Ox)n](3–2n), n = 1, 2, 3, 4) are identified, and their molar fractions are determined by peak deconvolution of the emission spectra. The conditional log K′n(T) values of the first three complexes are calculated and extrapolated to zero ionic strength with the specific ion interaction theory approach. The [Cm(Ox)4](5–) complex forms only at high temperatures. Thus, the log K4(0)(T) value was determined at T > 60 °C. The log K1(0)(25 °C) = 6.86 ± 0.02 decreases by 0.1 logarithmic units in the studied temperature range. The log K2(0)(25 °C) = 4.68 ± 0.09 increases by 0.35, and log K3(0)(25 °C) = 2.11 ± 0.05 increases by 0.37 orders of magnitude. The log Kn(0)(T) (n = 1, 2, 3) values are linearly correlated with the reciprocal temperature. Thus, their temperature dependencies are fitted with the linear Van’t Hoff equation yielding the standard reaction enthalpy (ΔrHm(0)) and standard reaction entropy (ΔrSm(0)) of the stepwise formation of the [Cm(Ox)n](3–2n) species (n = 1, 2, 3). Furthermore, the binary ion–ion interaction coefficients of the four Cm(III) oxalate species with Cl(–)/Na(+) are determined. The binding energies, bond lengths, and bond angles of the different Cm(III) oxalate complexes are calculated in the gas phase as well as in a box containing 1000 H2O molecules by ab inito calculations and molecular dynamics simulations, respectively. PMID:25646935

  7. Two-dimensional fluorescence spectroscopy of laser-produced plasmas.

    PubMed

    Harilal, S S; LaHaye, N L; Phillips, M C

    2016-08-01

    We use a two-dimensional laser-induced fluorescence spectroscopy technique to measure the coupled absorption and emission properties of atomic species in plasmas produced via laser ablation of a solid aluminum target at atmospheric pressure. Emission spectra from the Al I 394.4 nm and Al I 396.15 nm transitions are measured while a frequency-doubled, continuous wave (cw) Ti:sapphire laser is tuned across the Al I 396.15 nm transition. The resulting two-dimensional spectra show the energy coupling between the two transitions via increased emission intensity for both transitions during resonant absorption of the cw laser at one transition. Time-delayed, gated detection of the emission spectrum is used to isolate resonantly excited fluorescence emission from thermally excited emission from the plasma. In addition, the tunable cw laser measures the absorption spectrum of the Al transition with ultrahigh resolution after the plasma has cooled, resulting in narrower spectral linewidths than observed in emission spectra. Our results highlight that fluorescence spectroscopy employing cw laser re-excitation after pulsed laser ablation combines benefits of both traditional emission and absorption spectroscopic methods. PMID:27472615

  8. Coded spectroscopy for ethanol detection in diffuse, fluorescent media

    NASA Astrophysics Data System (ADS)

    McCain, Scott Thomas

    Optical sensing in the visible and near-infrared regions of the electromagnetic spectrum has many useful applications. One particularly interesting one is the non-invasive analysis of tissue since a high penetration depth is possible. With the use of Raman spectroscopy, a high degree of chemical specificity is available with laser powers that are harmless to living tissue. Such systems, however, are plagued by the low efficiency of the Raman scattering process by molecules and the intense background fluorescence from some biological materials. To address these drawbacks, we have investigated the use of coded spectroscopy to make Raman spectroscopy more feasible in routine use. By coding the input aperture of a dispersive spectrometer, throughput gains of 10-100 are possible over a traditional slit spectrometer. The theory, design, and performance characteristics of this static aperture coding will be discussed in this thesis. In addition, by coding the excitation light sources one can filter out the shifting Raman signals from the stationary fluorescent background. The theory and implementation of an expectation maximization algorithm for Raman signal reconstruction will be analyzed. In addition, the design of a multi-excitation, coded-aperture Raman spectrometer will be described, which uses both of the coding mechanisms described.

  9. The study of blue LED to induce fluorescence spectroscopy and fluorescence imaging for oral carcinoma detection

    NASA Astrophysics Data System (ADS)

    Zheng, Longjiang; Hu, Yuanting

    2009-07-01

    Fluorescence spectroscopy and fluorescence imaging diagnosis of malignant lesions provides us with a new method to diagnose diseases in precancerous stage. Early diagnosis of disease has significant importance in cancer treatment, because most cancers can be cured well in precancerous, especially when the diffusion of cancer is limited in a restricted region. In this study, Golden hamster models were applied to 5% 9, 10 dimethyl-1, 2-benzanthracene (DMBA) to induce hamster buccal cheek pouch carcinoma three times a week. Rose Bengal, which has been used in clinican for years and avoids visible side-effect to human was chosen as photosensitizer. 405 nm blue LED was used to induce the fluorescence of photosensitizer. After topical application of photosensitizer, characteristic red emission fluorescence peak was observed around 600nm. Similar, normal oral cavity has special luminescence around 480nm. Fluorescence spectroscopy technology is based on analysing emission peaks of photosensitizer in the areas of oral carcinoma, moreover, red-to-green (IR/IG) intensity ratio is also applied as a diagnostic algorithm. A CCD which is connected with a computer is used to take pictures at carcinoma areas through different filters. Fluorescence images from normal hamster buccal cheek pouch are compared with those from carcinogen-induced models of carcinoma, and morphological differences between normal and lesion tissue can be distinguished. The pictures are analyzed by Matlab and shown on the screen of computer. This paper demonstrates that Rose Bengal could be used as photosensitizer to detect oral carcinoma, and blue LED as excitation source could not only have a good effect to diagnose oral carcinoma, but also decrease cost greatly.

  10. Investigation of asphaltene association by front-face fluorescence spectroscopy.

    PubMed

    Albuquerque, Flávio Cortiñas; Nicodem, David E; Rajagopal, Krishnaswamy

    2003-07-01

    The tendency of asphaltenes to aggregate and form clusters in solvents was studied by fluorescence spectroscopy. This was done by evaluating the relative fluorescence quantum yield of asphaltenes diluted at several concentrations in toluene and by studying the changes in the fluorescence spectra of asphaltene solutions as the composition of the solvent, toluene and cyclohexane, is changed. The asphaltene fraction (heptane insoluble) was collected from a Brazilian heavy crude oil, and solutions of this material varying from 0.016 g/L up to 10 g/L were prepared in toluene. Front-face emission spectra were obtained in two wavelength ranges, from 310 to 710 nm, excited at 300 nm (short range), and from 410 to 710 nm, excited at 400 nm (long range). Severe quenching was observed at concentrations above about 0.1 g/L. Stern-Volmer plots (reciprocal of quantum yield against concentration) exhibited nonlinear, downward-curved behavior, indicating that a more complex suppression mechanism, probably influenced by the association of the asphaltene molecules, is taking place. The same asphaltenes were dissolved (0.1 g/L) in binary mixtures of toluene and cyclohexane, and emission spectra in both the short range and long range were obtained. Fluorescence was progressively quenched at longer wavelengths of the spectra as the proportion of cyclohexane in the solvent grew. Cyclohexane, a poor asphaltene solvent, is probably inducing static quenching through association of asphaltenes. PMID:14658659

  11. Single-molecule fluorescence spectroscopy in (bio)catalysis

    PubMed Central

    Roeffaers, Maarten B. J.; De Cremer, Gert; Uji-i, Hiroshi; Muls, Benîot; Sels, Bert F.; Jacobs, Pierre A.; De Schryver, Frans C.; De Vos, Dirk E.; Hofkens, Johan

    2007-01-01

    The ever-improving time and space resolution and molecular detection sensitivity of fluorescence microscopy offer unique opportunities to deepen our insights into the function of chemical and biological catalysts. Because single-molecule microscopy allows for counting the turnover events one by one, one can map the distribution of the catalytic activities of different sites in solid heterogeneous catalysts, or one can study time-dependent activity fluctuations of individual sites in enzymes or chemical catalysts. By experimentally monitoring individuals rather than populations, the origin of complex behavior, e.g., in kinetics or in deactivation processes, can be successfully elucidated. Recent progress of temporal and spatial resolution in single-molecule fluorescence microscopy is discussed in light of its impact on catalytic assays. Key concepts are illustrated regarding the use of fluorescent reporters in catalytic reactions. Future challenges comprising the integration of other techniques, such as diffraction, scanning probe, or vibrational methods in single-molecule fluorescence spectroscopy are suggested. PMID:17664433

  12. Cytoskeleton dynamics studied by dispersion-relation fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Wang, Ru; Lei, Lei; Wang, Yingxiao; Levine, Alex; Popescu, Gabriel

    2013-03-01

    Fluorescence is the most widely used microscopy technique for studying the dynamics and function in both medical and biological sciences due to its sensitivity and specificity. Inspired by the spirit of spatial fluorescence correlation spectroscopy, we propose a new method to study the transport dynamics over a broad range of spatial and temporal scales. The molecules of interest are labeled with a fluorophore whose motion gives rise to spontaneous fluorescence intensity fluctuations that can be further analyzed to quantify the governing molecular mass transport dynamics. We analyze these data by the dispersion relation in the form of a power law, Γ(q) ~qα , which describe the relaxation rate of fluorescence intensity fluctuations, Γ, vs. the wavenumber, q. We used this approach to study the interplay of various cytoskeletal components in intracellular transport under the influence of protein-motor inhibitors. We found that after actin is depolymerized, the transport becomes completely random for a few minutes and then it starts to organize deterministically again. We conclude that the disrupted cytoskeletal components first diffuse in the cytoplasm, but then become attached to microtubules and get transported deterministically.

  13. New approach on fluorescence spectroscopy for caries detection

    NASA Astrophysics Data System (ADS)

    Hibst, Raimund; Paulus, Robert

    1999-05-01

    Today the diagnosis of caries is based mainly on examinations by visual inspection, dental probe or by x- rays. All methods are very limited when either initial or undermining caries have to be found. For initial caries promising results have been demonstrated by fluorescence spectroscopy with excitation wavelengths in the (ultra-)violet to green spectral region, especially 406 nm or 488 nm. In our investigations, we extended the considered excitation wavelength range into red. As expected, total fluorescence yield is decreasing with increasing wavelength, but this decrease is much more pronounced for sound compared to carious enamel or dentin. For 640 nm or 655 nm excitation for example, integral (λ>680nm) fluorescence intensity of cares can exceed that of healthy tissue by about one order of magnitude. This allows to detect caries by fluorescence intensity rather than by spectral analysis. On the basis of these results we have built up a system using a diode laser as light source, and a photo diode combined with a long pass filter as detector. It provides quantitatively reproducible measurements and detection even through sound enamel of 1 mm thickness. Clinical applications include detection of undermining caries and monitoring of the decay process.

  14. Fluorescence spectroscopy for endogenous porphyrins in human facial skin

    NASA Astrophysics Data System (ADS)

    Seo, I.; Tseng, S. H.; Cula, G. O.; Bargo, P. R.; Kollias, N.

    2009-02-01

    The activity of certain bacteria in skin is known to correlate to the presence of porphyrins. In particular the presence of coproporphyrin produced by P.acnes inside plugged pores has been correlated to acne vulgaris. Another porphyrin encountered in skin is protoporphyrin IX, which is produced by the body in the pathway for production of heme. In the present work, a fluorescence spectroscopy system was developed to measure the characteristic spectrum and quantify the two types of porphyrins commonly present in human facial skin. The system is comprised of a Xe lamp both for fluorescence excitation and broadband light source for diffuse reflectance measurements. A computer-controlled filter wheel enables acquisition of sequential spectra, first excited by blue light at 405 nm then followed by the broadband light source, at the same location. The diffuse reflectance spectrum was used to correct the fluorescence spectrum due to the presence of skin chromophores, such as blood and melanin. The resulting fluorescence spectra were employed for the quantification of porphyrin concentration in a population of healthy subjects. The results show great variability on the concentration of these porphyrins and further studies are being conducted to correlate them with skin conditions such as inflammation and acne vulgaris.

  15. An analog filter approach to frequency domain fluorescence spectroscopy

    DOE PAGESBeta

    Trainham, Clifford P.; O'Neill, Mary D.; McKenna, Ian J.

    2015-10-01

    The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modeled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as Spicea can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modeling of the entiremore » system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. Furthermore, the techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response.« less

  16. An Analog Filter Approach to Frequency Domain Fluorescence Spectroscopy.

    PubMed

    Trainham, R; O'Neill, M; McKenna, I J

    2015-11-01

    The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modelled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as SPICE can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modelling of the entire system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. The techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response. The simplification of the analysis mathematics, and the ability to model the entire detection chain, make it possible to develop more compact instruments for remote sensing applications. PMID:26429345

  17. An analog filter approach to frequency domain fluorescence spectroscopy

    SciTech Connect

    Trainham, Clifford P.; O'Neill, Mary D.; McKenna, Ian J.

    2015-10-01

    The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modeled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as Spicea can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modeling of the entire system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. Furthermore, the techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response.

  18. 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

  19. Quantum process tomography by 2D fluorescence spectroscopy

    SciTech Connect

    Pachón, Leonardo A.; Marcus, Andrew H.; Aspuru-Guzik, Alán

    2015-06-07

    Reconstruction of the dynamics (quantum process tomography) of the single-exciton manifold in energy transfer systems is proposed here on the basis of two-dimensional fluorescence spectroscopy (2D-FS) with phase-modulation. The quantum-process-tomography protocol introduced here benefits from, e.g., the sensitivity enhancement ascribed to 2D-FS. Although the isotropically averaged spectroscopic signals depend on the quantum yield parameter Γ of the doubly excited-exciton manifold, it is shown that the reconstruction of the dynamics is insensitive to this parameter. Applications to foundational and applied problems, as well as further extensions, are discussed.

  20. Toward quantitative "in vivo biochemistry" with fluorescence fluctuation spectroscopy.

    PubMed

    Slaughter, Brian D; Li, Rong

    2010-12-01

    Quantitative description of protein dynamics and interactions in vivo with temporal and spatial resolution is a key step in dissecting molecular mechanisms in cell biology. Fluorescence fluctuation spectroscopy (FFS) has recently emerged as a powerful in vivo tool for assessing molecular concentration and movement and formation of hetero- and homo-oligomeric complexes. This article discusses point FFS-based analysis methods that have proven useful to cell biologists, focusing on the kinds of information they provide, their pros and cons, and the basic instrumentation required. Along the way, we describe briefly a few recent examples where these analyses have helped address important biological questions. PMID:21160072

  1. Stochastic fractal behavior in concentration fluctuation and fluorescence correlation spectroscopy

    PubMed Central

    Raymond, Gary M.; Bassingthwaighte, James B.

    2010-01-01

    Fluctuations in the concentration of Brownian particles in one and two dimensions, or any reasonable measurement of the concentration such as in fluorescence correlation spectroscopy, is shown to be a stochastic fractal with a long tail. Being singular at ω = 0, the power spectrum of the fluctuation S(ω) ~ ω −1/2 for diffusion in one dimension, ~ log ω in two dimensions, but non-singular in three dimensions. This discovery provides one simple physical mechanism for possible long-memory fractal behavior, and its implications to various biological processes are discussed. PMID:10457592

  2. Fluorescence-excitation and Emission Spectroscopy on Single FMO Complexes

    PubMed Central

    Löhner, Alexander; Ashraf , Khuram; Cogdell, Richard J.; Köhler, Jürgen

    2016-01-01

    In green-sulfur bacteria sunlight is absorbed by antenna structures termed chlorosomes, and transferred to the RC via the Fenna-Matthews-Olson (FMO) complex. FMO consists of three monomers arranged in C3 symmetry where each monomer accommodates eight Bacteriochlorophyll a (BChl a) molecules. It was the first pigment-protein complex for which the structure has been determined with high resolution and since then this complex has been the subject of numerous studies both experimentally and theoretically. Here we report about fluorescence-excitation spectroscopy as well as emission spectroscopy from individual FMO complexes at low temperatures. The individual FMO complexes are subjected to very fast spectral fluctuations smearing out any possible different information from the ensemble data that were recorded under the same experimental conditions. In other words, on the time scales that are experimentally accessible by single-molecule techniques, the FMO complex exhibits ergodic behaviour. PMID:27545197

  3. Detectors for single-molecule fluorescence imaging and spectroscopy

    PubMed Central

    MICHALET, X.; SIEGMUND, O.H.W.; VALLERGA, J.V.; JELINSKY, P.; MILLAUD, J.E.; WEISS, S.

    2010-01-01

    Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy. PMID:20157633

  4. Fluorescence-excitation and Emission Spectroscopy on Single FMO Complexes.

    PubMed

    Löhner, Alexander; Ashraf, Khuram; Cogdell, Richard J; Köhler, Jürgen

    2016-01-01

    In green-sulfur bacteria sunlight is absorbed by antenna structures termed chlorosomes, and transferred to the RC via the Fenna-Matthews-Olson (FMO) complex. FMO consists of three monomers arranged in C3 symmetry where each monomer accommodates eight Bacteriochlorophyll a (BChl a) molecules. It was the first pigment-protein complex for which the structure has been determined with high resolution and since then this complex has been the subject of numerous studies both experimentally and theoretically. Here we report about fluorescence-excitation spectroscopy as well as emission spectroscopy from individual FMO complexes at low temperatures. The individual FMO complexes are subjected to very fast spectral fluctuations smearing out any possible different information from the ensemble data that were recorded under the same experimental conditions. In other words, on the time scales that are experimentally accessible by single-molecule techniques, the FMO complex exhibits ergodic behaviour. PMID:27545197

  5. Identification of active fluorescence stained bacteria by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Krause, Mario; Beyer, Beatrice; Pietsch, Christian; Radt, Benno; Harz, Michaela; Rösch, Petra; Popp, Jürgen

    2008-04-01

    Microorganisms can be found everywhere e.g. in food both as useful ingredients or harmful contaminations causing food spoilage. Therefore, a fast and easy to handle analysis method is needed to detect bacteria in different kinds of samples like meat, juice or air to decide if the sample is contaminated by harmful microorganisms. Conventional identification methods in microbiology require always cultivation and therefore are time consuming. In this contribution we present an analysis approach to identify fluorescence stained bacteria on strain level by means of Raman spectroscopy. The stained bacteria are highlighted and can be localized easier against a complex sample environment e.g. in food. The use of Raman spectroscopy in combination with chemometrical methods allows the identification of single bacteria within minutes.

  6. Identification of Atherosclerotic Plaques in Carotid Artery by Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Rocha, Rick; Villaverde, Antonio Balbin; Silveira, Landulfo; Costa, Maricília Silva; Alves, Leandro Procópio; Pasqualucci, Carlos Augusto; Brugnera, Aldo

    2008-04-01

    The aim of this work was to identify the presence of atherosclerotic plaques in carotid artery using the Fluorescence Spectroscopy. The most important pathogeny in the cardiovascular disorders is the atherosclerosis, which may affect even younger individuals. With approximately 1.2 million heart attacks and 750,000 strokes afflicting an aging American population each year, cardiovascular disease remains the number one cause of death. Carotid artery samples were obtained from the Autopsy Service at the University of São Paulo (São Paulo, SP, Brazil) taken from cadavers. After a histopathological analysis the 60 carotid artery samples were divided into two groups: normal (26) and atherosclerotic plaques (34). Samples were irradiated with the wavelength of 488 nm from an Argon laser. A 600 μm core optical fiber, coupled to the Argon laser, was used for excitation of the sample, whereas another 600 optical fiber, coupled to the spectrograph entrance slit, was used for collecting the fluorescence from the sample. Measurements were taken at different points on each sample and then averaged. Fluorescence spectra showed a single broad line centered at 549 nm. The fluorescence intensity for each sample was calculated by subtracting the intensity at the peak (550 nm) and at the bottom (510 nm) and then data were statistically analyzed, looking for differences between both groups of samples. ANOVA statistical test showed a significant difference (p<0,05) between both types of tissues, with regard to the fluorescence peak intensities. Our results indicate that this technique could be used to detect the presence of the atherosclerotic in carotid tissue.

  7. Classification of plum spirit drinks by synchronous fluorescence spectroscopy.

    PubMed

    Sádecká, J; Jakubíková, M; Májek, P; Kleinová, A

    2016-04-01

    Synchronous fluorescence spectroscopy was used in combination with principal component analysis (PCA) and linear discriminant analysis (LDA) for the differentiation of plum spirits according to their geographical origin. A total of 14 Czech, 12 Hungarian and 18 Slovak plum spirit samples were used. The samples were divided in two categories: colorless (22 samples) and colored (22 samples). Synchronous fluorescence spectra (SFS) obtained at a wavelength difference of 60 nm provided the best results. Considering the PCA-LDA applied to the SFS of all samples, Czech, Hungarian and Slovak colorless samples were properly classified in both the calibration and prediction sets. 100% of correct classification was also obtained for Czech and Hungarian colored samples. However, one group of Slovak colored samples was classified as belonging to the Hungarian group in the calibration set. Thus, the total correct classifications obtained were 94% and 100% for the calibration and prediction steps, respectively. The results were compared with those obtained using near-infrared (NIR) spectroscopy. Applying PCA-LDA to NIR spectra (5500-6000 cm(-1)), the total correct classifications were 91% and 92% for the calibration and prediction steps, respectively, which were slightly lower than those obtained using SFS. PMID:26593555

  8. Laser-induced fluorescence and dispersed fluorescence spectroscopy of jet-cooled 1-phenylpropargyl radical

    NASA Astrophysics Data System (ADS)

    Reilly, Neil J.; Nakajima, Masakazu; Gibson, Bligh A.; Schmidt, Timothy W.; Kable, Scott H.

    2009-04-01

    The D1(A2″)-D0(A2″) electronic transition of the resonance-stabilized 1-phenylpropargyl radicalooled discharge of 3-phenyl-1-propyne, has been investigated in detail by laser-induced fluorescence excitation and dispersed single vibronic level fluorescence (SVLF) spectroscopy. The transition is dominated by the origin band at 21 007 cm-1, with weaker Franck-Condon activity observed in a' fundamentals and even overtones and combinations of a″ symmetry. Ab initio and density functional theory calculations of the D0 and D1 geometries and frequencies were performed to support and guide the experimental assignments throughout. Analysis of SVLF spectra from 16 D1 vibronic levels has led to the assignment of 15 fundamental frequencies in the excited state and 19 fundamental frequencies in the ground state; assignments for many more normal modes not probed directly by fluorescence spectroscopy are also suggested. Duschinsky mixing, in which the excited state normal modes are rotated with respect to the ground state modes, is prevalent throughout, in vibrations of both a' and a″ symmetry.

  9. Fluorescence spectroscopy and chemometric modeling for bioprocess monitoring.

    PubMed

    Faassen, Saskia M; Hitzmann, Bernd

    2015-01-01

    On-line sensors for the detection of crucial process parameters are desirable for the monitoring, control and automation of processes in the biotechnology, food and pharma industry. Fluorescence spectroscopy as a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states. During a cultivation process significant changes occur in the fluorescence spectra. By means of chemometric modeling, prediction models can be calculated and applied for process supervision and control to provide increased quality and the productivity of bioprocesses. A range of applications for different microorganisms and analytes has been proposed during the last years. This contribution provides an overview of different analysis methods for the measured fluorescence spectra and the model-building chemometric methods used for various microbial cultivations. Most of these processes are observed using the BioView® Sensor, thanks to its robustness and insensitivity to adverse process conditions. Beyond that, the PLS-method is the most frequently used chemometric method for the calculation of process models and prediction of process variables. PMID:25942644

  10. Single-Molecule Fluorescence Spectroscopy using Phospholipid Bilayer Nanodiscs

    PubMed Central

    Nath, Abhinav; Trexler, Adam J.; Koo, Peter; Miranker, Andrew D.; Atkins, William M.; Rhoades, Elizabeth

    2012-01-01

    Nanodiscs are a new class of model membranes that are being used to solubilize and study a range of integral membrane proteins and membrane-associated proteins. Unlike other model membranes, the Nanodisc bilayer is bounded by a scaffold protein coat that confers enhanced stability and a narrow particle size distribution. The bilayer diameter can be precisely controlled by changing the diameter of the protein coat. All these properties make Nanodiscs excellent model membranes for single molecule fluorescence applications. In this chapter, we describe our work using Nanodiscs to apply total internal reflection fluorescence microscopy (TIRFM), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) to study the integral membrane protein cytochrome P450 3A4 and the membrane-binding proteins islet amyloid popypeptide (IAPP) and α-synuclein, respectively. The monodisperse size distribution of Nanodiscs enhances control over the oligomeric state of the membrane protein of interest, and also facilitates accurate solution-based measurements. Nanodiscs also comprise an excellent system to stably immobilize integral membrane proteins in a bilayer without covalent modification, enabling a range of surface-based experiments where accurate localization of the protein of interest is required. PMID:20580961

  11. 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.

  12. Preparation, fluorescence spectroscopy, and AFM analysis of erbium oxide nanocolloid

    NASA Astrophysics Data System (ADS)

    Patel, Darayas; Vance, Calvin; King, Newton; Jessup, Malcolm; Sarkisov, Sergey

    2009-02-01

    Nanocolloids of compounds containing fluorescent rare earth ions have recently attracted significant attention as agents for biolabeling, bioimaging, bio- and chemical sensing, and other applications. Erbium oxide nanocolloids have been prepared for the first time in water and gammabutyrolactone. Optical dynamic scatterometry and atomic force microscopy determined an average size (average mean height) of erbium oxide nanoparticles to be 10-11 nm. Prominent optical absorption peaks of the nanocolloids at 442.5 nm, 450.0 nm, 487.2 nm (strong), 492.0 nm, 523.0 nm (strong), 541.6 nm, 548.6 nm, 652.6 nm, and 665.7 nm (strong) can be attributed to erbium ions hosted within nanoparticles. Laser fluorescence spectroscopy of the nanocolloids was conducted using excitations with the lines of argon-ion laser (514 nm, 488 nm, 476 nm, and 458 nm) and 980-nm semiconductor laser. Strong green emission at 571 nm is more likely from transition between 4S3/2 and 4I15/2 levels and relatively weak red emissions from transition between 4I9/2 and 4I15/2 level of erbium was observed at excitation with visible laser radiation 488 nm and 476 nm. The reported nanocolloids thus showed to be good candidates for fluorescent biosensing applications and also as a new lasing filling medium in fiber lasers.

  13. Fluorescence Spectroscopy and Chemometric Modeling for Bioprocess Monitoring

    PubMed Central

    Faassen, Saskia M.; Hitzmann, Bernd

    2015-01-01

    On-line sensors for the detection of crucial process parameters are desirable for the monitoring, control and automation of processes in the biotechnology, food and pharma industry. Fluorescence spectroscopy as a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states. During a cultivation process significant changes occur in the fluorescence spectra. By means of chemometric modeling, prediction models can be calculated and applied for process supervision and control to provide increased quality and the productivity of bioprocesses. A range of applications for different microorganisms and analytes has been proposed during the last years. This contribution provides an overview of different analysis methods for the measured fluorescence spectra and the model-building chemometric methods used for various microbial cultivations. Most of these processes are observed using the BioView® Sensor, thanks to its robustness and insensitivity to adverse process conditions. Beyond that, the PLS-method is the most frequently used chemometric method for the calculation of process models and prediction of process variables. PMID:25942644

  14. Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

    PubMed Central

    Guan, Yinghua; Meurer, Matthias; Raghavan, Sarada; Rebane, Aleksander; Lindquist, Jake R.; Santos, Sofia; Kats, Ilia; Davidson, Michael W.; Mazitschek, Ralph; Hughes, Thomas E.; Drobizhev, Mikhail; Knop, Michael; Shah, Jagesh V.

    2015-01-01

    We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells. PMID:25877871

  15. Studies in atomic-fluorescence spectroscopy-V The fluorescence characteristics and determination of antimony.

    PubMed

    Dagnall, R M; Thompson, K C; West, T S

    1967-10-01

    Atomic-fluorescence of antimony may be generated in an air-propane flame by nebulizing aqueous solutions of antimony salts whilst irradiating the flame by means of a microwave-excited electrode-less discharge tube operating at 30 W. The strongest fluorescence is exhibited by the (4)S(11 2 ) --> (4)P(1 3 ) 2311 A resonance line and weaker signals are observed at the 2068 and 2176 A resonance lines and at four intercombination lines, at 2598, 2671, 2770 and 2878 A. A process of thermally assisted direct-line fluorescence is postulated to account for the otherwise inexplicable intensity of the 2598 A line emission. Atomic-fluorescence spectroscopy at 2176 A permits the determination of antimony in the range 0.1-120 ppm with a detection limit of 0.05 ppm. With the same equipment and source, the range of measurement for atomic-absorption was 6-120 ppm and the detection limit was 1 ppm. No interferences were observed from 100-fold molar amounts of Cd, Co, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, NH(4), Pb and Zn or from arsenate, chloride, nitrate, phosphate and sulphate. PMID:18960212

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

    PubMed

    Ishii, Kunihiko; Tahara, Tahei

    2013-10-01

    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. PMID:23977902

  17. Sorption of Cm(III) and Eu(III) onto clay minerals under saline conditions: Batch adsorption, laser-fluorescence spectroscopy and modeling

    NASA Astrophysics Data System (ADS)

    Schnurr, Andreas; Marsac, Rémi; Rabung, Thomas; Lützenkirchen, Johannes; Geckeis, Horst

    2015-02-01

    The present work reports experimental data for trivalent metal cation (Cm/Eu) sorption onto illite (Illite du Puy) and montmorillonite (Na-SWy-2) in NaCl solutions up to 4.37 molal (m) in the absence of carbonate. Batch sorption experiments were carried out for a given ionic strength at fixed metal concentration (mEu = 2 × 10-7 m, labeled with 152Eu for γ-counting) and at a constant solid to liquid ratio (S:L = 2 g/L) for 3 < pHm < 12 (pHm = -log mH+). The amount of clay sorbed Eu approaches almost 100% (with log KD > 5) for pHm > 8, irrespective of the NaCl concentration. Variations in Eu uptake are minor at elevated NaCl concentrations. Time-resolved laser fluorescence spectroscopy (TRLFS) studies on Cm sorption covering a wide range of NaCl concentrations reveal nearly identical fluorescence emission spectra after peak deconvolution, i.e. no significant variation of Cm surface speciation with salinity. Beyond the three surface complexes already found in previous studies an additional inner-sphere surface species with a fluorescence peak maximum at higher wavelength (λ ∼ 610 nm) could be resolved. This new surface species appears in the high pH range and is assumed to correspond to a clay/curium/silicate complex as already postulated in the literature for kaolinite. The 2 site protolysis non-electrostatic surface complexation and cation exchange sorption model (2SPNE SC/CE) was applied to describe Eu sorption data by involving the Pitzer and SIT (specific ion interaction) formalism in the calculation of the activities of dissolved aqueous species. Good agreement of model and experiment is achieved for sorption data at pHm < 6 without the need of adjusting surface complexation constants. For pHm > 6 in case of illite and pHm > 8 in case of montmorillonite calculated sorption data systematically fall below experimental data with increasing ionic strength. Under those conditions sorption is almost quantitative and deviations must be discussed considering

  18. Fluorescence spectroscopy: considerations for highly absorbing dissolved organic matter samples

    NASA Astrophysics Data System (ADS)

    Simone, B. E.; Miller, M.; McKnight, D. M.

    2009-12-01

    Fluorescence spectroscopy is a robust method for characterizing organic matter (OM). However, proper collection and correction of spectra are necessary to provide useful data. One important correction is the inner-filter correction, which primarily accounts for the inner-filter effect by adjusting for the wavelength dependent attenuation of emitted light by the solution prior to detection by the fluorometer. The most commonly used correction is based on an assumption that light is emitted at the center of the pathlength. Thus, the inner-filter effect is more pronounced in highly absorbing samples, and has the potential to skew the fluorescence spectra. For this study, the terrestrially derived Suwannee River fulvic acid (SRFA) and microbially derived Pony Lake fulvic acid (PLFA), from the International Humic Substances Society (IHSS), were diluted to incremental absorbances at a wavelength of 254 nm from 0.05 to 1.0 at pH 4 and 7. Three dimensional fluorescence spectra were measured and modeled with the Cory and McKnight (2005) parallel factor analysis (PARAFAC) model which resolves the fluorescence spectra into 13 components, including quinone-like and protein-like components. In the absence of inner-filter effects, plots of absorbance vs. loadings should be linear. Using the data from absorbance of 0.05 to 0.3, where the inner-filter affect is least pronounced, a linear regression was created and used as a baseline to predict component loadings at higher absorbance values in the absence of inner-filter effects. Results indicate that at absorbance values greater than 0.3, the commonly-used inner-filter correction is not able to remove the inner-filter effect. Therefore, in order to obtain reliable component loadings and correctly interpret the spectra, samples should be diluted to absorbance values less than 0.3 at 254 nm prior to collection of three dimensional fluorescence scans. The recommendation of a maximum absorbance of 0.3 agrees with the results of a

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

    NASA Astrophysics Data System (ADS)

    Smith, Elizabeth Myhra

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

  20. Fluorescence and UV-vis Spectroscopy of Synovial Fluids

    NASA Astrophysics Data System (ADS)

    Pinti, Marie J.; Stojilovic, Nenad; Kovacik, Mark W.

    2009-10-01

    Total joint arthroplasty involves replacing the worn cartilaginous surfaces of the joint with man-made materials that are designed to be biocompatible and to withstand mechanical stresses. Commonly these bearing materials consist of metallic alloys (TiAlV or CoCrMo) and UHMWPE. Following joint arthroplasty, the normal generation of micro-metallic wear debris particles that dislodge from the prosthesis has been shown to cause inflammatory aseptic osteolysis (bone loss) that ultimately results in the failure of the implant. Here we report our results on the novel use of Fluorescence and UV-vis spectroscopy to investigate the metallic content of synovial fluid specimens taken from postoperative total knee arthroplasties. Preliminary finding showed presence of alumina and chromium is some specimens. The ability to detect and monitor the wear rate of these implants could have far reaching implications in the prevention of metallic wear-debris induced osteolysis and impending implant failure.

  1. Shedding light on azopolymer brush dynamics by fluorescence correlation spectroscopy.

    PubMed

    Kollarigowda, R H; De Santo, I; Rianna, C; Fedele, C; Manikas, A C; Cavalli, S; Netti, P A

    2016-09-14

    Understanding the response to illumination at a molecular level as well as characterising polymer brush dynamics are key features that guide the engineering of new light-stimuli responsive materials. Here, we report on the use of a confocal microscopy technique that was exploited to discern how a single molecular event such as the photoinduced isomerisation of azobenzene can affect an entire polymeric material at a macroscopic level leading to photodriven mass-migration. For this reason, a set of polymer brushes, containing azobenzene (Disperse Red 1, DR) on the side chains of poly(methacrylic acid), was synthesised and the influence of DR on the polymer brush dynamics was investigated for the first time by Fluorescence Correlation Spectroscopy (FCS). Briefly, two dynamics were observed, a short one coming from the isomerisation of DR and a long one related to the brush main chain. Interestingly, photoinduced polymer aggregation in the confocal volume was observed. PMID:27491890

  2. Fluorescence spectroscopy, exciton dynamics, and photochemistry of single allophycocyanin trimers

    SciTech Connect

    Ying, L.; Sie, X.S.

    1998-12-10

    The authors report a study of the allophycocyanin trimer (APC), a light-harvesting protein complex from cyanobacteria, by room-temperature single-molecule measurements of fluorescence spectra, lifetimes, intensity trajectories, and polarization modulation. Emission spectra of individual APC trimers are found to be homogeneous on the time scale of seconds. In contrast, their emission lifetimes are found to be widely distributed because of generation of long-lived exciton traps during the course of measurements. The intensity trajectories and polarization modulation experiments indicate reversible exciton trap formation within the three quasi-independent pairs of strong interacting {alpha}84 and {beta}84 chromophores in APC, as well as photobleaching of individual chromophores. Comparison experiments under continuous-wave and pulsed excitation reveal a two-photon mechanism for generating exciton traps and/or photobleaching, which involves exciton-exciton annihilation. These single-molecule experiments provide new insights into the spectroscopy, exciton dynamics, and photochemistry of light-harvesting complexes.

  3. Fluorescence correlation spectroscopy evidence for structural heterogeneity in ionic liquids

    SciTech Connect

    Guo, J C; Baker, G. A.; Hillesheim, P. C.; Dai, S.; Shaw, R. W.; Mahurin, S., M.

    2011-01-01

    In this work, we provide new experimental evidence for chain length-dependent self-aggregation in room temperature ionic liquids (RTILs) using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [C{sub n}MPy][Tf{sub 2}N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decreased with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chain [C{sub n}MPy][Tf{sub 2}N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [CnMPy]{sup +}.

  4. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    SciTech Connect

    Laurence, Ted A.; Ly, Sonny; Bourguet, Feliza; Fischer, Nicholas O.; Coleman, Matthew A.

    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 the 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.

  5. Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy.

    PubMed

    Park, Hweon; Han, Sung-Sik; Sako, Yasushi; Pack, Chan-Gi

    2015-03-01

    Organization and functions of the nucleolus is maintained by mobilities and interactions of nucleolar factors. Because the nucleolus is a densely packed structure, molecular crowding effects determined by the molecular concentrations and mobilities in the nucleolus should also be important for regulating nucleolar organization and functions. However, such molecular property of nucleolar organization is not fully understood. To understand the biophysical property of nucleolar organization, the diffusional behaviors of inert green fluorescent protein (GFP) oligomers with or without nuclear localization signals (NLSs) were analyzed under various conditions by fluorescence correlation spectroscopy. Our result demonstrates that the mobility of GFPs inside the nucleolus and the nucleoplasm can be represented by single free diffusion under normal conditions, even though the mobility in the nucleolus is considerably slower than that in the chromatin region. Moreover, the free diffusion of GFPs is found to be significantly size- and NLS-dependent only in the nucleolus. Interestingly, the mobility in the nucleolus is highly sensitive to ATP depletion, as well as actinomycin D (ActD) treatment. In contrast, the ultra-structure of the nucleolus was not significantly changed by ATP depletion but was changed by ActD treatment. These results suggest that the nucleolus behaves similarly to an open aqueous-phase medium with an increased molecular crowding effect that depends on both energy and transcription. PMID:25404711

  6. Diagnosis of meningioma by time-resolved fluorescence spectroscopy.

    PubMed

    Butte, Pramod V; Pikul, Brian K; Hever, Aviv; Yong, William H; Black, Keith L; Marcu, Laura

    2005-01-01

    We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors. PMID:16409091

  7. Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy.

    PubMed

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

    2007-12-01

    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 approximately 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

  8. Continuous-wave laser fluorescence spectroscopy of impurities in tokamaks

    SciTech Connect

    Young, C.E.; Pellin, M.J.; Gruen, D.M.; Norem, J.H.

    1982-07-01

    Laser-induced fluorescence spectroscopy has been applied as an in-situ diagnostic for impurity atoms in the edge region of the plasma in the Argonne Plasma Engineering Experiment (APEX) tokamak. Zirconium atoms introduced from a moveable probe were excited by a cw single-mode ring dye laser and monitored on lines of the a/sup 3/F-z/sup 3/F/sup 0/ manifold. The fluorescence signal from a 0.03 cm/sup 3/ volume was recorded at 1-ms intervals with a computer-controlled 4-channel 100-MHz scaler system. Acousto-optic modulation of the laser beam at 100 kHz allowed subtraction of plasma background light. Absolute calibration by Rayleigh scattering gave a detectability limit approx.10/sup 10/ Zr atoms/cm/sup 3/ in this apparatus. The detectability limit was determined by a detailed consideration of power and transit time broadening. The effects of several experimental parameters were examined and suggestions for increasing detection sensitivity are presented. Doppler-shift experiments indicated a thermal-velocity distribution for the detected Zr atoms. Intrinsic-velocity resolution of the experiments, calculated from effective excitation linewidths, was approx.25 m/s.

  9. Continuous-wave laser fluorescence spectroscopy of impurities in tokamaks

    NASA Astrophysics Data System (ADS)

    Young, C. E.; Pellin, M. J.; Gruen, D. M.; Norem, J. H.

    1982-07-01

    Laser-induced fluorescence spectroscopy has been applied as an in-situ diagnostic for impurity atoms in the edge region of the plasma in the Argonne Plasma Engineering Experiment (APEX) tokamak. Zirconium atoms introduced from a moveable probe were excited by a cw single-mode ring dye laser and monitored on lines of the a3F-z3F0 manifold. The fluorescence signal from a 0.03 cm3 volume was recorded at 1-ms intervals with a computer-controlled 4-channel 100-MHz scaler system. Acousto-optic modulation of the laser beam at 100 kHz allowed subtraction of plasma background light. Absolute calibration by Rayleigh scattering gave a detectability limit ˜1010 Zr atoms/cm3 in this apparatus. The detectability limit was determined by a detailed consideration of power and transit time broadening. The effects of several experimental parameters were examined and suggestions for increasing detection sensitivity are presented. Doppler-shift experiments indicated a thermal-velocity distribution for the detected Zr atoms. Intrinsic-velocity resolution of the experiments, calculated from effective excitation linewidths, was ˜25 m/s.

  10. 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.

  11. Fluorescence correlation spectroscopy in biology, chemistry, and medicine.

    PubMed

    Perevoshchikova, I V; Kotova, E A; Antonenko, Y N

    2011-05-01

    This review describes the method of fluorescence correlation spectroscopy (FCS) and its applications. FCS is used for investigating processes associated with changes in the mobility of molecules and complexes and allows researchers to study aggregation of particles, binding of fluorescent molecules with supramolecular complexes, lipid vesicles, etc. The size of objects under study varies from a few angstroms for dye molecules to hundreds of nanometers for nanoparticles. The described applications of FCS comprise various fields from simple chemical systems of solution/micelle to sophisticated regulations on the level of living cells. Both the methodical bases and the theoretical principles of FCS are simple and available. The present review is concentrated preferentially on FCS applications for studies on artificial and natural membranes. At present, in contrast to the related approach of dynamic light scattering, FCS is poorly known in Russia, although it is widely employed in laboratories of other countries. The goal of this review is to promote the development of FCS in Russia so that this technique could occupy the position it deserves in modern Russian science. PMID:21639831

  12. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  13. Fluorescence correlation spectroscopy: Statistical analysis and biological applications

    NASA Astrophysics Data System (ADS)

    Saffarian, Saveez

    2002-01-01

    The experimental design and realization of an apparatus which can be used both for single molecule fluorescence detection and also fluorescence correlation and cross correlation spectroscopy is presented. A thorough statistical analysis of the fluorescence correlation functions including the analysis of bias and errors based on analytical derivations has been carried out. Using the methods developed here, the mechanism of binding and cleavage site recognition of matrix metalloproteinases (MMP) for their substrates has been studied. We demonstrate that two of the MMP family members, Collagenase (MMP-1) and Gelatinase A (MMP-2) exhibit diffusion along their substrates, the importance of this diffusion process and its biological implications are discussed. We show through truncation mutants that the hemopexin domain of the MMP-2 plays and important role in the substrate diffusion of this enzyme. Single molecule diffusion of the collagenase MMP-1 has been observed on collagen fibrils and shown to be biased. The discovered biased diffusion would make the MMP-1 molecule an active motor, thus making it the first active motor that is not coupled to ATP hydrolysis. The possible sources of energy for this enzyme and their implications are discussed. We propose that a possible source of energy for the enzyme can be in the rearrangement of the structure of collagen fibrils. In a separate application, using the methods developed here, we have observed an intermediate in the intestinal fatty acid binding protein folding process through the changes in its hydrodynamic radius also the fluctuations in the structure of the IFABP in solution were measured using FCS.

  14. Dynamic nuclear protein interactions investigated using fluorescence lifetime and fluorescence fluctuation spectroscopy

    NASA Astrophysics Data System (ADS)

    Siegel, Amanda P.; Hays, Nicole M.; Day, Richard N.

    2012-03-01

    The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic interactions in pituitary specific gene regulation.

  15. Fluorescence Spectroscopy of Human Nonmalignant and Malignant Cells and Tissues.

    NASA Astrophysics Data System (ADS)

    Glassman, Wenling Sha

    This thesis explores steady state and time resolved fluorescence spectroscopy from human malignant and non -malignant cells and tissues. The focus of these studies are the analysis of the excitation spectra, emission spectra, and decay time based on the contribution from several key intrinsic fluorophors: NAD(P)H, flavins, tryptophan, elastin and collagen that exist in different amounts in the human tissues and cells. The comparison between the spectra from malignant and non-malignant cells and tissues gives information on the changes that occur from non-malignancy to malignancy in the cells and tissues. The spectra of tissues and cells are also compared to help in understanding what fluorophors are responsible for fluorescence spectral differences between the malignant and non-malignant tissues and cells. The results in this thesis show that the spectral differences between the normal and cancerous tissues and cells exist in various wavelength ranges. The experimental data from GYN tissues have shown with over 95% of the sensitivity and specificity to separate malignant from non-malignant tissues using 300nm excitation. The 340nm band, which is mostly in response to intrinsic fluorophor (amino acid tryptophan), from malignant tissues were relatively higher then that from the non-malignant tissues. This might have been caused by the higher concentration of free tryptophan in the malignant tumor when compared to that of the normal tissue. This has been found in medical clinical study. The experimental data in this thesis also show that the fluorescence intensities around 450nm-460nm, which are mostly due to the intrinsic fluorophor coenzyme NADH, from both malignant cells in vitro and tissues in vitro are relatively higher than from non-malignant cells in vitro and tissues in vitro. These findings are reinforced by the faster decay time of the NADH fluorescence from normal cells in vitro than from neoplasm cells in vitro. Thus, the NADH in the mitochondria might be

  16. Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy

    PubMed Central

    Sánchez, Susana A.; Brunet, Juan E.; Jameson, David M.; Lagos, Rosalba; Monasterio, Octavio

    2004-01-01

    The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5–1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two-photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady-state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems. PMID:14691224

  17. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Tsekouras, Konstantinos

    2015-03-01

    Fluctuations are an inherent part of single molecule or few particle biophysical data sets. Traditionally, ``noise'' fluctuations have been viewed as a nuisance, to be eliminated or minimized. Here we look on how statistical inference methods - that take explicit advantage of fluctuations - have allowed us to draw an unexpected picture of single molecule diffusional dynamics. Our focus is on the diffusion of proteins probed using fluorescence correlation spectroscopy (FCS). First, we discuss how - in collaboration with the Bustamante and Marqusee labs at UC Berkeley - we determined using FCS data that individual enzymes are perturbed by self-generated catalytic heat (Riedel et al, Nature, 2014). Using the tools of inference, we found how distributions of enzyme diffusion coefficients shift in the presence of substrate revealing that enzymes performing highly exothermic reactions dissipate heat by transiently accelerating their center of mass following a catalytic reaction. Next, when molecules diffuse in the cell nucleus they often appear to diffuse anomalously. We analyze FCS data - in collaboration with Rich Day at the IU Med School - to propose a simple model for transcription factor binding-unbinding in the nucleus to show that it may give rise to apparent anomalous diffusion. Here inference methods extract entire binding affinity distributions for the diffusing transcription factors, allowing us to precisely characterize their interactions with different components of the nuclear environment. From this analysis, we draw key mechanistic insight that goes beyond what is possible by simply fitting data to ``anomalous diffusion'' models.

  18. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    DOE PAGESBeta

    Laurence, Ted A.; Ly, Sonny; Bourguet, Feliza; Fischer, Nicholas O.; Coleman, Matthew A.

    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

  19. Fluorescence Correlation Spectroscopy and Nonlinear Stochastic Reaction-Diffusion

    SciTech Connect

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

    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 closed 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.

  20. Fluorescence Correlation Spectroscopy Evidence for Structural Heterogeneity in Ionic Liquids

    SciTech Connect

    Guo, Jianchang; Baker, Gary A; Hillesheim, Patrick C; Dai, Sheng; Shaw, Robert W; Mahurin, Shannon Mark

    2011-01-01

    Self-aggregation in room temperature ionic liquids (RTILs) has been a subject of intense interest in recent years. In this work, we provide new experimental evidence for chain length-dependent self-aggregation in RTILs using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [CnMPy][Tf2N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decrease with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chained [CnMPy][Tf2N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [CnMPy]+. The presence of this local liquid structuring provides important insight into the behavior of RTILs relevant to their application in photovoltaics, fuel cells, and batteries.

  1. Analyses of the Dynamic Properties of Nuclear Lamins by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS).

    PubMed

    Takeshi, Shimi; Pack, Chan-Gi; Goldman, Robert D

    2016-01-01

    The major structural components of the nuclear lamina are the A- and B-type nuclear lamin proteins which are also present in the nucleoplasm. Studies of molecular movements of the lamins in both the lamina and nucleoplasm of living cell nuclei have provided insights into their roles in maintaining nuclear architecture. In this chapter, we present protocols for quantitatively measuring the mobilities of lamin proteins by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) in mammalian cell nuclei. PMID:27147036

  2. Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics

    PubMed Central

    Jameson, David M.; Ross, Justin A.; Albanesi, Joseph P.

    2011-01-01

    Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming widely used in cell biology. This review traces the development of the method and describes some of the more important applications. Specifically, the methods discussed include fluorescence correlation spectroscopy (FCS), scanning FCS, dual color cross-correlation FCS, the photon counting histogram and fluorescence intensity distribution analysis approaches, the raster scanning image correlation spectroscopy method, and the Number and Brightness technique. The physical principles underlying these approaches will be delineated, and each of the methods will be illustrated using examples from the literature. PMID:21547245

  3. Poly(dimethylsiloxane) microlens array integrated with microfluidic channel for fluorescence spectroscopy detection

    NASA Astrophysics Data System (ADS)

    Rujihan, Suparat; Damrongsak, Badin; Kittidachachan, Pattareeya

    2013-06-01

    Fluorescence spectroscopy detection has been commonly used in chemical and biochemical applications as it provides a good reliability and high sensitivity. Commercially available fluorescence spectroscopy system is typically bulky and expensive, hence making it inconvenience for on-site measurement which requires portable systems. However, the drawback of small devices is that it has a low detection volume, resulting in low fluorescence signal. In this paper, we report a microfluidic channel implemented with a microlens array for enhancing the performance of fluorescence spectroscopy detection. The microlens array was used to focus an excitation light onto the microchannel, thus expecting the increase in fluorescence detection signal. Both microchannels and microlens arrays were individually fabricated from poly-dimethylsiloxane (PDMS) using low-cost printed-circuit-board master molds. The fabrication and characterization of PDMS-based microlens arrays are discussed. In short, the microlens in plano-convex shape was designed with diameters of 700, 800 and 900 microns. The fabricated microlens arrays were characterized for radius of curvatures, SAGs and focal lengths. The plano-convex microlens array was then integrated into a microfluidic system in order to investigate the overall performance of fluorescence spectroscopy detection. Experiments were conducted with two fluorescence dyes, i.e. Rhodamine 6G and Coumarin 153. The preliminary results revealed that the PDMS microlens array implemented on the designed system shows potential for improving excitation and emission light intensity and, as a consequence, signal to background ratio of the fluorescence spectroscopy detection.

  4. Multispectral scanning time-resolved fluorescence spectroscopy (TRFS) technique for intravascular diagnosis

    PubMed Central

    Xie, Hongtao; Bec, Julien; Liu, Jing; Sun, Yang; Lam, Matthew; Yankelevich, Diego R.; Marcu, Laura

    2012-01-01

    This study describes a scanning time-resolved fluorescence spectroscopy (TRFS) system designed to continuously acquire fluorescence emission and to reconstruct fluorescence lifetime images (FLIM) from a luminal surface by using a catheter-based optical probe with rotary joint and pull-back device. The ability of the system to temporally and spectrally resolve the fluorescence emission from tissue was validated using standard dyes and tissue phantoms (e.g., ex vivo pig aorta phantom). Current results demonstrate that this system is capable to reliably resolve the fluorescence emission of multiple fluorophores located in the lumen; and suggest its potential for intravascular detection of distinct biochemical features of atherosclerotic plaques. PMID:22808425

  5. Fluorescence lifetime spectroscopy for breast cancer margins assessment

    NASA Astrophysics Data System (ADS)

    Gorpas, Dimitris; Fatakdawala, Hussain; Zhang, Yanhong; Bold, Richard; Marcu, Laura

    2015-03-01

    During breast conserving surgery (BCS), which is the preferred approach to treat most early stage breast cancers, the surgeon attempts to excise the tumor volume, surrounded by thin margin of normal tissue. The intra-operative assessment of cancerous areas is a challenging procedure, with the surgeon usually relying on visual or tactile guidance. This study evaluates whether time-resolved fluorescence spectroscopy (TRFS) presents the potential to address this problem. Point TRFS measurements were obtained from 19 fresh tissue slices (7 patients) and parameters that characterize the transient signals were quantified via constrained least squares deconvolution scheme. Fibrotic tissue (FT, n=69), adipose tissue (AT, n=76), and invasive ductal carcinoma (IDC, n=27) were identified in histology and univariate statistical analysis, followed by multi-comparison test, was applied to the corresponding lifetime data. Significant differentiation between the three tissue types exists at 390 nm and 500 nm bands. The average lifetime is 3.23+/-0.74 ns for AT, 4.21+/-0.83 ns for FT and 4.71+/-0.35 ns (p<0.05) for IDC at 390 nm. Due to the smaller contribution of collagen in AT the average lifetime value is different from FT and IDC. Additionally, although intensity measurements do not show difference between FT and IDC, lifetime can distinguish them. Similarly, in 500 nm these values are 7.01+/-1.08 ns, 5.43+/-1.05 ns and 4.39+/-0.88 ns correspondingly (p<0.05) and this contrast is due to differentiation in retinol or flavins relative concentration, mostly contributing to AT. Results demonstrate the potential of TRFS to intra-operatively characterize BCS breast excised tissue in real-time and assess tumor margins.

  6. Simulation of autocorrelation function and photon counting distribution in fluorescence fluctuation spectroscopy.

    PubMed

    Shingaryov, Igor P; Skakun, Victor V; Apanasovich, Vladimir V

    2014-01-01

    In modern fluorescence fluctuation spectroscopy, the autocorrelation function and photon counting distribution are two widely used statistical characteristics of the measured fluctuating fluorescence intensity signal. Applying special analysis methods such as fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) to these properties, it is possible to recover values of different parameters of fluorescent molecules such as the concentration, diffusion coefficient, molecular brightness, and kinetic rate constants. The development of new analysis methods is senseless without testing their validity, accuracy, and robustness. The most appropriate check of a method is its application to experimental data. However, sometimes it is more convenient and easier to verify a method on simulated data. Simulation is also useful for better understanding the processes that were modeled during the development of analysis methods. Here, we present two simulation models providing an autocorrelation function and photon counting distribution of a sequence of photon arrival times detected in fluorescence fluctuation spectroscopy. PMID:24108653

  7. 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.

  8. Protein oligomerization monitored by fluorescence fluctuation spectroscopy: Self-assembly of Rubisco activase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A methodology is presented to characterize complex protein assembly pathways by fluorescence correlation spectroscopy. We have derived the total autocorrelation function describing the behavior of mixtures of labeled and unlabeled protein under equilibrium conditions. Our modeling approach allows us...

  9. Multiple stimulated emission fluorescence photoacoustic sensing and spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Gaoming; Gao, Fei; Qiu, Yishen; Feng, Xiaohua; Zheng, Yuanjin

    2016-07-01

    Multiple stimulated emission fluorescence photoacoustic (MSEF-PA) phenomenon is demonstrated in this letter. Under simultaneous illumination of pumping light and stimulated emission light, the fluorescence emission process is speeded up by the stimulated emission effect. This leads to nonlinear enhancement of photoacoustic signal while the quantity of absorbed photons is more than that of fluorescent molecules illuminated by pumping light. The electronic states' specificity of fluorescent molecular can also be labelled by the MSEF-PA signals, which can potentially be used to obtain fluorescence excitation spectrum in deep scattering tissue with nonlinearly enhanced photoacoustic detection. In this preliminary study, the fluorescence excitation spectrum is reconstructed by MSEF-PA signals through sweeping the wavelength of exciting light, which confirms the theoretical derivation well.

  10. Excitation–emission matrices and synchronous fluorescence spectroscopy for the diagnosis of gastrointestinal cancers

    NASA Astrophysics Data System (ADS)

    Genova, Ts; Borisova, E.; Penkov, N.; Vladimirov, B.; Zhelyazkova, A.; Avramov, L.

    2016-06-01

    We report the development of an improved fluorescence technique for cancer diagnostics in the gastrointestinal tract. We investigate the fluorescence of ex vivo colorectal (cancerous and healthy) tissue samples using excitation–emission matrix (EEM) and synchronous fluorescence spectroscopy (SFS) steady-state approaches. The obtained results are processed for revealing characteristic fluorescence spectral features with a valuable diagnostic meaning. The main tissue fluorophores, contributing to the observed fluorescence, are tyrosine, tryptophan, NADH, FAD, collagen and elastin. Based on the results of the Mann–Whitney test as useful parameters for differentiation of gastrointestinal cancer from normal mucosa, we suggest using excitation wavelengths in the range 300 – 360 nm for fluorescence spectroscopy and wavelengths intervals of 60 nm and 90 nm for SFS.

  11. Tryptophan content for monitoring breast cancer cell aggressiveness by native fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Lin; Pu, Yang; Xue, Jianpeng; Pratavieira, Sebastião.; Xu, Baogang; Achilefu, Samuel; Alfano, R. R.

    2014-03-01

    This study shows tryptophan as the key native marker in cells to determine the level of aggressive cancer in breast cell lines using native fluorescence spectroscopy. An algorithm based on the ratio of tryptophan fluorescence intensity at 340 nm to intensity at 460 nm is associated with aggressiveness of the cancer cells. The higher the ratio is, the more aggressive the tumor towards metastasis.

  12. Endogenous synchronous fluorescence spectroscopy (SFS) of basal cell carcinoma-initial study

    NASA Astrophysics Data System (ADS)

    Borisova, E.; Zhelyazkova, Al.; Keremedchiev, M.; Penkov, N.; Semyachkina-Glushkovskaya, O.; Avramov, L.

    2016-01-01

    The human skin is a complex, multilayered and inhomogeneous organ with spatially varying optical properties. Analysis of cutaneous fluorescence spectra could be a very complicated task; therefore researchers apply complex mathematical tools for data evaluation, or try to find some specific approaches, that would simplify the spectral analysis. Synchronous fluorescence spectroscopy (SFS) allows improving the spectral resolution, which could be useful for the biological tissue fluorescence characterization and could increase the tumour detection diagnostic accuracy.

  13. A fluorescence spectroscopy study of traditional Chinese medicine Angelica

    NASA Astrophysics Data System (ADS)

    Zhao, Hongyan; Song, Feng; Liu, Shujing; Chen, Guiyang; Wei, Chen; Liu, Yanling; Liu, Jiadong

    2013-10-01

    By measuring the fluorescence spectra of Chinese medicine (CM) Angelica water solutions with different concentrations from 0.025 to 2.5 mg/mL, results showed that the fluorescence intensity was proportional to the concentration. Through fluorescence spectra of Angelica solution under different pH values, results indicated coumarin compounds were the active ingredients of Angelica. We also observed fluorescence quenching of the Angelica solution in the presence of spherical silver nanoparticles with radius of 12 nm. Keeping a certain value for the volume of the silver nanoparticles, the fluorescence intensity at 402 nm was linearly proportional to the Angelica in the range of 1-3 mg/mL.

  14. Single gold nanoparticles to enhance the detection of single fluorescent molecules at micromolar concentration using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Punj, Deep; Rigneault, Hervé; Wenger, Jérôme

    2014-05-01

    Single nanoparticles made of noble metals are strongly appealing to develop practical applications to detect fluorescent molecules in solution. Here, we detail the use of a single gold nanoparticle of 100 nm diameter to enhance the detection of single Alex Fluor 647 fluorescent molecules at high concentrations of several micromolar. We discuss the implementation of fluorescence correlation spectroscopy, and provide a new method to reliably extract the enhanced fluorescence signal stemming from the nanoparticle near-field from the background generated in the confocal volume. The applicability of our method is checked by reporting the invariance of the single molecule results as function of the molecular concentration, and the experimental data is found in good agreement with numerical simulations.

  15. Fluorescence spectroscopy of individual semiconductor nanoparticles in different ethylene glycols.

    PubMed

    Flessau, Sandra; Wolter, Christopher; Pöselt, Elmar; Kröger, Elvira; Mews, Alf; Kipp, Tobias

    2014-06-14

    The optical properties of single colloidal semiconductor nanoparticles (NPs) are considerably influenced by the direct environment of the NPs. Here, the influence of different liquid and solid glycol matrices on CdSe-based NPs is investigated. Since the fluorescence of individual NPs varies from one NP to another, it is highly desirable to study the very same individual NPs in different matrices. This was accomplished by immobilizing NPs in a liquid cell sample holder or in microfluidic devices. The samples have been investigated by space-resolved wide-field fluorescence microscopy and energy- and time-resolved confocal scanning fluorescence microscopy with respect to fluorescence intensities, emission energies, blinking behavior, and fluorescence decay dynamics of individual NPs. During the measurements the NPs were exposed to air, to liquid ethylene glycols H(OCH2CH2)nOH (also called EGn) with different chain lengths (1 ≤ n ≤ 7), to liquid 2-methylpentane-2,3-diol, or to solid polyethylene oxide. It was found that EG6-7 (also known as PEG 300) is very well suited as a liquid matrix or solvent for experiments that correlate chemical and physical modifications of the surface and of the immediate environment of individual NPs to their fluorescence properties since it leads to intense and stable fluorescence emission of the NPs. PMID:24788878

  16. Fluorescence spectroscopy: A promising tool for carbonate petrology

    SciTech Connect

    Vice, M.A.; Bensley, D.F.; Utgaard, J.E. . Dept. of Geology)

    1992-01-01

    Responses of depositional and diagenetic components in samples of the Mission Canyon Limestone to blue-light excitation vary most noticeably with mineralogy and crystal size. The finely crystalline micrites, dolomicrites and argillaceous carbonates fluoresce more intensely than the more coarsely crystalline sparry calcite cements, dolospar cements and coarsely crystalline dolomites. Low intensity spectral analysis of cherts, anhydrites, and the carbonate phases provides an objective manner for quantifying fluorescence responses and for comparing them statistically. Nineteen of the optical parameters used in organic petrology are evaluated for their utility in carbonate petrology. Results of the discriminant function analysis suggest that red-weighted fluorescence chromaticity indices and yellow-weighted ones are more useful for mineral identification than the blue-weighted or equal-energy chromaticity indices. Statistical analysis of the optical data, mineralogy, and minor element compositions suggests correlations between the fluorescence responses and major minerals, carbonate diagenetic components, and the minor element geochemistry of carbonate components. Although no single element is identified as an activator of fluorescence in this study, the complex correlations of optical indices with Fe suggest that it does act to quench fluorescence. The four fluorescence cy chromaticity indices correlate significantly and positively with mineralogy and negatively with MgCo[sub 3]. In organic petrology, these indices are related to maceral content. The positive correlations of the four fluorescence cx chromaticity indices with Fe and Mn likely reflect fluorescence response to changes in compositions of pore fluids during diagenesis. This trend parallels the increase in cx indices with increasing maturation of organic materials.

  17. Optical spectroscopy of a highly fluorescent aggregate of bacteriochlorophyll c

    NASA Technical Reports Server (NTRS)

    Causgrove, T. P.; Cheng, P.; Brune, D. C.; Blankenship, R. E.

    1993-01-01

    Bacteriochlorophyll (BChl) c and a similar model compound, Mg-methyl bacteriopheophorbide d, form several types of aggregates in nonpolar solvents. One of these aggregates is highly fluorescent, with a quantum yield higher than that of the monomer. This aggregate is also unusual in that it shows a rise time in its fluorescence emission decay at certain wavelengths, which is ascribed to a change in conformation of the aggregate. An analysis of fluorescence depolarization data is consistent with either a linear aggregate of four or five monomers or preferably a cyclic arrangement of three dimers.

  18. Laser-induced fluorescence spectroscopy of the secondary cataract

    NASA Astrophysics Data System (ADS)

    Maslov, N. A.; Larionov, P. M.; Rozhin, I. A.; Druzhinin, I. B.; Chernykh, V. V.

    2016-06-01

    Excitation-emission matrices of laser-induced fluorescence of lens capsule epithelium, the lens nucleus, and the lens capsule are investigated. A solid-state laser in combination with an optical parametric generator tunable in the range from 210 to 350 nm was used for excitation of fluorescence. The spectra of fluorescence of all three types of tissues exhibit typical features that are specific to them and drastically differ from one another. This effect can be used for intrasurgical control of presence of residual lens capsule epithelium cells in the capsular bag after surgical treatment of a cataract.

  19. Detection of mechanical and disease stresses in citrus plants by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Belasque, J., Jr.; Gasparoto, M. C. G.; Marcassa, L. G.

    2008-04-01

    We have investigated the detection of mechanical and disease stresses in citrus plants (Citrus limonia [L.] Osbeck) using laser-induced fluorescence spectroscopy. Due to its economic importance we have chosen to investigate the citrus canker disease, which is caused by the Xanthomonas axonopodis pv. citri bacteria. Mechanical stress was also studied because it plays an important role in the plant's infection by such bacteria. A laser-induced fluorescence spectroscopy system, composed of a spectrometer and a 532 nm10 mW excitation laser was used to perform fluorescence spectroscopy. The ratio of two chlorophyll fluorescence bands allows us to detect and discriminate between mechanical and disease stresses. This ability to discriminate may have an important application in the field to detect citrus canker infected trees.

  20. Two-photon fluorescence correlation spectroscopy with high count rates and low background using dielectric microspheres

    PubMed Central

    Aouani, Heykel; Schön, Peter; Brasselet, Sophie; Rigneault, Hervé; Wenger, Jérôme

    2010-01-01

    Two-photon excitation fluorescence is a powerful technique commonly used for biological imaging. However, the low absorption cross section of this non-linear process is a critical issue for performing biomolecular spectroscopy at the single molecule level. Enhancing the two-photon fluorescence signal would greatly improve the effectiveness of this technique, yet current methods struggle with medium enhancement factors and/or high background noise. Here, we show that the two-photon fluorescence signal from single Alexa Fluor 488 molecules can be enhanced up to 10 times by using a 3 µm diameter latex sphere while adding almost no photoluminescence background. We report a full characterization of the two-photon fluorescence enhancement by a single microsphere using fluorescence correlation spectroscopy. This opens new routes to enhance non-linear optical signals and extend biophotonic applications. PMID:21258531

  1. Comparative studies on the interaction of cefixime with bovine serum albumin by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy.

    PubMed

    Zhang, Lihui; Liu, Baosheng; Li, Zhiyun; Guo, Ying

    2015-08-01

    Under simulated physiological conditions, the reaction mechanism between cefixime and bovine serum albumin at different temperatures (293, 303 and 310 K) was investigated using a fluorescence quenching method and synchronous fluorescence method, respectively. The results indicated that the fluorescence intensity and synchronous fluorescence intensity of bovine serum albumin decreased regularly on the addition of cefixime. In addition, the quenching mechanism, binding constants, number of binding sites, type of interaction force and energy-transfer parameters of cefixime with bovine serum albumin obtained from two methods using the same equation were consistent. The results indicated that the synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the conventional method. PMID:25351241

  2. Ultrafast Fluorescence Spectroscopy via Upconversion: Applications to Biophysics

    PubMed Central

    Xu, Jianhua; Knutson, Jay R.

    2012-01-01

    This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed. PMID:19152860

  3. Fluorescence spectroscopy of anisole at elevated temperatures and pressures

    NASA Astrophysics Data System (ADS)

    Tran, K. H.; Morin, C.; Kühni, M.; Guibert, P.

    2014-06-01

    Laser-induced fluorescence of anisole as tracer of isooctane at an excitation wavelength of 266 nm was investigated for conditions relevant to rapid compression machine studies and for more general application of internal combustion engines regarding temperature, pressure, and ambient gas composition. An optically accessible high pressure and high temperature chamber was operated by using different ambient gases (Ar, N2, CO2, air, and gas mixtures). Fluorescence experiments were investigated at a large range of pressure and temperature (0.2-4 MPa and 473-823 K). Anisole fluorescence quantum yield decreases strongly with temperature for every considered ambient gas, due to efficient radiative mechanisms of intersystem crossing. Concerning the pressure effect, the fluorescence signal decreases with increasing pressure, because increasing the collisional rate leads to more important non-radiative collisional relaxation. The quenching effect is strongly efficient in oxygen, with a fluorescence evolution described by Stern-Volmer relation. The dependence of anisole fluorescence versus thermodynamic parameters suggests the use of this tracer for temperature imaging in specific conditions detailed in this paper. The calibration procedure for temperature measurements is established for the single-excitation wavelength and two-color detection technique.

  4. [Laser-time-resolved fluorescence spectroscopy in immunoassays].

    PubMed

    Pan, L; Du, J; Xie, W; Du, Q; Yun, Q

    2000-06-01

    This paper described a laser-excited time-resolved fluoroimmunoassay set. It made lanthanide ion to couple the anhydrde of diethylenetriaminepentaacetic acid (DTPAA) for labeling antibodies. The experiment used polystyrene tap coated with HCV antigen as the solid phase and a chelate of the rare earth metal europium as fluorescent label. A nitrogen laser beam was used to excite the Eu3- chelates and after 60 microseconds delay time, the emission fluorescence was measured. Background fluorescence of short lifetimes caused by serum components and Raman scattering can be eliminated by set the delay time. In the system condition, fluorescent spectra and fluorescent lifetimes of Eu3+ beta-naphthoyltrifluroacetone (NTA) chelates were measured. The fluorescent lifetime value is 650 microseconds. The maximum emission wavelength is 613 nm. The linear range of europium ion concentration is 1 x 10(-7)-1 x 10(-11) g.mL-1 and the detection limit is 1 x 10(-13) g.mL-1. The relative standard deviation of determination (n = 12) for samples at 0.01 ng.mL-1 magnitude is 6.4%. Laser-TRFIA was also found to be suitable for diagnosis of HCV. The sensitivity and specificity were comparable to enzyme immunoassay. The result was obtained with laser-TRFIA for 29 human correlated well with enzyme immunoassay. PMID:12958930

  5. Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

    PubMed

    Hickey, James L; James, Janine L; Henderson, Clare A; Price, Katherine A; Mot, Alexandra I; Buncic, Gojko; Crouch, Peter J; White, Jonathan M; White, Anthony R; Smith, Trevor A; Donnelly, Paul S

    2015-10-01

    The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease. PMID:26397162

  6. Comparing Compositions of Modern Cast Bronze Sculptures: Optical Emission Spectroscopy Versus x-Ray Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Young, M. L.; Dunand, D. C.

    2015-07-01

    Bulk elemental compositions of 74 modern cast bronze sculptures from the collection at the Art Institute of Chicago, the Philadelphia Museum of Art, and the Rodin Museum (Philadelphia, PA) were determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and a handheld x-ray fluorescence (XRF) spectrometer. The elemental compositions of the cast sculptures as measured previously by ICP-OES and presently by XRF are compared: A good match is found between the two methods for the base metal (Cu) and the two majority alloying elements (Zn and Sn). For both ICP-OES and XRF data, when the Zn composition is plotted versus the Sn composition, three discernable clusters are found that are related to the artist, foundry, casting date, and casting method; they consist of (A) high-zinc brass, (B) low-zinc, low-tin brass, and (C) low-zinc, tin bronze. Thus, our study confirms that the relatively fast, nondestructive XRF spectrometry can be used effectively over slower and invasive, but more accurate, ICP-OES to help determine a sculpture's artist, foundry, date of creation, date of casting, and casting method.

  7. Compact point-detection fluorescence spectroscopy system for quantifying intrinsic fluorescence redox ratio in brain cancer diagnostics

    PubMed Central

    Liu, Quan; Grant, Gerald; Li, Jianjun; Zhang, Yan; Hu, Fangyao; Li, Shuqin; Wilson, Christy; Chen, Kui; Bigner, Darell; Vo-Dinh, Tuan

    2011-01-01

    We report the development of a compact point-detection fluorescence spectroscopy system and two data analysis methods to quantify the intrinsic fluorescence redox ratio and diagnose brain cancer in an orthotopic brain tumor rat model. Our system employs one compact cw diode laser (407 nm) to excite two primary endogenous fluorophores, reduced nicotinamide adenine dinucleotide, and flavin adenine dinucleotide. The spectra were first analyzed using a spectral filtering modulation method developed previously to derive the intrinsic fluorescence redox ratio, which has the advantages of insensitivty to optical coupling and rapid data acquisition and analysis. This method represents a convenient and rapid alternative for achieving intrinsic fluorescence-based redox measurements as compared to those complicated model-based methods. It is worth noting that the method can also extract total hemoglobin concentration at the same time but only if the emission path length of fluorescence light, which depends on the illumination and collection geometry of the optical probe, is long enough so that the effect of absorption on fluorescence intensity due to hemoglobin is significant. Then a multivariate method was used to statistically classify normal tissues and tumors. Although the first method offers quantitative tissue metabolism information, the second method provides high overall classification accuracy. The two methods provide complementary capabilities for understanding cancer development and noninvasively diagnosing brain cancer. The results of our study suggest that this portable system can be potentially used to demarcate the elusive boundary between a brain tumor and the surrounding normal tissue during surgical resection. PMID:21456877

  8. Compact point-detection fluorescence spectroscopy system for quantifying intrinsic fluorescence redox ratio in brain cancer diagnostics

    NASA Astrophysics Data System (ADS)

    Liu, Quan; Grant, Gerald; Li, Jianjun; Zhang, Yan; Hu, Fangyao; Li, Shuqin; Wilson, Christy; Chen, Kui; Bigner, Darell; Vo-Dinh, Tuan

    2011-03-01

    We report the development of a compact point-detection fluorescence spectroscopy system and two data analysis methods to quantify the intrinsic fluorescence redox ratio and diagnose brain cancer in an orthotopic brain tumor rat model. Our system employs one compact cw diode laser (407 nm) to excite two primary endogenous fluorophores, reduced nicotinamide adenine dinucleotide, and flavin adenine dinucleotide. The spectra were first analyzed using a spectral filtering modulation method developed previously to derive the intrinsic fluorescence redox ratio, which has the advantages of insensitivty to optical coupling and rapid data acquisition and analysis. This method represents a convenient and rapid alternative for achieving intrinsic fluorescence-based redox measurements as compared to those complicated model-based methods. It is worth noting that the method can also extract total hemoglobin concentration at the same time but only if the emission path length of fluorescence light, which depends on the illumination and collection geometry of the optical probe, is long enough so that the effect of absorption on fluorescence intensity due to hemoglobin is significant. Then a multivariate method was used to statistically classify normal tissues and tumors. Although the first method offers quantitative tissue metabolism information, the second method provides high overall classification accuracy. The two methods provide complementary capabilities for understanding cancer development and noninvasively diagnosing brain cancer. The results of our study suggest that this portable system can be potentially used to demarcate the elusive boundary between a brain tumor and the surrounding normal tissue during surgical resection.

  9. Compact point-detection fluorescence spectroscopy system for quantifying intrinsic fluorescence redox ratio in brain cancer diagnostics.

    PubMed

    Liu, Quan; Grant, Gerald; Li, Jianjun; Zhang, Yan; Hu, Fangyao; Li, Shuqin; Wilson, Christy; Chen, Kui; Bigner, Darell; Vo-Dinh, Tuan

    2011-03-01

    We report the development of a compact point-detection fluorescence spectroscopy system and two data analysis methods to quantify the intrinsic fluorescence redox ratio and diagnose brain cancer in an orthotopic brain tumor rat model. Our system employs one compact cw diode laser (407 nm) to excite two primary endogenous fluorophores, reduced nicotinamide adenine dinucleotide, and flavin adenine dinucleotide. The spectra were first analyzed using a spectral filtering modulation method developed previously to derive the intrinsic fluorescence redox ratio, which has the advantages of insensitivity to optical coupling and rapid data acquisition and analysis. This method represents a convenient and rapid alternative for achieving intrinsic fluorescence-based redox measurements as compared to those complicated model-based methods. It is worth noting that the method can also extract total hemoglobin concentration at the same time but only if the emission path length of fluorescence light, which depends on the illumination and collection geometry of the optical probe, is long enough so that the effect of absorption on fluorescence intensity due to hemoglobin is significant. Then a multivariate method was used to statistically classify normal tissues and tumors. Although the first method offers quantitative tissue metabolism information, the second method provides high overall classification accuracy. The two methods provide complementary capabilities for understanding cancer development and noninvasively diagnosing brain cancer. The results of our study suggest that this portable system can be potentially used to demarcate the elusive boundary between a brain tumor and the surrounding normal tissue during surgical resection. PMID:21456877

  10. Research of the interaction between kangai injection and human serum albumin by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Ye, Changbin; Lin, Xiaogang; Zhu, Hao; Li, Wenchao; Wu, Jie

    2015-10-01

    The interaction between drugs and serum albumin is the theoretical basis of pharmacology research. Kangai injection with invigorating Qi, enhancing the immune function, is widely used for a variety of malignant tumor treatment. Fluorescence spectroscopy was adopted due to its high sensitivity and other advantages. The interaction between kangai injection and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence spectroscopy and UV-Vis absorption spectroscopy. The results of fluorescence spectrum at three temperature (296K, 303K and 310K) showed the degree of binding at 310K is the highest. Also, the maximum emission peak has a slight blue shift, which indicates that the interaction between kangai injection and HSA has an effect on the conformation of HSA. That is, the microenvironment of tryptophan increase hydrophobic due to the increase of the concentration of kangai injection. Results obtained from analysis of fluorescence spectrum and fluorescence intensity indicated that kangai injection has a strong ability to quench the intrinsic fluorescence of HSA. And according to the Stern-Volume equation, the quenching mechanism is static quenching, which is further proved by the UV-Vis absorption spectroscopy.

  11. Time-resolved and steady-state fluorescence spectroscopy for the assessment of skin photoaging process

    NASA Astrophysics Data System (ADS)

    D´Almeida, Camila de Paula; Campos, Carolina; Saito Nogueira, Marcelo; Pratavieira, Sebastião.; Kurachi, Cristina

    2015-06-01

    pathology. The optical properties of these intrinsic fluorophores respond to the microenvironment and the metabolic status, thus making fluorescence spectroscopy a valuable tool to study the conditions of biological tissues. The purpose of this study is to investigate the hairless mice skin metabolic changes during the photoaging process through lifetime and fluorescence measurements targeting NADH and FAD. Two lasers centered at 378 nm and 445 nm, respectively, perform excitation of NADH and FAD. The fluorescence acquisition is carried out at mice dorsal and ventral regions throughout the photoaging protocol and aging process. Differences in fluorescence and lifetime data between young and photoaged mice measurements were observed. The endogenous fluorescence spectrum of photoaged dorsal skin showed an increase compared to young and aged skin. Lifetime of bound NADH and free FAD presented an increase in the first week that continued until the end of the protocol. Aging process is being investigated to complement the information obtained from fluorescence data and lifetime of photoaging process.

  12. Unravelling molecular mechanisms in the fluorescence spectra of doxorubicin in aqueous solution by femtosecond fluorescence spectroscopy.

    PubMed

    Changenet-Barret, Pascale; Gustavsson, Thomas; Markovitsi, Dimitra; Manet, Ilse; Monti, Sandra

    2013-02-28

    Doxorubicin (DOX) is a potent anti-tumoral agent widely used for cancer therapy. Despite numerous studies, the fluorescence properties of DOX, usually exploited for the characterization of the interaction with biological media, have until now led to controversial interpretations, mainly due to self-association of the drug in aqueous solution. We present here the first femtosecond study of DOX based on measurements with the fluorescence up-conversion technique in combination with time-correlated single photon counting using the same laser source. We provide evidence that fluorescence signals of DOX stem from monomers and dimers. DOX dimerization induces a dramatic decrease in the fluorescence quantum yield from 3.9 × 10(-2) to 10(-5) associated with the red shift of the fluorescence spectrum by ~25 nm. While the fluorescence lifetime of the monomer is 1 ns, the dimer fluorescence is found to decay with a lifetime of about 2 ps. In contrast to monomers, the fluorescence anisotropy of dimers is found to be negative. These experimental observations are consistent with an ultrafast internal conversion (<200 fs) between two exciton states, possibly followed by a charge separation process. PMID:23340955

  13. Diagnosis of corneal pathology by laser fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Salmin, V. V.; Lazarenko, V. I.; Salmina, A. B.; Hovalyg, M. Sh.; Vladimirova, E. S.

    2012-09-01

    We have studied the difference between the fluorescence spectra of the human cornea in vivo under normal conditions and after contact lenses have been worn for different lengths of time, with excitation by emission from a nitrogen laser (337 nm). The most significant sections of the difference spectrum were identified, corresponding to peaks for endogenous fluorophores (NADH and collagen). A high correlation was found between how long the contact lenses have been worn and the fluorescence intensity ratio for wavelengths 460 nm and 410 nm.

  14. Time-resolved Hyperspectral Fluorescence Spectroscopy using Frequency Modulated Excitation

    SciTech Connect

    ,; Neill, M

    2012-07-01

    An intensity-modulated excitation light source is used together with a micro channel plate intensified CCD (ICCD) detector gated at a slightly different frequency to generate a beat frequency from a fluorescent sample. The addition of a spectrograph produces a hyperspectral time-resolved data product where the resulting beat frequency is detected with a low frame rate camera. Measuring the beat frequency of the spectrum as a function of time allows separation of the excited fluorescence from ambient constant light sources. The excitation and detector repetition rates are varied over a range of discrete frequencies, and the phase shift of the beat wave maps out the emission decay rate(s).

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

    PubMed

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

    2015-11-10

    Laser-induced fluorescence and Raman spectroscopy are used for the investigation of different genotypes of Thymus daenensis native to the Ilam province of Iran. Different genotypes of T. daenensis essential oils, labeled T1 through T7, possess slight differences with regard to the composition of the thymol. The gas chromatography-mass spectrometry (GC-MS) method is performed to determine the concentration of each constituent as a reference method. The Raman spectra of different concentrations of pure thymol dissolved in hexane as standard samples are obtained via a laboratory prototype Raman spectroscopy setup for the calculation of the calibration curve. The regression coefficient and limit of detection are calculated. The possibility of the differentiation of different genotypes of T. daenensis is also examined by laser-induced fluorescence spectroscopy, although we do not know the exact amounts of their components. All the fluorescence spectral information is used jointly by cluster analysis to differentiate between 7 genotypes. Our results demonstrate the acceptable precision of Raman spectroscopy with GC-MS and corroborate the capacity of Raman spectroscopy in applications in the quantitative analysis field. Furthermore, the cluster analysis results show that laser-induced fluorescence spectroscopy is an acceptable technique for the rapid classification of different genotypes of T. daenensis without having any previous information of their exact amount of constituents. So, the ability to rapidly and nondestructively differentiate between genotypes makes it possible to efficiently select high-quality herbs from many samples. PMID:26560783

  16. A new incorporation mechanism for trivalent actinides into bioapatite: a TRLFS and EXAFS study.

    PubMed

    Holliday, Kiel; Handley-Sidhu, Stephanie; Dardenne, Kathy; Renshaw, Joanna; Macaskie, Lynne; Walther, Clemens; Stumpf, Thorsten

    2012-02-28

    One of the most toxic byproducts of nuclear power and weapons production is the transuranics, which have a high radiotoxicity and long biological half-life due to their tendency to accumulate in the skeletal system. This accumulation is inhomogeneous and has been associated with the chemical properties and structure of the bone material rather than its location or function. This suggests a chemical driving force to incorporation and requires an atomic scale mechanistic understanding of the incorporation process. Here we propose a new incorporation mechanism for trivalent actinides and lanthanides into synthetic and biologically produced hydroxyapatite. Time-resolved laser fluorescence spectroscopy and extended X-ray absorption fine structure have been used to demonstrate that trivalent actinides and lanthanides incorporate into the amorphous grain boundaries of apatite. This incorporation site can be used to explain patterns in uptake and distribution of radionuclides in the mammalian skeletal system. PMID:22313032

  17. Fluorescence spectroscopy of the retina from scrapie-infected mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated to the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model ...

  18. Variation of fluorescence spectroscopy during the menstrual cycle

    NASA Astrophysics Data System (ADS)

    Macaulay, Calum; Richards-Kortum, Rebecca; Utzinger, Urs; Fedyk, Amanda; Neely Atkinson, E.; Cox, Dennis; Follen, Michele

    2002-06-01

    Cervical autofluorescence has been demonstrated to have potential for real-time diagnosis. Inter-patient and intra-patient variations in fluorescence intensity have been measured. Inter-patient measurements may vary by a factor of ten, while intra-patient measurements may vary by a factor of two. Age and menopausal status have been demonstrated to account for some of the variations, while race and smoking have not. In order to explore in detail the role of the menstrual cycle in intra-patient variation, a study was designed to measure fluorescence excitation emission matrices (EEMs) in patients daily throughout one cycle. Ten patients with a history of normal menstrual cycles and normal Papanicolaou smears underwent daily measurements of fluorescence EEMs from three colposcopically normal sites throughout one menstrual cycle. Changes in signals from porphyrin, NADH, and FAD fluorescence and blood absorption were noted when the data was viewed in a graphical format. Visually interpreted features of the EEMs in this graphical format did not appear to correlate with the day of the menstrual cycle with the exception that blood absorption features were more prominent during the menstrual phase (during which bleeding occurs), suggesting that measurements during the menstrual phase should be avoided. Variations in cycle date likely do not account for inter- or intra-patient variations.

  19. Two-photon fluorescence excitation spectroscopy of biological molecules

    NASA Astrophysics Data System (ADS)

    Meshalkin, Yuri P.; Alfimov, E. E.; Groshev, D. E.; Makukha, V. K.

    1996-06-01

    The UV fluorescence spectra of aromatic amino-acids and some proteins at two photon excitation by second harmonic of Nd:YAG laser are received. Two-photon absorption cross sections of tryptophan, tyrosine, phenylalanine and proteins: bovine serum albumin, lysozyme, trypsin, (alpha) - chymotrypsinogen and pepsin at wavelength 532 nm were measured by means of the two-quantum standard method.

  20. Quantitative Tissue Spectroscopy of Near Infrared Fluorescent Nanosensor Implants.

    PubMed

    Iverson, Nicole M; Bisker, Gili; Farias, Edgardo; Ivanov, Vsevolod; Ahn, Jiyoung; Wogan, Gerald N; Strano, Michael S

    2016-05-01

    Implantable, near infrared (nIR) fluorescent nanosensors are advantageous for in vivo monitoring of biological analytes since they can be rendered selective for a particular target molecule while utilizing their unique optical properties and the nIR tissue transparency window for information transfer without an internal power source or telemetry. However, basic questions remain regarding the optimal encapsulation platform, geometrical properties, and concentration ranges required for high signal to noise ratio and effective detection through biological tissue. In this work, we systematically explore these variables quantitatively to optimize the performance of such optical nanosensors for biomedical applications. We investigate both alginate and polyethylene glycol (PEG) as model hydrogel systems, encapsulating d(GT)15 ssDNA-wrapped single-walled carbon nanotubes (SWNT) as model fluorescent nanoparticle sensors, responsive to riboflavin. Hydrogel sensors implanted 0.5 mm into thick tissue samples exhibit 50% reduction of initial fluorescence intensity, allowing an optical detection limit of 5.4 mm and 5.1 mm depth in tissue for alginate and PEG gels, respectively, at a SWNT concentration of 10 mg L(-1), and 785 nm laser excitation of 80 mW and 30 s exposure. These findings are supported with in vivo nIR fluorescent imaging of SWNT hydrogels implanted subcutaneously in mice. For the case of SWNT, we find that the alginate system is preferable in terms of emission intensity, sensor response, rheological properties, and shelf life. PMID:27305824

  1. Diagnosis of atherosclerotic tissue by resonance fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Neu, Walter; Haase, Karl K.; Tischler, Christian; Nyga, Ralf; Karsch, Karl R.

    1991-05-01

    Resonantly enhanced fluorescence emission induced by a tunable dye laser can be used for the identification of ablated atherosclerotic tissue. This method has been tested with anorganic samples exposed to air and to saline solution. A XeCl excimer laser pulse ((lambda) = 308 nm), delivered by a fused silica optical fiber, causes an efficient ablation of the irradiated samples. The wavelength of the narrow-band dye laser radiation is set to a strong transition of a specific species to be detected in the ablation plume. Taking into account the formation of the plume, the dye laser pulse is applied with a certain delay in order to excite resonantly the selected species in the plume. The resulting resonance fluorescence then is guided by optical fibers to an optical multi-channel analyzer system. Compared to the broad-band fluorescence during excimer laser ablation the resonance fluorescence signal shows a distinct and easily detectable sharp peak. The signal-to-background ratio is improved by one order of magnitude.

  2. New method for tissue indentification: resonance fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Neu, Walter

    1991-11-01

    The method proposed in this paper is based on the detection of resonantly enhanced fluorescence emission induced by a tunable dye laser. First test on anorganic samples exposed to air and to saline solution demonstrate the potential of this technique. A XeCl excimer-laser ((lambda) equals308 nm) pulse, guided by quartz fibers, causes an efficient ablation of the irradiated samples. The specific species to be detected in the ablation plume determines the wavelength of the narrow-band dye-laser radiation. Preferably, it is set to a strong transition of the selected ablation product. Taking into account the formation of the plume, the dye-laser pulse is applied with a certain delay in order to excite resonantly the chosen species in the plume. The resulting resonance fluorescence is then guided by optical fibers to an OMA system. Compared to the broad-band excimer-laser-indiced fluorescence during the ablation process, the resonance fluorescence signal shows a distinct and easily detectable sharp peak. The signal-to-background ratio is improved by one order of magnitude. The achieved increase in sensitivity as well as selectivity is for the benefit of a reliable identification of ablated tissue.

  3. Optical spectroscopy of the bladder washout fluid to optimize fluorescence cystoscopy with Hexvix®

    NASA Astrophysics Data System (ADS)

    Martoccia, Carla; Zellweger, Matthieu; Lovisa, Blaise; Jichlinski, Patrice; van den Bergh, Hubert; Wagnières, Georges

    2014-09-01

    Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate the bladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degrades fluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studied their fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C). A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320/420 nm, FWHM=50/100 nm) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455/525 nm, FWHM=80/50 nm). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine's main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370-430 nm to 395-415 nm would reduce the BWF background by a factor 2.

  4. Combining chemical sequential extractions with 3D fluorescence spectroscopy to characterize sludge organic matter.

    PubMed

    Muller, Mathieu; Jimenez, Julie; Antonini, Maxime; Dudal, Yves; Latrille, Eric; Vedrenne, Fabien; Steyer, Jean-Philippe; Patureau, Dominique

    2014-12-01

    The design and management of anaerobic digestion of sewage sludge (SS) require a relevant characterisation of the sludge organic matter (OM). Methods currently used are time-consuming and often insufficiently informative. A new method combining chemical sequential extractions (CSE) with 3D fluorescence spectroscopy was developed to provide a relevant SS characterisation to assess both OM bioaccessibility and complexity which govern SS biodegradability. CSE fractionates the sludge OM into 5 compartments of decreasing accessibility. First applied on three SS samples with different OM stability, fractionation profiles obtained were in accordance with the latter. 3D fluorescence spectroscopy revealed that the bioaccessible compartments were mainly constituted of simple and easily biodegradable OM while the unaccessible ones were largely made of complex and refractory OM. Then, primary, secondary and anaerobically digested sludge with different biodegradabilities were tested. Complexity revealed by 3D fluorescence spectroscopy was linked with biodegradability and chemical accessibility was correlated with sludge bioaccessibility. PMID:25223440

  5. Changes and characteristics of dissolved organic matter in a constructed wetland system using fluorescence spectroscopy.

    PubMed

    Yao, Yuan; Li, Yun-Zhen; Guo, Xu-Jing; Huang, Tao; Gao, Ping-Ping; Zhang, Ying-Pei; Yuan, Feng

    2016-06-01

    Domestic wastewater was treated by five constructed wetland beds in series. Dissolved organic matter (DOM) collected from influent and effluent samples from the constructed wetland was investigated using fluorescence spectroscopy combined with fluorescence regional integration (FRI), parallel factor (PARAFAC) analysis, and two-dimensional correlation spectroscopy (2D-COS). This study evaluates the capability of these methods in detecting the spectral characteristics of fluorescent DOM fractions and their changes in constructed wetlands. Fluorescence excitation-emission matrix (EEM) combined with FRI analysis showed that protein-like materials displayed a higher removal ratio compared to humic-like substances. The PARAFAC analysis of wastewater DOM indicated that six fluorescent components, i.e., two protein-like substances (C1 and C6), three humic-like substances (C2, C3 and C5), and one non-humic component (C4), could be identified. Tryptophan-like C1 was the dominant component in the influent DOM. The removal ratios of six fluorescent components (C1-C6) were 56.21, 32.05, 49.19, 39.90, 29.60, and 45.87 %, respectively, after the constructed wetland treatment. Furthermore, 2D-COS demonstrated that the sequencing of spectral changes for fluorescent DOM followed the order 298 nm → 403 nm → 283 nm (310-360 nm) in the constructed wetland, suggesting that the peak at 298 nm is associated with preferential tryptophan fluorescence removal. Variation of the fluorescence index (FI) and the ratio of fluorescence components indicated that the constructed wetland treatment resulted in the decrease of fluorescent organic pollutant with increasing the humification and chemical stability of the DOM. PMID:26976008

  6. Remote filament-induced fluorescence spectroscopy from thin clouds of smoke

    NASA Astrophysics Data System (ADS)

    Daigle, J.-F.; Kamali, Y.; Roy, G.; Chin, S. L.

    2008-12-01

    Remote filament-induced fluorescence spectroscopy is used to probe a cloud of smoke, produced from burning mosquito coils, located at a distance of 25 m from the laser source and LIDAR detector. CN, CH and C2 molecular fragments were identified in the sample. We demonstrate that temporally gated measurement is an efficient technique to easily suppress spectral contaminations, such as white light and atmospheric N2 fluorescence.

  7. Short communication: rapid detection of milk fat adulteration with vegetable oil by fluorescence spectroscopy.

    PubMed

    Ntakatsane, M P; Liu, X M; Zhou, P

    2013-04-01

    This study assessed the potential application of fluorescence spectroscopy in detecting adulteration of milk fat with vegetable oil and characterizing the samples according to the source of the fat. Pure butterfat was adulterated with different vegetable oils at various concentrations (0, 5, 10, 15, 20, 30, and 40%). Nonfat and reduced-fat milk were also adulterated with vegetable oils to simulate full-fat milk (3.2%). The 2- and 3-dimensional front-face fluorescence spectroscopy and gas chromatography were used to obtain the fluorescence spectra and fatty acid profile, respectively. Principal component analysis and 3-way partial least squares regression analysis were applied to analyze the data. The pure and adulterated samples were discriminated based on the total concentration of saturated fatty acids and unsaturated fatty acids, and also on the 3 major fluorophores: tryptophan, tocopherols, and riboflavin. Fluorescence spectroscopy was able to detect up to 5% of adulteration of vegetable oil into the butterfat. The saturated fatty acids showed higher predictability than the unsaturated fatty acids (R(2) = 0.73-0.92 vs. 0.20-0.65, respectively). The study demonstrated the high potential of fluorescence spectroscopy to rapidly detect adulteration of milk fat with vegetable oil, and discriminate commercial butter and milk according to the source of the fat. PMID:23415535

  8. Noncontact point spectroscopy guided by two-channel fluorescence imaging in a hamster cheek pouch model

    NASA Astrophysics Data System (ADS)

    Yang, Victor X.; Yeow, Jenny; Lilge, Lothar D.; Kost, James; Mang, Thomas S.; Wilson, Brian C.

    1999-07-01

    A system for in vivo, fluorescence image-guided, non-contact point fluorescence spectroscopy is presented. A 442 nm HeCd laser is used as the fluorescence excitation source. An intensified CCD serves as the detector for both imaging and spectroscopy, on which two regions of 300 X 300 pixels were used for green (500 +/- 18 nm) and red (630 +/- 18 nm) imaging channels, and a strip of 600 X 120 pixels are used for emission spectroscopy (450 - 750 nm). At a working distance of 40 mm, the system has a spatial resolution of 0.16 mm and a spectral resolution of 5 nm. System performance is demonstrated in a carcinogenesis model in hamsters, where tumors were induced by painting DMBA in the cheek pouch. Autofluorescence and Photofrin-induced fluorescence measurements were performed every 2 weeks during the 18 weeks of tumor induction. Punch biopsies on selected animals were taken for histological staging. The results show that autofluorescence fluorescence can distinguish dysplasia from normal mucosal tissue model, utilizing the peak red intensity (or the red-to-green intensity ratio). Photofrin-induced fluorescence was superior to autofluorescence for differentiating high grade dysplasia from invasive cancer.

  9. Time-resolved fluorescence spectroscopy for chemical sensors

    NASA Astrophysics Data System (ADS)

    Draxler, Sonja; Lippitsch, Max E.

    1996-07-01

    A family of sensors is presented with fluorescence decay-time measurements used as the sensing technique. The concept is to take a single fluorophore with a suitably long fluorescence decay time as the basic building block for numerous different sensors. Analyte recognition can be performed by different functional groups that are necessary for selective interaction with the analyte. To achieve this, the principle of excited-state electron transfer is applied with pyrene as the fluorophore. Therefore the same instrumentation based on a small, ambient air-nitrogen laser and solid-state electronics can be used to measure different analytes, for example, oxygen, pH, carbon dioxide, potassium, ammonium, lead, cadmium, zinc, and phosphate.

  10. Ultrafast fluorescence spectroscopy for axial resolution of flurorophore distributions

    NASA Astrophysics Data System (ADS)

    Gräfe, Maximilian G. O.; Hoffmann, Andreas; Spielmann, Christian

    2014-12-01

    A new method for determining the fluorophore distribution along the propagation axis of an ultrashort optical pulse is presented. The axial resolution is obtained by temporal gating of the backward emitted fluorescence via optical parametric amplification, and we demonstrated a resolution in the order of a few 100 μm. With this approach, sampling of the fluorophore concentration of thin layers without using optics with a large numerical aperture will be possible, such as investigating the human retina via time-resolved fluorescence measurements. Additionally, we verified the gain is orders of magnitude higher for coherent seeding, making optical parametric gating very interesting for discriminating between coherently and incoherently scattered light for other multimodal imaging applications.

  11. A scanning fluorescence spectroscopy of decorin under high pressure

    NASA Astrophysics Data System (ADS)

    Komoda, Takahito; Kim, Yun-Jung; Suzuki, Atsushi; Nishiumi, Tadayuki

    2013-06-01

    High pressure processing is able to tenderize not only meat but also intramuscular connective tissue, which is mainly composed of collagen. Decorin, one of the proteoglycans, binds to and stabilizes collagen fibrils. It has been suggested that structural weakening of intramuscular connective tissue may result from the disappearance of the decorin-collagen interaction. In this study, the fluorescence spectra and the surface hydrophobicity of decorin molecules were measured under high pressure in order to examine the resulting change in the tertiary structure. The fluorescence intensity and the surface hydrophobicity of decorin molecules both decreased with increasing applied pressure and with applied time at the constant applied pressure, respectively. The observations indicate that the native structure of decorin is maintained during 200 MPa pressurization for less than 30 min.

  12. Quantitative frequency-domain fluorescence spectroscopy in tissues and tissue-like media

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert Edward

    1999-09-01

    In the never-ending quest for improved medical technology at lower cost, modern near-infrared optical spectroscopy offers the possibility of inexpensive technology for quantitative and non-invasive diagnoses. Hemoglobin is the dominant chromophore in the 700-900 nm spectral region and as such it allows for the optical assessment of hemoglobin concentration and tissue oxygenation by absorption spectroscopy. However, there are many other important physiologically relevant compounds or physiological states that cannot be effectively sensed via optical methods because of poor optical contrast. In such cases, contrast enhancements are required. Fluorescence spectroscopy is an attractive component of optical tissue spectroscopy. Exogenous fluorophores, as well as some endogenous ones, may furnish the desperately needed sensitivity and specificity that is lacking in near-infrared optical tissue spectroscopy. The main focus of this thesis was to investigate the generation and propagation of fluorescence photons inside tissues and tissue-like media (i.e., scattering dominated media). The standard concepts of fluorescence spectroscopy have been incorporated into a diffusion-based picture that is sometimes referred to as photon migration. The novelty of this work lies in the successful quantitative recovery of fluorescence lifetimes, absolute fluorescence quantum yields, fluorophore concentrations, emission spectra, and both scattering and absorption coefficients at the emission wavelength from a tissue-like medium. All of these parameters are sensitive to the fluorophore local environment and hence are indicators of the tissue's physiological state. One application demonstrating the capabilities of frequency-domain lifetime spectroscopy in tissue-like media is a study of the binding of ethidium bromide to bovine leukocytes in fresh milk. Ethidium bromide is a fluorescent dye that is commonly used to label DNA, and hence visualize chromosomes in cells. The lifetime of

  13. Impurity studies in fusion devices using laser-fluorescence-spectroscopy

    SciTech Connect

    Husinsky, W.R.

    1980-08-01

    Resonance fluorescence excitation of neutral atoms using tunable radiation from dye lasers offers a number of unique advantages for impurity studies in fusion devices. Using this technique, it is possible to perform local, time-resolved measurements of the densities and velocity distributions of metallic impurities in fusion devices without disturbing the plasma. Velocities are measured by monitoring the fluorescence intensity while tuning narrow bandwidth laser radiation through the Doppler - broadened absorbtion spectrum of the transition. The knowledge of the velocity distribution of neutral impurities is particularly useful for the determination of impurity introduction mechanisms. The laser fluorescence technique will be described in terms of its application to metallic impurities in fusion devices and related laboratory experiments. Particular attention will be given to recent results from the ISX-B tokamak using pulsed dye lasers where detection sensitivities for neutral Fe of 10/sup 6/ atoms/cm/sup 3/ with a velocity resolution of 600 m/sec (0.1 eV) have been achieved. Techniques for exciting plasma particles (H,D) will also be discussed.

  14. Femtosecond broadband fluorescence upconversion spectroscopy: Spectral coverage versus efficiency.

    PubMed

    Gerecke, Mario; Bierhance, Genaro; Gutmann, Michael; Ernsting, Nikolaus P; Rosspeintner, Arnulf

    2016-05-01

    Sum frequency mixing of fluorescence and ∼1300 nm gate pulses, in a thin β-barium borate crystal and non-collinear type II geometry, is quantified as part of a femtosecond fluorimeter [X.-X. Zhang et al., Rev. Sci. Instrum. 82, 063108 (2011)]. For a series of fixed phasematching angles, the upconversion efficiency is measured depending on fluorescence wavelength. Two useful orientations of the crystal are related by rotation around the surface normal. Orientation A has higher efficiency (factor ∼3) compared to B at the cost of some loss of spectral coverage for a given crystal angle. It should be used when subtle changes of an otherwise stationary emission band are to be monitored. With orientation B, the fluorescence range λF > 420-750 nm is covered with a single setting of the crystal and less gate scatter around time zero. The accuracy of determining an instantaneous emission band shape is demonstrated by comparing results from two laboratories. PMID:27250400

  15. Using the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastichuman breast tissue.

    PubMed

    Hage, R; Galhanone, P R; Zângaro, R A; Rodrigues, K C; Pacheco, M T T; Martin, A A; Netto, M M; Soares, F A; da Cunha, I W

    2003-01-01

    This article reports results of the in vitro study for potential evaluation of the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastic human breast tissue. A coumarine dye laser pumped by nitrogen laser generated an excitation light centered at 458 nm. In order to collect the fluorescence signal was used an optical fiber catheter coupled to a spectrometer and CCD detector. Fluorescence spectra were recorded from normal and neoplastic (benign and malignant) human breast tissue, adding up 94 different areas. The discrimination between normal and neoplasm groups reach a sensitivity and specificity of 100%. PMID:14505202

  16. High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy.

    PubMed

    Czarnocki, Cyprian; Kerfoot, Mark L; Casara, Joshua; Jacobs, Andrew R; Jennings, Cameron; Scheibner, Michael

    2016-01-01

    High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we demonstrate an optical spectroscopy method that is capable of resolving spectral features beyond that of the spin fine structure and homogeneous linewidth of single quantum dots (QDs) using a standard, easy-to-use spectrometer setup. This method incorporates both laser and photoluminescence spectroscopy, combining the advantage of laser line-width limited resolution with multi-channel photoluminescence detection. Such a scheme allows for considerable improvement of resolution over that of a common single-stage spectrometer. The method uses phonons to assist in the measurement of the photoluminescence of a single quantum dot after resonant excitation of its ground state transition. The phonon's energy difference allows one to separate and filter out the laser light exciting the quantum dot. An advantageous feature of this method is its straight forward integration into standard spectroscopy setups, which are accessible to most researchers. PMID:27405015

  17. Determination of dissolved organic matter removal efficiency in wastewater treatment works using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Carstea, Elfrida M.; Bridgeman, John

    2015-04-01

    Fluorescence spectroscopy was used to investigate the removal efficiency of dissolved organic matter (DOM) in several wastewater treatment works, at different processing stages. The correlation between fluorescence values and biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total organic carbon (TOC) has been examined. Fluorescence was measured for unfiltered and filtered (0.45 and 0.20 μm) samples of crude, settled and secondary treated wastewater (activated sludge), and final effluent. Moreover, the potential of using portable fluorimeters has been explored in a laboratory scale activated sludge process. Good correlations were observed for filtered and unfiltered wastewater samples between protein-like fluorescence intensity (excitation 280 nm, emission 350 nm) and BOD (r = 0.78), COD (r = 0.90) and TOC (r = 0.79). BOD displayed a higher correlation at the 0.20 μm filtered samples compared to COD and TOC. Slightly better relation was seen between fluorescence and conventional parameters at the portable fluorimeters compared to laboratory-based instruments. The results indicated that fluorescence spectroscopy, in particular protein-like fluorescence, could be used for continuous, real-time assessment of DOM removal efficiency in wastewater treatment works.

  18. Excitation-emission matrices (EEMs) and synchronous fluorescence spectroscopy (SFS) investigations of gastrointestinal tissues

    NASA Astrophysics Data System (ADS)

    Genova, Ts.; Borisova, E.; Zhelyazkova, Al.; Semyachkina-Glushkovskaya, O.; Penkov, N.; Keremedchiev, M.; Vladimirov, B.; Avramov, L.

    2015-01-01

    In this report we will present our recent investigations of the fluorescence properties of lower part gastrointestinal tissues using excitation-emission matrix and synchronous fluorescence spectroscopy measurement modalities. The spectral peculiarities observed will be discussed and the endogenous sources of the fluorescence signal will be addressed. For these fluorescence spectroscopy measurements the FluoroLog 3 system (HORIBA Jobin Yvon, France) was used. It consists of a Xe lamp (300 W, 200-650 nm), a double mono-chromators, and a PMT detector with a work region at 220- 850 nm. Autofluorescence signals were detected in the form of excitation-emission matrices for the samples of normal mucosa, dysphasia and colon carcinoma and specific spectral features for each tissue were found. Autofluorescence signals from the same samples are observed through synchronous fluorescence spectroscopy, which is a novel promising modality for fluorescence spectroscopy measurements of bio-samples. It is one of the most powerful techniques for multicomponent analysis, because of its sensitivity. In the SFS regime, the fluorescence signal is recorded while both excitation λexc and emission wavelengths λem are simultaneously scanned. A constant wavelength interval is maintained between the λexc and λem wavelengths throughout the spectrum. The resulted fluorescence spectrum shows narrower peak widths, in comparison with EEMs, which are easier for identification and minimizes the chance for false determinations or pretermission of specific spectral feature. This modality is also faster, than EEMs, a much smaller number of data points are required.1 In our measurements we use constant wavelength interval Δλ in the region of 10-200 nm. Measurements are carried out in the terms of finding Δλ, which results in a spectrum with most specific spectral features for comparison with spectral characteristics observed in EEMs. Implementing synchronous fluorescence spectroscopy in optical

  19. Classification evaluation of tobaccos using LED-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhong, Weijia; Dong, Yongjiang; Liu, Xuan; Lin, Hongze; Mei, Liang; Yan, Chunsheng

    2014-02-01

    Tobacco is one of the most important economic crops in the world, assessment of its quality has a very important business significance. A compact, low-cost, and maneuverable optical sensor system for classification evaluation of different tobaccos was described in this paper using light-emitting-diodes (LEDs)-induced fluorescence. The principal components analysis (PCA) method is used to extract the dominant features of the tobaccos for identifying the classification of tobaccos. The technique is suitable for practical identification due to the use of a straightforward data evaluation method and compact system.

  20. 2D fluorescence spectroscopy for monitoring ion-exchange membrane based technologies - Reverse electrodialysis (RED).

    PubMed

    Pawlowski, Sylwin; Galinha, Claudia F; Crespo, João G; Velizarov, Svetlozar

    2016-01-01

    Reverse electrodialysis (RED) is one of the emerging, membrane-based technologies for harvesting salinity gradient energy. In RED process, fouling is an undesirable operation constraint since it leads to a decrease of the obtainable net power density due to increasing stack electric resistance and pressure drop. Therefore, early fouling detection is one of the main challenges for successful RED technology implementation. In the present study, two-dimensional (2D) fluorescence spectroscopy was used, for the first time, as a tool for fouling monitoring in RED. Fluorescence excitation-emission matrices (EEMs) of ion-exchange membrane surfaces and of natural aqueous streams were acquired during one month of a RED stack operation. Fouling evolvement on the ion-exchange membrane surfaces was successfully followed by 2D fluorescence spectroscopy and quantified using principal components analysis (PCA). Additionally, the efficiency of cleaning strategy was assessed by measuring the membrane fluorescence emission intensity before and after cleaning. The anion-exchange membrane (AEM) surface in contact with river water showed to be significantly affected due to fouling by humic compounds, which were found to cross through the membrane from the lower salinity (river water) to higher salinity (sea water) stream. The results obtained show that the combined approach of using 2D fluorescence spectroscopy and PCA has a high potential for studying fouling development and membrane cleaning efficiency in ion exchange membrane processes. PMID:26497936

  1. Near-Field Fluorescence Cross-Correlation Spectroscopy on Planar Membranes

    PubMed Central

    2015-01-01

    The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (FcεRI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods. PMID:25004429

  2. Fluorescence kinetics of Trp-Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy.

    PubMed

    Jia, Menghui; Yi, Hua; Chang, Mengfang; Cao, Xiaodan; Li, Lei; Zhou, Zhongneng; Pan, Haifeng; Chen, Yan; Zhang, Sanjun; Xu, Jianhua

    2015-08-01

    Ultrafast fluorescence dynamics of Tryptophan-Tryptophan (Trp-Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N'-aldehyde group-l-tryptophan-l-tryptophan (NBTrp2), l-tryptophan-l-tryptophan methyl ester (Trp2Me), and N-acetyl-l-tryptophan-l-tryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22ns, respectively. Due to the intramolecular interaction between two Trp residues, the "water relaxation" lifetime was observed around 4ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of ∼100ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20-30ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed. PMID:26111991

  3. Intrinsic photosensitizer fluorescence measured using multi-diameter single-fiber spectroscopy in vivo

    NASA Astrophysics Data System (ADS)

    van Leeuwen-van Zaane, Floor; Gamm, Ute A.; van Driel, Pieter B. A. A.; Snoeks, Thomas J.; de Bruijn, Henriette S.; van der Ploeg-van den Heuvel, Angelique; Sterenborg, Henricus J. C. M.; Löwik, Clemens W.; Amelink, Arjen; Robinson, Dominic J.

    2014-01-01

    Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48] h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.

  4. Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

    NASA Technical Reports Server (NTRS)

    Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

  5. Capillary Electrophoresis and Fluorescence Excitation-Emission Matrix Spectroscopy for Characterization of Humic Substances

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Capillary electrophoresis (CE) and fluorescence spectroscopy have been used in natural organic matter (NOM) studies. In this study, we characterized five fulvic acids, six humic acids and two unprocessed NOM samples obtained from the International Humic Substances Society (IHSS) using these two ana...

  6. A quantitative study of the intracellular dynamics of fluorescently labelled glyco-gold nanoparticles via fluorescence correlation spectroscopy.

    PubMed

    Murray, Richard A; Qiu, Yuan; Chiodo, Fabrizio; Marradi, Marco; Penadés, Soledad; Moya, Sergio E

    2014-07-01

    The dynamic behaviour of gold nanoparticles functionalised with glucose (Glc-Au NPs) has been studied here by means of fluorescence correlation spectroscopy (FCS). Meaningful data on the state of aggregation and dynamics of Glc-Au NPs fluorescently-labelled with HiLyte Fluor647 (Glc-Au-Hi NPs) in the intracellular environment were obtained. Moreover, the work presented here shows that FCS can be used to visualise the presence of single NPs or NP aggregates following uptake and to estimate, locally, NP concentrations within the cell. FCS measurements become possible after applying a "prebleaching" methodology, when the immobile NP fraction has been effectively removed and thus significant FCS data has been recorded. In this study, Glc-Au-Hi NPs have been incubated with HepG2 cells and their diffusion time in the intracellular environment has been measured and compared with their diffusion value in water and cell media. PMID:24639360

  7. Nonlinear Theory of Anomalous Diffusion and Application to Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Boon, Jean Pierre; Lutsko, James F.

    2015-12-01

    The nonlinear theory of anomalous diffusion is based on particle interactions giving an explicit microscopic description of diffusive processes leading to sub-, normal, or super-diffusion as a result of competitive effects between attractive and repulsive interactions. We present the explicit analytical solution to the nonlinear diffusion equation which we then use to compute the correlation function which is experimentally measured by correlation spectroscopy. The theoretical results are applicable in particular to the analysis of fluorescence correlation spectroscopy of marked molecules in biological systems. More specifically we consider the cases of fluorescently labeled lipids in the plasma membrane and of fluorescent apoferritin (a spherically shaped oligomer) in a crowded dextran solution and we find that the nonlinear correlation spectra reproduce very well the experimental data indicating sub-diffusive molecular motion.

  8. High-throughput single-molecule fluorescence spectroscopy using parallel detection

    PubMed Central

    Michalet, X.; Colyer, R. A.; Scalia, G.; Kim, T.; Levi, Moran; Aharoni, Daniel; Cheng, Adrian; Guerrieri, F.; Arisaka, Katsushi; Millaud, Jacques; Rech, I.; Resnati, D.; Marangoni, S.; Gulinatti, A.; Ghioni, M.; Tisa, S.; Zappa, F.; Cova, S.; Weiss, S.

    2011-01-01

    Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors. PMID:21625288

  9. Optofluidic jet waveguide for laser-induced fluorescence spectroscopy.

    PubMed

    Persichetti, Gianluca; Testa, Genni; Bernini, Romeo

    2012-12-15

    An optofluidic water-jet waveguide on chip for fluorescence analysis is presented. A high-speed water stream produced by means of a microchannel acts at the same time as the solution to analyze and as the collecting optical waveguide. The absence of solid walls and the very optically smooth surface of the liquid stream permits a strong increase of the signal-to-noise ratio. The device layout allows a self-alignment direct coupling of a water-jet waveguide with a multimode optical fiber connected to the detector. The performances of the integrated system are tested on Cy5 water solutions. For a 4.5 mm long water-jet waveguide, the measured limit of detection (LOD) is 2.56 nM and filter-free detection is possible with an LOD of 6.11 nM. PMID:23258023

  10. Characterising organic matter in recirculating aquaculture systems with fluorescence EEM spectroscopy.

    PubMed

    Hambly, A C; Arvin, E; Pedersen, L-F; Pedersen, P B; Seredyńska-Sobecka, B; Stedmon, C A

    2015-10-15

    The potential of recirculating aquaculture systems (RAS) in the aquaculture industry is increasingly being acknowledged. Along with intensified application, the need to better characterise and understand the accumulated dissolved organic matter (DOM) within these systems increases. Mature RASs, stocked with rainbow trout and operated at steady state at four feed loadings, were analysed by dissolved organic carbon (DOC) analysis and fluorescence excitation-emission matrix (EEM) spectroscopy. The fluorescence dataset was then decomposed by PARAFAC analysis using the drEEM toolbox. This revealed that the fluorescence character of the RAS water could be represented by five components, of which four have previously been identified in fresh water, coastal marine water, wetlands and drinking water. The fluorescence components as well as the DOC showed positive correlations with feed loading, however there was considerable variation between the five fluorescence components with respect to the degree of accumulation with feed loading. The five components were found to originate from three sources: the feed; the influent tap water (groundwater); and processes related to the fish and the water treatment system. This paper details the first application of fluorescence EEM spectroscopy to assess DOM in RAS, and highlights the potential applications of this technique within future RAS management strategies. PMID:26141427

  11. Fluorescence imaging and time-resolved spectroscopy of steroid using confocal synchrotron radiation microscopy

    NASA Astrophysics Data System (ADS)

    Gerritsen, Hans C.; van der Oord, C. J. R.; Levine, Yehudi K.; Munro, Ian H.; Jones, Gareth R.; Shaw, D. A.; Rommerts, Fokko F.

    1994-08-01

    The Confocal Synchrotron Radiation Microscope at Daresbury was used in a study of the transport and distribution of the steroid Coumestrol in single Leydig cells. The broad spectrum of synchrotron radiation in combination with UV compatible microscope optics affords the extension of confocal microscopy from the visible to the UV region down to about 200 nm. Consequently fluorescent molecules with absorption bands in the UV can be imaged. In addition the pulsed nature of the light source allows us to perform time-resolved fluorescence spectroscopy experiments on microscopic volumes. Coumestrol is a naturally fluorescing plant steroid exhibiting estrogenic activity. In physiological environments it has an absorption peak in the UV at 340 nm and it emits around 440 nm. First results indicate that the Coumestrol transport through the cell membrane is diffusion limited. The weak fluorescence observed in the nuclei of the Leydig cells may be due to fluorescence quenching arising from the interaction of the Coumesterol with nuclear components. However, micro-volume time-resolved fluorescence spectroscopy experiments on cell nuclei have revealed the same decay behavior for Coumesterol in both the cytoplasm and nucleus of the cells.

  12. Determination of changes in wastewater quality through a treatment works using fluorescence spectroscopy.

    PubMed

    Bridgeman, John; Baker, Andy; Carliell-Marquet, Cynthia; Carstea, Elfrida

    2013-01-01

    Fluorescence spectroscopy was used to characterize municipal wastewater at various stages of treatment in order to understand how its fluorescence signature changes with treatment and how the signal relates to biochemical oxygen demand (BOD) and chemical oxygen demand (COD). The impact of size fractionation on the fluorescence signal was also investigated. Fluorescence measurements were taken for unfiltered and filtered (0.45 and 0.20 microm) samples of crude, settled and secondary treated wastewater (activated sludge and trickling filter), and final effluent. Good correlations were observed for unfiltered, diluted wastewater samples between BOD and fluorescence intensity at excitation 280 nm, emission 350 nm (Peak T1) (r = 0.92) and between COD and Peak T1 intensity (r = 0.85). The majority of the T1 and T2 signal was found to be derived from the <0.20 microm fraction. Initial results indicate that fluorescence spectroscopy, and changes in Peak T1 intensity in particular, could be used for continuous, real-time wastewater quality assessment and process control of wastewater treatment works. PMID:24617065

  13. Fluorescence spectroscopy incorporating a ratiometric approach for the diagnosis and classification of urothelial carcinoma

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Crisci, Alfonso; Nesi, Gabriella; Carini, Marco; Pavone, Francesco S.

    2016-02-01

    The current most popular clinical method for the screening of urothelial carcinoma is white light cystoscopy. This method has inherent disadvantages making a strong genesis towards developing more powerful diagnostic techniques. Laser induced intrinsic fluorescence spectroscopy has been studied as an adjunct to current methods for the detection of tumors. This technique allows real time results based on the changes in spectral profile between normal and tumor tissues. We conducted a pilot study based on fluorescence spectroscopy at two wavelengths 378 and 445 nm excitation for the differentiation of urothelial carcinoma. At both the excitation wavelengths, the measured fluorescence signal showed an increased intensity at wavelengths greater than 520 nm. In addition, the emission profile showed modulation at 580 nm which is due to the reabsorption of emitted fluo- rescence due to hemoglobin. Additionally, we developed a tissue characterizing algorithm, based on fluorescence intensity ratios, F510/F600 and F520/F580 at 378 and 445 nm excitation wavelengths respectively. Further, the results were correlated with the pathologists assessment of urothelial carcinoma. This ratiometric classification algorithm yielded 81% sensitivity and 83% specificity at 378 nm and while at 445 nm excitation we achieved a sensitivity and specificity of 85% and 86% for classifying normal and tumor bladder tissues. In this study we have demonstrated the potential of a simple ratiometric algorithm based on fluorescence spectroscopy could be an alternative tool to tissue biopsy. Furthermore, this technique based fiber-based fluorescence spectroscopy could be integrated into an endoscopy system for use in the operating room.

  14. Combined fiber probe for fluorescence lifetime and Raman spectroscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Wachsmann-Hogiu, Sebastian; Marple, Eric; Urmey, Kirk; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-03-01

    Raman spectroscopy has been proven to have tremendous potential as biomedical analytical tool for spectroscopic disease diagnostics. The use of fiberoptic coupled Raman spectroscopy systems can enable in-vivo characterization of suspicious lesions. However, Raman spectroscopy has the drawback of rather long acquisition times of several hundreds of milliseconds which makes scanning of larger regions quite challenging. By combining Raman spectroscopy with a fast imaging technique this problem can be alleviate in part. Fluorescence lifetime imaging (FLIm) offers a great potential for such a combination. FLIm can allow for fast tissue area pre-segmentation and location of the points for Raman spectra acquisition. Here, we introduce an optical fiber probe combining FLIm and Raman spectroscopy with an outer diameter of 2 mm. Fluorescence is generated via excitation with a fiber laser at 355 nm. The fluorescence emission is spectrally resolved using a custom-made wavelength-selection module (WSM). The Raman excitation power at 785 nm was set to 50 mW for the in-vivo measurements to prevent sample drying. The lateral probe resolution was determined to be <250 μm for both modalities. This value was taken as step size for several raster scans of different tissue types which were conducted to show the overlap of both modalities under realistic conditions. Finally the probe was used for in vivo raster scans of a rat's brain and subsequently to acquire FLIm guided Raman spectra of several tissues in and around the craniotomy.

  15. Feasibility of Raman spectroscopy in vitro after 5-ALA-based fluorescence diagnosis in the bladder

    NASA Astrophysics Data System (ADS)

    Grimbergen, M. C. M.; van Swol, C. F. P.; van Moorselaar, R. J. A.; Mahadevan-Jansen, A.,; Stone, N.

    2006-02-01

    Photodynamic diagnosis (PDD) has become popular in bladder cancer detection. Several studies have however shown an increased false positive biopsies rate under PDD guidance compared to conventional cystoscopy. Raman spectroscopy is an optical technique that utilizes molecular specific, inelastic scattering of light photons to interrogate biological tissues, which can successfully differentiate epithelial neoplasia from normal tissue and inflammations in vitro. This investigation was performed to show the feasibility of NIR Raman spectroscopy in vitro on biopsies obtained under guidance of 5-ALA induced PPIX fluorescence imaging. Raman spectra of a PPIX solution was measured to obtain a characteristic signature for the photosensitzer without contributions from tissue constituents. Biopsies were obtained from patients with known bladder cancer instilled with 50ml, 5mg 5-ALA two hours prior to trans-urethral resection of tumor (TURT). Additional biopsies were obtained at a fluorescent and non-fluorescent area, snap-frozen in liquid nitrogen and stored at -80 °C. Each biopsy was thawed before measurements (10sec integration time) with a confocal Raman system (Renishaw Gloucestershire, UK). The 830 nm excitation (300mW) source is focused on the tissue by a 20X ultra-long-working-distance objective. Differences in fluorescence background between the two groups were removed by means of a special developed fluorescence subtraction algorithm. Raman spectra from ALA biopsies showed different fluorescence background which can be effectively removed by a fluorescence subtraction algorithm. This investigation shows that the interaction of the ALA induced PPIX with Raman spectroscopy in bladder samples. Combination of these techniques in-vivo may lead to a viable method of optical biopsies in bladder cancer detection.

  16. Laser-induced fluorescence-cued, laser-induced breakdown spectroscopy biological-agent detection

    SciTech Connect

    Hybl, John D.; Tysk, Shane M.; Berry, Shaun R.; Jordan, Michael P

    2006-12-01

    Methods for accurately characterizing aerosols are required for detecting biological warfare agents. Currently, fluorescence-based biological agent sensors provide adequate detection sensitivity but suffer from high false-alarm rates. Combining single-particle fluorescence analysis with laser-induced breakdown spectroscopy (LIBS) provides additional discrimination and potentially reduces false-alarm rates. A transportable UV laser-induced fluorescence-cued LIBS test bed has been developed and used to evaluate the utility of LIBS for biological-agent detection. Analysis of these data indicates that LIBS adds discrimination capability to fluorescence-based biological-agent detectors.However, the data also show that LIBS signatures of biological agent simulants are affected by washing. This may limit the specificity of LIBS and narrow the scope of its applicability in biological-agent detection.

  17. Depth-resolved fluorescence spectroscopy of normal and dysplastic cervical tissue

    NASA Astrophysics Data System (ADS)

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

    2005-01-01

    A portable confocal system with the excitations at 355nm and 457nm was instrumented to investigate the depth-resolved fluorescence of cervical tissue. The study focused on extracting biochemical and morphological information carried in the depth-resolved signals measured from the normal squamous epithelial tissue and squamous intraepithelial lesions. Strong keratin fluorescence with the spectral characteristics similar to collagen were observed from the topmost keratinizing layer of all tissue samples. It was found that NADH and FAD fluorescence 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 potentially provide more accurate diagnostic information for determining tissue pathology.

  18. 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.

  19. Assembly and characterization of a fluorescence lifetime spectroscopy system for skin lesions diagnostic

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Texiera Rosa, Ramon Gabriel; Pratavieira, Sebastião.; D´Almeida, Camila de Paula; Kurachi, Cristina

    2015-06-01

    The fluorescence spectra and fluorescence lifetime analysis in biological tissues has been presented as a technique of a great potential for tissue characterization for diagnostic purposes. The objective of this study is to assemble and characterize a fluorescence lifetime spectroscopy system for diagnostic of clinically similar skin lesions in vivo. The fluorescence lifetime measurements were performed using the Time Correlated Single Photon Counting (Becker & Hickl, Berlin, Germany) technique. Two lasers, one emitting at 378 nm and another at 445 nm, are used for excitation with 20, 50 and 80 MHz repetition rate. A bifurcated optical fiber probe conducts the excitation light to the sample, the collected light is transmitted through bandpass filters and delivered to a hybrid photomultiplier tube detector. The fluorescence spectra were obtained by using a portable spectrometer (Ocean Optics USB-2000-FLG) with the same excitation sources. An instrument response function of about 300 ps was obtained and the spectrum and fluorescence lifetime of a standard fluorescent molecule (Rhodamine 6G) was measured for the calibration of the system ((4.1 +/- 0.3) ns). The assembled system was considered robust, well calibrated and will be used for clinical measurements of skin lesions.

  20. Probing Ternary Complex Equilibria of Crown Ether Ligands by Time-Resolved Fluorescence Spectroscopy

    PubMed Central

    2015-01-01

    Ternary complex formation with solvent molecules and other adventitious ligands may compromise the performance of metal-ion-selective fluorescent probes. As Ca(II) can accommodate more than 6 donors in the first coordination sphere, commonly used crown ether ligands are prone to ternary complex formation with this cation. The steric strain imposed by auxiliary ligands, however, may result in an ensemble of rapidly equilibrating coordination species with varying degrees of interaction between the cation and the specific donor atoms mediating the fluorescence response, thus diminishing the change in fluorescence properties upon Ca(II) binding. To explore the influence of ligand architecture on these equilibria, we tethered two structurally distinct aza-15-crown-5 ligands to pyrazoline fluorophores as reporters. Due to ultrafast photoinduced electron-transfer (PET) quenching of the fluorophore by the ligand moiety, the fluorescence decay profile directly reflects the species composition in the ground state. By adjusting the PET driving force through electronic tuning of the pyrazoline fluorophores, we were able to differentiate between species with only subtle variations in PET donor abilities. Concluding from a global analysis of the corresponding fluorescence decay profiles, the coordination species composition was indeed strongly dependent on the ligand architecture. Altogether, the combination of time-resolved fluorescence spectroscopy with selective tuning of the PET driving force represents an effective analytical tool to study dynamic coordination equilibria and thus to optimize ligand architectures for the design of high-contrast cation-responsive fluorescence switches. PMID:25313708

  1. Fluorescence lifetime spectroscopy in multiple-scattering environments: an application to biotechnology

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert E.; Gratton, Enrico; Fantini, Sergio

    1999-07-01

    Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in the milk, which is an application vital to the tremendous economic burden of bovine subclinical mastitis detection. Furthermore, we may in principle use the spirit of this technique as a quantitative monitor of the binding of fluorescent drugs inside tissues. This is a first step towards lifetime spectroscopy in tissues.

  2. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    PubMed Central

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-01-01

    Abstract. Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations. PMID:26160347

  3. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    NASA Astrophysics Data System (ADS)

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  4. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds.

    PubMed

    Holt, David; Parthasarathy, Ashwin B; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16 . Ten animals showed no residual tumor cells in the wound bed (mean SBR<2 , P<0.001 ). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15 , and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations. PMID:26160347

  5. Cy3 in AOT reverse micelles II. Probing intermicellar interactions using fluorescence correlation spectroscopy.

    PubMed

    McPhee, Jeffrey T; Scott, Eric; Levinger, Nancy E; Van Orden, Alan

    2011-08-11

    Cyanine-3 (Cy3) fluorescent dye molecules confined in sodium di-2-ethylhexyl sulfosuccinate (AOT) reverse micelles were examined using dynamic light scattering and fluorescence correlation spectroscopy to probe the kinetics of Cy3 dye and reverse micelle aggregation. This study explored a range of reverse micelle sizes, defined as w(0) = [H(2)O]/[AOT], in which the occupation number ranged from one Cy3 molecule per ∼10(5) to ∼10(6) reverse micelles. These measurements reveal that in the smallest reverse micelle, w(0) = 1, the Cy3 molecules aggregate to form H-aggregate dimers, and the Cy3 dimerization is accompanied by the formation of a transient dimer between reverse micelles. Transient reverse micelle dimer particles are only observed in the small fraction of Cy3-labeled reverse micelles probed by fluorescence correlation spectroscopy and are not observed in the bulk solution probed by dynamic light scattering. Furthermore, fluorescence correlation spectroscopy makes it possible to probe the size and shape of these dimers, revealing prolate ellipsoid-shaped particles with twice the volume and surface area of a single reverse micelle. PMID:21761943

  6. Applicability of Fluorescence and Absorbance Spectroscopy to Estimate Organic Pollution in Rivers

    PubMed Central

    Knapik, Heloise Garcia; Fernandes, Cristovão Vicente Scapulatempo; de Azevedo, Júlio Cesar Rodrigues; do Amaral Porto, Monica Ferreira

    2014-01-01

    Abstract This article explores the applicability of fluorescence and absorbance spectroscopy for estimating organic pollution in polluted rivers. The relationship between absorbance, fluorescence intensity, dissolved organic carbon, biochemical oxygen demand (BOD), chemical oxygen demand (COD), and other water quality parameters were used to characterize and identify the origin and the spatial variability of the organic pollution in a highly polluted watershed. Analyses were performed for the Iguassu River, located in southern Brazil, with area about 2,700 km2 and ∼3 million inhabitants. Samples were collect at six monitoring sites covering 107 km of the main river. BOD, COD, nitrogen, and phosphorus concentration indicates a high input of sewage to the river. Specific absorbance at 254 and 285 nm (SUVA254 and A285/COD) did not show significant variation between sites monitored, indicating the presence of both dissolved compounds found in domestic effluents and humic and fulvic compounds derived from allochthonous organic matter. Correlations between BOD and tryptophan-like fluorescence peak (peak T2, r=0.7560, and peak T1, r=0.6949) and tyrosine-like fluorescence peak (peak B, r=0.7321) indicated the presence of labile organic matter and thus confirmed the presence of sewage in the river. Results showed that fluorescence and absorbance spectroscopy provide useful information on pollution in rivers from critical watersheds and together are a robust method that is simpler and more rapid than traditional methods employed by regulatory agencies. PMID:25469076

  7. Optical fluorescence spectroscopy to detect hepatic necrosis after normothermic ischemia: animal model

    NASA Astrophysics Data System (ADS)

    Romano, Renan A.; Vollet-Filho, Jose D.; Pratavieira, Sebastião.; Fernandez, Jorge L.; Kurachi, Cristina; Bagnato, Vanderlei S.; Castro-e-Silva, Orlando; Sankarankutty, Ajith K.

    2015-06-01

    Liver transplantation is a well-established treatment for liver failure. However, the success of the transplantation procedure depends on liver graft conditions. The tissue function evaluation during the several transplantation stages is relevant, in particular during the organ harvesting, when a decision is made concerning the viability of the graft. Optical fluorescence spectroscopy is a good option because it is a noninvasive and fast technique. A partial normothermic hepatic ischemia was performed in rat livers, with a vascular occlusion of both median and left lateral lobes, allowing circulation only for the right lateral lobe and the caudate lobe. Fluorescence spectra under excitation at 532 nm (doubled frequency Nd:YAG laser) were collected using a portable spectrometer (USB2000, Ocean Optics, USA). The fluorescence emission was collected before vascular occlusion, after ischemia, and 24 hours after reperfusion. A morphometric histology analysis was performed as the gold standard evaluation - liver samples were analyzed, and the percentage of necrotic tissue was obtained. The results showed that changes in the fluorescence emission after ischemia can be correlated with the amount of necrosis evaluated by a morphometric analysis, the Pearson correlation coefficient of the generated model was 0.90 and the root mean square error was around 20%. In this context, the laser-induced fluorescence spectroscopy technique after normothermic ischemia showed to be a fast and efficient method to differentiate ischemic injury from viable tissues.

  8. Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis

    NASA Astrophysics Data System (ADS)

    Praveen, Bavishna B.; Ashok, Praveen C.; Mazilu, Michael; Riches, Andrew; Herrington, Simon; Dholakia, Kishan

    2012-07-01

    In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (~30 s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

  9. Assessing the photoaging process at sun exposed and non-exposed skin using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Kurachi, Cristina

    2016-03-01

    Photoaging is the skin premature aging due to exposure to ultraviolet light, which damage the collagen, elastin and can induce alterations on the skin cells DNA, and, then, it may evolve to precancerous lesions, which are widely investigated by fluorescence spectroscopy and lifetime. The fluorescence spectra and fluorescence lifetime analysis has been presented as a technique of great potential for biological tissue characterization at optical diagnostics. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and may contribute to a non-invasive clinical investigation of injuries such as skin lesions. These lesions and the possible areas where they may develop can be interrogated using fluorescence lifetime spectroscopy taking into account the variability of skin phototypes and the changes related to melanin, collagen and elastin, endogenous fluorophores which have emissions that spectrally overlap to the NADH and FAD emission. The objective of this study is to assess the variation on fluorescence lifetimes of normal skin at sun exposed and non-exposed areas and associate this variation to the photoaging process.

  10. Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics.

    PubMed

    Franco, Juliana C; Gonçalves, Grasiele; Souza, Monique S; Rosa, Samantha B C; Thiegue, Larissa M; Atvars, Teresa D Z; Rosa, Paulo T V; Nome, René A

    2013-12-16

    We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy. PMID:24514660

  11. Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

    PubMed

    Rezgui, Rachid; Blumer, Katy; Yeoh-Tan, Gilbert; Trexler, Adam J; Magzoub, Mazin

    2016-07-01

    Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane. PMID:27033412

  12. Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

    PubMed Central

    Siegel, Amanda P.; Baird, Michelle A.; Davidson, Michael W.; Day, Richard N.

    2013-01-01

    The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy. PMID:24129172

  13. Rapid detection of authenticity and adulteration of walnut oil by FTIR and fluorescence spectroscopy: a comparative study.

    PubMed

    Li, Bingning; Wang, Haixia; Zhao, Qiaojiao; Ouyang, Jie; Wu, Yanwen

    2015-08-15

    Fourier transform infrared (FTIR) and fluorescence spectroscopy combined with soft independent modeling of class analogies (SIMCA) and partial least square (PLS) were used to detect the authenticity of walnut oil and adulteration amount of soybean oil in walnut oil. A SIMCA model of FTIR spectra could differentiate walnut oil and other oils into separate categories; the classification limit of soybean oil in walnut oil was 10%. Fluorescence spectroscopy could differentiate oil composition by the peak position and intensity of emission spectrum without multivariate analysis. The classification limit of soybean oil adulterated in walnut oil by fluorescence spectroscopy was below 5%. The deviation of the prediction model for fluorescence spectra was lower than that for FTIR spectra. Fluorescence spectroscopy was more applicable than FTIR in the adulteration detection of walnut oil, both from the determination limit and prediction deviation. PMID:25794716

  14. Portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection

    NASA Astrophysics Data System (ADS)

    Mota, Alessandro D.; Rossi, Giuliano; de Castro, Guilherme Cunha; Ortega, Tiago A.; de Castro N., Jarbas C.

    2014-02-01

    In this work, the development of a portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection is presented. The equipment consists of an excitation blue LED light source, a commercial miniature spectrometer and embedded software. Measurements of healthy, HLB-symptomatic and HLB-asymptomatic citrus leafs were performed. Leafs were excited with the blue LED and their fluorescence spectra collected. Embedded electronics and software were responsible for the spectrum processing and classification via partial least squares regression. Global success rates above 80% and 100% distinction of healthy and HLB-symptomatic leafs were obtained.

  15. Two-photon-excited fluorescence spectroscopy of atomic fluorine at 170 nm

    NASA Technical Reports Server (NTRS)

    Herring, G. C.; Dyer, Mark J.; Jusinski, Leonard E.; Bischel, William K.

    1988-01-01

    Two-photon-excited fluorescence spectroscopy of atomic fluorine is reported. A doubled dye laser at 286-nm is Raman shifted in H2 to 170 nm (sixth anti-Stokes order) to excite ground-state 2P(0)J fluorine atoms to the 2D(0)J level. The fluorine atoms are detected by one of two methods: observing the fluorescence decay to the 2PJ level or observing F(+) production through the absorption of an additional photon by the excited atoms. Relative two-photon absorption cross sections to and the radiative lifetimes of the 2D(0)J states are measured.

  16. Far-field infrared super-resolution microscopy using picosecond time-resolved transient fluorescence detected IR spectroscopy

    NASA Astrophysics Data System (ADS)

    Sakai, Makoto; Kawashima, Yasutake; Takeda, Akihiro; Ohmori, Tsutomu; Fujii, Masaaki

    2007-05-01

    A new far-field infrared super-resolution microscopy combining laser fluorescence microscope and picosecond time-resolved transient fluorescence detected IR (TFD-IR) spectroscopy is proposed. TFD-IR spectroscopy is a kind of IR-visible/UV double resonance spectroscopy, and detects IR transitions by the transient fluorescence due to electronic transition originating from vibrationally excited level populated by IR light. IR images of rhodamine-6G solution and of fluorescent beads were clearly observed by monitoring the transient fluorescence. Super-resolution twice higher than the diffraction limit for IR light was achieved. The IR spectrum due to the transient fluorescence was also measured from spatial domains smaller than the diffraction limit.

  17. Laser-induced fluorescence spectroscopy of benign and malignant cutaneous lesions

    NASA Astrophysics Data System (ADS)

    Borisova, Ekaterina G.; Troyanova, P. P.; Stoyanova, V. P.; Avramov, Lachezar A.

    2005-04-01

    The goals of this work were investigation of pigmented skin lesions by the method of laser-induced fluorescence spectroscopy. Fluorescence spectra were obtained from malignant and benign skin lesions after excitation with nitrogen laser at 337 nm, namely: benign nevi, dysplastic nevi, malignant melanoma (MM), keratopapilloma, base-cell papilloma and base-cell carcinoma, as well as from healthy skin areas near to the lesion that were used posteriori to reveal changes between healthy and lesion skin spectra. Initially lesions were classified by ABCD-dermatscopic method. All suspicious lesions were excised and were investigated histologically. Spectrum of healthy skin consists of one main maximum at 470-500 nm spectral region and secondary maxima at in the regions round 400 and 440 nm. In the cases of nevi and melanoma significant decrease of fluorescence intensity, which correlated with the type of pigment lesion was observed. This reduction of the signal is related to the accumulation of melanin in the lesions that re-absorb strongly the fluorescence from native skin fluorophores in whole visible spectral region. In cases of papilloma and base-cell carcinoma an intensity decrease was also observed, related to accumulation of pigments in these cutaneous lesions. An relative increase of the fluorescence peak at 440 nm were registered in the case of base-cell carcinoma, and appearance of green fluorescence, related to increase of keratin content in benign papilloma lesions were detected. The results, obtained in this investigation of the different pigment lesions could be used for better comprehension of the skin optical properties. The fluorescence spectroscopy of the human skin are very prominent for early diagnosis and differentiation of cutaneous diseases and gives a wide range of possibilities related to real-time determination of existing pathological condition.

  18. Fluorescence spectroscopy: a rapid, noninvasive method for measurement of skin surface thickness of topical agents.

    PubMed

    Rhodes, L E; Diffey, B L

    1997-01-01

    We report the quantification of skin surface thickness of topical agents by in vivo fluorescence spectroscopy, and demonstrate its potential uses for assessment of application technique and substantivity. A series of studies were performed on forearm skin of eight normal subjects using three creams which have intrinsic fluorescence: a sunscreen (Neutrogena SPF15 waterproof cream), an antiseptic (Hewlett's cream) and a steroid (Trimovate (clobetasone butyrate) cream). Initially, the dose-response relationship was established for each agent by applying a series of five doses (0.5-8 microliters/cm2) and measuring cream fluorescence using appropriate excitation and emission wavelengths. Next, the influence of application technique was examined by comparing light application of cream with firm rubbing. Substantivity of the three creams was assessed on dry skin by taking fluorescence measurements over 8 h. Finally, water resistance of 2 microliters/cm2 of sunscreen and antiseptic cream were compared by measuring fluorescence after each of four water immersions. The fluorescence intensity was strongly correlated with the logarithm of surface density. r = 1.0, 0.92 and 0.98 for sunscreen, antiseptic and steroid creams, respectively, allowing derivation of a simple expression for equivalent thickness. Surface thickness of each cream was lower following firm rubbing compared with light application (P < 0.01). The rate constants for reduction of surface density of the three creams with time on dry skin were not significantly different. However, on washed skin, the rate constant was higher for Hewlett's than Neutrogena cream (0.503 and 0.243 h. respectively, P = 0.02), with a higher rate for each cream on wet compared with dry skin (P < 0.001). Hence, fluorescence spectroscopy is a simple, rapid method for measurement of cream thickness in vivo. The many potential applications in dermatology include quantitative assessment of application technique and substantivity of topical

  19. Fluorescence excitation-emission matrix spectroscopy of vitiligo skin in vivo (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Zhao, Jianhua; Richer, Vincent; Al Jasser, Mohammed; Zandi, Soodabeh; Kollias, Nikiforos; Kalia, Sunil; Zeng, Haishan; Lui, Harvey

    2016-02-01

    Fluorescence signals depend on the intensity of the exciting light, the absorption properties of the constituent molecules, and the efficiency with which the absorbed photons are converted to fluorescence emission. The optical features and appearance of vitiligo have been explained primarily on the basis of reduced epidermal pigmentation, which results in abnormal white patches on the skin. The objective of this study is to explore the fluorescence properties of vitiligo and its adjacent normal skin using fluorescence excitation-emission matrix (EEM) spectroscopy. Thirty five (35) volunteers with vitiligo were acquired using a double-grating spectrofluorometer with excitation and emission wavelengths of 260-450 nm and 300-700 nm respectively. As expected, the most pronounced difference between the spectra obtained from vitiligo lesions compared to normally pigmented skin was that the overall fluorescence was much higher in vitiligo; these differences increased at shorter wavelengths, thus matching the characteristic spectral absorption of epidermal melanin. When comparing the fluorescence spectra from vitiligo to normal skin we detected three distinct spectral bands centered at 280nm, 310nm, and 335nm. The 280nm band may possibly be related to inflammation, whereas the 335 nm band may arise from collagen or keratin cross links. The source of the 310 nm band is uncertain; it is interesting to note its proximity to the 311 nm UV lamps used for vitiligo phototherapy. These differences are accounted for not only by changes in epidermal pigment content, but also by other optically active cutaneous biomolecules.

  20. Moving in on the Action: An Experimental Comparison of Fluorescence Excitation and Photodissociation Action Spectroscopy.

    PubMed

    Wellman, Sydney M J; Jockusch, Rebecca A

    2015-06-18

    Photodissociation action spectroscopy is often used as a proxy for measuring gas-phase absorption spectra of ions in a mass spectrometer. Although the potential discrepancy between linear optical and photodissociation spectra is generally acknowledged, direct experimental comparisons are lacking. In this work, we use a quadrupole ion trap that has been modified to enable both photodissociation and laser-induced fluorescence to assess how closely the visible photodissociation action spectrum of a fluorescent dye reflects its fluorescence excitation spectrum. Our results show the photodissociation action spectrum of gaseous rhodamine 110 is both substantially narrower and slightly red-shifted (∼120 cm(-1)) compared to its fluorescence excitation spectrum. Power dependence measurements reveal that the photodissociation of rhodamine 110 requires, on average, the absorption of three photons whereas fluorescence is a single-photon process. These differing power dependences are the key to interpreting the differences in the measured spectra. The experimental results provide much-needed quantification and insight into the differences between action spectra and linear optical spectra, and emphasize the utility of fluorescence excitation spectra to provide a more reliable benchmark for comparison with theory. PMID:26020810

  1. Electronic excited states of guanine-cytosine hairpins and duplexes studied by fluorescence spectroscopy.

    PubMed

    Brazard, Johanna; Thazhathveetil, Arun K; Vayá, Ignacio; Lewis, Frederick D; Gustavsson, Thomas; Markovitsi, Dimitra

    2013-08-01

    Guanine-cytosine hairpins, containing a hexaethylene glycol bridge, are studied by steady-state fluorescence spectroscopy and time-correlated single photon counting; their properties are compared to those of duplexes with the same sequence. It is shown that, both in hairpins and in duplexes, base pairing induces quenching of the ππ* fluorescence, the quantum yield decreasing by at least two orders of magnitude. When the size of the systems increases from two to ten base pairs, a fluorescent component decaying on the nanosecond time-scale appears at energy higher than that stemming from the bright states of non-interacting mono-nucleotides (ca. 330 nm). For ten base pairs, this new fluorescence forms a well-defined band peaking at 305 nm. Its intensity is about 20% higher for the hairpin compared to the duplex. Its position (red-shifted by 1600 cm(-1)) and width (broader by 1800 cm(-1) FWHM) differ from those observed for large duplexes containing 1000 base pairs, suggesting the involvement of electronic coupling. Fluorescence anisotropy reveals that the excited states responsible for high energy emission are not populated directly upon photon absorption but are reached during a relaxation process. They are assigned to charge transfer states. According to the emerging picture, the amplitude of conformational motions determines whether instantaneous deactivation to the ground state or emission from charge transfer states will take place, while ππ* fluorescence is associated to imperfect base-pairing. PMID:23736116

  2. Laser-induced fluorescence spectroscopy of colonic dysplasia: prospects for optical histological analysis

    NASA Astrophysics Data System (ADS)

    Manoharan, Ramasamy; Zonios, George I.; Cothren, Robert M., Jr.; Arendt, Joseph; Van Dam, Jacques; Feld, Michael S.

    1995-05-01

    Several groups have shown that laser-induced fluorescence spectroscopy can detect dysplastic changes in human colon tissues. We present an approach based on analysis of the underlying tissue microstructure for extracting histological information from such spectral signals. The method employs fluorescence microscopy and tissue optics to model the `bulk' fluorescence collected with an optical fiber probe in a clinical setting. For both colonic normal and adenoma, we measured the intrinsic fluorescence lineshapes, the spatial distributions of the fluorophores, and optical parameters of tissue. Numerical and analytical solutions to the radiative transfer equation were then used to compute fluorescence spectra. The results of the model were in excellent agreement with clinical spectra collected during colonoscopy, using 370 nm excitation. Four factors were found to be responsible for the spectral differences between normal tissue and adenoma: fluorescence of mucosal collagen, dysplastic cell, and submucosa, and hemoglobin attenuation. Preliminary results indicate that these parameters can be extracted from individual clinical spectra by reversing the modeling procedure.

  3. Laser-excitation atomic fluorescence spectroscopy in a helium microwave-induced plasma

    NASA Astrophysics Data System (ADS)

    Schroeder, Timothy S.

    The focus of this dissertation is to report the first documented coupling of helium microwave induced plasmas (MIPs) to laser excitation atomic fluorescence spectroscopy. The ability to effectively produce intense atomic emission from both metal and nonmetal analytes gives helium microwave induced plasmas a greater flexibility than the more commonly utilized argon inductively coupled plasma (ICP). Originally designed as an element selective detector for non-aqueous chromatography applications at low applied powers (<100W), the helium microwave plasma has been applied to aqueous sample determinations at higher applied powers (>500 W). The helium MIP has been shown to be a very powerful analytical atomic spectroscopy tool. The development of the pulsed dye laser offered an improved method of excitation in the field of atomic fluorescence. The use of laser excitation for atomic fluorescence was a logical successor to the conventional excitation methods involving hollow cathode lamps and continuum sources. The highly intense, directional, and monochromatic nature of laser radiation results in an increased population of atomic species in excited electronic states where atomic fluorescence can occur. The application of laser excitation atomic fluorescence to the analysis of metals in a helium microwave induced plasma with ultrasonic sample nebulization was the initial focus of this work. Experimental conditions and results are included for the aqueous characterization of manganese, lead, thallium, and iron in the helium MIP- LEAFS system. These results are compared to previous laser excitation atomic fluorescence experimentation. The effect of matrix interferences on the analytical fluorescence signal was also investigated for each element. The advantage of helium MIPs over argon ICPs in the determination of nonmetals in solution indicates that the helium MIP is an excellent candidate for laser excitation atomic fluorescence experiments involving nonmetals such as

  4. Spot Variation Fluorescence Correlation Spectroscopy Allows for Superresolution Chronoscopy of Confinement Times in Membranes

    PubMed Central

    Ruprecht, Verena; Wieser, Stefan; Marguet, Didier; Schütz, Gerhard J.

    2011-01-01

    Resolving the dynamical interplay of proteins and lipids in the live-cell plasma membrane represents a central goal in current cell biology. Superresolution concepts have introduced a means of capturing spatial heterogeneity at a nanoscopic length scale. Similar concepts for detecting dynamical transitions (superresolution chronoscopy) are still lacking. Here, we show that recently introduced spot-variation fluorescence correlation spectroscopy allows for sensing transient confinement times of membrane constituents at dramatically improved resolution. Using standard diffraction-limited optics, spot-variation fluorescence correlation spectroscopy captures signatures of single retardation events far below the transit time of the tracer through the focal spot. We provide an analytical description of special cases of transient binding of a tracer to pointlike traps, or association of a tracer with nanodomains. The influence of trap mobility and the underlying binding kinetics are quantified. Experimental approaches are suggested that allow for gaining quantitative mechanistic insights into the interaction processes of membrane constituents. PMID:21641330

  5. Electronic Resonance Enhancement in Raman and CARS Spectroscopy: Surface Enhanced Scattering of Highly Fluorescent Molecules

    NASA Astrophysics Data System (ADS)

    Lawhead, Carlos; Ujj, Laszlo

    2015-03-01

    Surface enhanced Raman spectroscopy (SERS) is an extremely useful tool in increasing sensitivity of Raman spectroscopy; this technique significantly increases the signal from vibrational resonances which can overcome background fluoresces. Silver nanoparticles coated substrates and the silver nanoparticles in solution were used on a variety of fluorescent molecules in order to overcome sample complexities and measure the vibrational spectra. The possible enhancement of SERS using a coherent Raman (CARS) method was investigated, but enhancement factors due to Surface Enhanced CARS have yet to be verified. The instrument used was developed in the University of West Florida Physics Department utilized the second harmonic of a Nd:YAG laser to provide the excitation wavelength at 532 nm and is capable of both transmission and reflection Raman measurements. Special thanks to the UWF Office of Undergraduate Research.

  6. Visible-super-resolution infrared microscopy using saturated transient fluorescence detected infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Bokor, Nándor; Inoue, Keiichi; Kogure, Satoshi; Fujii, Masaaki; Sakai, Makoto

    2010-02-01

    A scanning visible-super-resolution microscope based on the saturation behaviour of transient fluorescence detected infrared (TFD-IR) spectroscopy is proposed. A Gaussian IR beam, a Gaussian visible beam and a Laguerre-Gaussian (LG) visible beam are used to obtain two separate two-color excitation fluorescence (2CF) images of the sample. The final image is obtained as the difference between the two recorded images. If the peak intensity of the LG beam is high enough to induce saturation in the fluorescence signal, the image can, in principle, have unlimited spatial resolution. A ˜3-fold improvement in transverse resolution over the visible diffraction limit (and far exceeding the IR diffraction limit) is easily achievable in present experimental setups.

  7. Fluorescence spectroscopy for assessment of liver transplantation grafts concerning graft viability and patient survival

    NASA Astrophysics Data System (ADS)

    Vollet Filho, José D.; da Silveira, Marina R.; Castro-e-Silva, Orlando; Bagnato, Vanderlei S.; Kurachi, Cristina

    2015-06-01

    Evaluating transplantation grafts at harvest is essential for its success. Laser-induced fluorescence spectroscopy (LIFS) can help monitoring changes in metabolic/structural conditions of tissue during transplantation. The aim of the present study is to correlate LIFSobtained spectra of human hepatic grafts during liver transplantation with post-operative patients' mortality rate and biochemical parameters, establishing a method to exclude nonviable grafts before implantation. Orthotopic liver transplantation, piggyback technique was performed in 15 patients. LIFS was performed under 408nm excitation. Collection was performed immediately after opening donor's abdominal cavity, after cold perfusion, end of back-table period, and 5 min and 1 h after warm perfusion at recipient. Fluorescence information was compared to lactate, creatinine, bilirubin and INR levels and to survival status. LIFS was sensitive to liver changes during transplantation stages. Study-in-progress; initial results indicate correlation between fluorescence and life/death status of patients.

  8. Polarization-dependent fluorescence correlation spectroscopy for studying structural properties of proteins in living cell

    PubMed Central

    Oura, Makoto; Yamamoto, Johtaro; Ishikawa, Hideto; Mikuni, Shintaro; Fukushima, Ryousuke; Kinjo, Masataka

    2016-01-01

    Rotational diffusion measurement is predicted as an important method in cell biology because the rotational properties directly reflect molecular interactions and environment in the cell. To prove this concept, polarization-dependent fluorescence correlation spectroscopy (pol-FCS) measurements of purified fluorescent proteins were conducted in viscous solution. With the comparison between the translational and rotational diffusion coefficients obtained from pol-FCS measurements, the hydrodynamic radius of an enhanced green fluorescent protein (EGFP) was estimated as a control measurement. The orientation of oligomer EGFP in living cells was also estimated by pol-FCS and compared with Monte Carlo simulations. The results of this pol-FCS experiment indicate that this method allows an estimation of the molecular orientation using the characteristics of rotational diffusion. Further, it can be applied to analyze the degree of molecular orientation and multimerization or detection of tiny aggregation of aggregate-prone proteins. PMID:27489044

  9. In vivo detection of epileptic brain tissue using static fluorescence and diffuse reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    Yadav, Nitin; Bhatia, Sanjiv; Ragheb, John; Mehta, Rupal; Jayakar, Prasanna; Yong, William; Lin, Wei-Chiang

    2013-02-01

    Diffuse reflectance and fluorescence spectroscopy are used to detect histopathological abnormalities of an epileptic brain in a human subject study. Static diffuse reflectance and fluorescence spectra are acquired from normal and epileptic brain areas, defined by electrocorticography (ECoG), from pediatric patients undergoing epilepsy surgery. Biopsy specimens are taken from the investigated sites within an abnormal brain. Spectral analysis reveals significant differences in diffuse reflectance spectra and the ratio of fluorescence and diffuse reflectance spectra from normal and epileptic brain areas defined by ECoG and histology. Using these spectral differences, tissue classification models with accuracy above 80% are developed based on linear discriminant analysis. The differences between the diffuse reflectance spectra from the normal and epileptic brain areas observed in this study are attributed to alterations in the static hemodynamic characteristics of an epileptic brain, suggesting a unique association between the histopathological and the hemodynamic abnormalities in an epileptic brain.

  10. Ultra-sensitive fluorescence spectroscopy of isolated surface-adsorbed molecules using an optical nanofiber.

    PubMed

    Stiebeiner, A; Rehband, O; Garcia-Fernandez, R; Rauschenbeutel, A

    2009-11-23

    The strong radial confinement and the pronounced evanescent field of the guided light in optical nanofibers yield favorable conditions for ultra-sensitive surface spectroscopy of molecules deposited on the fiber. Using the guided mode of the nanofiber for both excitation and fluorescence collection, we present spectroscopic measurements on 3,4,9,10-perylenetetracarboxylic dianhydride molecules (PTCDA) at ambient conditions. Surface coverages as small as 1 per thousand of a compact monolayer still give rise to fluorescence spectra with a good signal to noise ratio. Moreover, we analyze and quantify the self-absorption effects due to reabsorption of the emitted fluorescence light by circumjacent surface-adsorbed molecules distributed along the fiber waist. PMID:19997412

  11. Revealing the photophysics of gold-nanobeacons via time-resolved fluorescence spectroscopy.

    PubMed

    Wei, Guoke; Simionesie, Dorin; Sefcik, Jan; Sutter, Jens U; Xue, Qingjiang; Yu, Jun; Wang, Jinliang; Birch, David J S; Chen, Yu

    2015-12-15

    We demonstrate that time-resolved fluorescence spectroscopy is a powerful tool to investigate the conformation states of hairpin DNA on the surface of gold nanoparticles (AuNPs) and energy transfer processes in Au-nanobeacons. Long-range fluorescence quenching of Cy5 by AuNPs has been found to be in good agreement with electrodynamics modeling. Moreover, time-correlated single-photon counting (TCSPC) is shown to be promising for real-time monitoring of the hybridization kinetics of Au-nanobeacons, with up to 60% increase in decay time component and 300% increase in component fluorescence fraction observed. Our results also indicate the importance of the stem and spacer designs for the performance of Au-nanobeacons. PMID:26670500

  12. Transition probability of the 5971-A line in neutral uranium from collision-induced fluorescence spectroscopy

    SciTech Connect

    Gagne, J.M.; Mongeau, B.; Demers, Y.; Pianarosa, P.

    1981-09-01

    From collision-induced fluorescence spectroscopy measurements, we have determined the transition probability Aof the 5971-A transition in neutral uranium. Our value, A/sub 5971/ = (5.9 +- 1.8) x 10/sup 5/ sec/sup -1/, is, within experimental error, in good agreement with the previous determination of Corliss, A/sub 5971/ = (7.3 +- 3.0) x 10/sup 5/ sec/sup -1/ (J. Res. Nat. Bur. Stand. Sect. A 80,1 (1976)).

  13. TOTAL INTERNAL REFLECTION WITH FLUORESCENCE CORRELATION SPECTROSCOPY: APPLICATIONS TO SUBSTRATE-SUPPORTED PLANAR MEMBRANES

    PubMed Central

    Thompson, Nancy L.; Wang, Xiang; Navaratnarajah, Punya

    2009-01-01

    In this review paper, the conceptual basis and experimental design of total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is described. The few applications to date of TIR-FCS to supported membranes are discussed, in addition to a variety of applications not directly involving supported membranes. Methods related, but not technically equivalent, to TIR-FCS are also summarized. Future directions for TIR-FCS are outlined. PMID:19269331

  14. Spoilage of foods monitored by native fluorescence spectroscopy with selective excitation wavelength

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Wang, Wubao; Alfano, Robert R.

    2015-03-01

    The modern food processing and storage environments require the real-time monitoring and rapid microbiological testing. Optical spectroscopy with selective excitation wavelengths can be the basis of a novel, rapid, reagent less, noncontact and non-destructive technique for monitoring the food spoilage. The native fluorescence spectra of muscle foods stored at 2-4°C (in refrigerator) and 20-24°C (in room temperature) were measured as a function of time with a selective excitation wavelength of 340nm. The contributions of the principal molecular components to the native fluorescence spectra of meat were measured spectra of each fluorophore: collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin. The responsible components were extracted using a method namely Multivariate Curve Resolution with Alternating Least-Squares (MCR-ALS). The native fluorescence combined with MCR-ALS can be used directly on the surface of meat to produce biochemically interpretable "fingerprints", which reflects the microbial spoilage of foods involved with the metabolic processes. The results show that with time elapse, the emission from NADH in meat stored at 24°C increases much faster than that at 4°C. This is because multiplying of microorganisms and catabolism are accompanied by the generation of NADH. This study presents changes of relative content of NADH may be used as criterion for detection of spoilage degree of meat using native fluorescence spectroscopy.

  15. Freshness estimation of intact frozen fish using fluorescence spectroscopy and chemometrics of excitation-emission matrix.

    PubMed

    ElMasry, Gamal; Nagai, Hiroto; Moria, Keisuke; Nakazawa, Naho; Tsuta, Mizuki; Sugiyama, Junichi; Okazaki, Emiko; Nakauchi, Shigeki

    2015-10-01

    The current study attempted to provide a convenient, non-invasive and time-saving method to estimate the freshness of intact horse mackerel (Trachurus japonicus) fish in a frozen state using autofluorescence spectroscopy in tandem with multivariate analysis of fluorescence excitation-emission matrices (EEM). The extracted fluorescence data from different freshness conditions were pretreated, masked and reorganized to resolve fish fluorescence spectra from overlapping signals and scattering profiles for detecting and characterizing freshness changes. The real freshness values of the examined fish samples were then traditionally determined by the hard chemical analysis using the high performance liquid chromatography (HPLC) method and expressed as K-values. The fluorescence EEM data and the real freshness values were modeled using partial least square (PLS) regression and a novel algorithm was proposed to identify the ideal combinations of excitation and emission wavelengths being used as perfect predictors. The results revealed that freshness of frozen fish could be accurately predicted with R(2) of 0.89 and root mean square error estimated by cross validation (RMSECV) of 9.66%. This work substantially demonstrated that the autofluorescence spectroscopy associated with the proposed technical approaches has a high potential in non-destructive sensing of fish freshness in the frozen state. PMID:26078142

  16. Blood perfusion and pH monitoring in organs by laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Vari, Sandor G.; Papazoglou, Theodore G.; Pergadia, Vani R.; Stavridi, Marigo; Snyder, Wendy J.; Papaioannou, Thanassis; Duffy, J. T.; Weiss, Andrew B.; Thomas, Reem; Grundfest, Warren S.

    1994-01-01

    Sensitivity of laser-induced fluorescence spectroscopy (LIFS) in detecting a change in tissue pH, and blood perfusion was determined. Rabbits were anesthetized, paralyzed, and mechanically ventilated. The arterial and venous blood supplies of the kidney were isolated and ligated to alter the perfusion. The femoral artery was cannulated to extract samples for blood gas analysis. A 308-nm XeCl was used as an excitation source. A 600 micrometers core diameter fiber was used for fluorescence acquisition, and the spectra analyzed by an optical multichannel analyzer (EG & G, OMA III). the corresponding intensity ratio R equals INADH / ICOLL was used as an index for respiratory acidosis. Blood perfusion was assessed using the following algorithm: (IELAS minus ICOLL) divided by (INADH minus ICOLL). The intensity ratio linearly decreased with the reduction of blood perfusion. When we totally occluded the artery the ratio decreased tenfold when compared to the ratio of a fully perfused kidney. Results of monitoring blood acidosis by laser-induced fluorescence spectroscopy shows a significant trend between pH and intensity ratio. Since all the slopes were negative, there is an obvious significant correlation between the pH and NADH.COLLAGEN RATIO. Blue-light-induced fluorescence measurements and ratio fluorometry is a sensitive method for monitoring blood perfusion and acidity or alkalinity of an organ.

  17. Applications of Fluorescence Spectroscopy for dissolved organic matter characterization in wastewater treatment plants

    NASA Astrophysics Data System (ADS)

    Goffin, Angélique; Guérin, Sabrina; Rocher, Vincent; Varrault, Gilles

    2016-04-01

    Dissolved organic matter (DOM) influences wastewater treatment plants efficiency (WTTP): variations in its quality and quantity can induce a foaming phenomenon and a fouling event inside biofiltration processes. Moreover, in order to manage denitrification step (control and optimization of the nitrate recirculation), it is important to be able to estimate biodegradable organic matter quantity before biological treatment. But the current methods used to characterize organic matter quality, like biological oxygen demand are laborious, time consuming and sometimes not applicable to directly monitor organic matter in situ. In the context of MOCOPEE research program (www.mocopee.com), this study aims to assess the use of optical techniques, such as UV-Visible absorbance and more specifically fluorescence spectroscopy in order to monitor and to optimize process efficiency in WWTP. Fluorescence excitation-emission matrix (EEM) spectroscopy was employed to prospect the possibility of using this technology online and in real time to characterize dissolved organic matter in different effluents of the WWTP Seine Centre (240,000 m3/day) in Paris, France. 35 sewage water influent samples were collected on 10 days at different hours. Data treatment were performed by two methods: peak picking and parallel factor analysis (PARAFAC). An evolution of DOM quality (position of excitation - emission peaks) and quantity (intensity of fluorescence) was observed between the different treatment steps (influent, primary treatment, biological treatment, effluent). Correlations were found between fluorescence indicators and different water quality key parameters in the sewage influents. We developed different multivariate linear regression models in order to predict a variety of water quality parameters by fluorescence intensity at specific excitation-emission wavelengths. For example dissolved biological oxygen demand (r2=0,900; p<0,0001) and ammonium concentration (r2=0,898; p<0

  18. Native fluorescence spectroscopy of cervical tissues: classification by different statistical methods

    NASA Astrophysics Data System (ADS)

    Ganesan, Singaravelu; Vengadesan, Nammalver; Anbupalam, Thalaimuthu; Hemamalini, Srinivasan; Aruna, Prakasa R.; Karkuzhali, P.

    2002-05-01

    Optical Spectroscopy in the diagnosis of diseases has attracted the medical community due to their minimally invasive nature. Among various optical spectroscopic techniques, native fluorescence spectroscopy has emerged as a potential tool in diagnostic oncology. However, still the reasons for the altered spectral signatures between normal and cancer tissues not yet completely understood. Recently, data reported that emission due to the alteration of some proteins is responsible for the transformation of normal in to malignant one. In this regard, the present study is aimed to characterize the native fluorescence spectroscopy of abnormal and normal cervical tissues, at 280nm excitation. From the study, it is observed that the normal and pathologically diseased cervical tissues have their peak emission around 339 and 336nm respectively with a secondary peak around 440nm. The FWHM value of emission spectra of abnormal tissues is lower than that of normal tissues. The fluorescence spectra of normal and various pathological conditions of cancerous tissues were analyzed by various empirical and statistical methods. Among various type of discriminant analysis, combination of ratio values and linear discrimination method provides better discrimination of normal from pre-malignant and malignant tissues.

  19. Identifying the origins of microbially derived aquatic DOM using fluorescence spectroscopy.

    NASA Astrophysics Data System (ADS)

    Fox, Bethany; Thorn, Robin; Anesio, Alexandre; Reynolds, Darren

    2016-04-01

    Dissolved organic matter (DOM) in aquatic systems is an essential support of the microbial population and, therefore, of the entire aquatic ecosystem. Aquatic DOM is also key for global biogeochemical cycling of nutrients and connects land processes to the marine environment via hydrological transportation. There have been multiple advances in technological assessments of the characteristics of aquatic DOM, with spectroscopy becoming widely used. The extensive use of benchtop spectroscopic instruments has led to the development of in situ sensors, improving the spatiotemporal scale of data acquisition. Whilst this has greatly improved understanding of DOM characteristics and patterns, there are still unknown variables, parameters and interactions of DOM within the aquatic environment. In particular, the interactions of aquatic DOM with the microbial population is still mostly unidentified. It is generally accepted that certain DOM fluorescence regions are autochthonous and microbially derived, such as "peak T" fluorescence. However, the origins and metabolic pathways involved in the production and release of these fluorescent molecules is, as yet, not definitively known. Our work focuses on the identification of these metabolic pathways from whence this microbially derived DOM originates. The most recent stage of the research has utilised traditional microbiological techniques, such as growth curves and chemostat experiments, alongside DOM fluorescence spectroscopic analysis and flow cytometry. The information gained regarding the microbial production and processing of DOM is central for the development of novel in situ fluorescence technology, the ultimate aim of this project.

  20. Time-resolved fluorescence polarization spectroscopy of visible and near infrared dyes in picosecond dynamics

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Alfano, Robert R.

    2015-03-01

    Near-infrared (NIR) dyes absorb and emit light within the range from 700 to 900 nm have several benefits in biological studies for one- and/or two-photon excitation for deeper penetration of tissues. These molecules undergo vibrational and rotational motion in the relaxation of the excited electronic states, Due to the less than ideal anisotropy behavior of NIR dyes stemming from the fluorophores elongated structures and short fluorescence lifetime in picosecond range, no significant efforts have been made to recognize the theory of these dyes in time-resolved polarization dynamics. In this study, the depolarization of the fluorescence due to emission from rotational deactivation in solution will be measured with the excitation of a linearly polarized femtosecond laser pulse and a streak camera. The theory, experiment and application of the ultrafast fluorescence polarization dynamics and anisotropy are illustrated with examples of two of the most important medical based dyes. One is NIR dye, namely Indocyanine Green (ICG) and is compared with Fluorescein which is in visible range with much longer lifetime. A set of first-order linear differential equations was developed to model fluorescence polarization dynamics of NIR dye in picosecond range. Using this model, the important parameters of ultrafast polarization spectroscopy were identified: risetime, initial time, fluorescence lifetime, and rotation times.

  1. Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system

    PubMed Central

    Hill, Laura S.; Richardson, Tammi L.; Profeta, Luisa T. M.; Shaw, Timothy J.; Hintz, Christopher J.; Twining, Benjamin S.; Lawrenz, Evelyn; Myrick, Michael L.

    2010-01-01

    Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 106 photons∕s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter. PMID:20113077

  2. Interaction Studies of Greenly Synthesized Gold Nanoparticles with Bovine Serum Albumin (BSA) Using Fluorescence Spectroscopy.

    PubMed

    Ravikumar, Sambandam; Sreekanth, T V M; Eom, In-Yong

    2015-12-01

    In the present study, gold nanoparticles (AuNPs) with an average particle size of -41.23 nm were synthesized using eco-friendly reducing material (i.e., aqueous Nelumbo nucifera root extract). Rapid reduction results in the formation of polydispersed nanoparticles. The formation of AuNPs was characterized by surface plasmon resonance (SPR) which was determined by UV-Vis spectra (band at 544 nm), FTIR, SEM-EDX, TEM, HR-TEM, and XRD. This study aims to investigate the interaction between AuNPs and Bovine Serum Albumin (BSA) using fluorescence spectroscopy. The analysis of fluorescence spectra and intensity at physiological pH in an aqueous solution indicates that AuNPs have a potent ability to quench the BSA fluorescence by both quenching mechanisms. Resonance light scattering spectra indicated the formation of BSA-AuNPs complex. The number of binding sites and binding constants were determined based on fluorescence quenching at different temperatures. The thermodynamic parameters were also calculated at various temperatures that indicate that hydrophobic forces are abundant in the AuNPs-BSA complex. Negative ΔG degrees values suggest that the binding process is spontaneous. Synchronous fluorescence spectra showed a blue shift and CD spectra showed an increase in a-helicity content which is an indication of increasing hydrophobicity. PMID:26682387

  3. Enhanced energy transfer in respiratory-deficient endothelial cells probed by microscopic fluorescence excitation spectroscopy

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Gschwend, Michael H.; Bauer, Manfred; Strauss, Wolfgang S. L.; Steiner, Rudolf W.

    1996-12-01

    Mitochondrial malfunction may be concomitant with changes of the redox states of the coenzymes nicotinamide adenine dinucleotide (NAD+/NADH), as well as flavin.mononucleotide or dinucleotide. The intrinsic fluorescence of these coenzymes was therefore proposed to be a measure of malfunction. Since mitochondrial fluorescence is strongly superposed by autofluorescence from various cytoplasmatic fluorophores, cultivated endothelial cells were incubated with the mitochondrial marker rhodamine 123 (R123), and after excitation of flavin molecules, energy transfer to R123 was investigated. Due to spectral overlap of flavin and R123 fluorescence, energy transfer flavin yields R123 could not be detected from their emission spectra. Therefore, the method of microscopic fluorescence excitation spectroscopy was established. When detecting R123 fluorescence, excitation maxima at 370 - 390 nm and 420-460 nm were assigned to flavins, whereas a pronounced excitation band at 465 - 490 nm was attributed to R123. Therefore, excitation at 475 nm reflected the intracellular concentration of R123, whereas excitation at 385 nm reflected flavin excitation with a subsequent energy transfer to R123 molecules. An enhanced energy transfer after inhibition of specific enzyme complexes of the respiratory chain is discussed in the present article.

  4. Applications of fluorescence spectroscopy for predicting percent wastewater in an urban stream

    USGS Publications Warehouse

    Goldman, Jami H.; Rounds, Stewart A.; Needoba, Joseph A.

    2012-01-01

    Dissolved organic carbon (DOC) is a significant organic carbon reservoir in many ecosystems, and its characteristics and sources determine many aspects of ecosystem health and water quality. Fluorescence spectroscopy methods can quantify and characterize the subset of the DOC pool that can absorb and re-emit electromagnetic energy as fluorescence and thus provide a rapid technique for environmental monitoring of DOC in lakes and rivers. Using high resolution fluorescence techniques, we characterized DOC in the Tualatin River watershed near Portland, Oregon, and identified fluorescence parameters associated with effluent from two wastewater treatment plants and samples from sites within and outside the urban region. Using a variety of statistical approaches, we developed and validated a multivariate linear regression model to predict the amount of wastewater in the river as a function of the relative abundance of specific fluorescence excitation/emission pairs. The model was tested with independent data and predicts the percentage of wastewater in a sample within 80% confidence. Model results can be used to develop in situ instrumentation, inform monitoring programs, and develop additional water quality indicators for aquatic systems.

  5. Characterization of dissolved organic matter in fogwater by excitation-emission matrix fluorescence spectroscopy

    USGS Publications Warehouse

    Birdwell, J.E.; Valsaraj, K.T.

    2010-01-01

    Dissolved organic matter (DOM) present in fogwater samples collected in southeastern Louisiana and central-eastern China has been characterized using excitation-emission matrix fluorescence spectroscopy. The goal of the study was to illustrate the utility of fluorescence for obtaining information on the large fraction of organic carbon in fogwaters (typically >40% by weight) that defies characterization in terms of specific chemical compounds without the difficulty inherent in obtaining sufficient fogwater volume to isolate DOM for assessment using other spectroscopic and chemical analyses. Based on the findings of previous studies using other characterization methods, it was anticipated that the unidentified organic carbon fraction would have characteristic peaks associated with humic substances and fluorescent amino acids. Both humic- and protein-like fluorophores were observed in the fogwater spectra and fluorescence-derived indices for the fogwater had similar values to those of soil and sediment porewater. Greater biological character was observed in samples with higher organic carbon concentrations. Fogwaters are shown to contain a mixture of terrestrially- and microbially-derived fluorescent organic material, which is expected to be derived from an array of different sources, such as suspended soil and dust particles, biogenic emissions and organic substances generated by atmospheric processes. The fluorescence results indicate that much of the unidentified organic carbon present in fogwater can be represented by humic-like and biologically-derived substances similar to those present in other aquatic systems, though it should be noted that fluorescent signatures representative of DOM produced by atmospheric processing of organic aerosols may be contributing to or masked by humic-like fluorophores. ?? 2010.

  6. Characterization of dissolved organic matter in fogwater by excitation-emission matrix fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Birdwell, Justin E.; Valsaraj, Kalliat T.

    Dissolved organic matter (DOM) present in fogwater samples collected in southeastern Louisiana and central-eastern China has been characterized using excitation-emission matrix fluorescence spectroscopy. The goal of the study was to illustrate the utility of fluorescence for obtaining information on the large fraction of organic carbon in fogwaters (typically >40% by weight) that defies characterization in terms of specific chemical compounds without the difficulty inherent in obtaining sufficient fogwater volume to isolate DOM for assessment using other spectroscopic and chemical analyses. Based on the findings of previous studies using other characterization methods, it was anticipated that the unidentified organic carbon fraction would have characteristic peaks associated with humic substances and fluorescent amino acids. Both humic- and protein-like fluorophores were observed in the fogwater spectra and fluorescence-derived indices for the fogwater had similar values to those of soil and sediment porewater. Greater biological character was observed in samples with higher organic carbon concentrations. Fogwaters are shown to contain a mixture of terrestrially- and microbially-derived fluorescent organic material, which is expected to be derived from an array of different sources, such as suspended soil and dust particles, biogenic emissions and organic substances generated by atmospheric processes. The fluorescence results indicate that much of the unidentified organic carbon present in fogwater can be represented by humic-like and biologically-derived substances similar to those present in other aquatic systems, though it should be noted that fluorescent signatures representative of DOM produced by atmospheric processing of organic aerosols may be contributing to or masked by humic-like fluorophores.

  7. Fluorescence and UV/VIS absorption spectroscopy studies on polymer blend films for photovoltaics

    NASA Astrophysics Data System (ADS)

    van Stam, Jan; Lindqvist, Camilla; Hansson, Rickard; Ericsson, Leif; Moons, Ellen

    2015-08-01

    The quinoxaline-based polymer TQ1 (poly[2,3-bis-(3-octyloxyphenyl)quinoxaline-5,8-diyl-alt-thiophene-2,5- diyl]) is a promising candidate as electron donor in organic solar cells. In combination with the electron acceptor [6,6]- phenyl-C71- butyric acid methyl ester (PC70BM), TQ1 has resulted in solar cells with power conversion efficiencies of 7 %. We have studied TQ1 films, with and without PC70BM, spin-casted from different solvents, by fluorescence spectroscopy and UV/VIS absorption spectroscopy. We used chloroform (CF), chlorobenzene (CB), and odichlorobenzene (o-DCB) as solvents for the coating solutions and 1-chloronaphthalene (CN) as solvent additive. CN addition has been shown to enhance photo-conversion efficiency of these solar cells. Phase-separation causes lateral domain formation in the films and the domain size depends on the solvent . These morphological differences coincide with changes in the spectroscopic patterns of the films. From a spectroscopic point of view, TQ1 acts as fluorescent probe and PC70BM as quencher. The degree of fluorescence quenching is coupled to the morphology through the distance between TQ1 and PC70BM. Furthermore, if using a bad solvent for PC70BM, morphological regions rich in the fullerene yield emission characteristic for aggregated PC70BM. Clear differences were found, comparing the TQ1:PC70BM blend films casted from different solvents and at different ratios between the donor and acceptor. The morphology also influences the UV/VIS absorption spectra, yielding further information on the composition. The results show that fluorescence and UV/VIS absorption spectroscopy can be used to detect aggregation in blended films and that these methods extend the morphological information beyond the scale accessible with microscopy.

  8. A comparative evaluation of Raman and fluorescence spectroscopy for optical diagnosis of oral neoplasia

    NASA Astrophysics Data System (ADS)

    Majumder, S. K.; Krishna, H.; Sidramesh, M.; Chaturvedi, P.; Gupta, P. K.

    2011-08-01

    We report the results of a comparative evaluation of in vivo fluorescence and Raman spectroscopy for diagnosis of oral neoplasia. The study carried out at Tata Memorial Hospital, Mumbai, involved 26 healthy volunteers and 138 patients being screened for neoplasm of oral cavity. Spectral measurements were taken from multiple sites of abnormal as well as apparently uninvolved contra-lateral regions of the oral cavity in each patient. The different tissue sites investigated belonged to one of the four histopathology categories: 1) squamous cell carcinoma (SCC), 2) oral sub-mucous fibrosis (OSMF), 3) leukoplakia (LP) and 4) normal squamous tissue. A probability based multivariate statistical algorithm utilizing nonlinear Maximum Representation and Discrimination Feature for feature extraction and Sparse Multinomial Logistic Regression for classification was developed for direct multi-class classification in a leave-one-patient-out cross validation mode. The results reveal that the performance of Raman spectroscopy is considerably superior to that of fluorescence in stratifying the oral tissues into respective histopathologic categories. The best classification accuracy was observed to be 90%, 93%, 94%, and 89% for SCC, SMF, leukoplakia, and normal oral tissues, respectively, on the basis of leave-one-patient-out cross-validation, with an overall accuracy of 91%. However, when a binary classification was employed to distinguish spectra from all the SCC, SMF and leukoplakik tissue sites together from normal, fluorescence and Raman spectroscopy were seen to have almost comparable performances with Raman yielding marginally better classification accuracy of 98.5% as compared to 94% of fluorescence.

  9. A comparative evaluation of Raman and fluorescence spectroscopy for optical diagnosis of oral neoplasia

    NASA Astrophysics Data System (ADS)

    Majumder, S. K.; Krishna, H.; Sidramesh, M.; Chaturvedi, P.; Gupta, P. K.

    2010-12-01

    We report the results of a comparative evaluation of in vivo fluorescence and Raman spectroscopy for diagnosis of oral neoplasia. The study carried out at Tata Memorial Hospital, Mumbai, involved 26 healthy volunteers and 138 patients being screened for neoplasm of oral cavity. Spectral measurements were taken from multiple sites of abnormal as well as apparently uninvolved contra-lateral regions of the oral cavity in each patient. The different tissue sites investigated belonged to one of the four histopathology categories: 1) squamous cell carcinoma (SCC), 2) oral sub-mucous fibrosis (OSMF), 3) leukoplakia (LP) and 4) normal squamous tissue. A probability based multivariate statistical algorithm utilizing nonlinear Maximum Representation and Discrimination Feature for feature extraction and Sparse Multinomial Logistic Regression for classification was developed for direct multi-class classification in a leave-one-patient-out cross validation mode. The results reveal that the performance of Raman spectroscopy is considerably superior to that of fluorescence in stratifying the oral tissues into respective histopathologic categories. The best classification accuracy was observed to be 90%, 93%, 94%, and 89% for SCC, SMF, leukoplakia, and normal oral tissues, respectively, on the basis of leave-one-patient-out cross-validation, with an overall accuracy of 91%. However, when a binary classification was employed to distinguish spectra from all the SCC, SMF and leukoplakik tissue sites together from normal, fluorescence and Raman spectroscopy were seen to have almost comparable performances with Raman yielding marginally better classification accuracy of 98.5% as compared to 94% of fluorescence.

  10. Multiphoton microscopy, fluorescence lifetime imaging and optical spectroscopy for the diagnosis of neoplasia

    NASA Astrophysics Data System (ADS)

    Skala, Melissa Caroline

    2007-12-01

    the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology.

  11. A simple preparation of Ag@graphene nanocomposites for surface-enhanced Raman spectroscopy of fluorescent anticancer drug

    NASA Astrophysics Data System (ADS)

    Meng, Ying; Yan, Xueying; Wang, Yi

    2016-05-01

    A simple method was developed to synthesize Ag@graphene nanocomposites with rough Ag nanoparticles (AgNPs) conjugated with graphene nanosheets, and the nanocomposites could be used as substrates for effective surface-enhanced Raman spectroscopy (SERS) of fluorescent anticancer drug (Dox) since they could not only enhance the Raman signals but also suppress the fluorescent signals.

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  13. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

    NASA Astrophysics Data System (ADS)

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

  14. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

    SciTech Connect

    Yankelevich, Diego R.; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Marcu, Laura; Elson, Daniel S.

    2014-03-15

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8–7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence

  15. Fluorescence spectroscopy of soil pellets : The use of CP/PARAFAC.

    NASA Astrophysics Data System (ADS)

    Mounier, Stéphane; Nicolodeli, Gustavo; Redon, Roland; Hacherouf, Kalhed; Milori, Debora M. B. P.

    2014-05-01

    Fluorescence spectroscopy is one of the most sensitive techniques available for analytical purposes. It is relatively easy to implement, phenomenologically straightforward and well investigated. Largely non-invasive and fast, so that it can be useful for environmental applications. Fluorescence phenomenon is highly probable in molecular systems containing atoms with lone pairs of electrons such as C=O, aromatic, phenolic, quinone and more rigid unsaturated conjugated systems. These functional groups are present in humic substances (HS) from soils (Senesi, 1990; N. Senesi et al., 1991) and represent the main fluorophors of Soil Organic Matter (SOM). The extension of the conjugated electronic system, the level of heteroatom substitution and type and number of substituting groups under the aromatic rings strongly affect the intensity and wavelength of molecular fluorescence. However, to analyse the SOM it is generally done a chemical extraction that allows measuring the fluorescence response of the liquid extract. To avoid this fractionation of the SOM, Milori et al. (2006) proposed the application of laser induced fluorescence spectroscopy (LIFS) in whole soil. This work intends to assess the technical feasibility of 3D fluorescence spectroscopy using lamp for excitation to analyse solids opaque samples prepared with different substances. Seventy four (74) solid samples were prepared from different mixtures of boric acid (BA), humic substance acid and tryptophan (TRP) powder. The compounds were mixture and a pellet was done by using pressure (8 ton). The pellets were measured using a spectrofluorimeter HITACHI F4500, and a 3D fluorescence tensor was done from emission spectra (200-600 nm) with excitation range from 200 to 500 nm. The acquisition parameters were: step at 5 nm, scan speed at 2400 nm.min-1, response time at 0.1 s, excitation and emission slits at 5 nm and photomultiplier voltage at 700 V. Furthermore, measures of Laser-induced Fluorescence were

  16. Fluorescence excitation-emission matrix spectroscopy as a tool for determining quality of sparkling wines.

    PubMed

    Elcoroaristizabal, Saioa; Callejón, Raquel M; Amigo, Jose M; Ocaña-González, Juan A; Morales, M Lourdes; Ubeda, Cristina

    2016-09-01

    Browning in sparkling wines was assessed by the use of excitation-emission fluorescence spectroscopy combined with PARAllel FACtor analysis (PARAFAC). Four different cava sparkling wines were monitored during an accelerated browning process and subsequently storage. Fluorescence changes observed during the accelerated browning process were monitored and compared with other conventional parameters: absorbance at 420nm (A420) and the content of 5-hydroxymethyl-2-furfural (5-HMF). A high similarity of the spectral profiles for all sparkling wines analyzed was observed, being explained by a four component PARAFAC model. A high correlation between the third PARAFAC factor (465/530nm) and the commonly used non-enzymatic browning indicators was observed. The fourth PARAFAC factor (280/380nm) gives us also information about the browning process following a first order kinetic reaction. Hence, excitation-emission fluorescence spectroscopy, together with PARAFAC, provides a faster alternative for browning monitoring to conventional methods, as well as useful key indicators for quality control. PMID:27041327

  17. Tissue classification and diagnostics using a fiber probe for combined Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Crisci, Alfonso; Giordano, Flavio; Rossari, Susanna; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2015-07-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnostic purposes on various tissues in a good agreement with common routine histology. This study included skin, brain and bladder tissues and in particular the classification of: malignant melanoma against melanocytic lesions and healthy skin; urothelial carcinoma against healthy bladder mucosa; brain tumor against dysplastic brain tissue. The diagnostic capabilities were determined using a cross-validation method with a leave-one-out approach, finding very high sensitivity and specificity for all the examined tissues. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  18. Cure Monitoring of an Unsaturated Polyester Resin Using Near-Infrared and Fluorescence Spectroscopies

    NASA Astrophysics Data System (ADS)

    Sung, Chong S. P.; Grunden, Bradley L.

    1998-03-01

    The applicability of both near-infrared (NIR) and fluorescence spectroscopy for the purpose of cure monitoring an unsaturated polyester (UPE) resin was investigated. Based on standard reference mixtures, peak assignments in the NIR region of the spectrum were made. It was determined that the peak at 1629 nm was due to the first overtone band of RHC=CH2 stretching modes in styrene, while a combination of RHC=CHR and -C=C- stretching modes in diethyl fumarate were responsible for the peak observed at 2087 nm. NIR spectra of the UPE resin during isothermal cure at 75 C exhibited decreases in peak absorbance at 1629 and 2087 nm due to conversion of styrene and vinylene bonds, respectively. Conversion of styrene and vinylene with time calculated using NIR spectra showed similar trends found with FTIR analysis throughout the entire conversion range. Fluorescence spectroscopy was used to monitor the isothermal curing reaction of the UPE resin by exciting styrene at 250 nm. Emission intensity at ca. 306 nm remained unchanged for the initial 60-80 minutes then increased with cure time due to a reduced self-quenching effect as cure proceeded. The increase in fluorescence intensity was concurrent with an increase in styrene conversion up to 93% styrene conversion.

  19. The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

    PubMed Central

    Kim, Jiho; Doose, Sören; Neuweiler, Hannes; Sauer, Markus

    2006-01-01

    Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides. PMID:16687657

  20. The modified fluorescence based vesicle fluctuation spectroscopy technique for determination of lipid bilayer bending properties.

    PubMed

    Drabik, Dominik; Przybyło, Magda; Chodaczek, Grzegorz; Iglič, Aleš; Langner, Marek

    2016-02-01

    Lipid bilayer is the main constitutive element of biological membrane, which confines intracellular space. The mechanical properties of biological membranes may be characterized by various parameters including membrane stiffness or membrane bending rigidity, which can be measured using flicker noise spectroscopy. The flicker noise spectroscopy exploits the spontaneous thermal undulations of the membrane. The method is based on the quantitative analysis of a series of microscopic images captured during thermal membrane fluctuations. Thus, measured bending rigidity coefficient depends on the image quality as well as the selection of computational tools for image processing and mathematical model used. In this work scanning and spinning disc confocal microscopies were used to visualize fluctuating membranes of giant unilamellar vesicles. The bending rigidity coefficient was calculated for different acquisition modes, using different fluorescent probes and different image processing methods. It was shown that both imaging approaches gave similar bending coefficient values regardless of acquisition time. Using the developed methodology the effect of fluorescent probe type and aqueous phase composition on the value of the membrane bending rigidity coefficient was measured. Specifically it was found that the bending rigidity coefficient of DOPC bilayer in water is smaller than that determined for POPC membrane. It has been found that the POPC and DOPC bending rigidities coefficient in sucrose solution was lower than that in water. Fluorescence imaging makes possible the quantitative analysis of membrane mechanical properties of inhomogeneous membrane. PMID:26615919

  1. Tissue classification and diagnostics using a fiber probe for combined Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Rossari, Susanna; Sturiale, Alessandro; Giordano, Flavio; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Tonelli, Francesco; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2015-03-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnostic purposes on various tissues in a good agreement with common routine histology. This study included skin, brain and bladder tissues and in particular the classification of: malignant melanoma against melanocytic lesions and healthy skin; urothelial carcinoma against healthy bladder mucosa; brain tumor against dysplastic brain tissue. The diagnostic capabilities were determined using a cross-validation method with a leave-one-out approach, finding very high sensitivity and specificity for all the examined tissues. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  2. Quantitative Fluorescence Correlation Spectroscopy Reveals a 1000-Fold Increase in Lifetime of Protein Functionality

    PubMed Central

    Zhang, Dianwen; Lans, Hannes; Vermeulen, Wim; Lenferink, Aufried; Otto, Cees

    2008-01-01

    We have investigated dilute protein solutions with fluorescence correlation spectroscopy (FCS) and have observed that a rapid loss of proteins occurs from solution. It is commonly assumed that such a loss is the result of protein adsorption to interfaces. A protocol was developed in which this mode of protein loss can be prevented. However, FCS on fluorescent protein (enhanced green fluorescent protein, mCherry, and mStrawberry) solutions enclosed by adsorption-protected interfaces still reveals a decrease of the fluorescent protein concentration, while the diffusion time is stable over long periods of time. We interpret this decay as a loss of protein functionality, probably caused by denaturation of the fluorescent proteins. We show that the typical lifetime of protein functionality in highly dilute, approximately single molecule per femtoliter solutions can be extended more than 1000-fold (typically from a few hours to >40 days) by adding compounds with surfactant behavior. No direct interactions between the surfactant and the fluorescent proteins were observed from the diffusion time measured by FCS. A critical surfactant concentration of more than 23 μM was required to achieve the desired protein stabilization for Triton X-100. The surfactant does not interfere with DNA-protein binding, because similar observations were made using DNA-cutting restriction enzymes. We associate the occurrence of denaturation of proteins with the activity of water at the water-protein interface, which was recently proposed in terms of the “water attack model”. Our observations suggest that soluble biomolecules can extend an influence over much larger distances than suggested by their actual volume. PMID:18586843

  3. Histologic differences between orthotopic xenograft pancreas models affect Verteporfin uptake measured by fluorescence microscopy and spectroscopy

    NASA Astrophysics Data System (ADS)

    O'Hara, Julia A.; Samkoe, Kimberley S.; Chen, Alina; Isabelle, Martin; Hoopes, P. J.; Hasan, Tayyaba; Pogue, Brian W.

    2012-02-01

    Photodynamic therapy (PDT) that uses the second generation photosensitizer, verteporfin (VP), is a developing therapy for pancreatic cancer. The optimal timing of light delivery related to VP uptake and distribution in pancreatic tumors will be important information to obtain to improve treatment for this intractable disease. In this work we examined uptake and distribution of VP in two orthotopic pancreatic tumors with different histological structure. ASPC-1 (fast-growing) and Panc-1 (slower growing) tumors were implanted in SCID mice and studied when tumors were approximately 100mm3. In a pilot study, these tumors had been shown to differ in uptake of VP using lightinduced fluorescence spectroscopy (LIFS) in vivo and fluorescence imaging ex vivo and that work is extended here. In vivo fluorescence mean readings of tumor and liver increased rapidly up to 15 minutes after photosensitizer injection for both tumor types, and then continued to increase up to 60 minutes post injection to a higher level in ASPC-1 than in Panc-1. There was variability among animals with the same tumor type, in both liver and tumor uptake and no selectivity of tumor over liver. In this work we further examined VP uptake at multiple time points in relation to microvascular density and perfusion, using DiOC7 (to mark blood vessels) and VP fluorescence in the same tissue slices. Analysis of DiOC7 fluorescence indicates that AsPC-1 and Panc-1 have different vascular densities but AsPC-1 vasculature is more perfusive. Analysis of colocalized DiOC7 and VP fluorescence showed ASPC-1 with higher accumulation of VP 3 hrs after injection and more VP at a distance from blood vessels compared to Panc-1. This work shows the need for techniques to analyze photosensitizer distribution in order to optimize photodynamic therapy as an effective treatment for pancreatic tumors.

  4. Substrate-Supported Phospholipid Membranes Studied by Surface Plasmon Resonance and Surface Plasmon Fluorescence Spectroscopy

    PubMed Central

    Tawa, Keiko; Morigaki, Kenichi

    2005-01-01

    Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates. PMID:16040759

  5. 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. PMID:27041337

  6. Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP.

    PubMed

    Colletier, Jacques-Philippe; Sliwa, Michel; Gallat, François-Xavier; Sugahara, Michihiro; Guillon, Virginia; Schirò, Giorgio; Coquelle, Nicolas; Woodhouse, Joyce; Roux, Laure; Gotthard, Guillaume; Royant, Antoine; Uriarte, Lucas Martinez; Ruckebusch, Cyril; Joti, Yasumasa; Byrdin, Martin; Mizohata, Eiichi; Nango, Eriko; Tanaka, Tomoyuki; Tono, Kensuke; Yabashi, Makina; Adam, Virgile; Cammarata, Marco; Schlichting, Ilme; Bourgeois, Dominique; Weik, Martin

    2016-03-01

    Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins. PMID:26866390

  7. Multicolor whole-cell bacterial sensing using a synchronous fluorescence spectroscopy-based approach.

    PubMed

    Parrello, Damien; Mustin, Christian; Brie, David; Miron, Sebastian; Billard, Patrick

    2015-01-01

    The wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems. PMID:25822488

  8. Derivative Synchronous Fluorescence Spectroscopy for the Simultaneous Determination of Dapoxetine Hydrochloride and Vardenafil in Binary Mixtures

    NASA Astrophysics Data System (ADS)

    Soliman, S. M.; El-Agizy, H. M. Y.; El Bayoumi, Abd El Aziz

    2014-07-01

    The first and second derivative synchronous fluorescence spectroscopy (FDSFS&SDSFS) methods have been developed and validated for the simultaneous analysis of a binary mixture of dapoxetine hydrochloride and vardenafil. Method 1A describes a measurement of the normal synchronous fluorescence intensity of these drugs at Δλ = 35 nm using sodium dodecyl sulfate as the fluorescence enhancer in aqueous solutions. This method was extended (Method 1B) to the use of FDSFS&SDSFS for the determination of both drugs. The fluorescence concentration plots were linear over the range of 1-10 and 0.2-2 μg/ml for dapoxetine hydrochloride and vardenafil, respectively, with lower detection limits of 290 and 62.5 ng/ml, and quantification limits of 890 and 190 ng/ml for dapoxetine hydrochloride and vardenafil, respectively. The proposed method was applied for the simultaneous determination of DAP and VAR in different synthetic mixtures and in co-formulated pharmaceutical preparation. The results obtained were in good agreement with those obtained using a reference method.

  9. Multicolor Whole-Cell Bacterial Sensing Using a Synchronous Fluorescence Spectroscopy-Based Approach

    PubMed Central

    Parrello, Damien; Mustin, Christian; Brie, David; Miron, Sebastian; Billard, Patrick

    2015-01-01

    The wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems. PMID:25822488

  10. Evaluation of a fiber-optic fluorescence spectroscopy system to assist neurosurgical tumor resections

    NASA Astrophysics Data System (ADS)

    Ilias, Michail A.; Richter, Johan; Westermark, Frida; Brantmark, Martin; Andersson-Engels, Stefan; Wårdell, Karin

    2007-07-01

    The highly malignant brain tumor, glioblastoma multiforme, is difficult to totally resect without aid due to its infiltrative way of growing and its morphological similarities to surrounding functioning brain under direct vision in the operating field. The need for an inexpensive and robust real-time visualizing system for resection guiding in neurosurgery has been formulated by research groups all over the world. The main goal is to develop a system that helps the neurosurgeon to make decisions during the surgical procedure. A compact fiber optic system using fluorescence spectroscopy has been developed for guiding neurosurgical resections. The system is based on a high power light emitting diode at 395 nm and a spectrometer. A fiber bundle arrangement is used to guide the excitation light and fluorescence light between the instrument and the tissue target. The system is controlled through a computer interface and software package especially developed for the application. This robust and simple instrument has been evaluated in vivo both on healthy skin but also during a neurosurgical resection procedure. Before surgery the patient received orally a low dose of 5-aminolevulinic acid, converted to the fluorescence tumor marker protoporphyrin IX in the malignant cells. Preliminary results indicate that PpIX fluorescence and brain tissue autofluorescence can be recorded with the help of the developed system intraoperatively during resection of glioblastoma multiforme.

  11. Fluorescence spectroscopy of collagen crosslinking: non-invasive and in situ evaluation of corneal stiffness

    NASA Astrophysics Data System (ADS)

    Franco, Walfre; Ortega-Martinez, Antonio; Zhu, Hong; Wang, Ruisheng; Kochevar, Irene E.

    2015-03-01

    Collagen is a long fibrous structural protein that imparts mechanical support, strength and elasticity to many tissues. The state of the tissue mechanical environment is related to tissue physiology, disease and function. In the cornea, the collagen network is responsible for its shape and clarity; disruption of this network results in degradation of visual acuity, for example in the keratoconus eye disease. The objective of the present study is to investigate the feasibility of using the endogenous fluorescence of collagen crosslinks to evaluate variations in the mechanical state of tissue, in particular, the stiffness of cornea in response to different degrees of photo-crosslinking or RGX treatment—a novel keratoconus treatment. After removing the epithelium, rabbit corneas were stained with Rose Bengal and then irradiated with a 532 nm solid-state laser. Analysis of the excitation spectra obtained by fluorescence spectroscopy shows a correlation between the fluorescence intensity at 370/460 nm excitation/emission wavelengths and the mechanical properties. In principle, it may be feasible to use the endogenous fluorescence of collagen crosslinks to evaluate the mechanical stiffness of cornea non-invasively and in situ.

  12. Fluorescence spectroscopy as a tool for quality assessment of humic substances

    NASA Astrophysics Data System (ADS)

    Boguta, Patrycja

    2016-04-01

    *The studies were partly carried out within the framework of a research project. The project was financed from funds of National Science Center on the base of decision number DEC-2013/11/D/NZ9/02545. Fluorescence spectroscopy belongs to modern, non-destructive, rapid and relatively cheap methods, as well as for many years it was successfully used in studies of organic compounds in the fields of medicine, biology and chemistry. On the other hand, soil organic matter is a group of compounds with a complex spatial structure showing a large number of groups with different kinds of fluorophores. This could suggest the possibility of application of fluorescence spectroscopy in assessing the quality of humic substances as well as in monitoring of their chemical transformations. The aim of study was chemical description of humic and fulvic acids based on fluorescence spectra, as well as an attempt of evaluation of changes occurring under the influence of different pH and during interactions with various concentrations of metal. The humic and fulvic acids were isolated from chemically different soils. The measurements were carried out on Hitachi fluorescence spectrometer in solutions with a concentration of humic acids 40mg dm-3, at pH from 3 to 7, and for the evaluation of the metal impact: with increasing Zn concentrations (0-50mg dm-3). The fluorescence spectra were recorded in the form of synchronous and emission-excitation matrices (EEM). Studies have shown the presence of different groups of fluorophores. Synchronous spectra were characterized by a well-separated bands showing fluorescence in the area of low, medium and high wavelengths, suggesting the presence of structures, both weakly and strongly humified. EEM spectra revealed map of fluorophores within wide ranges of emission and excitation. Fluorophores differed in both position and intensity. The highest intensity was observed for compounds with the lowest humification degree which might be due to high amount

  13. 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.

  14. X-ray fluorescence/Auger-electron coincidence spectroscopy of vacancy cascades in atomic argon

    SciTech Connect

    Arp, U.; LeBrun, T.; Southworth, S.H.; Jung, M.; MacDonald, M.A.

    1996-12-01

    Argon L{sub 2.3}-M{sub 2.3}M{sub 2.3} Auger-electron spectra were measured in coincidence with K{alpha} fluorescent x-rays in studies of Ar K-shell vacancy decays at several photon energies above the K-threshold and on the 1s-4p resonance in atomic argon. The complex spectra recorded by conventional electron spectroscopy are greatly simplified when recorded in coincidence with fluorescent x-rays, allowing a more detailed analysis of the vacancy cascade process. The resulting coincidence spectra are compared with Hartree-Fock calculations which include shake-up transitions in the resonant case. Small energy shifts of the coincidence electron spectra are attributed to post-collision interaction with 1s photoelectrons.

  15. Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

    PubMed

    Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

    2015-01-27

    Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy. PMID:25506739

  16. Synthesis of Ag clusters in microemulsions: A time-resolved UV vis and fluorescence spectroscopy study

    NASA Astrophysics Data System (ADS)

    Ledo, Ana; Martínez, F.; López-Quintela, M. A.; Rivas, J.

    2007-09-01

    The combined use of the microemulsion technique and the kinetic control allows the preparation of small silver clusters. By using UV-vis and fluorescence spectroscopy the main stages by which the clusters grow, before the formation of nanoparticles, were elucidated. Transmission electron microscopy (TEM) and scanning tunnelling microscopy (STM) were used to further characterize the samples. Two main stages were clearly identified, which are associated with: (1) the formation of Ag n clusters with n<10, which self-aggregate into one atom high 2D nanodiscs of 3.2 nm size and (2) Ag n clusters, which self-aggregate into 3D nanostructures of 1.5 nm in size. The fluorescence properties observed with both stages show that the formed clusters are small enough to display a molecule-like behaviour.

  17. Ultrafast Dynamics of Polythiophene with Phenyl Vinylene Branches Studied by Femtosecond Fluorescence Spectroscopy in Solution

    NASA Astrophysics Data System (ADS)

    Chu, Sai-Sai; Gao, Chao; Wang, Shu-Feng; Gong, Qi-Huang

    2011-11-01

    Two polythiophene based polymers, poly[(3-[2-[4-(2-ethyl-hexyloxy)-phenyl]-vinyl]-thiophene)-co-thiophene] (PT1) and poly(3-[2-[4-(2-ethyl-hexyloxy)-phenyl]-vinyl]-thiophene) (PT2), are synthesized and investigated by static, picosecond fluorescence spectroscopies and the femtosecond up-conversion technique in solution. Compared with pristine poly(3-hexylthiophene) (P3HT), PT1 and PT2, in which the main chains are decorated with phenyl vinylene present a ‘camel back’ structure in the absorption spectra. Phenyl vinylene side chains induce a new process of charge transfer, chain twisting motion and defect-induced fluorescence quenching at time scales of 1 ps, 10 ps and 150 ps, respectively.

  18. Fluorescence spectroscopy to study dissolved organic matter interactions with agrochemicals applied in Swiss vineyards.

    PubMed

    Daouk, Silwan; Frege, Carla; Blanc, Nicolas; Mounier, Stéphane; Redon, Roland; Merdy, Patricia; Lucas, Yves; Pfeifer, Hans-Rudolf

    2015-06-01

    UV/Vis fluorescence spectroscopy was used to study the possible interactions of dissolved organic matter (DOM) with the herbicide glyphosate and copper-based fungicide used in vineyards. The study focused on the role of DOM in the transport of these micropollutants from parcels to surface waters (river, lake). Soil solution and river water samples were collected in the Lavaux vineyard area, western Switzerland. Their fluorescence excitation emission matrices (EEM) were decomposed using parallel factor (PARAFAC) analysis, and compared to their content in glyphosate and copper. PARAFAC analysis of EEM of both types of samples showed the contribution of protein-like and humic-like fluorophores. In soil water samples, complexes between fulvic-like and humic-like fluorophores of DOM, copper, and glyphosate were likely formed. In surface water, DOM-copper and glyphosate-copper interactions were observed, but not between glyphosate and DOM. PMID:25592914

  19. Fluorescence Spectroscopy of the Retina for the Screening of Bovine Spongiform Encephalopathy.

    PubMed

    Bhattacharjee, Ujjal; Graham, Catherine; Czub, Stefanie; Dudas, Sandor; Rasmussen, Mark A; Casey, Thomas A; Petrich, Jacob W

    2016-01-13

    Transmissible spongiform encephalopathies (TSE) are progressive, neurodegenerative disorders, of which bovine spongiform encephalopathy (BSE) is of special concern because it is infectious and debilitating to humans. The possibility of using fluorescence spectroscopy to screen for BSE in cattle was explored. Fluorescence spectra from the retinas of experimentally infected BSE-positive cattle with clinical disease were compared with those from both sham-inoculated and non-inoculated BSE-negative cattle. The distinct intensity difference of about 4-10-fold between the spectra of the BSE-positive and the BSE-negative (sham-inoculated and non-inoculated) eyes suggests the basis for a means of developing a rapid, noninvasive examination of BSE in particular and TSEs in general. PMID:26623498

  20. Application of fluorescence spectroscopy for on-line bioprocess monitoring and control

    NASA Astrophysics Data System (ADS)

    Boehl, Daniela; Solle, D.; Toussaint, Hans J.; Menge, M.; Renemann, G.; Lindemann, Carsten; Hitzmann, Bernd; Scheper, Thomas-Helmut

    2001-02-01

    12 Modern bioprocess control requires fast data acquisition and in-time evaluation of bioprocess variables. On-line fluorescence spectroscopy for data acquisition and the use of chemometric methods accomplish these requirements. The presented investigations were performed with fluorescence spectrometers with wide ranges of excitation and emission wavelength. By detection of several biogenic fluorophors (amino acids, coenzymes and vitamins) a large amount of information about the state of the bioprocess are obtained. For the evaluation of the process variables partial least squares regression is used. This technique was applied to several bioprocesses: the production of ergotamine by Claviceps purpurea, the production of t-PA (tissue plasminogen activator) by animal cells and brewing processes. The main point of monitoring the brewing processes was to determine the process variables cell count and extract concentration.

  1. Absolute OH density determination by laser induced fluorescence spectroscopy in an atmospheric pressure RF plasma jet

    NASA Astrophysics Data System (ADS)

    Xiong, Q.; Nikiforov, A. Yu.; Li, L.; Vanraes, P.; Britun, N.; Snyders, R.; Lu, X. P.; Leys, C.

    2012-11-01

    In this paper, the ground state OH density is measured in high pressure plasma by laser-induced fluorescence (LIF) spectroscopy. The OH density determination is based on the simulation of the intensity fraction of fluorescence from the laser-excited level of OH (A) in the total detected LIF signal. The validity of this approach is verified in an atmospheric pressure Ar + H2O plasma jet sustained by a 13.56 MHz power supply. The transition line P1 (4) from OH (A, v' = 1, J' = 3) → OH (X, v'' = 0, J'' = 4) is used for the LIF excitation. The absolute OH density is determined to be 2.5 × 1019 m-3 at 1 mm away from the jet nozzle. It corresponds to a dissociation of 0.06% of the water vapor in the working gas. Different mechanisms of OH (X) production in the core of the plasma jet are discussed and analyzed.

  2. Measurement of the temperature-dependent diffusion properties of nanoparticles by using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

    Changes in the diffusion properties of three kinds of fluorescent particles, Alexa Fluor 647, Q-dots (quantum dots), and beads, with temperature were investigated with a home-built fluorescence correlation spectroscopy (FCS) system based on a confocal microscope. In all samples, as the temperature was increased, the diffusion times were reduced, indicating an increase in the diffusion coefficient. In particular, of all the particles, Alexa Fluor 647 having the smallest size of ˜1 nm, showed a hydrodynamic radius that increased with increasing temperature of the solvent. However, for the Q-dots and beads with larger sizes, the hydrodynamic radius of the particles was inversely proportional to the temperature. These results show that diffusion coefficient obtained by changing the temperature has an influence on the hydrodynamic radius of the particles.

  3. Artificial neural networks for processing fluorescence spectroscopy data in skin cancer diagnostics

    NASA Astrophysics Data System (ADS)

    Lenhardt, L.; Zeković, I.; Dramićanin, T.; Dramićanin, M. D.

    2013-11-01

    Over the years various optical spectroscopic techniques have been widely used as diagnostic tools in the discrimination of many types of malignant diseases. Recently, synchronous fluorescent spectroscopy (SFS) coupled with chemometrics has been applied in cancer diagnostics. The SFS method involves simultaneous scanning of both emission and excitation wavelengths while keeping the interval of wavelengths (constant-wavelength mode) or frequencies (constant-energy mode) between them constant. This method is fast, relatively inexpensive, sensitive and non-invasive. Total synchronous fluorescence spectra of normal skin, nevus and melanoma samples were used as input for training of artificial neural networks. Two different types of artificial neural networks were trained, the self-organizing map and the feed-forward neural network. Histopathology results of investigated skin samples were used as the gold standard for network output. Based on the obtained classification success rate of neural networks, we concluded that both networks provided high sensitivity with classification errors between 2 and 4%.

  4. Probe diffusion in polymer solutions and hydrogels using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Michelman-Ribeiro, Ariel; Boukari, Hacene; Horkay, Ferenc; Nossal, Ralph

    2006-03-01

    We apply fluorescence correlation spectroscopy (FCS) to measure the diffusion of small fluorescent probes (TAMRA, Mw = 430 Da; dextran, Mw = 10 kDa) in poly(vinyl alcohol) (PVA) solutions and hydrogels. PVA is a linear, neutral, biocompatible polymer, whose hydrogels have many biotechnology applications, such as drug-delivery devices and tissue scaffolds. The FCS measurements indicate that the probe diffusion decreases when the polymer solution is cross-linked. Further, the more the polymer chains are cross-linked, the slower the particles diffuse. These results suggest that the cross-link density, which is often ignored in the analysis of probe diffusion data in gels, must be taken into account. Remarkably, we find that the apparent diffusion time and the elastic modulus of the gels show a linear correlation.

  5. What information is contained in the fluorescence correlation spectroscopy curves, and where

    NASA Astrophysics Data System (ADS)

    Khadem, S. M. J.; Hille, C.; Löhmannsröben, H.-G.; Sokolov, I. M.

    2016-08-01

    We discuss the application of fluorescence correlation spectroscopy (FCS) for characterization of anomalous diffusion of tracer particles in crowded environments. While the fact of anomaly may be detected by the standard fitting procedure, the value of the exponent α of anomalous diffusion may be not reproduced correctly for non-Gaussian anomalous diffusion processes. The important information is however contained in the asymptotic behavior of the fluorescence autocorrelation function at long and at short times. Thus, analysis of the short-time behavior gives reliable values of α and of lower moments of the distribution of particles' displacement, which allows us to confirm or reject its Gaussian nature. The method proposed was tested on the FCS data obtained in artificial crowded fluids and in living cells.

  6. Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

    PubMed Central

    Qian, H; Elson, E L; Frieden, C

    1992-01-01

    Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316). PMID:1420920

  7. Fluorescence spectroscopy of soil pellets : The use of CP/PARAFAC.

    NASA Astrophysics Data System (ADS)

    Mounier, Stéphane; Nicolodeli, Gustavo; Redon, Roland; Hacherouf, Kalhed; Milori, Debora M. B. P.

    2014-05-01

    Fluorescence spectroscopy is one of the most sensitive techniques available for analytical purposes. It is relatively easy to implement, phenomenologically straightforward and well investigated. Largely non-invasive and fast, so that it can be useful for environmental applications. Fluorescence phenomenon is highly probable in molecular systems containing atoms with lone pairs of electrons such as C=O, aromatic, phenolic, quinone and more rigid unsaturated conjugated systems. These functional groups are present in humic substances (HS) from soils (Senesi, 1990; N. Senesi et al., 1991) and represent the main fluorophors of Soil Organic Matter (SOM). The extension of the conjugated electronic system, the level of heteroatom substitution and type and number of substituting groups under the aromatic rings strongly affect the intensity and wavelength of molecular fluorescence. However, to analyse the SOM it is generally done a chemical extraction that allows measuring the fluorescence response of the liquid extract. To avoid this fractionation of the SOM, Milori et al. (2006) proposed the application of laser induced fluorescence spectroscopy (LIFS) in whole soil. This work intends to assess the technical feasibility of 3D fluorescence spectroscopy using lamp for excitation to analyse solids opaque samples prepared with different substances. Seventy four (74) solid samples were prepared from different mixtures of boric acid (BA), humic substance acid and tryptophan (TRP) powder. The compounds were mixture and a pellet was done by using pressure (8 ton). The pellets were measured using a spectrofluorimeter HITACHI F4500, and a 3D fluorescence tensor was done from emission spectra (200-600 nm) with excitation range from 200 to 500 nm. The acquisition parameters were: step at 5 nm, scan speed at 2400 nm.min-1, response time at 0.1 s, excitation and emission slits at 5 nm and photomultiplier voltage at 700 V. Furthermore, measures of Laser-induced Fluorescence were

  8. Detection of citrus canker and Huanglongbing using fluorescence imaging spectroscopy and support vector machine technique.

    PubMed

    Wetterich, Caio Bruno; Felipe de Oliveira Neves, Ruan; Belasque, José; Marcassa, Luis Gustavo

    2016-01-10

    Citrus canker and Huanglongbing (HLB) are citrus diseases that represent a serious threat to the citrus production worldwide and may cause large economic losses. In this work, we combined fluorescence imaging spectroscopy (FIS) and a machine learning technique to discriminate between these diseases and other ordinary citrus conditions that may be present at citrus orchards, such as citrus scab and zinc deficiency. Our classification results are highly accurate when discriminating citrus canker from citrus scab (97.8%), and HLB from zinc deficiency (95%). These results show that it is possible to accurately identify citrus diseases that present similar symptoms. PMID:26835778

  9. Effect of different agents onto multidrug resistant cells revealed by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Boutin, C.; Roche, Y.; Jaffiol, R.; Millot, J.-M.; Millot, C.; Plain, J.; Deturche, R.; Jeannesson, P.; Manfait, M.; Royer, P.

    Fluorescence correlation spectroscopy (FCS), which is a sensitive and non invasive technique, has been used to characterize the plasma membrane fluidity and heterogeneity of multidrug resistant living cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been analyzed. Firstly, we reveal a modification of plasma membrane microviscosity according to the addition of a fluidity modulator, benzyl alcohol. In the other hand, revertant such as verapamil and cyclosporin-A appears to act more specifically on the slow diffusion sites as microdomains.

  10. Study on the interaction of anticancer drug mitoxantrone with DNA by fluorescence and Raman spectroscopies

    NASA Astrophysics Data System (ADS)

    Tang, Lingjuan; Sun, Zhenrong; Guo, Jianyu; Wang, Zugeng

    2006-02-01

    Mitoxantrone, a clinically useful antitumour antibiotic for leukaemia and breast cancer, has received more attentions. In this paper, the interaction between mitoxantrone and calf thymus DNA is investigated by Raman and fluorescence spectroscopies, and the binding site of mitoxantrone to calf thymus DNA is explored. The results showed that mitoxantrone interacts with calf thymus DNA bases by the intercalation of anthracycline into the base pair plane of adenine (A) and thymine (T), and it results in the disruption of the hydrogen bonds between calf thymus DNA bases, and thus the calf thymus DNA double-strand can be disrupted into the B-form DNA double-strand segments.

  11. Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy.

    PubMed

    Bixler, Joel N; Cone, Michael T; Hokr, Brett H; Mason, John D; Figueroa, Eleonora; Fry, Edward S; Yakovlev, Vladislav V; Scully, Marlan O

    2014-05-20

    Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment. PMID:24799690

  12. Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy

    PubMed Central

    Bixler, Joel N.; Cone, Michael T.; Hokr, Brett H.; Mason, John D.; Figueroa, Eleonora; Fry, Edward S.; Yakovlev, Vladislav V.; Scully, Marlan O.

    2014-01-01

    Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment. PMID:24799690

  13. On the measurement of particle number and mobility in nonideal solutions by fluorescence correlation spectroscopy.

    PubMed Central

    Abney, J R; Scalettar, B A; Hackenbrock, C R

    1990-01-01

    Interparticle interactions are incorporated into the theoretical description of the initial amplitude, G(0), of the normalized fluorescence correlation spectroscopy autocorrelation function. Measurements of particle number, aggregate size, and interaction-dependent diffusion are then analyzed in the context of this generalized theory. It is shown that the neglect of interactions can introduce order-of-magnitude errors into estimates of particle number and aggregate size. It is also shown that measurement of G(0) provides an essentially unique method for testing the validity of theories of interaction-dependent membrane protein diffusion. PMID:2383634

  14. Transient absorption spectroscopy detection of sensitized delayed fluorescence in chiral benzophenone/naphthalene systems

    NASA Astrophysics Data System (ADS)

    Bonancía, Paula; Jiménez, M. Consuelo; Miranda, Miguel A.

    2011-10-01

    Transient absorption spectroscopy has proven to be a powerful tool to investigate the formation and decay of excited singlet states upon triplet-triplet annihilation, following T-T energy transfer from a selectively excited sensitizer. Thus, upon selective excitation of benzophenone (BZP) by laser flash photolysis (LFP) at λ = 355 nm in the presence of naphthalene (NPT), a negative band centered at 340 nm has been detected, with growth and decay in the microsecond timescale. It has been assigned to the P-type NPT delayed-fluorescence. In the case of chiral BZP/NPT systems, stereodifferentiation has been observed in the kinetics of the involved photophysical processes.

  15. 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

  16. Tissue diagnosis using power-sharing multifocal Raman micro-spectroscopy and auto-fluorescence imaging

    PubMed Central

    Sinjab, Faris; Kong, Kenny; Gibson, Graham; Varma, Sandeep; Williams, Hywel; Padgett, Miles; Notingher, Ioan

    2016-01-01

    We describe a multifocal Raman micro-spectroscopy detection method based on a digital micromirror device, which allows for simultaneous “power-sharing” acquisition of Raman spectra from ad hoc sampling points. As the locations of the points can be rapidly updated in real-time via software control of a liquid-crystal spatial light modulator (LC-SLM), this technique is compatible with automated adaptive- and selective-sampling Raman spectroscopy techniques, the latter of which has previously been demonstrated for fast diagnosis of skin cancer tissue resections. We describe the performance of this instrument and show examples of multiplexed measurements on a range of test samples. Following this, we show the feasibility of reducing measurement time for power-shared multifocal Raman measurements combined with confocal auto-fluorescence imaging to provide guided diagnosis of tumours in human skin samples. PMID:27570692

  17. Tissue diagnosis using power-sharing multifocal Raman micro-spectroscopy and auto-fluorescence imaging.

    PubMed

    Sinjab, Faris; Kong, Kenny; Gibson, Graham; Varma, Sandeep; Williams, Hywel; Padgett, Miles; Notingher, Ioan

    2016-08-01

    We describe a multifocal Raman micro-spectroscopy detection method based on a digital micromirror device, which allows for simultaneous "power-sharing" acquisition of Raman spectra from ad hoc sampling points. As the locations of the points can be rapidly updated in real-time via software control of a liquid-crystal spatial light modulator (LC-SLM), this technique is compatible with automated adaptive- and selective-sampling Raman spectroscopy techniques, the latter of which has previously been demonstrated for fast diagnosis of skin cancer tissue resections. We describe the performance of this instrument and show examples of multiplexed measurements on a range of test samples. Following this, we show the feasibility of reducing measurement time for power-shared multifocal Raman measurements combined with confocal auto-fluorescence imaging to provide guided diagnosis of tumours in human skin samples. PMID:27570692

  18. Fluorescent properties and spontaneous Raman spectroscopy of new ketocyanine probes in organic solvents

    NASA Astrophysics Data System (ADS)

    Nemkovich, N. A.; Sobchuk, A. N.; Khodasevich, I. A.

    2006-11-01

    We have used fluorescence spectroscopy and spontaneous Raman spectroscopy to study the characteristics of two ketocyanine dyes: 2,5-di[(E)-1-(4-diethylaminophenyl)methylidene]-1-cyclopentanone (CPET) and 2-[(E)-1-(4-diethylaminophenyl)methylidene]-5-{(E)-1-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecalin) phenyl]methylidene}-1-cyclopentanone (CPMR) in organic solvents. The position of their electronic spectra depends strongly on the polarity of the solvent. We measured the dipole moments of the dyes in the equilibrium ground state and the Franck-Condon excited state. In mixtures of neutral nonpolar toluene with aprotic polar dimethylsulfoxide, we observe inhomogeneous broadening of the electronic spectra for the indicated compounds, due to fluctuations in solution of the intermolecular interaction energy. The time-resolved characteristics of fluorescence obtained suggest formation of an intermolecular hydrogen bond between the dye and the surrounding medium in a toluene-ethanol mixture. We measured the Raman spectra of CPET and CPMR in different organic solvents. The most intense lines in the 1582 1591 cm-1 region can be assigned to stretching of the phenyl rings of the molecules; the lines in the 831 842 cm-1 region can be assigned to a cyclopentanone ring mode; the lines at 1186 1195 cm-1 can be assigned to stretching of the =C-C-bond of the phenyl ring and rocking of the H atoms of the phenyl ring. We have observed that the position and width of the lines for the stretching vibrations of the ketocyanines depend substantially on the polarity of the surrounding medium. The studied dyes can be used as probes for studying different biological systems by site-selective laser spectroscopy and Raman spectroscopy. The fact that these two methods can be used simultaneously for diagnostics of biosystems is an important advantage of ketocyanine dyes compared with other known probes.

  19. Highly Sensitive Determination of Hydrogen Peroxide and Glucose by Fluorescence Correlation Spectroscopy

    PubMed Central

    Watabe, Satoshi; Sakamoto, Yuki; Morikawa, Mika; Okada, Ryuichi; Miura, Toshiaki; Ito, Etsuro

    2011-01-01

    Background Because H2O2 is generated by various oxidase-catalyzed reactions, a highly sensitive determination method of H2O2 is applicable to measurements of low levels of various oxidases and their substrates such as glucose, lactate, glutamate, urate, xanthine, choline, cholesterol and NADPH. We propose herein a new, highly sensitive method for the measurement of H2O2 and glucose using fluorescence correlation spectroscopy (FCS). Methodology/Principal Findings FCS has the advantage of allowing us to determine the number of fluorescent molecules. FCS measures the fluctuations in fluorescence intensity caused by fluorescent probe movement in a small light cavity with a defined volume generated by confocal illumination. We thus developed a highly sensitive determination system of H2O2 by FCS, where horseradish peroxidase (HRP) catalyzes the formation of a covalent bond between fluorescent molecules and proteins in the presence of H2O2. Our developed system gave a linear calibration curve for H2O2 in the range of 28 to 300 nM with the detection limit of 8 nM. In addition, by coupling with glucose oxidase (GOD)-catalyzed reaction, the method allows to measure glucose in the range of 80 nM to 1.5 µM with detection limit of 24 nM. The method was applicable to the assay of glucose in blood plasma. The mean concentration of glucose in normal human blood plasma was determined to be 4.9 mM. Conclusions/Significance In comparison with commercial available methods, the detection limit and the minimum value of determination for glucose are at least 2 orders of magnitude more sensitive in our system. Such a highly sensitive method leads the fact that only a very small amount of plasma (20 nL) is needed for the determination of glucose concentration in blood plasma. PMID:21850246

  20. A study of diagnostic criteria established for two oral mucous diseases by HMME-fluorescence spectroscopy.

    PubMed

    Lv, Moyang; Qin, Feng; Mao, Limin; Zhang, Lei; Lv, Shaohua; Jin, Jian; Zhang, Zhiguo

    2015-11-01

    Malignant oral ulcers are common pathological occurrence in oral and maxillofacial tumors. A noninvasive method for diagnosis of malignant oral ulcers was developed in the study, which is based on hematoporphyrin monomethylether (HMME) fluorescence spectroscopy. The objective of this work is to determine the feasibility of this method in differentiating the malignant tissues from the inflammatory ones in the hamster cheek pouch model. Adult hamsters were used for the study and a cheek pouch model was established. For the malignant model, the 9, 10-dimethyl-1, 2-benzanthracene carcinogenesis was applied to one cheek pouch for 10 weeks (N = 35). The simple ulcers were created on buccal cheek mucosa in a simple manner (N = 10). Prior to sacrifice, HMME solution was injected into the tissues. The induced fluorescence spectra of the cheek tissues were recorded by a fiber spectrometer with excitation at 405 nm. A spectral algorithm was used to eliminate the effect of autofluorescence, and a spectral parameter S was selected as diagnostic criterion. After fluorescence measurement, the animals were sacrificed and the measured tissues were collected. Histological staining was performed and the results of histopathological evaluation were documented. The diagnostic criteria that reflected the fluorescence intensity were set as follows: normal, S ≤ 10; simple ulcer, 230 ≤ S ≤ 290; and malignant ulcer, 140 ≤ S ≤ 200. The sensitivity and specificity of this detection method was verified by scalpel biopsy, and the overall accuracy was over 90%. The results of this study showed that the fluorescence spectroscopic method implemented by HMME can accurately differentiate the two kinds of clinically indistinguishable diseases. PMID:26071098

  1. Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Siegel, Amanda P.; Hays, Nicole M.; Day, Richard N.

    2013-02-01

    The epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by the heterochromatin protein 1 (HP1). In earlier studies, we showed that the CCAAT/enhancer-binding protein alpha (C/EBPα), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1α. Here, deletion and mutagenesis are combined with live-cell imaging approaches to characterize these protein interactions. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1α and the BZip protein, and co-expression studies indicated that the mobile fractions of these nuclear proteins diffuse independently of one another. The steady-state interactions of these proteins in regions of heterochromatin were monitored using Förster resonance energy transfer (FRET). A point mutation in HP1α, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1α W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1α or HP1αW174A. The functional significance of these interactions is discussed.

  2. Photon and fluorescence correlation spectroscopy and light scattering of eye-lens proteins at moderate concentrations.

    PubMed Central

    Andries, C; Guedens, W; Clauwaert, J; Geerts, H

    1983-01-01

    The bovine eye-lens protein, alpha L-crystallin, has been studied with photon correlation spectroscopy to obtain the mutual diffusion coefficient, Dm, with fluorescence correlation spectroscopy to determine the tracer diffusion coefficient, DT, and with light scattering to get the isothermal osmotic compressibility (delta pi/delta c) P,T. The concentration dependence of Dm, DT, and (delta pi/delta c) P,T up to a volume fraction phi of the protein of 2.5 x 10(-2) has been interpreted on the basis of four different interaction potentials: (a) an extended hard-sphere potential; (b) a shielded Coulomb potential; (c) a shielded Coulomb interaction where the effect of counterions is included; (d) a simple mixed potential. The three parameters Dm, DT, and (delta pi/delta c) P,T have also been combined in the generalized Stokes-Einstein equation, Dm = [(delta pi/delta c)P,T . (1--phi) . (DT)]/(kappa B . T). Our results indicate that, in the case that photon correlation spectroscopy gives the mutual diffusion coefficient Dm, the applicability of the Stokes-Einstein equation can be questioned; or that, when one assumes the Stokes-Einstein equation to be valid, there is significant discrepancy between the result of photon correlation spectroscopy and Dm. PMID:6626672

  3. Technique for real-time tissue characterization based on scanning multispectral fluorescence lifetime spectroscopy (ms-TRFS)

    PubMed Central

    Ma, Dinglong; Bec, Julien; Gorpas, Dimitris; Yankelevich, Diego; Marcu, Laura

    2015-01-01

    We report a novel technique for continuous acquisition, processing and display of fluorescence lifetimes enabling real-time tissue diagnosis through a single hand held or biopsy fiber-optic probe. A scanning multispectral time-resolved fluorescence spectroscopy (ms-TRFS) with self-adjustable photon detection range was developed to account for the dynamic changes of fluorescence intensity typically encountered in clinical application. A fast algorithm was implemented in the ms-TRFS software platform, providing up to 15 Hz continuous display of fluorescence lifetime values. Potential applications of this technique, including biopsy guidance, and surgical margins delineation were demonstrated in proof-of-concept experiments. Current results showed accurate display of fluorescence lifetimes values and discrimination of distinct fluorescence markers and tissue types in real-time (< 100 ms per data point). PMID:25798320

  4. Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy

    PubMed Central

    Michalet, Xavier; Ingargiola, Antonino; Colyer, Ryan A.; Scalia, Giuseppe; Weiss, Shimon; Maccagnani, Piera; Gulinatti, Angelo; Rech, Ivan; Ghioni, Massimo

    2014-01-01

    Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements. PMID:25309114

  5. The use of solid-phase fluorescence spectroscopy in the characterisation of organic matter transformations.

    PubMed

    Albrecht, R; Verrecchia, E; Pfeifer, H-R

    2015-03-01

    Given its high sensitivity and non-destructive nature, fluorescence excitation-emission matrix (EEM) spectroscopy is widely used to differentiate changes and transformations of dissolved or water-extracted organic matter (OM) in natural environments. The same technique applied directly on solid samples (solid-phase fluorescence spectroscopy, SPF-EEM) provides accurate results when used with pharmaceutical products or food samples, but only a few studies have considered natural OM. This study reports on the use of SPF-EEM on solid compost samples and emphasises the way the different maturation phases can be distinguished with fluorophores closely resembling those found in dissolved samples. A very good correlation has been found with data from Rock-Eval pyrolysis, nuclear magnetic resonance ((13)C CPMAS NMR), and humic-fulvic acid ratios determined by conventional NaOH-extraction. SPF-EEM appears as a much simpler method than the conventional ones to detect transformations in natural OM samples with low mineral contents. However, direct application to soil samples requires some additional studies. PMID:25618693

  6. Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy.

    PubMed

    Michalet, Xavier; Ingargiola, Antonino; Colyer, Ryan A; Scalia, Giuseppe; Weiss, Shimon; Maccagnani, Piera; Gulinatti, Angelo; Rech, Ivan; Ghioni, Massimo

    2014-11-01

    Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements. PMID:25309114

  7. UV laser-induced fluorescence spectroscopy and laser Doppler flowmetry in the diagnostics of alopecia

    NASA Astrophysics Data System (ADS)

    Skomorokha, Diana P.; Pigoreva, Yulia N.; Salmin, Vladimir V.

    2016-04-01

    Development of optical biopsy methods has a great interest for medical diagnostics. In clinical and experimental studies it is very important to analyze blood circulation quickly and accurately, thereby laser Doppler flowmetry (LDF) is widely used. UV laser-induced fluorescence spectroscopy (UV LIFS) is express highly sensitive and widely-spread method with no destructive impact, high excitation selectivity and the possibility to use in highly scattering media. The goal of this work was to assess a correlation of UV laser-induced fluorescence spectroscopy and laser Doppler flowmetry parameters, and a possibility to identify or to differentiate various types of pathological changes in tissues according to their autofluorescence spectra. Three groups of patients with diffuse (symptomatic) alopecia, androgenic alopecia, and focal alopecia have been tested. Each groups consisted of not less than 20 persons. The measurements have been done in the parietal and occipital regions of the sculls. We used the original automated spectrofluorimeter to record autofluorescence spectra, and standard laser Doppler flowmeter BLF-21 (Transonic Systems, Inc., USA) to analyze the basal levels of blood circulation. Our results show that UV LIFS accurately distinguishes the zones with different types of alopecia. We found high correlation of the basal levels of blood circulation and the integrated intensity of autofluorescence in the affected tissue.

  8. 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

  9. Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy.

    PubMed

    Sankaran, Jagadish; Manna, Manoj; Guo, Lin; Kraut, Rachel; Wohland, Thorsten

    2009-11-01

    Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid "platforms"). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), a widely used spectroscopic technique to measure membrane dynamics, has the required time resolution but lacks imaging capabilities. A promising solution is the recently introduced method known as imaging total internal reflection (ITIR)-FCS, which can probe diffusion phenomena in lipid membranes with good temporal and spatial resolution. In this work, we extend ITIR-FCS to perform ITIR fluorescence cross-correlation spectroscopy (ITIR-FCCS) between pixel areas of arbitrary shape and derive a generalized expression that is applicable to active transport and diffusion. ITIR-FCCS is applied to model systems exhibiting diffusion, active transport, or a combination of the two. To demonstrate its applicability to live cells, we observe the diffusion of a marker, the sphingolipid-binding domain (SBD) derived from the amyloid peptide Abeta, on live neuroblastoma cells. We investigate the organization and dynamics of SBD-bound lipid microdomains under the conditions of cholesterol removal and cytoskeleton disruption. PMID:19883607

  10. Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

    NASA Astrophysics Data System (ADS)

    Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

    2001-05-01

    In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

  11. Use of time-resolved fluorescence spectroscopy to evaluate diagnostic value of collagen degradation products.

    PubMed

    Sikora, Joanna; Cyrankiewicz, Michał; Wybranowski, Tomasz; Ziomkowska, Blanka; Ośmiałowski, Borys; Obońska, Ewa; Augustyńska, Beata; Kruszewski, Stefan; Kubica, Jacek

    2015-05-01

    The concentration of collagen degradation products (CDPs) may reflect the process of left ventricular remodeling (LVR). The aim of this study was to evaluate the potential diagnostic usefulness of time-resolved fluorescence spectroscopy (TRFS) in assessment of CDPs. The preliminary experiment was designed to establish if CDPs’ characteristics might be visible by mean fluorescence lifetime (FLT) in determined conditions. The in vitro model of CDPs was prepared by conducting the hydrolysis of type III collagen. The FLT of samples was measured by the time-resolved spectrometer Life Spec II with the subnanosecond pulsed 360-nm EPLED diode. The FLTs were obtained by deconvolution analysis of the data using a multiexponential model of fluorescence decay. In order to determine the limit of traceability of CDPs, a comparison of different collagen/plasma ratio in samples was performed. The results of our study showed that the increase of added plasma to hydrolyzed collagen extended the mean FLT. Thus, the diagnosis of LVR based on measurements using TRFS is possible. However, it is important to point out the experiment was preliminary and further investigation in this field of research is crucial. PMID:25764396

  12. Two-Dimensional Fluorescence Difference Spectroscopy to Characterize Nanoparticles and their Interactions.

    PubMed

    Hurst, Miranda N; DeLong, Robert K

    2016-01-01

    Two dimensional fluorescence difference spectroscopy (2D FDS) detects nanoparticle interactions following surface functionalization and biomolecule loading by generating a spectral signature of the fluorescent intensity per excitation and emission wavelengths. Comparing metal oxide nanoparticles revealed a unique spectral signature per material composition. 2D FDS showed to be sensitive to changes in surface properties between ZnO NPs synthesized by different methods. ZnO NP loaded with glycol chitosan, polyacrylic acid (PAA), or methoxy polyethylene glycol (mPEG) exhibited a distinct spectral signature shift. ZnO NP loaded with Torula Yeast RNA (TYRNA)(640 nm), polyinosinic: polycytidylic acid (pIC)(680 nm), or splice switching oligonucleotide (SSO)(650 nm) each revealed a shift in emission. Ras-Binding domain (RBD) at three concentrations (25, 37.5, 50 μg/mL) showed that fluorescent intensity was inversely related to the concentration of protein loaded. These data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions as a quality assurance tool. PMID:27624316

  13. 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 (Ka ) 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. PMID:26377428

  14. Confined diffusion in tubular structures analyzed by fluorescence correlation spectroscopy on a mirror

    NASA Astrophysics Data System (ADS)

    Etienne, Emilien; Lenne, Pierre-François; Sturgis, James N.; Rigneault, Hervé

    2006-06-01

    In fluorescence correlation spectroscopy (FCS) analysis it is generally assumed that molecular species diffuse freely in volumes much larger than the three-dimensional FCS observation volume. However, this standard assumption is not valid in many measurement conditions, particularly in tubular structures with diameters in the micrometer range, such as those found in living cells (organelles, dendrites) and microfluidic devices (capillaries, reaction chambers). As a result the measured autocorrelation functions (ACFs) deviate from those predicted for free diffusion, and this can shift the measured diffusion coefficient by as much as ~50% when the tube diameter is comparable with the axial extension of the FCS observation volume. We show that the range of validity of the FCS measurements can be drastically improved if the tubular structures are located in the close vicinity of a mirror on which FCS is performed. In this case a new fluctuation time in the ACF, arising from the diffusion of fluorescent probes in optical fringes, permits measurement of the real diffusion coefficient within the tubular structure without assumptions about either the confined geometry or the FCS observation volume geometry. We show that such a measurement can be done when the tubular structure contains at least one pair of dark and bright fringes resulting from interference between the incoming and the reflected excitation beams on the mirror surface. Measurement of the diffusion coefficient of the enhanced green fluorescent protein (EGFP) and IscS-EGFP in the cytoplasm of living Escherichia coli illustrates the capabilities of the technique.

  15. Confined diffusion in tubular structures analyzed by fluorescence correlation spectroscopy on a mirror

    SciTech Connect

    Etienne, Emilien; Lenne, Pierre-Francois; Sturgis, James N.; Rigneault, Herve

    2006-06-20

    In fluorescence correlation spectroscopy (FCS) analysis it is generally assumed that molecular species diffuse freely in volumes much larger than the three-dimensional FCS observation volume. However, this standard assumption is not valid in many measurement conditions, particularly in tubular structures with diameters in the micrometer range, such as those found in living cells (organelles, dendrites) and microfluidic devices (capillaries,reaction chambers). As a result the measured autocorrelation functions (ACFs) deviate from those predicted for free diffusion, and this can shift the measured diffusion coefficient by as much as {approx}50% when the tube diameter is comparable with the axial extension of the FCS observation volume. We show that the range of validity of the FCS measurements can be drastically improved if the tubular structures are located in the close vicinity of a mirror on which FCS is performed. In this case a new fluctuation time in the ACF, arising from the diffusion of fluorescent probes in optical fringes,permits measurement of the real diffusion coefficient within the tubular structure without assumptions about either the confined geometry orthe FCS observation volume geometry. We show that such a measurement can be done when the tubular structure contains at least one pair of dark and bright fringes resulting from interference between the incoming and the reflected excitation beams on the mirror surface. Measurement of the diffusion coefficient of the enhanced green fluorescent protein (EGFP) and IscS-EGFP in the cytoplasm of living Escherichiacoli illustrates the capabilities of the technique.

  16. Plasmon-Enhanced Raman and Fluorescence Spectroscopy with Gold and Silver Nanoparticles

    NASA Astrophysics Data System (ADS)

    Guerrero Hernandez, Ariel Rodrigo

    This thesis contains five major contributions to the field of plasmon-enhanced spectroscopy. We start with the report of a unique SERS study of the amino acid hydroxyproline and a deuterated analogue. Later, we move on to the exploration of a major new research path known as shell-isolated nanoparticle-enhanced fluorescence (SHINEF), consisting in the application of silica-shelled noble metal nanoparticles to achieve surface-enhanced fluorescence. The proof of concept of this technique is explained in one chapter. The two following chapters are devoted to the exploration of the plasmonic properties of SHINEF: spectral profile modification showing the close relationship between the observed enhanced fluorescence and the nanoparticle scattering. The SHIN particles are employed to experimentally prove the relationship between the SEF and SERS enhancement factors, theoretically predicted before, but never verified experimentally until now. The thesis ends with an investigation, in aqueous solutions, of several different factors that play a role in the origin of SEF, showing greater enhancement for SHINEF after inducing nanoparticle aggregation.

  17. Probing the conformation of protein (bFGF) precipitates by fluorescence spectroscopy.

    PubMed

    Shahrokh, Z; Eberlein, G; Wang, Y J

    1994-08-01

    Aggregation and precipitation are major events in the handling and aging of most protein pharmaceuticals. We demonstrate the utility of fluorescence spectroscopy in determining protein conformation in precipitates using basic fibroblast growth factor (bFGF) as an example. Conversion of the native to the soluble denatured from by chaotropes was accompanied by an increase in tryptophan emission. The emission spectra of resuspended precipitates were as reproducible as the spectra of the soluble form. The sum of emission spectra of native soluble bFGF and denatured precipitated bFGF was superimposable on the spectrum of the unfractionated suspension, suggesting that quantitative analysis of denatured aggregates in turbid protein formulations is possible. The ratio of tryptophan to tyrosine emissions increased with increasing extent of denaturation both in solution and in suspension. For example, salting out by ammonium sulphate increased the fluorescence index (indicative of denaturation) which was reversible upon dissolution. In addition, aging (35 degrees C) of bFGF in the presence of sulphated ligands produced precipitates with native-like fluorescence index, in contrast to denatured precipitates formed without ligands. PMID:7819377

  18. Characterization of simian virus 40 on its infectious entry pathway in cells using fluorescence correlation spectroscopy.

    PubMed

    Bernacchi, S; Mueller, G; Langowski, J; Waldeck, W

    2004-11-01

    SV40 (simian virus 40) is a double-stranded DNA virus and is strongly oncogenic in experimental animals. SV40 enters cells by atypical endocytosis mediated by caveolae, transporting the virus to its usual destination, namely the endoplasmic reticulum. The cellular mechanisms of capsid disassembly (uncoating) and deliverance of the viral genome into the cellular nucleus remain unknown. Here, we study (i) the formation of caveolae after viral infection and the diffusion of caveosome vesicles in the cytoplasm and (ii) the capsid disassembly and the mobility of the viral genome on its way to the nucleus, using fluorescence correlation spectroscopy. To follow the viral genome and capsids separately, the histone components of SV40 minichromosomes were labelled with enhanced yellow fluorescent protein and the capsid was labelled with a fluorescent red dye, Alexa568. We characterized the diffusion of caveosomes, the capsid disassembly process in the cytoplasm and the mobility of the viral genome in the nucleus, using two kinds of permissive cells. PMID:15494004

  19. Pancreatic tumor detection using hypericin-based fluorescence spectroscopy and cytology

    NASA Astrophysics Data System (ADS)

    Lavu, Harish; Geary, Kevin; Fetterman, Harold R.; Saxton, Romaine E.

    2005-04-01

    Hypericin is a novel, highly fluorescent photosensitizer that exhibits selective tumor cell uptake properties and is particularly resistant to photobleaching. In this study, we have characterized hypericin uptake in human pancreatic tumor cells with relation to incubation time, cell number, and drug concentration. Ex vivo hypericin based fluorescence spectroscopy was performed to detect the presence of MIA PaCa-2 pancreatic tumor cells in the peritoneal cavity of BALB/c nude mice, as well as to quantify gross tumor burden. Hypericin based cytology of peritoneal lavage samples, using both one and two photon laser confocal microscopy, demonstrated more than a two-fold increase in fluorescence emission of pancreatic tumor cells as compared to control samples. In vitro treatment of pancreatic cancer cells with hypericin based photodynamic therapy showed tumor cell cytotoxicity in a drug dose, incident laser power, and time dependent manner. For these experiments, a continuous wavelength solid-state laser source (532 nm) was operated at power levels in the range of 100-400 mW. Potential applications of hypericin in tumor diagnosis, staging, and therapy will be presented.

  20. Detection of polycyclic aromatic hydrocarbons (PAHs) in raw menhaden fish oil using fluorescence spectroscopy: Method development.

    PubMed

    Pena, Edwin A; Ridley, Lauren M; Murphy, Wyatt R; Sowa, John R; Bentivegna, Carolyn S

    2015-09-01

    Raw menhaden fish oil was developed for biomonitoring polycyclic aromatic hydrocarbons (PAHs) using fluorescence spectroscopy. Menhaden (Genus Brevoortia) were collected in 2010 and/or 2011 from Delaware Bay, New Jersey, USA; James River, Virginia, USA; Vermillion Bay, Louisiana, USA (VBLA); and Barataria Bay, Louisiana, USA (BBLA). Barataria Bay, Louisiana received heavy oiling from the Deepwater Horizon oil spill. Method development included determining optimal wavelengths for PAH detection, fish oil matrix interferences, and influence of solvent concentration on extraction. Results showed that some fish oils contained high molecular weight PAH-like compounds in addition to other fluorescent compounds such as albumin and vitamin A and vitamin E. None of these naturally occurring compounds interfered with detection of high molecular weight PAHs. However, data suggested that the lipid component of fish oil was altering fluorescence spectra by supporting the formation of PAH excimers. For example, the most intense excitation wavelength for hydroxypyrene shifted from Ex285/Em430 to Ex340/Em430. Comparison of Deepwater Horizon crude oil and fish oil spectra indicated that some fish oils contained crude oil-like PAHs. Using wavelengths of Ex360/Em430, fish oil concentrations were calculated as 3.92 μg/g, 0.61 μg/g, and 0.14 μg/g for a Delaware Bay sample, BBLA 2011, and VBLA 2011, respectively. Overall, these results supported using menhaden fish oil to track PAH exposures spatially and temporally. PMID:25867932

  1. Quantitative determination of the surfactant-induced split ratio of influenza virus by fluorescence spectroscopy.

    PubMed

    Lee, Kenny Kwon Ho; Sahin, Yusuf Ziya; Neeleman, Ronald; Trout, Bernhardt L; Kayser, Veysel

    2016-07-01

    The majority of marketed seasonal influenza vaccines are prepared using viruses that are chemically inactivated and treated with a surfactant. Treating with surfactants has important consequences: it produces 'split viruses' by solubilizing viral membranes, stabilizes free membrane proteins and ensures a low level of reactogenicity while retaining high vaccine potency. The formulation stability and potency of split influenza vaccines are largely determined by the specifics of this 'splitting' process; namely, the consequent conformational changes of proteins and interactions of solubilized particles, which may form aggregates. Robust methods to quantitatively determine the split ratio need to be developed before optimal splitting conditions can be investigated to streamline production of superior influenza vaccines. Here, we present a quantitative method, based on both steady-state and time-resolved fluorescence spectroscopy, to calculate the split ratio of the virus after surfactant treatment. We use the lipophilic dye Nile Red (NR) as a probe to elucidate molecular interactions and track changes in molecular environments. Inactivated whole influenza viruses obtained from a sucrose gradient were incubated with NR and subsequently treated with increasing concentrations of the surfactant Triton X-100 (TX-100) to induce virus splitting. NR's emission spectra showed that the addition of TX-100 caused ˜27 nm red-shifts in the emission peak, indicative of increasingly hydrophilic environments surrounding NR. The emission spectra of NR at different surfactant concentrations were analyzed with multi-peak fitting to ascertain the number of different micro-environments surrounding NR and track its population change in these different environments. Results from both the emission spectra and fluorescence lifetime spectroscopy revealed that NR showed presence in 3 distinct molecular environments. The split ratio of the virus was then calculated from the percentages of NR in

  2. A system for endoscopic mechanically scanned localized proton MR and light-induced fluorescence emission spectroscopies

    NASA Astrophysics Data System (ADS)

    Sonmez, Ahmet E.; Webb, Andrew G.; Spees, William M.; Ozcan, Alpay; Tsekos, Nikolaos V.

    2012-09-01

    Molecular and near-cellular modalities offer new opportunities in assessing living tissue in situ, and multimodality approaches, which offer complementary information, may lead to improved characterization of tissue pathophysiology benefiting diagnosis and focal therapy. However, many such modalities are limited by their low penetration through tissue, which has led to minimally invasive trans-cannula approaches to place the corresponding sensors locally at the area of interest. This work presents a system for performing localized fluorescence emission and proton magnetic resonance (MR) spectroscopies via endoscopic access. The in-house developed side-firing 1.9-mm wide dual-sensor integrates a three-fiber optical sensor for fluorescence emission optical spectroscopy and a 1-mm circular radiofrequency (RF) coil for localized MR proton spectroscopy. An MR-compatible manipulator was developed for carrying and mechanically translating the dual-sensor along a linear access channel. The hardware and software control of the system allows reconfigurable synchronization of the manipulator-assisted translation of the sensor, and MR and optical data collection. The manipulator serves as the mechanical link for the three modalities and MR images, MR spectra and optical spectra are inherently co-registered to the MR scanner coordinate system. These spectra were then used to generate spatio-spectral maps of the fluorophores and proton MR-signal sources in three-compartment phantoms with optically- and MR-visible, and distinguishable, materials. These data demonstrate a good spatial match between MR images, MR spectra and optical spectra along the scanned path. In addition to basic research, such a system may have clinical applications for assessing and characterizing cancer in situ, as well as guiding focal therapies.

  3. 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. PMID:20459282

  4. 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.

  5. Immunoglobulin surface-binding kinetics studied by total internal reflection with fluorescence correlation spectroscopy.

    PubMed Central

    Thompson, N L; Axelrod, D

    1983-01-01

    An experimental application of total internal reflection with fluorescence correlation spectroscopy (TIR/FCS) is presented. TIR/FCS is a new technique for measuring the binding and unbinding rates and surface diffusion coefficient of fluorescent-labeled solute molecules in equilibrium at a surface. A laser beam totally internally reflects at the solid-liquid interface, selectively exciting surface-adsorbed molecules. Fluorescence collected by a microscope from a small, well-defined surface area approximately 5 micron2 spontaneously fluctuates as solute molecules randomly bind to, unbind from, and/or diffuse along the surface in chemical equilibrium. The fluorescence is detected by a photomultiplier and autocorrelated on-line by a minicomputer. The shape of the autocorrelation function depends on the bulk and surface diffusion coefficients, the binding rate constants, and the shape of the illuminated and observed region. The normalized amplitude of the autocorrelation function depends on the average number of molecules bound within the observed area. TIR/FCS requires no spectroscopic or thermodynamic change between dissociated and complexed states and no extrinsic perturbation from equilibrium. Using TIR/FCS, we determine that rhodamine-labeled immunoglobulin and insulin each nonspecifically adsorb to serum albumin-coated fused silica with both reversible and irreversible components. The characteristic time of the most rapidly reversible component measured is approximately 5 ms and is limited by the rate of bulk diffusion. Rhodamine-labeled bivalent antibodies to dinitrophenyl (DNP) bind to DNP-coated fused silica virtually irreversibly. Univalent Fab fragments of these same antibodies appear to specifically bind to DNP-coated fused silica, accompanied by a large amount of nonspecific binding. TIR/FCS is shown to be a feasible technique for measuring absorption/desorption kinetic rates at equilibrium. In suitable systems where nonspecific binding is low, TIR

  6. Chemometric classification of Chinese lager beers according to manufacturer based on data fusion of fluorescence, UV and visible spectroscopies.

    PubMed

    Tan, Jin; Li, Rong; Jiang, Zi-Tao

    2015-10-01

    We report an application of data fusion for chemometric classification of 135 canned samples of Chinese lager beers by manufacturer based on the combination of fluorescence, UV and visible spectroscopies. Right-angle synchronous fluorescence spectra (SFS) at three wavelength difference Δλ=30, 60 and 80 nm and visible spectra in the range 380-700 nm of undiluted beers were recorded. UV spectra in the range 240-400 nm of diluted beers were measured. A classification model was built using principal component analysis (PCA) and linear discriminant analysis (LDA). LDA with cross-validation showed that the data fusion could achieve 78.5-86.7% correct classification (sensitivity), while those rates using individual spectroscopies ranged from 42.2% to 70.4%. The results demonstrated that the fluorescence, UV and visible spectroscopies complemented each other, yielding higher synergic effect. PMID:25872423

  7. Microneedles rollers as a potential device to increase ALA diffusion and PpIX production: evaluations by wide-field fluorescence imaging and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Gracielli Sousa, R. Phamilla; de Menezes, Priscila F. C.; Fujita, Alessandra K. L.; Requena, Michelle B.; Govone, Angelo Biassi; Escobar, André; de Nardi, Andrigo B.; Kurachi, Cristina; Bagnato, Vanderlei Salvador

    2014-03-01

    One of the limitations of topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is the poor ability to penetrate biological barriers of skin and the recurrence rates in treatments. This study aimed to identify possible signs of increased diffusion of ALA-induced PpIX by fluorescence images and fluorescence spectroscopy. The research was done using in vivo porcine skin model. Before the cream application, microholes was performed with microneedles rollers in only one direction, afterward the ALA cream was applied at a 2.5cm2 area in triplicate and an occlusive dressing was placed. PpIX production was monitored using fluorescence spectroscopy collected at skin surface after 70, 100, 140, and 180 minutes of ALA incubation. About 100 fluorescence spectra of each treatment were collected, distributed by about five points for each site. Wide-field fluorescence imaging was made after 70, 90, and 170 minutes after treatment. The results obtained by imaging analysis indicated increase of the PpIX diffusion in the skin surface using the microneedles rollers (MNs) before ALA application. Circular regions of red fluorescence around the microholes were observed. In addition, the fluorescence spectra showed a greater intensity (2 times as many) in groups microneedles rollers associated. In conclusion, our data shown greater homogeneity and PpIX production in the groups pre-treated with microneedles indicating that the technique can be used to greater uniformity of PpIX production throughout the area to be treated reducing the chances of recurrent tumor as well as has potential for decreasing the time of therapy. (FUNDING SUPPORT:CAPES, CNPq and FAPESP)

  8. 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. PMID:26695945

  9. Towards Environmental Microbial Analysis with Deep UV fluorescence and Raman Spectroscopy

    NASA Astrophysics Data System (ADS)

    Wanger, G.; Bhartia, R.; Orphan, V. J.; Rowe, A. R.

    2015-12-01

    The study of microbes from the environment is often facilitated by the fixation of samples prior to analyses in the laboratory. Samples not appropriately preserved can show dramatic changes e.g. unwanted growth, loss of biomass and sample degradation between collection and analysis. To move Deep-UV Raman analyses from model lab organisms to environmental samples the effect of preservation must be evaluated. Deep UV Raman and Fluorescence (i.e. excitation <250 nm) has been shown capable discriminating various types of microbes as well as giving some information on the growth stage of the culture. The fluorescence signal is typically 3-4 orders of magnitude more intense than the Raman signal and enables rapid location of bacteria on a surface and crudely split them into categories. However it suffers from broad spectral features making discrete classification of bacteria problematic. While a far weaker phenomenon, the chemical specificity of Raman spectroscopy has been shown capable of discriminating between different bacterial species and has even shown spectral variation in same species under differing growth conditions or growth stages and has even been used to measure microbial activity by measuring the incorporation of stable isotope labeled substrates. Typically these analyses are carried out on well-studied, lab-grown model organisms and while relatively easy, these analyses are performed on cells grow under non-environmentally relevant conditions using rich media types not often found in nature. Here we show the effect on the Raman and fluorescence signal (248 nm Deep-UV excitation) from E. coli and other bacteria, grown in more nutrient limited environments, and fixed/preserved in ethanol, PFA and formalin. These fixatives not only preserve the cells for spectroscopic analysis but are compatible with many common techniques that can be used for further characterization of environmental microbial samples. Ethanol appears to heavily degrade the signals from

  10. The use of one- and two- photon induced fluorescence spectroscopy for the optical characterization of carcinogenic aflatoxins

    NASA Astrophysics Data System (ADS)

    Smeesters, L.; Meulebroeck, W.; Raeymaekers, S.; Thienpont, H.

    2014-09-01

    Carcinogenic and toxic contaminants in food and feed products are nowadays mostly detected by destructive, time-consuming chemical analyses, like HPLC and LC-MS/MS methods. However, as a consequence of the severe and growing regulations on food products by the European Union, there arose an increased demand for the ultra-fast, high-sensitive and non-destructive detection of contaminants in food and feed products. Therefore, we have investigated fluorescence spectroscopy for the characterization of carcinogenic aflatoxins. With the use of a tunable titanium-sapphire laser in combination with second and third harmonic wavelength generation, both one- and two-photon induced fluorescence excitation wavelengths could be generated using the same setup. We characterized and compared the one- and two-photon induced fluorescence spectra of pure aflatoxin powder, after excitation with 365nm and 730nm respectively. Moreover, we investigated the absolute fluorescence intensity as function of the excitation power density. Afterwards, we applied our characterization setup to the detection of aflatoxins in maize grains. The fluorescence spectra of both healthy and contaminated maize samples were experimentally characterized. In addition to the fluorescence spectrum of the pure aflatoxin, we observed an unwanted influence of the intrinsic fluorescence of the maize. Depending on the excitation wavelength, a varying contrast between the fluorescence spectra of the healthy and contaminated samples was obtained. After a comparison of the measured fluorescence signals, a detection criterion for the optical identification of the contaminated maize samples could be defined. As a result, this illustrates the use of fluorescence spectroscopy as a valuable tool for the non-destructive, real-time and high-sensitive detection of aflatoxins in maize.

  11. Detection of lead in soil with excimer laser fragmentation fluorescence spectroscopy (ELFFS)

    SciTech Connect

    Choi, J.H.; Damm, C.J.; O'Donovan, N.J.; Sawyer, R.F.; Koshland, C.P.; Lucas, D.

    2004-03-01

    Excimer laser fragmentation fluorescence spectroscopy (ELFFS) is used to monitor lead in soil sample and investigate laser-solid interactions. Pure lead nitrate salt and soil doped with lead nitrate are photolyzed with 193 nm light from an ArF excimer at fluences from 0.4 to 4 J/cm{sup 2}. Lead emission is observed at 357.2, 364.0, 368.3, 373.9 and 405.8 nm. Time-resolved data show the decay time of the lead emission at 405.8 nm grows with increasing fluence, and a plasma is formed above fluences of 2 J/cm{sup 2}, where a strong continuum emission interferes with the analyte signal. Fluences below this threshold allow us to achieve a detection limit of approximately 200 ppm in soil.

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

    PubMed Central

    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. PMID:24049685

  13. Photosystem II antenna phosphorylation-dependent protein diffusion determined by fluorescence correlation spectroscopy.

    PubMed

    Iwai, Masakazu; Pack, Chan-Gi; Takenaka, Yoshiko; Sako, Yasushi; Nakano, Akihiko

    2013-01-01

    Flexibility of chloroplast thylakoid membrane proteins is essential for plant fitness and survival under fluctuating light environments. Phosphorylation of light-harvesting antenna complex II (LHCII) is known to induce dynamic protein reorganization that fine-tunes the rate of energy conversion in each photosystem. However, molecular details of how LHCII phosphorylation causes light energy redistribution throughout thylakoid membranes still remain unclear. By using fluorescence correlation spectroscopy, we here determined the LHCII phosphorylation-dependent protein diffusion in thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. As compared to the LHCII dephosphorylation-induced condition, the diffusion coefficient of LHCII increased nearly twofold under the LHCII phosphorylation-induced condition. We also verified the results by using the LHCII phosphorylation-deficient mutant. Our observation suggests that LHCII phosphorylation-dependent protein reorganization occurs along with the changes in the rate of protein diffusion, which would have an important role in mediating light energy redistribution throughout thylakoid membranes. PMID:24088948

  14. Fluorescence spectroscopy coupled with PARAFAC and PLS DA for characterization and classification of honey.

    PubMed

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

    2015-05-15

    Fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC) and Partial least squares Discriminant Analysis (PLS DA) were used for characterization and classification of honey. Excitation emission spectra were obtained for 95 honey samples of different botanical origin (acacia, sunflower, linden, meadow, and fake honey) by recording emission from 270 to 640 nm with excitation in the range of 240-500 nm. The number of fluorophores present in honey, excitation and emission spectra of each fluorophore, and their relative concentration are determined using a six-component PARAFAC model. Emissions from phenolic compounds and Maillard reaction products exhibited the largest difference among classes of honey of different botanical origin. The PLS DA classification model, constructed from PARAFAC model scores, detected fake honey samples with 100% sensitivity and specificity. Honey samples were also classified using PLS DA with errors of 0.5% for linden, 10% for acacia, and about 20% for both sunflower and meadow mix. PMID:25577082

  15. Probing the photoluminescence properties of gold nanoclusters by fluorescence lifetime correlation spectroscopy

    SciTech Connect

    Yuan, C. T. Lin, T. N.; Shen, J. L.; Lin, C. A.; Chang, W. H.; Cheng, H. W.; Tang, J.

    2013-12-21

    Gold nanoclusters (Au NCs) have attracted much attention for promising applications in biological imaging owing to their tiny sizes and biocompatibility. So far, most efforts have been focused on the strategies for fabricating high-quality Au NCs and then characterized by conventional ensemble measurement. Here, a fusion single-molecule technique combining fluorescence correlation spectroscopy and time-correlated single-photon counting can be successfully applied to probe the photoluminescence (PL) properties for sparse Au NCs. In this case, the triplet-state dynamics and diffusion process can be observed simultaneously and the relevant time constants can be derived. This work provides a complementary insight into the PL mechanism at the molecular levels for Au NCs in solution.

  16. Experimental study and verification of the residence time distribution using fluorescence spectroscopy and color measurement

    NASA Astrophysics Data System (ADS)

    Aigner, Michael; Lepschi, Alexander; Aigner, Jakob; Garmendia, Izaro; Miethlinger, Jürgen

    2015-05-01

    We report on the inline measurement of residence time (RT) and residence time distribution (RTD) by means of fluorescence spectroscopy [1] and optical color measurements [2]. Measurements of thermoplastics in a variety of single-screw extruders were conducted. To assess the influence of screw configurations, screw speeds and mass throughput on the RT and RTD, tracer particles were introduced into the feeding section and the RT was measured inline in the plasticization unit. Using special measurement probes that can be inserted into 1/2″ - 20 UNF (unified fine thread) bore holes, the mixing ability of either the whole plasticization unit or selected screw regions, e.g., mixing parts, can be validated during the extrusion process. The measurement setups complement each other well, and their combined use can provide further insights into the mixing behavior of single-screw plasticization units.

  17. Determination of the biodiesel content in diesel/biodiesel blends: a method based on fluorescence spectroscopy.

    PubMed

    Scherer, Marisa D; Oliveira, Samuel L; Lima, Sandro M; Andrade, Luis H C; Caires, Anderson R L

    2011-05-01

    Blends of biodiesel and diesel are being used increasingly worldwide because of environmental, economic, and social considerations. Several countries use biodiesel blends with different blending limits. Therefore, it is necessary to develop or improve methods to quantify the biodiesel level in a diesel/biodiesel blend, to ensure compliance with legislation. The optical technique based on the absorption of light in the mid-infrared has been successful for this application. However, this method presents some challenges that must be overcome. In this paper, we propose a novel method, based on fluorescence spectroscopy, to determine the biodiesel content in the diesel/biodiesel blend, which allows in loco measurements by using portable systems. The results showed that this method is both practical and more sensitive than the standard optical method. PMID:21213028

  18. Simultaneous Characterization of Lateral Lipid and Prothrombin Diffusion Coefficients by Z-Scan Fluorescence Correlation Spectroscopy

    PubMed Central

    Štefl, Martin; Kułakowska, Anna; Hof, Martin

    2009-01-01

    Abstract 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. PMID:19651025

  19. Investigation of the interaction between amodiaquine and human serum albumin by fluorescence spectroscopy and molecular modeling.

    PubMed

    Samari, Fayezeh; Shamsipur, Mojtaba; Hemmateenejad, Bahram; Khayamian, Taghi; Gharaghani, Sajjad

    2012-08-01

    The interaction of amodiaquine (AQ) with human serum albumin (HSA) has been studied by fluorescence spectroscopy. Based on the sign and magnitude of the enthalpy and entropy changes (ΔH(0) = -43.27 kJ mol(-1) and ΔS(0) = -50.03 J mol(-1) K(-1)), hydrogen bond and van der Waals forces were suggested as the main interacting forces. Moreover, the efficiency of energy transfer and distance between HSA and acceptor AQ was calculated. Finally, the binding of AQ to HSA was modeled by molecular docking and molecular dynamic simulation methods. Excellent agreement was found between the experimental and theoretical results. Both experimental results and modeling methods suggested that AQ binds mainly to the sub-domain IIA of HSA. PMID:22658498

  20. Study of mechanical properties of DNA in E. coli cells by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Kafle, Rudra; Liebeskind, Molly; Meiners, Jens-Christian

    Mechanical quantities like the elasticity of cells are conventionally measured by directly probing them mechanically. Measurements of these quantities for subcellular structures in living cells are almost impossible this way. We use fluorescence correlation spectroscopy (FCS) to measure such mechanical quantities in chromosomal DNA in E. coli cells. We present methods to address complexities of live-cell FCS such as photobleaching, and calculate the viscoelastic moduli from the FCS data. We compare the measured viscoelastic moduli of live cells with those that are ATP-depleted to stop all molecular motor action and find substantial differences. Active processes are stopped in ATP-depleted cells and hence the bacterial DNA appears to become stiffer and the surrounding intracellular medium more viscous. We also compare our results with the FCS data obtained from the lambda DNA solution in various concentrations to mimic the cellular environment.

  1. Ultrasensitive detection of genetically modified plants by fluorescence cross-correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Junfeng; Xing, Da; Chen, Tongsheng; Liu, Jinfeng

    2006-09-01

    In this study, a novel method for the direct detection of GMP without amplified by the general method of PCR is firstly presented and proved by experiments. In our method, fluorescence correlation spectroscopy, cleaving nucleic acid by restriction endonuclease and two nucleic acid probe hybridization techniques are combined to distinguish the caulifiower mosaic virus (CaMV) 35S promoter and determine whether samples contain genetically modified components. The detection principle is as follows: firstly two restriction endonucleases FOKI and BsrDlare used to cleave the genomic DNA and the 169bp fragments of CaMV 35S promoter are retrieved; secondly, two nucleic acid probes labeled by Rhodamine Green and y5 dyes respectively hybridize with cleaved 169bp fragments of CaMV 35S promoter; thirdly, the hybridization products simultaneously with two dye-labeled probes are detected by fluorescence cross-correlation spectroscopy and GMP is distinguished. As the detection and analysis by FCS can be performed at the level of single molecule, there is no need for any type of amplification. Genetically modified tobaccos are measured by this method. The results indicate this method can detect CaMV 35S promoter of GMP exactly and the sensitivity can be down to 3.47X10 -10M. Because no any type of amplification is involved, this method can avoid the non-specffic amplification and false-positive problems of PCR, Due to its high-sensitivity, simplicity, reliability and little need for sample amounts, this method promises to be a highly effective detection method for GMP.

  2. Cellular response to heat shock studied by multiconfocal fluorescence correlation spectroscopy.

    PubMed

    Kloster-Landsberg, Meike; Herbomel, Gaëtan; Wang, Irène; Derouard, Jacques; Vourc'h, Claire; Usson, Yves; Souchier, Catherine; Delon, Antoine

    2012-09-19

    Heat shock triggers a transient and ubiquitous response, the function of which is to protect cells against stress-induced damage. The heat-shock response is controlled by a key transcription factor known as heat shock factor 1 (HSF1). We have developed a multiconfocal fluorescence correlation spectroscopy setup to measure the dynamics of HSF1 during the course of the heat-shock response. The system combines a spatial light modulator, to address several points of interest, and an electron-multiplying charge-coupled camera for fast multiconfocal recording of the photon streams. Autocorrelation curves with a temporal resolution of 14 μs were analyzed before and after heat shock on eGFP and HSF1-eGFP-expressing cells. Evaluation of the dynamic parameters of a diffusion-and-binding model showed a slower HSF1 diffusion after heat shock. It is also observed that the dissociation rate decreases after heat shock, whereas the association rate is not affected. In addition, thanks to the multiconfocal fluorescence correlation spectroscopy system, up to five spots could be simultaneously located in each cell nucleus. This made it possible to quantify the intracellular variability of the diffusion constant of HSF1, which is higher than that of inert eGFP molecules and increases after heat shock. This finding is consistent with the fact that heat-shock response is associated with an increase of HSF1 interactions with DNA and cannot be explained even partially by heat-induced modifications of nuclear organization. PMID:22995483

  3. Fluorescence spectroscopy of single molecules at room temperature and its applications

    SciTech Connect

    Ha, Taekjip

    1996-12-01

    We performed fluorescence spectroscopy of single and pairs of dye molecules on a surface at room temperature. Near field scanning optical microscope (NSOM) and far field scanning optical microscope with multi-color excitation/detection capability were built. The instrument is capable of optical imaging with 100nm resolution and has the sensitivity necessary for single molecule detection. A variety of dynamic events which cannot be observed from an ensemble of molecules is revealed when the molecules are probed one at a time. They include (1) spectral jumps correlated with dark states, (2) individually resolved quantum jumps to and from the meta-stable triplet state, (3) rotational jumps due to desorption/readsorption events of single molecules on the surface. For these studies, a computer controlled optical system which automatically and rapidly locates and performs spectroscopic measurements on single molecules was developed. We also studied the interaction between closely spaced pairs of molecules. In particular, fluorescence resonance energy transfer between a single resonant pair of donor and acceptor molecules was measured. Photodestruction dynamics of the donor or acceptor were used to determine the presence and efficiency of energy transfer Dual molecule spectroscopy was extended to a non-resonant pair of molecules to obtain high resolution differential distance information. By combining NSOM and dual color scheme, we studied the co-localization of parasite proteins and host proteins on a human red blood cell membrane infected with malaria. These dual-molecule techniques can be used to measure distances, relative orientations, and changes in distances/orientations of biological macromolecules with very good spatial, angular and temporal resolutions, hence opening new capabilities in the study of such systems.

  4. Evaluating fluorescence spectroscopy as a tool to characterize cyanobacteria intracellular organic matter upon simulated release and oxidation in natural water.

    PubMed

    Korak, Julie A; Wert, Eric C; Rosario-Ortiz, Fernando L

    2015-01-01

    Intracellular organic matter (IOM) from cyanobacteria may be released into natural waters following cell death in aquatic ecosystems and during oxidation processes in drinking water treatment plants. Fluorescence spectroscopy was evaluated to identify the presence of IOM from three cyanobacteria species during simulated release into natural water and following oxidation processes (i.e. ozone, free chlorine, chloramine, chlorine dioxide). Peak picking and the fluorescence index (FI) were explored to determine which IOM components (e.g., pigments) provide unique and persistent fluorescence signatures with minimal interferences from the background dissolved organic matter (DOM) found in Colorado River water (CRW). When IOM was added to ultrapure water, the fluorescence signature of the three cyanobacteria species showed similarities to each other. Each IOM exhibited a strong protein-like fluorescence and fluorescence at Ex 370 nm and Em 460 nm (FDOM), where commercial fluorescence sensors monitor. All species also had strong phycobiliprotein fluorescence (i.e. phycocyanin or phycoerythrin) in the higher excitation range (500-650 nm). All three IOM isolates had FI values greater than 2. When IOM was added to CRW, phycobiliprotein fluorescence was quenched through interactions between IOM and CRW-DOM. Mixing IOM and CRW demonstrated that protein-like and FDOM intensity responses were not a simple superposition of the starting material intensities, indicating that interactions between IOM and CRW-DOM fluorescing moieties were important. Fluorescence intensity in all regions decreased with exposure to ozone, free chlorine, and chlorine dioxide, but the FI still indicated compositional differences compared to CRW-DOM. The phycobiliproteins in IOM are not promising as a surrogate for IOM release, because their fluorescence intensity is quenched by interactions with DOM and decreased during oxidation processes. Increases in both FDOM intensity and FI are viable qualitative

  5. Fluorescence spectroscopy for monitoring reduction of natural organic matter and halogenated furanone precursors by biofiltration.

    PubMed

    Peleato, Nicolás M; McKie, Michael; Taylor-Edmonds, Lizbeth; Andrews, Susan A; Legge, Raymond L; Andrews, Robert C

    2016-06-01

    The application of fluorescence spectroscopy to monitor natural organic matter (NOM) reduction as a function of biofiltration performance was investigated. This study was conducted at pilot-scale where a conventional media filter was compared to six biofilters employing varying enhancement strategies. Overall reductions of NOM were identified by measuring dissolved organic carbon (DOC), and UV absorbance at 254 nm, as well as characterization of organic sub-fractions by liquid chromatography-organic carbon detection (LC-OCD) and parallel factors analysis (PARAFAC) of fluorescence excitation-emission matrices (FEEM). The biofilter using granular activated carbon media, with exhausted absorptive capacity, was found to provide the highest removal of all identified PARAFAC components. A microbial or processed humic-like component was found to be most amenable to biodegradation by biofilters and removal by conventional treatment. One refractory humic-like component, detectable only by FEEM-PARAFAC, was not well removed by biofiltration or conventional treatment. All biofilters removed protein-like material to a high degree relative to conventional treatment. The formation potential of two halogenated furanones, 3-chloro-4(dichloromethyl)-2(5H)-furanone (MX) and mucochloric acid (MCA), as well as overall treated water genotoxicity are also reported. Using the organic characterization results possible halogenated furanone and genotoxicity precursors are identified. Comparison of FEEM-PARAFAC and LC-OCD results revealed polysaccharides as potential MX/MCA precursors. PMID:27016810

  6. Dynamics of water-in-oil nanoemulsions revealed by fluorescence lifetime correlation spectroscopy.

    PubMed

    Orte, Angel; Ruedas-Rama, Maria J; Paredes, Jose M; Crovetto, Luis; Alvarez-Pez, Jose M

    2011-11-01

    The size, diffusional properties, and dynamics of reverse water-in-oil nanoemulsions, or reverse micelles (RMs), have been widely investigated because of interest in this system as a model for biological compartmentalization. Here, we have employed fluorescence lifetime correlation spectroscopy (FLCS) to reveal the dynamics and sizes of aerosol-OT (AOT)/isooctane RMs using a fluorescent xanthene derivative called Tokyo Green II (TG-II). The dye undergoes a partition and a shift in its tautomeric equilibrium such that the TG-II anion remains in the inner micellar aqueous core, and the neutral quinoid form lies in the interfacial region. By applying FLCS, we specifically obtained the lifetime filtered autocorrelation curves of the anionic TG-II, which shows a characteristic lifetime of approximately 4 ns. Analysis of the FLCS curves provides the diffusion coefficient and hydrodynamic radius of the RMs as well as micelle dynamics in the same experiment. The FLCS curves show dynamics in the microsecond time range, which represents an interconversion rate that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core. PMID:21913723

  7. Detection and characterization of stomach cancer and atrophic gastritis with fluorescence and Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Xiaozhou; Lin, Junxiu; Jia, Chunde; Wang, Rong

    2003-12-01

    In this paper, we attempt to find a valid method to distinguish gastric cancer and atrophic gastritis. Auto-fluorescence and Raman spectroscopy of laser induced (514.5 nm and 488.0 nm) was measured. The serum spectrum is different between normal and cancer. Average value of diagnosis parameter for normal serum, red shift is less than 12 nm and Raman relative intensity of peak C by 514.5 nm excited is stronger than that of 488.0 nm. To gastric cancer, its red shift of average is bigger than 12 nm and relative intensity of Raman peak C by 514.5 nm excited is weaker than that by 488.0 nm. To atrophic gastritis, the distribution state of Raman peaks is similar with normal serum and auto-fluorescence spectrum's shape is similar to that of gastric cancer. Its average Raman peak red shift is bigger than 12 nm and the relative intensity of peak C by 514.5 excited is stronger than that of by 488.0. We considered it as a criterion and got an accuracy of 85.6% for diagnosis of gastric cancer compared with the result of clinical diagnosis.

  8. Ablation plume structure and dynamics in ambient gas observed by laser-induced fluorescence imaging spectroscopy

    NASA Astrophysics Data System (ADS)

    Miyabe, M.; Oba, M.; Iimura, H.; Akaoka, K.; Khumaeni, A.; Kato, M.; Wakaida, I.

    2015-08-01

    The dynamic behavior of an ablation plume in ambient gas has been investigated by laser-induced fluorescence imaging spectroscopy. The second harmonic beam from an Nd:YAG laser (0.5-6 J/cm2) was focused on a sintered oxide pellet or a metal chip of gadolinium. The produced plume was subsequently intersected with a sheet-shaped UV beam from a dye laser so that time-resolved fluorescence images were acquired with an intensified CCD camera at various delay times. The obtained cross-sectional images of the plume indicate that the ablated ground state atoms and ions of gadolinium accumulate in a hemispherical contact layer between the plume and the ambient gas, and a cavity containing a smaller density of ablated species is formed near the center of the plume. At earlier expansion stage, another luminous component also expands in the cavity so that it coalesces into the hemispherical layer. The splitting and coalescence for atomic plume occur later than those for ionic plume. Furthermore, the hemispherical layer of neutral atoms appears later than that of ions; however, the locations of the layers are nearly identical. This coincidence of the appearance locations of the layers strongly suggests that the neutral atoms in the hemispherical layer are produced as a consequence of three-body recombination of ions through collisions with gas atoms. The obtained knowledge regarding plume expansion dynamics and detailed plume structure is useful for optimizing the experimental conditions for ablation-based spectroscopic analysis.

  9. Fluorescence Correlation Spectroscopy to Monitor Kai Protein-based Circadian Oscillations in Real Time*

    PubMed Central

    Goda, Kazuhito; Ito, Hiroshi; Kondo, Takao; Oyama, Tokitaka

    2012-01-01

    Dynamic protein-protein interactions play an essential role in cellular regulatory systems. The cyanobacterial circadian clock is an oscillatory system that can be reconstituted in vitro by mixing ATP and three clock proteins: KaiA, KaiB, and KaiC. Association and dissociation of KaiB from KaiC-containing complexes are critical to circadian phosphorylation and dephosphorylation of KaiC. We developed an automated and noninvasive method to monitor dynamic complex formation in real time using confocal fluorescence correlation spectroscopy (FCS) and uniformly labeled KaiB as a probe. A nanomolar concentration of the labeled KaiB for FCS measurement did not interfere with the oscillatory system but behaved similarly to the wild-type one during the measurement period (>5 days). The fluorescent probe was stable against repeated laser exposure. As an application, we show that this detection system allowed analysis of the dynamics of both long term circadian oscillations and short term responses to temperature changes (∼10 min) in the same sample. This suggested that a phase shift of the clock with a high temperature pulse occurred just after the stimulus through dissociation of KaiB from the KaiC complex. This monitoring method should improve our understanding of the mechanisms underlying this cellular circadian oscillator and provide a means to assess dynamic protein interactions in biological systems characterized by rates similar to those observed with the Kai proteins. PMID:22157012

  10. 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.

  11. Heterogeneity in binary mixtures of dimethyl sulfoxide and glycerol: fluorescence correlation spectroscopy.

    PubMed

    Chattoraj, Shyamtanu; Chowdhury, Rajdeep; Ghosh, Shirsendu; Bhattacharyya, Kankan

    2013-06-01

    Diffusion of four coumarin dyes in a binary mixture of dimethyl sulfoxide (DMSO) and glycerol is studied using fluorescence correlation spectroscopy (FCS). The coumarin dyes are C151, C152, C480, and C481. In pure DMSO, all the four dyes exhibit a very narrow (almost uni-modal) distribution of diffusion coefficient (Dt). In contrast, in the binary mixtures all of them display a bimodal distribution of Dt with broadly two components. One of the components of D(t) corresponds to the bulk viscosity. The other one is similar to that in pure DMSO. This clearly indicates the presence of two distinctly different nano-domains inside the binary mixture. In the first, the micro-environment of the solute consists of both DMSO and glycerol approximately at the bulk composition. The other corresponds to a situation where the first layer of the solute consists of DMSO only. The burst integrated fluorescence lifetime (BIFL) analysis also indicates presence of two micro-environments one of which resembles DMSO. The relative contribution of the DMSO-like environment obtained from the BIFL analysis is much larger than that obtained from FCS measurements. It is proposed that BIFL corresponds to an instantaneous environment in a small region (a few nm) around the probe. FCS, on the contrary, describes the long time trajectory of the probes in a region of dimension ~200 nm. The results are explained in terms of the theory of binary mixtures and recent simulations of binary mixtures containing DMSO. PMID:23758388

  12. Heterogeneity in binary mixtures of dimethyl sulfoxide and glycerol: Fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Chattoraj, Shyamtanu; Chowdhury, Rajdeep; Ghosh, Shirsendu; Bhattacharyya, Kankan

    2013-06-01

    Diffusion of four coumarin dyes in a binary mixture of dimethyl sulfoxide (DMSO) and glycerol is studied using fluorescence correlation spectroscopy (FCS). The coumarin dyes are C151, C152, C480, and C481. In pure DMSO, all the four dyes exhibit a very narrow (almost uni-modal) distribution of diffusion coefficient (Dt). In contrast, in the binary mixtures all of them display a bimodal distribution of Dt with broadly two components. One of the components of Dt corresponds to the bulk viscosity. The other one is similar to that in pure DMSO. This clearly indicates the presence of two distinctly different nano-domains inside the binary mixture. In the first, the micro-environment of the solute consists of both DMSO and glycerol approximately at the bulk composition. The other corresponds to a situation where the first layer of the solute consists of DMSO only. The burst integrated fluorescence lifetime (BIFL) analysis also indicates presence of two micro-environments one of which resembles DMSO. The relative contribution of the DMSO-like environment obtained from the BIFL analysis is much larger than that obtained from FCS measurements. It is proposed that BIFL corresponds to an instantaneous environment in a small region (a few nm) around the probe. FCS, on the contrary, describes the long time trajectory of the probes in a region of dimension ˜200 nm. The results are explained in terms of the theory of binary mixtures and recent simulations of binary mixtures containing DMSO.

  13. Light adaptation of the unicellular red alga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.

    PubMed

    Ueno, Yoshifumi; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

    2015-08-01

    Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed. PMID:25577254

  14. Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

    PubMed Central

    Kekic, M; Huang, W; Moens, P D; Hambly, B D; dos Remedios, C G

    1996-01-01

    We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region. Images FIGURE 4 FIGURE 5 PMID:8804587

  15. The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy.

    PubMed

    Singh, Anand Pratap; Krieger, Jan Wolfgang; Buchholz, Jan; Charbon, Edoardo; Langowski, Jörg; Wohland, Thorsten

    2013-04-01

    Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, transport, flow velocities and concentrations in an imaging mode. SPIM-FCS records correlation functions over a whole plane in a sample, which requires array detectors for recording the fluorescence signal. Several types of image sensors are suitable for FCS. They differ in properties such as effective area per pixel, quantum efficiency, noise level and read-out speed. Here we compare the performance of several low light array detectors based on three different technologies: (1) Single-photon avalanche diode (SPAD) arrays, (2) passive-pixel electron multiplying charge coupled device (EMCCD) and (3) active-pixel scientific-grade complementary metal oxide semiconductor cameras (sCMOS). We discuss the influence of the detector characteristics on the effective FCS observation volume, and demonstrate that light sheet based SPIM-FCS provides absolute diffusion coefficients. This is verified by parallel measurements with confocal FCS, single particle tracking (SPT), and the determination of concentration gradients in space and time. While EMCCD cameras have a temporal resolution in the millisecond range, sCMOS cameras and SPAD arrays can extend the time resolution of SPIM-FCS down to 10 μs or lower. PMID:23571955

  16. Quantitative analysis of cotton (Gossypium hirsutum) lint trash by fluorescence spectroscopy.

    PubMed

    Gamble, Gary R; Foulk, Jonn A

    2007-06-27

    The presence of cotton plant botanical components, or trash, embedded in lint subsequent to harvesting and ginning is an important criterion in the classification of baled cotton by the U.S. Department of Agriculture Agricultural Marketing Service. The trash particles may be reduced in size to the point that specific trash types are not identifiable by image or gravimetric analysis, and it is desirable to quantify different trash types so that processing lines may be optimized for removal of the most problematic trash to enhance processing performance and cotton lint quality. Currently, there are no methods available to adequately quantify cotton lint trash based on botanical origin. The present work attempts to address this issue through the analysis by fluorescence spectroscopy of dimethyl sulfoxide extracts of mixtures of six botanical trash types. The fluorescence data are subsequently subjected to chemometric analysis. The resulting 6 partial least-squares calibration models obtained from 128 mixtures are demonstrated in the case of leaf and hull to be capable of predicting individual trash component concentrations with a high degree of confidence. PMID:17536818

  17. THE USE OF FLUORESCENCE CORRELATION SPECTROSCOPY TO PROBE CHROMATIN IN THE CELL NUCLEUS

    SciTech Connect

    Sorscher, Stanley M.; Bartholemew, James C.; Klein, Melvin P.

    1980-03-01

    All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small systems, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well characterized DNA preparations, including calf thymus DNA, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and decreases as the cells leave the resting state, and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.

  18. Trimodal detection of early childhood caries using laser light scanning and fluorescence spectroscopy: clinical prototype

    PubMed Central

    Kim, Amy S.; Ridge, Jeremy S.; Nelson, Leonard Y.; Berg, Joel H.; Seibel, Eric J.

    2013-01-01

    Abstract. There is currently a need for a safe and effective way to detect and diagnose early stages of childhood caries. A multimodal optical clinical prototype for diagnosing caries demineralization in vivo has been developed. The device can be used to quickly image and screen for any signs of demineralized enamel by obtaining high-resolution and high-contrast surface images using a 405-nm laser as the illumination source, as well as obtaining autofluorescence and bacterial fluorescence images. When a suspicious region of demineralization is located, the device also performs dual laser fluorescence spectroscopy using 405- and 532-nm laser excitation. An autofluorescence ratio of the two excitation lasers is computed and used to quantitatively diagnose enamel health. The device was tested on five patients in vivo as well as on 28 extracted teeth with clinically diagnosed carious lesions. The device was able to provide detailed images that highlighted the lesions identified by the clinicians. The autofluorescence spectroscopic ratios obtained from the extracted teeth successfully quantitatively discriminated between sound and demineralized enamel. PMID:23986369

  19. Short communication: Suitability of fluorescence spectroscopy for characterization of commercial milk of different composition and origin.

    PubMed

    Ntakatsane, M P; Yang, X Q; Lin, M; Liu, X M; Zhou, P

    2011-11-01

    Thirteen milk brands comprising 76 pasteurized and UHT milk samples of various compositions (whole, reduced fat, skimmed, low lactose, and high protein) were obtained from local supermarkets, and milk samples manufactured in various countries were discriminated using front-face fluorescence spectroscopy (FFFS) coupled with chemometric tools. The emission spectra of Maillard reaction products and riboflavin (MRP/RF; 400 to 600 nm) and tryptophan (300 to 400 nm) were recorded using FFFS, and the excitation wavelengths were set at 360 nm for MRP/RF and 290 nm for tryptophan. Principal component analysis (PCA) was applied to analyze the normalized spectra. The PCA of spectral information from MRP/RF discriminated the milk samples originating in different countries, and PCA of spectral information from tryptophan discriminated the samples according to composition. The fluorescence spectral data were compared with liquid chromatography-mass spectrometry results for the glycation extent of the milk samples, and a positive association (R(2)=0.84) was found between the degree of glycation of α-lactalbumin and the MRP/RF spectral data. This study demonstrates the ability and sensitivity of FFFS to rapidly discriminate and classify commercial milk with various compositions and processing conditions. PMID:22032360

  20. Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy*S⃞

    PubMed Central

    Laurence, Ted A.; Kwon, Youngeun; Johnson, Aaron; Hollars, Christopher W.; O'Donnell, Mike; Camarero, Julio A.; Barsky, Daniel

    2008-01-01

    DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III β subunit or β clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the β clamp diffusing along DNA is on the order of 10–14 m2/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the β clamp remains at the 3′ end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA. PMID:18556658

  1. Dynamic imaging by fluorescence correlation spectroscopy identifies diverse populations of polyglutamine oligomers formed in vivo.

    PubMed

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

    2012-07-27

    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

  2. Characterization of the binding of paylean and DNA by fluorescence, UV spectroscopy and molecular docking techniques.

    PubMed

    Zhou, Huifeng; Bi, Shuyun; Wang, Yu; Zhao, Tingting

    2016-06-01

    The interaction of paylean (PL) with calf thymus DNA (ctDNA) was investigated using fluorescence spectroscopy, UV absorption, melting studies, ionic strength, viscosity experiments and molecular docking under simulated physiological conditions. Values for the binding constant Ka between PL and DNA were 5.11 × 10(3) , 2.74 × 10(3) and 1.74 × 10(3)  L mol(-1) at 19, 29 and 39°C respectively. DNA quenched the intrinsic fluorescence of PL via a static quenching procedure as shown from Stern-Volmer plots. The relative viscosity and the melting temperature of DNA were basically unchanged in the presence of PL. The fluorescence intensity of PL-DNA decreased with increasing ionic strength. The value of Ka for PL with double-stranded DNA (dsDNA) was larger than that for PL with single-stranded DNA (ssDNA). All the results revealed that the binding mode was groove binding, and molecular docking further indicated that PL was preferentially bonded to A-T-rich regions of DNA. The values for ΔH, ΔS and ΔG suggested that van der Waals forces or hydrogen bonding might be the main acting forces between PL and DNA. The binding distance was determined to be 3.37 nm based on the theory of Förster energy transference, which indicated that a non-radiation energy transfer process occurred. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26597997

  3. High resolution x-ray fluorescence spectroscopy - a new technique for site- and spin-selectivity

    SciTech Connect

    Wang, Xin

    1996-12-01

    X-ray spectroscopy has long been used to elucidate electronic and structural information of molecules. One of the weaknesses of x-ray absorption is its sensitivity to all of the atoms of a particular element in a sample. Through out this thesis, a new technique for enhancing the site- and spin-selectivity of the x-ray absorption has been developed. By high resolution fluorescence detection, the chemical sensitivity of K emission spectra can be used to identify oxidation and spin states; it can also be used to facilitate site-selective X-ray Absorption Near Edge Structure (XANES) and site-selective Extended X-ray Absorption Fine Structure (EXAFS). The spin polarization in K fluorescence could be used to generate spin selective XANES or spin-polarized EXAFS, which provides a new measure of the spin density, or the nature of magnetic neighboring atoms. Finally, dramatic line-sharpening effects by the combination of absorption and emission processes allow observation of structure that is normally unobservable. All these unique characters can enormously simplify a complex x-ray spectrum. Applications of this novel technique have generated information from various transition-metal model compounds to metalloproteins. The absorption and emission spectra by high resolution fluorescence detection are interdependent. The ligand field multiplet model has been used for the analysis of K{alpha} and K{beta} emission spectra. First demonstration on different chemical states of Fe compounds has shown the applicability of site selectivity and spin polarization. Different interatomic distances of the same element in different chemical forms have been detected using site-selective EXAFS.

  4. Laser Induced Fluorescence Spectroscopy of Neutral and Ionized Polycyclic Aromatic Hydrocarbons in a Cosmic Simulation Chamber

    NASA Astrophysics Data System (ADS)

    Bejaoui, Salma; Salama, Farid

    2015-08-01

    Polycyclic aromatic hydrocarbon (PAH) molecules are considered the best carriers to account for the ubiquitous infrared emission bands. PAHs have also been proposed as candidates to explain the diffuse interstellar bands (DIBs), a series of absorption features seen on the interstellar extinction curve and are plausible carriers for the extended red emission (ERE), a photoluminescent process associated with a wide variety of interstellar environments. Extensive efforts have been devoted over the past two decades to characterize the physical and chemical properties of PAH molecules and ions in space. Absorption spectra of PAH molecules and ions trapped in solid matrices have been compared to the DIBs [1, 2]. Absorption spectra of several cold, isolated gas-phase PAHs have also been measured under experimental conditions that mimic the interstellar conditions [see 3 for a review]. The purpose of this study is to provide a new dimension to the existing spectroscopic database of neutral and single ionized PAHs that is largely based on absorption spectra by adding emission spectroscopy data. The measurements are based on the laser-induced fluorescence (LIF) technique [4] and are performed with the Pulsed Discharge Nozzle (PDN) of the COSmIC laboratory facility at NASA Ames laboratory. The PDN generates plasma in a free supersonic jet expansion to simulate the physical and the chemical conditions in interstellar environments. We focus, here, on the fluorescence spectra of large neutral PAHs and their cations where there is a lack of fluorescence spectroscopy data. The astronomical implications of the data (e.g., ERE) are examinedReferences[1] F. Salama, E. Bakes, L.J. Allamandola, A.G.G.M. Tielens, Astrophys. J., 458 (1996) p.621[2] F. Salama, The ISO Revolution, EDP Sciences, Les Ulis, France (1999) p.65[3] Salama F., In Organic Matter in Space, IAU Symposium 251, Kwok & Sandford Eds.Cambridge University Press,4, S251,(2008), p. 357 (2008) and references therein.[4

  5. Detailed evaluation of the performance of microfluidic T mixers using fluorescence and ultraviolet resonance Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Masca, Sergiu I.; Rodriguez-Mendieta, Iñigo R.; Friel, Claire T.; Radford, Sheena E.; Smith, D. Alastair

    2006-05-01

    A reliable device that produces efficient mixing with a short dead time has enormous utility in the kinetic analysis of biochemical and chemical processes. We have designed two different T mixers that use moderate flow rates (0.2-0.4ml/s), can monitor reactions up to several milliseconds, and achieve mixing times as low as 20μs. The two mixers are easy to build and dismantle, reliable, and can perform hundreds of experiments without blocking. The first mixer comprises a stainless steel block, containing a microchannel, glued to a quartz cuvette, containing a 200×200μm2 observation channel defining a conventional T mixer. The reactions are monitored by imaging the length of the observation channel onto a charge-coupled device camera. In the second mixer the entire T (200×200μm2 internal cross section) is contained within a 40-mm-long quartz cuvette. We have adopted a novel approach to controlling the entrance channel bore by inserting a stainless steel wire in order to increase the linear speed of the impinging fluids. Using a dye to visualize the flow profile inside the second T mixer, it was shown that in this T geometry segregation of the reactants is observed in the junction between the inlet channels and the observation channel (T junction) and mixing occurs entirely in the observation channel. We thoroughly tested the two mixers through several kinetic reactions using both fluorescence and ultraviolet resonance Raman spectroscopy measurements. We show that both mixers provide efficient mixing with nominal dead times (using 1:10 v /v dilution), calculated using the quenching of the fluorescence of N-acetyl-L-tryptophanamide by N-bromosuccinimide, of 200±20 and 100±10μs, for each mixer, respectively. However, the ability to monitor within the inlet channels and the entire observation channel of the second mixer shows that this standard approach to estimating the dead time is artifactual, since it relies on assuming a constant flow speed throughout the

  6. Characteristics of dissolved organic carbon revealed by ultraviolet/visible absorbance and fluorescence spectroscopy: The current status and future exploration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dissolved organic carbon (DOC) is an important research subject for various disciplines. The objectives of this chapter are to review and summarize recent advancement in characterization of DOC by ultraviolet/visible (UV/Vis) and fluorescence spectroscopies and to identify the information gaps for ...

  7. Estimation of AOT and SDS CMC in a methanol using conductometry, viscometry and pyrene fluorescence spectroscopy methods

    NASA Astrophysics Data System (ADS)

    Mitsionis, Anastasios I.; Vaimakis, Tiverios C.

    2012-09-01

    Critical micelle concentration (CMC) of two anionic surfactants in methanol was estimated using conductometry, viscometry and pyrene fluorescence spectroscopy methods. The surfactants used, were sodium bis(2-ethylhexyl) sulfosuccinate (Aerosol-OT, AOT) and sodium dodecyl sulfate (SDS) dispersed in pure methanol. The CMC determination was evaluated in room temperature. The results have shown nearly similar concentrations.

  8. Organic Light Emitting Device as a fluorescence spectroscopy's light source : one step towards the lab-on-a-chip device

    NASA Astrophysics Data System (ADS)

    Camou, S.; Kitamura, M.; Gouy, Jean-Philippe; Fujita, Hiroyuki; Arakawa, Yasuhiko; Fujii, Teruo

    2003-02-01

    Many papers were recently dedicated to the lab-on-a-chip applications, where all the basic elements should be integrated directly onto the microchip. The fluorescence spectroscopy is mostly used as a detection method due to its high reliability and sensitivity, but requires light source and photo-detector. For the first time, we then propose to use Organic material Light Emitting Diode (OLED) to supply a light source for the optical detection based on fluorescence spectroscopy. By combining this OLED with micro-fluidic channels patterned in PDMS layer, the integration of light source on the chip is then achieved. First, the ability of Organic Material to excite fluorescent response from dye is demonstrated. Then, some configurations are described in order to decrease the major drawbacks that have to be solved before applying such kind of devices.

  9. Linking fluorescence spectroscopy to diffuse soil source for dissolved humic substances in the Daning River, China.

    PubMed

    Chen, Hao; Zheng, Bing-Hui; Zhang, Lei

    2013-02-01

    Dissolved organic matter collected in Daning River (China) in July 2009 was investigated with parallel factor analysis (PARAFAC) and fluorescence spectroscopy with the aim of identifying the origin of dissolved humic substance (HS) components. Two HS-like fluorescence components (peak M and C) with excitation/emission (ex/em) maxima at 305/406 nm and 360/464 nm showed relatively uniform distribution in the vertical direction for each sampling site but a trend of accumulation down the river, independent of the highly heterogeneous water environment as implicated by water quality parameters (i.e., water temperature, algae density, chlorophyll a, dissolved oxygen, dissolved organic carbon, pH, conductivity and turbidity), while an amino acid/protein-like component (peak T; ex/em = 280/334 nm) was quite variable in its spatial distribution, implying strong influence from point sources (e.g. sewage discharge) and local microbial activities. The fluorescence intensity (F max in Raman units) at these ex/em wavelength pairs fell in the range of 0.031-0.358, 0.051-0.224 and 0.026-0.115 for peak T, M and C, respectively. In addition, the F max values of peak C covaried with M (i.e. C = 0.503 ×M, p < 0.01, R (2) = 0.973). Taken together, these results indicate that peak M and C originated primarily and directly from the same soil sources that were diffusive in the catchment, but peak T was more influenced by local point sources (e.g. wastewater discharge) and in situ microbial activities. This study presents new insights into the currently controversial origin of some HS components (e.g."peak M", as commonly referred to in the literature). This study highlights that natural water samples should be collected at various depths in addition to along a river/stream flow path so as to better evaluate the origin of HS fluorescence components. PMID:25208714

  10. A fluorescence spectroscopic study on the speciation of Cm(III) and Eu(III) in the presence of organic chelates in highly basic solutions

    SciTech Connect

    Wang, Zheming; Felmy, Andrew R.; Xia, Yuanxian; Mason, Marvin J.

    2003-07-15

    The speciation of Eu(III) and Cm(III) was investigated by time resolved laser fluorescence spectroscopy (TRLFS) over a range of base concentrations ranging from 0.01m NaOH to 7.5M NaOH and in the presence of several organic chelates including EDTA, HEDTA, NTA, and oxalate. The results of this work suggest that both Eu(III) and Cm(III) form strong mixed ligand complexes with organic chelates and the hydroxyl groups(s) in dilute NaOH solutions. However, in concentrated NaOH solutions, Eu(III)-/Cm(III)-containing colloidal nanoparticles are the primary cause for the measured Eu(III)/Cm(III) in the aqueous solutions. Therefore, the interpretation of these data solely in terms of the formation of amphoteric hydroxyl species (e.g. Eu(OH)4-) would appear to be inappropriate. The organic chelating ligands form strong complexes with surface Cm(III)/Eu(III) sites of the colloidal nanoparticles. For Cm(III), such surface complexes show largely red-shifted fluorescence spectra as compared with the aqueous complexes and unusually short fluorescence lifetimes. The decreased fluorescence lifetimes are likely due to the presence of transition metal ions, such as Fe3+, in the nanoparticle as well as reduced inter-nuclear distance between neighboring Cm(III) centers.

  11. First fluorescence spectroscopic investigation of Am(III) complexation with an organic carboxylic ligand, pyromellitic acid.

    PubMed

    Barkleit, Astrid; Geipel, Gerhard; Acker, Margret; Taut, Steffen; Bernhard, Gert

    2011-01-01

    For the first time Am(III) complexation with a small organic ligand could be identified and characterized with time-resolved laser-induced fluorescence spectroscopy (TRLFS) at room temperature and trace metal concentration. With pyromellitic acid (1,2,4,5-benzene-tetracarboxylic acid, BTC) as ligand spectroscopic characteristics for the Am-BTC complex system were determined at pH 5.0, an ionic strength of 0.1 M (NaClO4) and room temperature. The fluorescence lifetimes were determined to be 23.2±2.2 ns for Am3+(aq) and 27.2±1.2 ns for the Am-BTC 1:1 complex; the emission maximum for the 5D1-(7)F1 transition is 691 nm for both species. The complex stability constant for the Am-BTC 1:1 complex was calculated to be logβ110=5.42±0.16. PMID:20943431

  12. Fluorescence Spectroscopy as a Rapid, High-Resolution Tool for Detecting Biomolecules in Glacial Ice

    NASA Astrophysics Data System (ADS)

    Rohde, R. A.; Price, P. B.; Bramall, N.; Bay, R.

    2007-12-01

    We have developed new instruments utilizing the intrinsic fluorescence of specific biomolecules as a sensitive, non-destructive tool for detecting microorganisms. Using a 224-nm excitation, we detect protein-bound tryptophan (an amino acid present in all cells) at a detection threshold of approximately 1 cell per laser excitation volume and a duty cycle of 100 ms per measurement. Tryptophan is easily distinguished from inorganic backgrounds due to its characteristic spectral shape and ~300 times higher intensity per unit volume than typical inorganic compounds. A different excitation was also used to detect coenzyme F420, a characteristic marker for viable methanogenic cells. At the National Ice Core Laboratory, systematic scans of a 1 meter core sections took about 15 minutes and generated ~5000 measurements per meter. The high-resolution of this work revealed strong variability of microbial content on a scale of cm within individual cores, which suggests that microbial deposition at polar sites is strongly influenced by meteorological events (e.g. storms) on subannual and interannual scales. In addition, high levels of microbes are found to correlate with anomalously high concentrations of metabolic gases (e.g. methane, nitrous oxide, and 18O/16O of O2), suggesting that many of the isolated "gas artifacts" identified in deep ice cores are the accumulated waste products of in situ metabolism. This means that fluorescence spectroscopy may be a useful tool for identifying regions where high microbial concentrations have contaminated gas records. The existing instrumentation is suitcase portable and could be easily deployed in a variety of environments. Future versions of these instruments may be practical for continuous, rapid scans of entire cores, as an on-site deployable technique for characterizing microbial abundances in ice, and for searching for as few as 1 microbe per cm3 in ice-bound planets. This work was supported by NSF grant ANT-0440609.

  13. Noninvasive fluorescence excitation spectroscopy for the diagnosis of oral neoplasia in vivo

    NASA Astrophysics Data System (ADS)

    Ebenezar, Jeyasingh; Ganesan, Singaravelu; Aruna, Prakasarao; Muralinaidu, Radhakrishnan; Renganathan, Kannan; Saraswathy, Thillai Rajasekaran

    2012-09-01

    Fluorescence excitation spectroscopy (FES) is an emerging approach to cancer detection. The goal of this pilot study is to evaluate the diagnostic potential of FES technique for the detection and characterization of normal and cancerous oral lesions in vivo. Fluorescence excitation (FE) spectra from oral mucosa were recorded in the spectral range of 340 to 600 nm at 635 nm emission using a fiberoptic probe spectrofluorometer to obtain spectra from the buccal mucosa of 30 sites of 15 healthy volunteers and 15 sites of 10 cancerous patients. Significant FE spectral differences were observed between normal and well differentiated squamous cell carcinoma (WDSCC) oral lesions. The FE spectra of healthy volunteers consists of a broad emission band around 440 to 470 nm, whereas in WDSCC lesions, a new primary peak was seen at 410 nm with secondary peaks observed at 505, 540, and 580 nm due to the accumulation of porphyrins in oral lesions. The FE spectral bands of the WDSCC lesions resemble the typical absorption spectra of a porphyrin. Three potential ratios (I410/I505, I410/I540, and I410/I580) were calculated from the FE spectra and used as input variables for a stepwise linear discriminant analysis (SLDA) for normal and WDSCC groups. Leave-one-out (LOO) method of cross-validation was performed to check the reliability on spectral data for tissue characterization. The diagnostic sensitivity and specificity were determined for normal and WDSCC lesions from the scatter plot of the discriminant function scores. It was observed that diagnostic algorithm based on discriminant function scores obtained by SLDA-LOO method was able to distinguish WDSCC from normal lesions with a sensitivity of 100% and specificity of 100%. Results of the pilot study demonstrate that the FE spectral changes due to porphyrin have a good diagnostic potential; therefore, porphyrin can be used as a native tumor marker.

  14. Studies of cytochrome c-551 unfolding using fluorescence correlation spectroscopy and other biophysical techniques.

    PubMed

    Sil, Pallabi; Paul, Simanta Sarani; Silvio, Eva Di; Travaglini-Allocatelli, Carlo; Chattopadhyay, Krishnananda

    2016-09-21

    In this paper, we have studied the equilibrium unfolding transitions of cytochrome c from Pseudomonas aeruginosa (cytc551), a small bacterial protein. Similar to eukaryotic cytochrome c, cytc551 folds sequentially, although significant differences exist in the order of folding units (foldons). There are two regions of cytc551 (N-terminal helix with residue number 3 to 10 and the loop 2 region containing residues 34 to 45), in which no foldon unit could be assigned. In addition, the helix containing the Cys-X-X-Cys-His motif, adjacent to the N-terminal helix (residue number 3 to 10), shows unexplained ultra-fast collapse. To obtain further insights, we have studied cytc551 site-directed mutants using fluorescence correlation spectroscopy (FCS) and molecular dynamics simulation. We have found out that cytc551 unfolds through the formation of a fluorescently dark intermediate state and the amplitude of the dark component depends on the position of labeling. We have utilized this position dependence to propose a shape change model during the unfolding of cytc551. The present results show that the N-terminal helix remains in a collapsed position even in the completely unfolded state and this helix may act as a rigid support to guide the folding of its adjacent helix. This rigid support may be responsible for the ultra-fast collapse of the adjacent helix region, which occurs during the initial events of folding. The present results also show that the C-terminal end of loop 2 traverses a large distance during unfolding compared to the N-terminal end, which justifies the observed flexibility of the loop 2 region. PMID:27538920

  15. Velocity of movement of actin filaments in in vitro motility assay. Measured by fluorescence correlation spectroscopy.

    PubMed Central

    Borejdo, J; Burlacu, S

    1992-01-01

    We have measured the velocity of actin filaments in in vitro motility assay by fluorescence correlation spectroscopy. In this method, one measures fluctuations in the number of filaments in an open sample volume. The number of filaments was calculated from measurements of fluorescence of rhodamine-phalloidin bound to F-actin. Sample volume was defined by a diaphragm placed in front of the photomultiplier. Fluctuations arise when actin filaments enter and leave the sample volume due to translations driven by mechanochemical interactions with myosin heads which are immobilized on a glass surface. The average velocity of the translation of filaments determined by the correlation method, (Vc), was equal to the diameter of the diaphragm divided by the half-time of the relaxation of fluctuations. The average number of moving filaments determined by correlation method, (Nc), was inversely proportional to the relative fluctuations. By the fluctuation method it was possible to determine the average velocity of over 800 moving filaments in less than 4 min. There was good agreement between (Vc) and (Nc) and the average velocity and the average number of moving filaments determined manually. To be able to apply correlation measurements to an experimental problem, neither (Vc) nor (Nc) must depend on the position of observation of filaments. We first confirmed that this was indeed the case. We then applied the method to investigate the dependence of motility on the ATPase activity of myosin heads. ATPase activity was varied by mixing intact heads with heads which were labeled with different thiol reagents. It was found that the motion was drastically influenced by the reagent used for modification. When the reagent was N-ethyl-maleimide, 1.5% modification was sufficient to completely inhibit the motion. When the reagent was 5-iodoacetamidofluorescein, motion declined hyperbolically with the fraction of modified heads. Images FIGURE 2 FIGURE 4 FIGURE 11 PMID:1534696

  16. Dopamine Receptor Signaling in MIN6 β-Cells Revealed by Fluorescence Fluctuation Spectroscopy.

    PubMed

    Caldwell, Brittany; Ustione, Alessandro; Piston, David W

    2016-08-01

    Insulin secretion defects are central to the development of type II diabetes mellitus. Glucose stimulation of insulin secretion has been extensively studied, but its regulation by other stimuli such as incretins and neurotransmitters is not as well understood. We investigated the mechanisms underlying the inhibition of insulin secretion by dopamine, which is synthesized in pancreatic β-cells from circulating L-dopa. Previous research has shown that this inhibition is mediated primarily by activation of the dopamine receptor D3 subtype (DRD3), even though both DRD2 and DRD3 are expressed in β-cells. To understand this dichotomy, we investigated the dynamic interactions between the dopamine receptor subtypes and their G-proteins using two-color fluorescence fluctuation spectroscopy (FFS) of mouse MIN6 β-cells. We show that proper membrane localization of exogenous G-proteins depends on both the Gβ and Gγ subunits being overexpressed in the cell. Triple transfections of the dopamine receptor subtype and Gβ and Gγ subunits, each labeled with a different-colored fluorescent protein (FP), yielded plasma membrane expression of all three FPs and permitted an FFS evaluation of interactions between the dopamine receptors and the Gβγ complex. Upon dopamine stimulation, we measured a significant decrease in interactions between DRD3 and the Gβγ complex, which is consistent with receptor activation. In contrast, dopamine stimulation did not cause significant changes in the interactions between DRD2 and the Gβγ complex. These results demonstrate that two-color FFS is a powerful tool for measuring dynamic protein interactions in living cells, and show that preferential DRD3 signaling in β-cells occurs at the level of G-protein release. PMID:27508444

  17. Imaging fluorescence correlation spectroscopy studies of dye diffusion in self-assembled organic nanotubes.

    PubMed

    Xu, Hao; Nagasaka, Shinobu; Kameta, Naohiro; Masuda, Mitsutoshi; Ito, Takashi; Higgins, Daniel A

    2016-06-22

    The rate and mechanism of diffusion by anionic sulforhodamine B (SRB) dye molecules within organic nanotubes self-assembled from bolaamphiphile surfactants were investigated by imaging fluorescence correlation spectroscopy (imaging-FCS). The inner and outer surfaces of the nanotubes are terminated with amine and glucose groups, respectively; the former allow for pH-dependent manipulation of nanotube surface charge while the latter enhance their biocompatibility. Wide-field fluorescence video microscopy was used to locate and image dye-doped nanotubes dispersed on a glass surface. Imaging-FCS was then used to spatially resolve the SRB transport dynamics. Mobilization of the dye molecules was achieved by immersion of the nanotubes in buffer solution. Experiments were performed in pH 6.4, 7.4 and 8.4 buffers, at ionic strengths ranging from 1.73 mM to 520 mM. The results show that coulombic interactions between cationic ammonium ions on the inner nanotube surface and the anionic SRB molecules play a critical role in governing mass transport of the dye. The apparent dye diffusion coefficient, D, was found to generally increase with increasing ionic strength and with increasing pH. The D values obtained were found to be invariant along the nanotube length. Mass transport of the SRB molecules within the nanotubes is concluded to occur by a desorption-mediated Fickian diffusion mechanism in which dye motion is slowed by its coulombic interactions with the inner surfaces of the nanotubes. The results of these studies afford information essential to the use of organic nanotubes in controlled drug release applications. PMID:27271313

  18. Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy.

    PubMed

    Bismuto, E; Gratton, E; Lamb, D C

    2001-12-01

    The dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough to be observed in tuna apomyoglobin, whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyoglobin at pH 7 and room temperature the on rate is 2200 microM(-1) s(-1) and the off rate is 5900 s(-1), in comparison with k(on) = 640 microM(-1) s(-1) and k(off) = 560 s(-1) for horse myoglobin as measured previously. The independence of the reaction rate from the ANS concentration indicates that the reaction rate is dominated by the off rate. The temperature dependence of the on-rate shows that this rate is diffusion limited. The temperature dependence of the off rates analyzed by Arrhenius and Ferry models indicates that the off rate depends on the dynamics of the protein. The differences between horse and tuna apomyoglobins in the ANS binding rate can be explained in terms of the three-dimensional apoprotein structures obtained by energy minimization after heme removal starting from crystallographic coordinates. The comparison of the calculated apomyoglobin surfaces shows a 15% smaller cavity for tuna apomyoglobin. Furthermore, a negative charge (D44) is present in the heme cavity of tuna apomyoglobin that could decrease the strength of ANS binding. At pH 5 the fluorescence lifetime distribution of ANS-apomyoglobin is bimodal, suggesting the presence of an additional binding site in the protein. The binding rates determined by FCS under these conditions show that the protein is either in the open configuration or is more flexible, making it much easier to bind. At pH 3, the

  19. Diagnostics of a see-through hollow cathode discharge by emission, absorption, and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Taylor, Nicholas

    Atomic line filters have been suggested to be attractive in areas of Doppler velocimetry, resonance fluorescence detection, and resonance ionization detection. They are based on the resonant absorption of photons by an atomic vapor, and allow all other radiation to pass. This allows the detection of very low levels of light superimposed on a large optical background. Several elements have been studied for use as atomic line filters, such as the alkali metals, alkaline earths, and thallium. As previously recognized, thallium is especially attractive since the 535.046 nm metastable transition overlaps with the second harmonic output of an Nd:La2Be2O 5 (BEL) laser (1070 nm). This makes thallium ideal for certain applications as an atomic line filter. Recently a see-through hollow cathode lamp, or galvatron (Hamamatsu), was made commercially available. The galvatron geometry is unique compared to traditional hollow cathode lamps since the cathode and cell are oriented in a T-shape, with the cathode bored completely through to allow the propagation of a light source through the cathode. This allows multi-step excitation of the atomic vapor, not easily accomplished with a traditional hollow cathode lamp. The advantages that a galvatron offers over conventional atomic reservoirs make it an attractive candidate for the application as an atomic line filter; however, little spectroscopic data have been found in the literature. For this reason, Doppler temperatures, number densities, quantum efficiencies, and lifetimes have been determined in order to characterize this atomic reservoir as a potential atomic line filter. These parameters are determined by use of various spectroscopic techniques which include emission, absorption, time-resolved fluorescence, and time-resolved laser-induced saturated fluorescence spectroscopy. From these measurements, it has been demonstrated that a galvatron is an attractive atomic reservoir for applications as an atomic line filter. The

  20. Laser-induced Fluorescence Spectroscopy for applications in chemical sensing and optical refrigeration

    NASA Astrophysics Data System (ADS)

    Kumi Barimah, Eric

    Laser-induced breakdown spectroscopy (LIBS) is an innovative technique that has been used as a method for fast elemental analysis in real time. Conventional ultraviolet-visible (UV-VIS) LIBS has been applied to detect the elemental composition of different materials, including explosives, pharmaceutical drugs, and biological samples. The extension of conventional LIBS to the infrared region (˜1-12 mum) promises to provide additional information on molecular emission signatures due to rotational-vibrational transitions. In this research, a pulsed Nd: YAG laser operating at 1064 nm was focused onto several sodium compounds (NaCl, NaClO3, Na2CO3 and NaClO4) and potassium compounds (KCl, KClO3, K2CO3 and KClO4) to produce an intense plasma at the target surface. Several distinct infrared (IR) atomic emission signatures were observed from all sodium and potassium containing compounds. The atomic emission lines observed from the investigated samples matched assigned transitions of neutral sodium and potassium atoms published in the National Institute of Standards and Technology (NIST) atomic database. In addition to the intense atomic lines, the rst evidence of molecular LIBS emission structures were observed at ˜10.0 m in KClO3 and NaClO3 for the chlorate anion (ClO3 --1), at ˜6.7 to 8.0 mum in KNO3 and NaNO 3 for the nitrate anion (NO3--1 ), ˜8.0 to 10.0 mum in KClO4 and NaClO4 for perchlorate anion (ClO4--1 ), and ˜6.88 mum and 11.53 mum in Na2CO3 for the carbonate anion (CO3--1 ). The observed molecular emission showed strong correlation with the conventional Fourier Transform Infrared Spectrometry (FTIR) absorption spectra of the investigated samples. IR LIBS was also applied to determine the limit of detection (LOD) for the perchlorate anion in KClO4 using the 8.0 -11.0 mum IR-LIBS emission band. The calibration curve of ClO4 in KClO4 was constructed using peak and integrated emission intensities for known concentrations of mixed KClO4/NH4NO3 samples. The

  1. Solution conformation of 2-aminopurine (2-AP) dinucleotide determined by ultraviolet 2D fluorescence spectroscopy (UV-2D FS)

    PubMed Central

    Widom, Julia R.; Johnson, Neil P.; von Hippel, Peter H.; Marcus, Andrew H.

    2013-01-01

    We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) – a fluorescence-detected variation of 2D electronic spectroscopy – to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R12 = 3.5 Å ± 0.5 Å, twist angle θ12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes. PMID:24223491

  2. Time-resolved fluorescence spectroscopy of matrix-isolated silver atoms after pulsed excitation of inner-shell transitions

    NASA Astrophysics Data System (ADS)

    Hebert, T.; Wiggenhauser, H.; Schriever, U.; Kolb, D. M.

    1990-02-01

    The energy dissipation in matrix-isolated silver atoms after pulsed vacuum ultraviolet (VUV) excitation of 4d-5p transitions has been studied by time-resolved fluorescence spectroscopy. The decay behavior of the various fluorescence bands has been analyzed and a model for the relaxation process proposed within the framework of a two-dimensional configuration-coordinate diagram. If minute quantities of Ag2 are present in the matrix, the analysis requires consideration of energy transfer between silver atoms and dimers.

  3. Fluorescence polarization spectroscopy and time-resolved fluorescence kinetics of native cancerous and normal rat kidney tissues.

    PubMed Central

    Tata, D B; Foresti, M; Cordero, J; Tomashefsky, P; Alfano, M A; Alfano, R R

    1986-01-01

    Steady state fluorescence polarization spectra and time-resolved emission decay kinetics have been measured in vitro from malignant and normal rat kidney tissue. The degrees of polarization and emission lifetimes from the cancerous and normal systems are different. The spectroscopic differences are attributed to environmental transformations local to the native flavin and porphyrin fluorophors' binding sites. PMID:3489490

  4. Conformation and dynamics of nucleotides in bulges and symmetric internal loops in duplex DNA studied by EPR and fluorescence spectroscopies

    SciTech Connect

    Cekan, Pavol; Sigurdsson, Snorri Th.

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Bulges and loops were studied by both EPR and fluorescence spectroscopies using the probe C/C{sup f}. Black-Right-Pointing-Pointer One-base bulge was in a temperature-dependent equilibrium between looped-out and stacked states. Black-Right-Pointing-Pointer Bases in two- and three-base bulges were stacked at all temperatures, resulting in DNA bending. Black-Right-Pointing-Pointer Bases were stacked in symmetrical two- to five-base internal loops, according to EPR data. Black-Right-Pointing-Pointer Unexpectedly high fluorescence for the smaller loops indicated local structural perturbations. -- Abstract: The dynamics and conformation of base bulges and internal loops in duplex DNA were studied using the bifunctional spectroscopic probe C, which becomes fluorescent (C{sup f}) upon reduction of the nitroxide functional group, along with EPR and fluorescence spectroscopies. A one-base bulge was in a conformational equilibrium between looped-out and stacked states, the former favored at higher temperature and the latter at lower temperature. Stacking of bulge bases was favored in two- and three-base bulges, independent of temperature, resulting in DNA bending as evidenced by increased fluorescence of C{sup f}. EPR spectra of C-labeled three-, four- and five-base symmetrical interior DNA bulges at 20 Degree-Sign C showed low mobility, indicating that the spin-label was stacked within the loop. The spin-label mobility at 37 Degree-Sign C increased as the loops became larger. A considerable variation in fluorescence between different loops was observed, as well as a temperature-dependence within constructs. Fluorescence unexpectedly increased as the size of the loop decreased at 2 Degree-Sign C. Fluorescence of the smallest loops, where a single T{center_dot}T mismatch was located between the stem region and the probe, was even larger than for the single strand, indicating a considerable local structural deformation of these loops

  5. Effects of multiple scattering on fluorescence correlation spectroscopy measurements of particles moving within optically dense media

    PubMed Central

    Riley, Jason; Boukari, Hacène; Gandjbakhche, Amir; Nossal, Ralph

    2012-01-01

    Abstract. Fluorescence correlation spectroscopy (FCS) is increasingly being used to assess the movement of particles diffusing in complex, optically dense surroundings, in which case measurement conditions may complicate data interpretation. It is considered how a single-photon FCS measurement can be affected if the sample properties result in scattering of the incident light. FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads are measured, which acted as scatterers. Data indicated that a scattering-linked increase in the illuminated volume, as much as two fold, resulted in minimal increase in diffusivity. To analyze the illuminated beam profile, Monte-Carlo simulations were employed, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, results indicate that multiple scattering does not result in FCS measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media. PMID:23208294

  6. Phospholipid Diffusion Coefficients of Cushioned Model Membranes determined via Z-Scan Fluorescence Correlation Spectroscopy

    PubMed Central

    Sterling, Sarah M.; Allgeyer, Edward S.; Fick, Jörg; Prudovsky, Igor; Mason, Michael D.; Neivandt, David J.

    2013-01-01

    Model cellular membranes enable the study of biological processes in a controlled environment and reduce the traditional challenges associated with live or fixed cell studies. However, model membrane systems based on the air/water or oil/solution interface do not allow for incorporation of transmembrane proteins, or for the study of protein transport mechanisms. Conversely, a phospholipid bilayer deposited via the Langmuir-Blodgett/Langmuir Schaefer method on a hydrogel layer is potentially an effective mimic of the cross-section of a biological membrane, and facilitates both protein incorporation and transport studies. Prior to application, however, such membranes must be fully characterized, particularly with respect to the phospholipid bilayer phase transition temperature. Here we present a detailed characterization of the phase transition temperature of the inner and outer leaflets of a chitosan supported model membrane system. Specifically, the lateral diffusion coefficient of each individual leaflet has been determined as a function of temperature. Measurements were performed utilizing z-scan fluorescence correlation spectroscopy (FCS), a technique that yields calibration-free diffusion information. Analysis via the method of Wawrezinieck and coworkers, revealed that phospholipid diffusion changes from raft-like to free diffusion as the temperature is increased; an insight into the dynamic behavior of hydrogel supported membranes not previously reported. PMID:23705855

  7. Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy

    PubMed Central

    Clark, Natalie M; Hinde, Elizabeth; Winter, Cara M; Fisher, Adam P; Crosti, Giuseppe; Blilou, Ikram; Gratton, Enrico; Benfey, Philip N; Sozzani, Rosangela

    2016-01-01

    To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development. DOI: http://dx.doi.org/10.7554/eLife.14770.001 PMID:27288545

  8. A multi-channel monolithic Ge detector system for fluorescence x-ray absorption spectroscopy

    SciTech Connect

    Bucher, J.J.; Allen, P.G.; Edelstein, N.M.; Shuh, D.K.; Madden, N.W.; Cork, C.; Luke, P.; Pehl, D.; Malone, D.

    1995-03-01

    Construction and performance of a monolithic quad-pixel Ge detector for fluorescence x-ray absorption spectroscopy (XAS) at synchrotron radiation sources are described. The detector semiconductor element has an active surface area of 4.0 cm{sup 2} which is electrically separated into four 1.0 cm{sup 2} pixels, with little interfacial dead volume. Spatial response of the array shows that cross-talk between adjacent pixels is < 10% for 5.9 keV photons that fall within 0.5 mm of the pixel boundaries. The detector electronics system uses pre-amplifiers built at LBNL with commercial Tennelec Model TC 244 amplifiers. Using an {sup 55}Fe test source (MnK{sub {alpha}}, 5.9 keV), energy resolution of better than 200 eV is achieved with a 4 {mu}sec peaking time. At 0.5 {mu}sec peaking time, pulse pileup results in a 75% throughput efficiency for an incoming count rate of 100 kHz. Initial XAS fluoresncece measurements at the beamline 4 wiggler end stations at SSRL show that the detector system has several advantages over commercial x-ray spectrometers for low-concentration counting.

  9. Distinction of brain tissue, low grade and high grade glioma with time-resolved fluorescence spectroscopy.

    PubMed

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

    2006-01-01

    Neuropathology frozen section diagnoses are difficult in part because of the small tissue samples and the paucity of adjunctive rapid intraoperative stains. This study aims to explore the use of time-resolved laser-induced fluorescence spectroscopy as a rapid adjunctive tool for the diagnosis of glioma specimens and for distinction of glioma from normal tissues intraoperatively. Ten low grade gliomas, 15 high grade gliomas without necrosis, 6 high grade gliomas with necrosis and/or radiation effect, and 14 histologically uninvolved "normal" brain specimens are spectroscopicaly analyzed and contrasted. Tissue autofluorescence was induced with a pulsed Nitrogen laser (337 nm, 1.2 ns) and the transient intensity decay profiles were recorded in the 370-500 nm spectral range with a fast digitized (0.2 ns time resolution). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site were used for tissue characterization. A linear discriminant analysis diagnostic algorithm was used for tissue classification. Both low and high grade gliomas can be distinguished from histologically uninvolved cerebral cortex and white matter with high accuracy (above 90%). In addition, the presence or absence of treatment effect and/or necrosis can be identified in high grade gliomas. Taking advantage of tissue autofluorescence, this technique facilitates a direct and rapid investigation of surgically obtained tissue. PMID:16368511

  10. Mapping Liquid-liquid protein phase separation using ultra-fast-scanning fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Wei, Ming-Tzo; Elbaum-Garfinkle, Shana; Arnold, Craig B.; Priestley, Rodney D.; Brangwynne, Clifford P.

    Intrinsically disordered proteins (IDPs) are an understudied class of proteins that play important roles in a wide variety of biological processes in cells. We've previously shown that the C. elegans IDP LAF-1 phase separates into P granule-like droplets in vitro. However, the physics of the condensed phase remains poorly understood. Here, we use a novel technique, ultra-fast-scanning fluorescence correlation spectroscopy, to study the nano-scale rheological properties of LAF-1 droplets. Ultra-fast-scanning FCS uses a tunable acoustic gradient index of refraction (TAG) lens with an oil immersion objective to control axial movement of the focal point over a length of several micrometers at frequencies of 70kHz. Using ultra-fast-scanning FCS allows for the accurate determination of molecular concentrations and their diffusion coefficient, when the particle is passing through an excitation volume. Our work reveals an asymmetric LAF-1 phase diagram, and demonstrates that LAF-1 droplets are purely viscous phases which are highly tunable by salt concentration.

  11. Effects of multiple scattering on fluorescence correlation spectroscopy measurements of particles moving within optically dense media.

    PubMed

    Zustiak, Silviya; Riley, Jason; Boukari, Hacène; Gandjbakhche, Amir; Nossal, Ralph

    2012-12-01

    Fluorescence correlation spectroscopy (FCS) is increasingly being used to assess the movement of particles diffusing in complex, optically dense surroundings, in which case measurement conditions may complicate data interpretation. It is considered how a single-photon FCS measurement can be affected if the sample properties result in scattering of the incident light. FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads are measured, which acted as scatterers. Data indicated that a scattering-linked increase in the illuminated volume, as much as two fold, resulted in minimal increase in diffusivity. To analyze the illuminated beam profile, Monte-Carlo simulations were employed, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, results indicate that multiple scattering does not result in FCS measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media. PMID:23208294

  12. X-ray fluorescence spectroscopy and Monte Carlo characterization of a unique nuragic artifact (Sardinia, Italy)

    NASA Astrophysics Data System (ADS)

    Brunetti, Antonio; Depalmas, Anna; di Gennaro, Francesco; Serges, Alessandra; Schiavon, Nicola

    2016-07-01

    The chemical composition of a unique bronze artifact known as the "Cesta" ("Basket") belonging to the ancient Nuragic civilization of the Island of Sardinia, Italy has been analyzed by combining X-ray Fluorescence Spectroscopy (XRF) with Monte Carlo simulations using the XRMC code. The "Cesta" had been discovered probably in the XVIII century with the first graphic representation reported around 1761. In a later draft (dated 1764), the basket has been depicted as being carried upside-down on the shoulder of a large bronze warrior Barthélemy (1761), Pinza (1901), Winckelmann (1776) . The two pictorial representations differed only by the presence of handles in the most recent one. XRF measurements revealed that the handles of the object are composed by brass while the other parts are composed by bronze suggesting the handles as being a later addition to the original object. The artifact is covered at its surface by a fairly thick corrosion patina. In order to determine the bronze bulk composition without the need for removing the outer patina, the artifact has been modeled as a two layer object in Monte Carlo simulations.

  13. Lasing dynamics study by femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy

    NASA Astrophysics Data System (ADS)

    Wei, Dang; Qing, Liao; Peng-Cheng, Mao; Hong-Bing, Fu; Yu-Xiang, Weng

    2016-05-01

    Femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy (FNOPAS) is a versatile technique with advantages of high sensitivity, broad detection bandwidth, and intrinsic spectrum correction function. These advantages should benefit the study of coherent emission, such as measurement of lasing dynamics. In this letter, the FNOPAS was used to trace the lasing process in Rhodamine 6G (R6G) solution and organic semiconductor nano-wires. High-quality transient emission spectra and lasing dynamic traces were acquired, which demonstrates the applicability of FNOPAS in the study of lasing dynamics. Our work extends the application scope of the FNOPAS technique. Project supported by the National Natural Science Foundation of China (Grant Nos. 20925313 and 21503066), the Innovation Program of Chinese Academy of Sciences (Grant No. KJCX2-YW-W25), the Postdoctoral Project of Hebei University, China, and the Project of Science and Technology Bureau of Baoding City, China (Grant No. 15ZG029).

  14. X-ray Fluorescence Spectroscopy Study of Coating Thickness and Base Metal Composition

    NASA Technical Reports Server (NTRS)

    Rolin, T. D.; Leszczuk, Y.

    2008-01-01

    For electrical, electronic, and electromechanical (EEE) parts to be approved for space use, they must be able to meet safety standards approved by NASA. A fast, reliable, and precise method is needed to make sure these standards are met. Many EEE parts are coated in gold (Au) and nickel (Ni), and the thickness coating is crucial to a part s performance. A nondestructive method that is efficient in measuring coating thickness is x-ray fluorescence (XRF) spectroscopy. The XRF spectrometer is a machine designed to measure layer thickness and composition of single or multilayered samples. By understanding the limitations in the collection of the data by this method, accurate composition and thickness measurements can be obtained for samples with Au and Ni coatings. To understand the limitations of data found, measurements were taken with the XRF spectrometer and compared to true values of standard reference materials (SRM) that were National Institute of Standards and Technology (NIST) traceable. For every sample, six different parameters were varied to understand measurement error: coating/substrate combination, number of layers, counting interval, collimator size, coating thickness, and test area location. Each measurement was taken in accordance with standards set by the American Society for Testing and Materials (ASTM) International Standard B 568.

  15. Investigation of metal ions binding of humic substances using fluorescence emission and synchronous-scan spectroscopy.

    PubMed

    Piana, M J; Zahir, K O

    2000-01-01

    The binding site interactions of IHSS humic substances, Suwannee River Humic Acid, Suwannee River Fulvic Acid, Nordic Fulvic Acid, and Aldrich Humic Acid with various metals ions and a herbicide, methyl viologen were investigated using fluorescence emission and synchronous-scan spectroscopy. The metal ions used were, Fe(III), Cr(III), Cr(VI), Pb(II), Cu(II) and Ni(II). Stern-Volmer constants, Ksv for these quenchers were determined at pH 4 and 8 using an ionic strength of 0.1 M. For all four humic substances, and at both pH studied, Fe(III) was found to be the most efficient quencher. Quenching efficiency was found to be 3-10 times higher at pH 8. The bimolecular quenching rate constants were found to exceed the maximum considered for diffusion controlled interactions, and indicate that the fluorophore and quencher are in close physical association. Synchronous-scan spectra were found to change with pH and provided useful information on binding site interactions between humic substances and these quenchers. PMID:10693057

  16. Green direct determination of mineral elements in artichokes by infrared spectroscopy and X-ray fluorescence.

    PubMed

    Mir-Marqués, Alba; Martínez-García, Maria; Garrigues, Salvador; Cervera, M Luisa; de la Guardia, Miguel

    2016-04-01

    Near infrared (NIR) and X-ray fluorescence (XRF) spectroscopy were investigated to predict the concentration of calcium, potassium, iron, magnesium, manganese and zinc in artichoke samples. Sixty artichokes were purchased from different Spanish areas (Benicarló, Valencia and Murcia). NIR and XRF spectra, combined with partial least squares (PLS) data treatment, were used to develop chemometric models for the prediction of mineral concentration. To obtain reference data, samples were mineralised and analysed by inductively coupled plasma optical emission spectrometry (ICP-OES). Coefficients of determination obtained for the regression between predicted values and reference ones for calcium, potassium, magnesium, iron, manganese and zinc were 0.61, 0.79, 0.53, 0.77, 0.54 and 0.60 for NIR and 0.96, 0.93, 0.80, 0.79, 0.76 and 0.90 for XRF, respectively. Both assayed methodologies, offer green alternatives to classical mineral analysis, but XRF provided the best results in order to be used as a quantitative screening method. PMID:26593585

  17. Electronically excited states of DNA oligonucleotides with disordered base sequences studied by fluorescence spectroscopy.

    PubMed

    Vayá, Ignacio; Brazard, Johanna; Gustavsson, Thomas; Markovitsi, Dimitra

    2012-11-01

    DNA double-stranded oligomers are studied by steady-state and time-resolved fluorescence spectroscopy from the femtosecond to the nanosecond time-scale, following excitation at 267 nm. It is shown that emission arises from three types of excited states. (i) Bright ππ* states emitting around 330 nm and decaying on the sub-picosecond time-scale with an average lifetime of ca. 0.4 ps and a quantum yield lower than 4 × 10(-6). (ii) Excimers/exciplexes emitting around 430 nm and decaying on the sub-nanosecond time-scale. (iii) Excited states emitting mainly at short wavelengths (λ < 330 nm) and decaying on the nanosecond time-scale, possibly correlated to GC pairs. The properties of the examined duplexes, exhibiting significant disorder with respect to the nearest neighbour base sequence, are radically different than those of the much longer and disordered calf thymus DNA. Such behaviour suggests that long range and/or sequence effects play a key role in the fate of excitation energy. PMID:23034563

  18. Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy.

    PubMed

    Clark, Natalie M; Hinde, Elizabeth; Winter, Cara M; Fisher, Adam P; Crosti, Giuseppe; Blilou, Ikram; Gratton, Enrico; Benfey, Philip N; Sozzani, Rosangela

    2016-01-01

    To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development. PMID:27288545

  19. Adsorption of Uranyl on Gibbsite: A Time-Resolved Laser-Induced Fluorescence Spectroscopy Study

    SciTech Connect

    Chang, Hyun-shik; Korshin, Gregory V.; Wang, Zheming; Zachara, John M.

    2006-02-15

    Uranyl adsorbed on gibbsite at pH 4.0-8.0 and ionic strengths (ISs) 0.001-0.4 M (NaClO4) in the absence of carbonate was studied using time-resolved laser-induced fluorescence spectroscopy (TRLIFS) under cryogenic conditions. TRLIFS data showed the presence of several distinct emission components. Their contributions were determined using the evolving factor analysis approach. Four components denoted as species A, B, C, and D were discerned. Each of them was characterized by a characteristic response to pH and IS changes and also by a unique combination of the values of the fundamental transition energy E0,0, vibronic spacing E, and half-width of the vibronic lines W. Species A and B were major contributors to the overall emission. They were mainly affected by the pH and predominated below and above pH 5.0, respectively. In contrast with that, the contribution of species C was noticeable only at IS = 0.001 M, while it was suppressed or absent at high IS values. It was concluded that species A and B are likely to correspond to inner-sphere surface aluminol complexes AlO-(UO2)+ and AlO-(UO2)OH, while species C was hypothesized to correspond to electrostatically bound uranyl complexes (predominantly [UO2(OH)3]-).

  20. Americium characterization by X-ray fluorescence and absorption spectroscopy in plutonium uranium mixed oxide

    NASA Astrophysics Data System (ADS)

    Degueldre, Claude; Cozzo, Cedric; Martin, Matthias; Grolimund, Daniel; Mieszczynski, Cyprian

    2013-06-01

    Plutonium uranium mixed oxide (MOX) fuels are currently used in nuclear reactors. The actinides in these fuels need to be analyzed after irradiation for assessing their behaviour with regard to their environment and the coolant. In this work the study of the atomic structure and next-neighbour environment of Am in the (Pu,U)O2 lattice in an irradiated (60 MW d kg-1) MOX sample was performed employing micro-X-ray fluorescence (µ-XRF) and micro-X-ray absorption fine structure (µ-XAFS) spectroscopy. The chemical bonds, valences and stoichiometry of Am (˜0.66 wt%) are determined from the experimental data gained for the irradiated fuel material examined in its peripheral zone (rim) of the fuel. In the irradiated sample Am builds up as Am3+ species within an [AmO8]13- coordination environment (e.g. >90%) and no (<10%) Am(IV) or (V) can be detected in the rim zone. The occurrence of americium dioxide is avoided by the redox buffering activity of the uranium dioxide matrix.

  1. Characterizing natural organic material from the Occoquan Watershed (Northern Virginia, US) using fluorescence spectroscopy and PARAFAC.

    PubMed

    Holbrook, R David; Yen, James H; Grizzard, Thomas J

    2006-05-15

    Surface water samples from the Occoquan Watershed (Virginia, US) were collected during summer baseflow conditions and characterized by excitation-emission matrix (EEM) fluorescence spectroscopy and parallel factor analysis (PARAFAC). Based on 55 samples, PARAFAC identified three individual fluorophore moieties, which were attributed to humic-like, fulvic-like, and protein-like materials. The categorization of these seemingly universal fluorophore moieties in aqueous systems was consistent with expected analyte concentrations that were independently measured by traditional wet chemistry techniques. The relative distribution of the three fluorophore fractions varied among the different land use catchments, especially in locations of known anthropogenic activity. Distinctive relationships between the fulvic-like and protein-like materials were observed for catchments known to be influenced by anthropogenic activity and those believed to reflect more natural environments, suggesting that this technique could be used to monitor human impact on aquatic systems. Finally, PARAFAC can be used to provide estimates (+/-30%) of select analyte concentrations in surface water. The results of this study support the use of EEM and PARAFAC analyses by watershed managers and other personnel interested in understanding organic matter behavior in aquatic systems. PMID:16442152

  2. Fluorescence Spectroscopy in Thermodynamic and Kinetic Analysis of pH-Dependent Membrane Protein Insertion

    PubMed Central

    Ladokhin, Alexey S.

    2016-01-01

    Experimental determination of the free energy stabilizing the structure of membrane proteins in their native lipid environment is undermined by a lack of appropriate methods and suitable model systems. Here, we demonstrate how fluorescence correlation spectroscopy can be used to characterize thermodynamics of pH-triggered bilayer insertion of nonconstitutive membrane proteins (e.g., bacterial toxins, colicins). The experimental design is guided by the appropriate thermodynamic scheme which considers two independent processes: pH-dependent formation of a membrane-competent form and its insertion into the lipid bilayer. Measurements of a model protein annexin B12 under conditions of lipid saturation demonstrate that protonation leading to the formation of the membrane-competent state occurs near membrane interface. Lipid titration experiments demonstrate that the free energy of transfer to the intermediate interfacial state is especially favorable, while the free energy of final insertion is modulated by interplay of hydrophobic and electrostatic interactions on the bilayer interface. The general principles of kinetic measurements along the insertion pathway containing interfacial intermediate are discussed and practical examples emphasizing appropriate fitting and normalization procedures are presented. PMID:21609856

  3. A Fluorescence Correlation Spectroscopy Study of the Cryoprotective Mechanism of Glucose on Hemocyanin

    NASA Astrophysics Data System (ADS)

    Hauger, Eric J.

    Cryopreservation is the method of preserving biomaterials by cooling and storing them at very low temperatures. In order to prevent the damaging effects of cooling, cryoprotectants are used to inhibit ice formation. Common cryoprotectants used today include ethylene glycol, propylene glycol, dimethyl sulfoxide, glycerol, and sugars. However, the mechanism responsible for the effectiveness of these cryoprotectants is poorly understood on the molecular level. The water replacement model predicts that water molecules around the surfaces of proteins are replaced with sugar molecules, forming a protective layer against the denaturing ice formation. Under this scheme, one would expect an increase in the hydrodynamic radius with increasing sugar concentration. In order to test this hypothesis, two-photon fluorescence correlation spectroscopy (FCS) was used to measure the hydrodynamic radius of hemocyanin (Hc), an oxygen-carrying protein found in arthropods, in glucose solutions up to 20wt%. FCS found that the hydrodynamic radius was invariant with increasing glucose concentration. Dynamic light scattering (DLS) results verified the hydrodynamic radius of hemocyanin in the absence of glucose. Although this invariant trend seems to indicate that the water replacement hypothesis is invalid the expected glucose layer around the Hc is smaller than the error in the hydrodynamic radius measurements for FCS. The expected change in the hydrodynamic radius with an additional layer of glucose is 1nm, however, the FCS standard error is +/-3.61nm. Therefore, the water replacement model cannot be confirmed nor refuted as a possible explanation for the cryoprotective effects of glucose on Hc.

  4. Heat shock-induced interactions among nuclear HSFs detected by fluorescence cross-correlation spectroscopy

    SciTech Connect

    Pack, Chan-Gi; Ahn, Sang-Gun

    2015-07-31

    The cellular response to stress is primarily controlled in cells via transcriptional activation by heat shock factor 1 (HSF1). HSF1 is well-known to form homotrimers for activation upon heat shock and subsequently bind to target DNAs, such as heat-shock elements, by forming stress granules. A previous study demonstrated that nuclear HSF1 and HSF2 molecules in live cells interacted with target DNAs on the stress granules. However, the process underlying the binding interactions of HSF family in cells upon heat shock remains unclear. This study demonstrate for the first time that the interaction kinetics among nuclear HSF1, HSF2, and HSF4 upon heat shock can be detected directly in live cells using dual color fluorescence cross-correlation spectroscopy (FCCS). FCCS analyses indicated that the binding between HSFs was dramatically changed by heat shock. Interestingly, the recovery kinetics of interaction between HSF1 molecules after heat shock could be represented by changes in the relative interaction amplitude and mobility. - Highlights: • The binding interactions among nuclear HSFs were successfully detected. • The binding kinetics between HSF1s during recovery was quantified. • HSF2 and HSF4 strongly formed hetero-complex, even before heat shock. • Nuclear HSF2 and HSF4 bound to HSF1 only after heat shock.

  5. Photolithography and Fluorescence Correlation Spectroscopy used to examine the rates of exchange in reverse micelle systems

    NASA Astrophysics Data System (ADS)

    Norris, Zach; Mawson, Cara; Johnson, Kyron; Kessler, Sarah; Rebecca, Anne; Wolf, Nathan; Lim, Michael; Nucci, Nathaniel

    Reverse micelles are molecular complexes that encapsulate a nanoscale pool of water in a surfactant shell dissolved in non-polar solvent. These complexes have a wide range of applications, and in all cases, the degree to which reverse micelles (RM) exchange their contents is relevant for their use. Despite its importance, this aspect of RM behavior is poorly understood. Photolithography is employed here to create micro and nano scale fluidic systems in which mixing rates can be precisely measured using fluorescence correlation spectroscopy (FCS). Micro-channel patterns are etched using reactive ion etching process into a layer of silicon dioxide on crystalline silicon substrates. Solutions containing mixtures of reverse micelles, proteins, and fluorophores are placed into reservoirs in the patterns, while diffusion and exchange between RMs is monitored using a FCS system built from a modified confocal Raman spectrometer. Using this approach, the diffusion and exchange rates for RM systems are measured as a function of the components of the RM mixture. Funding provided by Rowan University.

  6. Investigating axial diffusion in cylindrical pores using confocal single-particle fluorescence correlation spectroscopy.

    PubMed

    Chen, Fang; Neupane, Bhanu; Li, Peiyuan; Su, Wei; Wang, Gufeng

    2016-08-01

    We explored the feasibility of using confocal fluorescence correlation spectroscopy to study small nanoparticle diffusion in hundred-nanometer-sized cylindrical pores. By modeling single particle diffusion in tube-like confined three-dimensional space aligned parallel to the confocal optical axis, we showed that two diffusion dynamics can be observed in both original intensity traces and the autocorrelation functions (ACFs): the confined two-dimensional lateral diffusion and the unconfined one-dimensional (1D) axial diffusion. The separation of the axial and confined lateral diffusion dynamics provides an opportunity to study diffusions in different dimensions separately. We further experimentally studied 45 nm carboxylated polystyrene particles diffusing in 300 nm alumina pores. The experimental data showed consistency with the simulation. To extract the accurate axial diffusion coefficient, we found that a 1D diffusion model with a Lorentzian axial collection profile needs to be used to analyze the experimental ACFs. The diffusion of the 45 nm nanoparticles in polyethyleneglycol-passivated 300 nm pores slowed down by a factor of ∼2, which can be satisfactorily explained by hydrodynamic frictions. PMID:27196052

  7. Characterization of Diblock Copolymer Order-Order Transitions in Semidilute Aqueous Solution Using Fluorescence Correlation Spectroscopy.

    PubMed

    Clarkson, Christopher G; Lovett, Joseph R; Madsen, Jeppe; Armes, Steven P; Geoghegan, Mark

    2015-09-01

    The temperature and pH-dependent diffusion of poly(glycerol monomethacrylate)-block-poly(2-hydroxypropyl methacrylate) nanoparticles prepared via polymerization-induced self-assembly in water is characterized using fluorescence correlation spectroscopy (FCS). Lowering the solution temperature or raising the solution pH induces a worm-to-sphere transition and hence an increase in diffusion coefficient by a factor of between four and eight. FCS enables morphological transitions to be monitored at relatively high copolymer concentrations (10% w/w) compared to those required for dynamic light scattering (0.1% w/w). This is important because such transitions are reversible at the former concentration, whereas they are irreversible at the latter. Furthermore, the FCS data suggest that the thermal transition takes place over a very narrow temperature range (less than 2 °C). These results demonstrate the application of FCS to characterize order-order transitions, as opposed to order-disorder transitions. PMID:26096738

  8. Tar analysis from biomass gasification by means of online fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Baumhakl, Christoph; Karellas, Sotirios

    2011-07-01

    Optical methods in gas analysis are very valuable mainly due to their non-intrusive character. That gives the possibility to use them for in-situ or online measurements with only optical intervention in the measurement volume. In processes like the gasification of biomass, it is of high importance to monitor the gas quality in order to use the product gas in proper machines for energy production following the restrictions in the gas composition but also improving its quality, which leads to high efficient systems. One of the main problems in the biomass gasification process is the formation of tars. These higher hydrocarbons can lead to problems in the operation of the energy system. Up to date, the state of the art method used widely for the determination of tars is a standardized offline measurement system, the so-called "Tar Protocol". The aim of this work is to describe an innovative, online, optical method for determining the tar content of the product gas by means of fluorescence spectroscopy. This method uses optical sources and detectors that can be found in the market at low cost and therefore it is very attractive, especially for industrial applications where cost efficiency followed by medium to high precision are of high importance.

  9. Perturbation Facilitated Dispersed Fluorescence and Stimulated Emission Pumping Spectroscopies of HCP

    NASA Astrophysics Data System (ADS)

    Ishikawa, Haruki; Muramoto, Yasuhiko; Namai, Masahito; Mikami, Naohiko

    2011-06-01

    Perturbations among molecular rovibronic levels provide us with mainly two benefits. Perturbations themselves are characteristic features of structure and dynamics of molecules. We have been investigating dynamics of highly excited vibrational levels of HCP in the tilde{X} ^1Σ^+ state by dispersed fluorescence (DF) and stimulated emission pumping (SEP) spectroscopies of the tilde{C} ^1A^' - tilde{X} ^1Σ^+ transition. In the case of tilde{X} ^1Σ^+ HCP, its vibrational dynamics is well described by the Fermi resonance between the bend and the CP stretch modes. Based on the analysis of the Fermi resonance, we have succeeded in revealing the change in character of the bending motion in highly excited vibrational levels. In addition, perturbations enable us to explore rovibrational levels into much wider region that cannot be accessed under limits of selection rules. Jacobson and Child showed that the Coriolis interaction becomes very strong in the highly excited levels near and the above the CPH barrier. For the experimental confirmation of their prediction, the observation of the VCH≠0 and the ℓ'' ≠ 0 levels are necessary. However, due to the selection rules and the Franck-Condon selectivity, only the VCH=0 and the ℓ''=0 levels had been observed. In the course of our study, we have found a perturbed level in the tilde{C} state. In general, a very clear even-v_2 progression appears in the DF spectra of HCP. However, in the DF spectra measured by using the perturbed level as the intermediate both the odd- and even-v_2 levels are observed. Moreover, several VCH=1 levels are observed in the spectra. The perturbation-facilitated DF and SEP spectroscopies are very powerful tools to exploring the highly excited vibrational levels of HCP. Details of the perturbation-facilitated DF and SEP spectroscopies are presented in the paper. H. Ishikawa, et al. J. Chem. Phys. 109, 492 (1998); H. Ishikawa, et al. Annu. Rev. Phys. Chem. 50, 443 (1999). M. P. Jacobson and M. S

  10. Online identification of the fluorescent whitening agent 4,4-bis(2-sulfostyrol)biphenyl using a sweeping technique combined with capillary electrophoresis/77 K fluorescence spectroscopy.

    PubMed

    Kuo, Ting-Yang; Wang, Shu-Ping; Lin, Cheng-Huang

    2005-11-01

    The feasibility of combining the techniques of online concentration and CE/low-temperature fluorescence spectroscopy in the detection and identification of E,E-4,4'-bis(2-sulfostyryl)biphenyl (DSBP) in synthetic detergents at 77 K is demonstrated. The technique involves the use of sweeping-MEKC, and was used for the initial online concentration and separation, after which a cryogenic molecular fluorescence experiment was performed at 77 K. The proposed method not only permits the separation and detection of E,E-DSBP in a synthetic detergent sample, but also ensures that the online spectrum is readily distinguishable and can be unambiguously assigned at 77 K. The photoconversion and isomer separation of DSBP are also described. PMID:16240298

  11. Assessment of the quality attributes of cod caviar paste by means of front-face fluorescence spectroscopy.

    PubMed

    Airado-Rodríguez, Diego; Skaret, Josefine; Wold, Jens Petter

    2010-05-12

    This paper describes the fluorescent behavior of cod caviar paste, stored under different conditions, in terms of light exposure and concentration of oxygen in the headspace. Multivariate curve resolution was employed to decompose the overall fluorescence spectra into pure fluorescent components and calculate the relative concentrations of these components in the different samples. Profiles corresponding to protoporphyrin IX, photoprotoporphyrin, and fluorescent oxidation products were identified. Sensory evaluation, TBARS, and analysis of volatiles are typical methods employed in the routine analysis and quality control of such food. Successful calibration models were established between fluorescence and those routine methods. Correlation coefficients higher than 0.80 were found for 79% and higher than 0.90 for 50% of the assessed odors and flavors. For instance, R values of 0.94, and 0.96 were obtained for fresh and rancid flavors respectively, and 0.89 for TBARS. On the basis of these data, it can be argued that front-face fluorescence spectroscopy can substitute all of these expensive and tedious methodologies. PMID:20373814

  12. Effect of ethanol-water mixture on the structure and dynamics of lysozyme: A fluorescence correlation spectroscopy study

    NASA Astrophysics Data System (ADS)

    Chattoraj, Shyamtanu; Mandal, Amit Kumar; Bhattacharyya, Kankan

    2014-03-01

    Effect of ethanol-water mixture on the hydrodynamic radius (rH) and conformational dynamics of lysozyme has been studied by circular dichroism, emission spectra, and fluorescence correlation spectroscopy. For this purpose, the protein lysozyme is covalently labeled near the active site with a fluorescent probe, alexa 488. The ethanol molecules are sequestered near the hydrophobic tryptophan residues as indicated by the blue shift of the emission maximum of tryptophan. It is observed that both size (rH) and time constant of conformational relaxation (τR) of lysozyme oscillate with increase in ethanol concentration. The rH of the protein fluctuates from 19 Å in the native state, to a minimum of 13 Å, and a maximum of 29 Å. It is proposed that the oscillating behavior arises from competition between mutual interaction among protein, ethanol, and water. The fluorescence intensity fluctuates because of quenching of the fluorescence of the probe (alexa) by the free amino group of certain residues (e.g., tryptophan). Rate of inter-conversion (folding dynamics) between the open (fluorescent) and closed (non-fluorescent) form has been determined and is found to exhibit similar oscillation with variation in ethanol content.

  13. Time-resolved spectroscopy of charge-transfer fluorescent molecules in polymer matrices

    NASA Astrophysics Data System (ADS)

    Hofstraat, Johannes W.; Verhey, H. J.; Verhoeven, Jan W.; Kuemke, M.; McGown, Linda B.; Novikov, Eugene G.; van Hoek, Arie; Visser, Antonie J. W. G.

    1996-03-01

    Time-resolved fluorescence measurements have been carried out on charge-transfer fluorescent molecules incorporated in polymeric lattices, consisting of polystyrene cores and polyglycidylmethacrylate shells, and in polymethylmethacrylate thin films. New approaches to the analysis of fluorescence lifetime data obtained for molecules in polymer matrices had to be applied, since conventional analysis methods appeared not suitable for such strongly heterogeneous systems. The polymer lattices could be characterized by application of phase- resolved fluorescence lifetime measurements followed by maximum-entropy methods for data analysis. The thin films were studied using time-correlated single photon counting fluorescence lifetime measurements and data analysis with a home-built program based on stretched exponential decays. Interactions of the fluorescent guest molecules could be established by combined fluorescence lifetime and depolarization measurements. Suggestions for further improvements in fluorescence lifetime methods for characterization of polymeric materials have been made.

  14. Monitoring the accumulation of lipofuscin in aging murine eyes by fluorescence spectroscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The integrated fluorescence of murine eyes is collected as a function of age. This fluorescence is attributed to pigments generally referred to as lipofuscin and is observed to increase with age. No difference in fluorescence intensity is observed between the eyes of males or females. This work p...

  15. A Nanoplasmonic Strategy for Precision in-situ Measurements of Tip-enhanced Raman and Fluorescence Spectroscopy.

    PubMed

    Meng, Lingyan; Sun, Mengtao; Chen, Jianing; Yang, Zhilin

    2016-01-01

    We theoretically investigate an optimized tip-film system that supports in-situ measurement of tip-enhanced Raman spectroscopy (TERS) and tip-enhanced fluorescence (TEF) of dye molecules. A scanning tunneling microscope (STM) is proposed to precisely control the tip-film distance, and thus in-situ measurement of TERS and TEF can be realized utilizing the specific surface plasmon resonance (SPR) properties of the tip-film system. Our calculations show that the optimized tip-film distance of 2 nm suggests a possibility of efficient acquisition of TERS and TEF in-situ. The calculated spatial resolution of TERS and spectral resolution of TEF can be down to 6.5 nm and 10 nm, respectively. Our theoretical results may find promising application in developing multiple functional nano-spectroscopy through which Raman and fluorescence can be measured in-situ at the nanoscale level. PMID:26780882

  16. A Nanoplasmonic Strategy for Precision in-situ Measurements of Tip-enhanced Raman and Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Meng, Lingyan; Sun, Mengtao; Chen, Jianing; Yang, Zhilin

    2016-01-01

    We theoretically investigate an optimized tip-film system that supports in-situ measurement of tip-enhanced Raman spectroscopy (TERS) and tip-enhanced fluorescence (TEF) of dye molecules. A scanning tunneling microscope (STM) is proposed to precisely control the tip-film distance, and thus in-situ measurement of TERS and TEF can be realized utilizing the specific surface plasmon resonance (SPR) properties of the tip-film system. Our calculations show that the optimized tip-film distance of 2 nm suggests a possibility of efficient acquisition of TERS and TEF in-situ. The calculated spatial resolution of TERS and spectral resolution of TEF can be down to 6.5 nm and 10 nm, respectively. Our theoretical results may find promising application in developing multiple functional nano-spectroscopy through which Raman and fluorescence can be measured in-situ at the nanoscale level.

  17. A Nanoplasmonic Strategy for Precision in-situ Measurements of Tip-enhanced Raman and Fluorescence Spectroscopy

    PubMed Central

    Meng, Lingyan; Sun, Mengtao; Chen, Jianing; Yang, Zhilin

    2016-01-01

    We theoretically investigate an optimized tip-film system that supports in-situ measurement of tip-enhanced Raman spectroscopy (TERS) and tip-enhanced fluorescence (TEF) of dye molecules. A scanning tunneling microscope (STM) is proposed to precisely control the tip-film distance, and thus in-situ measurement of TERS and TEF can be realized utilizing the specific surface plasmon resonance (SPR) properties of the tip-film system. Our calculations show that the optimized tip-film distance of 2 nm suggests a possibility of efficient acquisition of TERS and TEF in-situ. The calculated spatial resolution of TERS and spectral resolution of TEF can be down to 6.5 nm and 10 nm, respectively. Our theoretical results may find promising application in developing multiple functional nano-spectroscopy through which Raman and fluorescence can be measured in-situ at the nanoscale level. PMID:26780882

  18. Development of fiber optic spectroscopy for in-vitro and in-planta detection of fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Liew, Oi Wah; Chen, Jun-Wei; Asundi, Anand K.

    2001-10-01

    The objective of this project is to apply photonics technology to bio-safety management of genetically modified (GM) plants. The conventional method for screening GM plants is through selection using antibiotic resistance markers. There is public concern with such approaches and these are associated with food safety issues, escape of antibiotic resistance genes to pathogenic microorganisms and interference with antibiotic therapy. Thus, the strategy taken in this project is to replace antibiotic resistance markers with fluorescent protein markers that allow for rapid and non-invasive optical screening of genetically modified plants. In this paper, fibre optic spectroscopy was developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in planta. In vitro detection was first carried out to optimize the sensitivity of the optical system. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fibre optic spectroscopy using different light sources, namely, blue LED (475 nm), tungsten halogen (350 - 1000 nm) and double frequency Nd:YAG green laser (532 nm). Fluorescence near the expected emission wavelengths could be detected up to 320X dilution for EGFP and DsRED with blue LED and 532 nm green laser, respectively, as the excitation source. Tungsten halogen was found to be unsuitable for excitation of both EGFP and DsRED. EGFP was successfully purified by size separation under non-denaturing electrophoretic conditions and quantified. The minimum concentration of EGFP detectable with blue LED excitation was 5 mg/ml. To determine the capability of spectroscopy detection in planta, transgenic potato hairy roots and whole modified plant lines expressing the

  19. Coherent photon interference elimination and spectral correction in femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy

    NASA Astrophysics Data System (ADS)

    Dang, Wei; Mao, Pengcheng; Weng, Yuxiang

    2013-07-01

    We report an improved setup of femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy (FNOPAS) with a 210 fs temporal response. The system employs a Cassegrain objective to collect and focus fluorescence photons, which eliminates the interference from the coherent photons in the fluorescence amplification by temporal separation of the coherent photons and the fluorescence photons. The gain factor of the Cassegrain objective-assisted FNOPAS is characterized as 1.24 × 105 for Rhodamine 6G. Spectral corrections have been performed on the transient fluorescence spectra of Rhodamine 6G and Rhodamine 640 in ethanol by using an intrinsic calibration curve derived from the spectrum of superfluorescence, which is generated from the amplification of the vacuum quantum noise. The validity of spectral correction is illustrated by comparisons of spectral shape and peak wavelength between the corrected transient fluorescence spectra of these two dyes acquired by FNOPAS and their corresponding standard reference spectra collected by the commercial streak camera. The transient fluorescence spectra of the Rhodamine 6G were acquired in an optimized phase match condition, which gives a deviation in the peak wavelength between the retrieved spectrum and the reference spectrum of 1.0 nm, while those of Rhodamine 640 were collected in a non-optimized phase match condition, leading to a deviation in a range of 1.0-3.0 nm. Our results indicate that the improved FNOPAS can be a reliable tool in the measurement of transient fluorescence spectrum for its high temporal resolution and faithfully corrected spectrum.

  20. Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule

    NASA Astrophysics Data System (ADS)

    Hassler, Kai; Leutenegger, Marcel; Rigler, Per; Rao, Ramachandra; Rigler, Rudolf; Gösch, Michael; Lasser, Theo

    2005-09-01

    We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores.

  1. Kinesin-1 inhibits the aggregation of amyloid-β peptide as detected by fluorescence cross-correlation spectroscopy.

    PubMed

    Zheng, Yanpeng; Tian, Shijun; Peng, Xianglei; Yang, Jingfa; Fu, Yuanhui; Jiao, Yueying; Zhao, Jiang; He, Jinsheng; Hong, Tao

    2016-04-01

    Although the exact etiology and pathogenesis of Alzheimer's disease (AD) are still unclear, amyloid-β (Aβ) generated by the proteolytic processing of amyloid-β precursor protein (APP) aggregate to form toxic amyloid species. Kinesin-1 is the first identified ATP-dependent axonal transport motor protein that has been proven to affect Aβ generation and deposition. In this paper, we applied dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) to investigate the direct interaction of Aβ with kinesin-1 at the single-molecule fluorescence level in vitro. The results showed that two kinds of enhanced green fluorescent protein (EGFP)-tagged kinesin light-chain subunits of kinesin-1(KLCs), KLC-E and E-KLC inhibited the aggregation of Aβ over a period of time, providing additional insight into the mechanism of axonal transport deficits in AD. PMID:26991731

  2. Multi-wavelength fluorescence lifetime spectroscopy: a new approach to the study of endogenous fluorescence in living cells and tissues

    NASA Astrophysics Data System (ADS)

    Chorvat, D., Jr.; Chorvatova, A.

    2009-03-01

    The study of biological systems in their real environmental conditions is crucial to decipher the true image of structures and processes underlying their functionality. In this regard, development of non-invasive optical techniques that do not require labelling, such as the investigation of tissue endogenous fluorescence, is particularly important and, as reflected in the increasing number of contributions published recently on this subject, was recognized by many leading groups. Multi-spectral and lifetime detection of fluorescence provides an effective experimental tool to discriminate between multiple naturally-occurring fluorophores in living tissues. At the same time, however, data analysis allowing us to understand the spectral, temporal and spatial information gathered, describing individual molecules involved in the autofluorescence of intact biological systems, represents a tough scientific challenge that has not yet been fully resolved. In this review, we discuss the latest advances in technologies that record and assess spectrally-resolved fluorescence lifetime data as well as their biological and clinical applications. We show how these methods provide efficient sensing of molecules correlated with changes in the mitochondrial metabolic redox state in pathological conditions and/or of cell ultrastructures in diseased tissue, based on the presence of oxidation/reduction-sensitive fluorophores and/or cell-specific chromophores. Future directions are also outlined.

  3. Excitation energy dependence of excited states dynamics in all- trans-carotenes determined by femtosecond absorption and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Kosumi, Daisuke; Yanagi, Kazuhiro; Nishio, Tomohiro; Hashimoto, Hideki; Yoshizawa, Masayuki

    2005-06-01

    Ultrafast relaxation kinetics in β-carotene and lycopene has been investigated by femtosecond absorption and fluorescence spectroscopies using tunable excitation pulses. The transient signals induced by the photoexcitation with larger excess energy have broader bands and longer lifetimes both in the 11Bu+and21Ag- excited states. The excess vibrational energy remains longer than several picoseconds and slows the relaxation kinetics in carotenoids.

  4. An analysis of lead, bromine, zinc and sulphur in airborne participate matter by x-ray fluorescence spectroscopy

    SciTech Connect

    McKee, J.S.C.; Lapointe, C.; Wise, M.L.H.

    1980-01-01

    Research report: Atmospheric concentrations of lead, bromine, zinc, and sulfur were measured daily for 7 mo at two sites in Birmingham, England. Samples collected at a highway location and at an inland site were analyzed by X-ray fluorescence spectroscopy. Atmospheric levels of the pollutants are higher at the highway site than at the inland site, demonstrating the polluting effects of vehicular traffic. Particulate sizes were also determined. (1 map, 4 photos, 19 references, 1 table)

  5. Cure characterization of an unsaturated polyester resin using near-infrared, fluorescence and UV/visible reflection spectroscopies

    NASA Astrophysics Data System (ADS)

    Grunden, Bradley Lyn

    This dissertation seeks to characterize the cure reaction of an unsaturated polyester resin using near-infrared, fluorescence and UV/Visible reflection spectroscopies. The results will provide a foundation for developing fiber-optic in-situ cure monitoring techniques based on near-infrared, fluorescence, and UV/Visible reflection spectroscopies for an unsaturated polyester resin system. Near-infrared spectra of the unsaturated polyester resin during cure showed a decrease in absorption at 1629, 2087, 2117, and 2227 nm. Model compounds representing the reactants and products of the cure reaction were characterized, and assignment of peaks in the NIR were made. Conversion of styrene and vinylene, determined from NIR measurements, were compared with values obtained using conventional FTIR measurements. Discrepancies between conversion values determined from NIR and FTIR measurements were attributed to a difference in sample sizes used for measurement. Using a microgel based reaction mechanism, the effects of temperature on the conversion of styrene and vinylene was discussed. A strong fluorescence emission was found during cure of the unsaturated polyester resin. As the reaction proceeded, the emission intensity at 306 nm increased. Model compound studies confirmed that the unsaturated polyester vinylene component exhibits negligible fluorescence when excited at 250 nm. The fluorescence emission at 306 nm was attributed to a reduced self-quenching effect of styrene monomer. In-situ fluorescence characterization of the cure reaction was also attempted. Fiber-optic fluorescence measurements taken in-situ at 75°C were found to be higher than those taken by fiber-optics at room temperature, indicating a temperature effect on the fluorescence emission. These results may be a consequence of the static quenching behavior of styrene monomer. UV/Visible reflection spectra of styrene showed a decrease in the % Reflectance at 255 nm with reaction time. This decrease was

  6. Time-Resolved Light Scattering and Fluorescence Spectroscopy in Biomedical and Model Random Media

    NASA Astrophysics Data System (ADS)

    Das, Bidyut Baran

    Optical spectroscopy, light scattering and ultrafast time-gated imaging have been shown to offer novel approaches to study the optical characteristics of various biomedical and other random media. Fluorescence spectra from human malignant and nonmalignant breast tissues were measured at 300 nm excitation and a significant spectral difference was found between the two tissue types by using the ratio of fluorescence intensities at 340 and 440 nm. Optical density measurements on thin breast tissues show that the scattering cross-sections of breast tissues are relatively constant over the visible and the uv region. Transport mean free paths and the absorption lengths for various tissues and model random media were measured using time-resolved transmission. The scattering coefficients for human breast and chicken tissues were found to remain relatively constant in 570-630 nm wavelength region while they change significantly at 1064 nm. Chicken breast and fat tissues were found to be good models for human breast tissues as the values of the optical parameters of the two tissue types are about the same. The less scattering observed at 1064 nm makes tissues more transparent in the NIR region making it easier to image in thick tissues. Time-resolved backscattering measurements show that the scattering and the absorption parameters of a random medium can be obtained accurately in a two-fiber configuration as long as the radial distance is more than about seven times the transport mean free path of the sample. The single point source-detection configuration provides a tool to diagnose breast malignancy though it fails to give accurate values of the optical parameters of tissues. This failure is attributed to the invalidity of the diffusion approximation in this experimental configuration. A 2.5 mm thin chicken fat strip was imaged inside a 40 mm thick chicken breast tissue using snake photons at 625 nm with ultrafast time-gated detection. A simple model to describe the effect

  7. Dynamics and Flexibility of Human Aromatase Probed by FTIR and Time Resolved Fluorescence Spectroscopy

    PubMed Central

    Sadeghi, Sheila J.; Castrignanò, Silvia; Mei, Giampiero; Di Venere, Almerinda; Nicolai, Eleonora; Allegra, Paola; Gilardi, Gianfranco

    2013-01-01

    Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min−1 for the inhibitor-bound enzyme to 0.12±0.02 min−1 for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor

  8. Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Elmalk, Abdalmohsen T.; Salverda, Jante M.; Tabares, Leandro C.; Canters, Gerard W.; Aartsma, Thijs J.

    2012-06-01

    The interaction between the fluorescently labeled redox protein, azurin, and a thin gold film is characterized using single-molecule fluorescence intensity and lifetime measurements. Fluorescence quenching starts at distances below 2.3 nm from the gold surface. At shorter distances the quantum yield may decrease down to fourfold for direct attachment of the protein to bare gold. Outside of the quenching range, up to fivefold enhancement of the fluorescence is observed on average with increasing roughness of the gold layer. Fluorescence-detected redox activity of individual azurin molecules, with a lifetime switching ratio of 0.4, is demonstrated for the first time close to a gold surface.

  9. Multimodal Raman-fluorescence spectroscopy of formalin fixed samples is able to discriminate brain tumors from dysplastic tissue

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Giordano, Flavio; Buccoliero, Anna Maria; Pavone, Francesco Saverio

    2014-05-01

    In the recent years, there has been a considerable surge in the application of spectroscopy for disease diagnosis. Raman and fluorescence spectra provide characteristic spectral profile related to biochemical and morphological changes when tissues progress from normal state towards malignancy. Spectroscopic techniques offer the advantage of being minimally invasive compared to traditional histopathology, real time and quantitative. In biomedical optical diagnostics, freshly excised specimens are preferred for making ex-vivo spectroscopic measurements. With regard to fresh tissues, if the lab is located far away from the clinic it could pose a problem as spectral measurements have to be performed immediately after dissection. Tissue samples are usually placed in a fixative agent such as 4% formaldehyde to preserve the samples before processing them for routine histopathological studies. Fixation prevents the tissues from decomposition by arresting autolysis. In the present study, we intend to investigate the possibility of using formalin fixed samples for discrimination of brain tumours from dysplastic tissue using Raman and fluorescence spectroscopy. Formalin fixed samples were washed with phosphate buffered saline for about 5 minutes in order to remove the effects of formalin during spectroscopic measurements. In case of fluorescence spectroscopy, changes in spectral profile have been observed in the region between 550-670 nm between dysplastic and tumor samples. For Raman measurements, we found significant differences in the spectral profiles between dysplasia and tumor. In conclusion, formalin fixed samples can be potentially used for the spectroscopic discrimination of tumor against dysplastic tissue in brain samples.

  10. Fluorescence spectroscopy to discriminate neoplastic human brain lesions: a study using the spectral intensity ratio and multivariate linear discriminant analysis

    NASA Astrophysics Data System (ADS)

    Nazeer, Shaiju S.; Saraswathy, Ariya; Gupta, Arun Kumar; Jayasree, Ramapurath S.

    2014-02-01

    Fluorescence spectroscopy is an emerging tool used to differentiate normal and malignant tissue based on the emission spectral profile from endogenous fluorophores. The goal of this study is to estimate the concentration of fluorophores using autofluorescence spectroscopy and try to utilize its diagnostic potential on samples of clinical importance. Brain tumor tissues from patients who received craniotomy for the removal of astrocytoma, glioma, meningioma and schwannoma were utilized in this study. Fluorescence emissions of the formalin fixed samples were recorded at excitation wavelengths of 320 and 410 nm. The emission characteristics of fluorophores such as collagen, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), phospholipids and porphyrins of tumor tissue and adjacent normal tissue were elicited. Exact tissue classification was carried out using the spectral intensity ratio (SIR) and multivariate principal component analysis-linear discriminant analysis (PCA-LDA). The diagnostic algorithm based on PCA-LDA provided better classification efficiency than SIR. Moreover, the spectral data based on an excitation wavelength of 410 nm are found to be more efficient in the classification than 320 nm excitation, using PCA-LDA. Better efficacy of PCA-LDA in tissue classification was further confirmed by the receiver operator characteristic (ROC) curve method. The results of this study establish the feasibility of using fluorescence spectroscopy based real time tools for the discrimination of brain tumors from the adjacent normal tissue during craniotomies, which at present faces a huge challenge.

  11. Fluorescence spectroscopy of PTCDA molecules on the KCl(100) surface in the limit of low coverages: site selection and diffusion.

    PubMed

    Paulheim, Alexander; Müller, Mathias; Marquardt, Christian; Sokolowski, Moritz

    2013-04-14

    We performed fluorescence (FL) and fluorescence excitation (FLE) spectroscopy on the model molecule perylene-3,4,9,10-tetracarboxyl acid dianhydride (PTCDA) for very low coverages (below 1% of a monolayer) on thin (100) oriented KCl films. Two different states of PTCDA molecules can be distinguished in the spectra: an initial state, which is observed directly after deposition of the molecules onto the cold sample at 20 K, and a final state, which is found after intensive optical excitation or thermal annealing of the sample. The spectrum of the final state is blue-shifted with respect to that of the initial state by 130 ± 15 cm(-1) and exhibits lines with significantly reduced widths. This can be explained by diffusion of molecules from initially populated terrace sites to energetically favoured step edge sites. Polarization dependent spectroscopy reveals the same azimuthal orientation of the molecules on both adsorption sites and leads to a model of the adsorption geometry of PTCDA at the KCl step sites. Our experiment demonstrates how optical spectroscopy can be used to investigate kinetic processes of fluorescent molecules on surfaces. PMID:23435674

  12. Americium characterization by X-ray fluorescence and absorption spectroscopy in plutonium uranium mixed oxide

    SciTech Connect

    Degueldre, Claude Cozzo, Cedric; Martin, Matthias; Grolimund, Daniel; Mieszczynski, Cyprian

    2013-06-01

    Plutonium uranium mixed oxide (MOX) fuels are currently used in nuclear reactors. The actinides in these fuels need to be analyzed after irradiation for assessing their behaviour with regard to their environment and the coolant. In this work the study of the atomic structure and next-neighbour environment of Am in the (Pu,U)O₂ lattice in an irradiated (60 MW d kg⁻¹) MOX sample was performed employing micro-X-ray fluorescence (µ-XRF) and micro-X-ray absorption fine structure (µ-XAFS) spectroscopy. The chemical bonds, valences and stoichiometry of Am (~0.66 wt%) are determined from the experimental data gained for the irradiated fuel material examined in its peripheral zone (rim) of the fuel. In the irradiated sample Am builds up as Am³⁺ species within an [AmO₈]¹³⁻ coordination environment (e.g. >90%) and no (<10%) Am(IV) or (V) can be detected in the rim zone. The occurrence of americium dioxide is avoided by the redox buffering activity of the uranium dioxide matrix. - Graphical abstract: Americium LIII XAFS spectra recorded for the irradiated MOX sub-sample in the rim zone for a 300 μm×300 μm beam size area investigated over six scans of 4 h. The records remain constant during multi-scan. The analysis of the XAFS signal shows that Am is found as trivalent in the UO₂ matrix. This analytical work shall open the door of very challenging analysis (speciation of fission product and actinides) in irradiated nuclear fuels. - Highlights: • Americium was characterized by microX-ray absorption spectroscopy in irradiated MOX fuel. • The americium redox state as determined from XAS data of irradiated fuel material was Am(III). • In the sample, the Am³⁺ face an AmO₈¹³⁻coordination environment in the (Pu,U)O₂ matrix. • The americium dioxide is reduced by the uranium dioxide matrix.

  13. Combining mid infrared and total X-ray fluorescence spectroscopy for prediction of soil properties

    NASA Astrophysics Data System (ADS)

    Towett, Erick; Shepherd, Keith; Sila, Andrew; Aynekulu, Ermias; Cadisch, Georg

    2015-04-01

    Mid-infrared diffuse reflectance spectroscopy (MIR) can predict many soil properties but extractable nutrients are often predicted poorly. We evaluated the potential of MIR and total elemental analysis using total X-ray fluorescence spectroscopy (TXRF), both individually and combined, to predict results of conventional soil tests. Total multi-elemental analysis provides a fingerprint of soil mineralogy and could predict some soil properties and help improve MIR predictions. A set of 700 georeferenced soil samples associated with the Africa Soil Information Service (AfSIS) (www.africasoils.net) from 44 stratified randomly-located 100-km2 sentinel sites distributed across sub-Saharan Africa were analysed for physico-chemical composition using conventional reference methods, and compared to MIR and TXRF spectra using the Random Forests regression algorithm and an internal out-of-bag validation. MIR spectra resulted in good prediction models (R2 >0.80) for organic C and total N, Mehlich-3 Ca and Al, and pH. To test the combined spectroscopic approach, TXRF element concentration data was included as a property predictor along with the first derivative of MIR spectral data using the RF algorithm. Including TXRF did not improve prediction of these properties. TXRF was poorer (R2 0.86) as these elements are not directly determined with TXRF, however the variance explained is still quite high and may be attributable to TXRF signatures relating to mineralogy correlated with protection of soil organic matter. TXRF model for Mehlich-3 Al had excellent prediction capability explaining 81% of the observed variation in extractable Al content and was comparable to that of MIR (R2 = 0.86). However, models for pH and Mehlich-3 exchangeable Ca exhibited R2 values of 0.74 and 0.79 respectively and thus had moderate predictive accuracy, compared to MIR alone with R2 values of 0.82 and 0.84 respectively. Both MIR and TXRF methods predicted soil properties that relate to nutrient

  14. Study of Organic Matter in Soils of the Amazon Region Employing Laser Induced Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Tadini, Amanda Maria; Nicolodelli, Gustavo; Mounier, Stéphane; Montes, Célia Regina; Marcondes Bastos Pereira Milori, Débora

    2014-05-01

    In the face of climate change and increasing CO2 levels in the atmosphere, the global carbon cycle, soil organic carbon (SOC) sequestration, and the role of different world biomes as potential sources and sinks of carbon are receiving increasing attention. Carbon quantification is an important environmental indicator, but the structure of organic matter is also important because is related to carbon stability. The synthesis of soil organic matter (SOM), as presented in soils of forest vegetation, can be originated from condensation polymeric polyphenols and quinones that are responsible for controlling the main physical-chemical properties of soils. These systems are present in humic substances, representing the major fluorophore of SOM[1-3]. Abiotic factors, such as soil texture, use and occupation of soil, can influence on the process of SOM formation, molecular structure and in its humification index[4]. Laser Induced Fluorescence Spectroscopy (LIFS) have become a promising technique for assessing humification index of SOM (HLIFS). In this context, the aim of this study was to analyze the humification index of the SOM in the region of Barcelos (Amazon) employing LIFS. The study area was the region of Barcelos, close the river Demeni. The whose vegetation distribution in this area, is two biomes the Dense Ombrophylous Forest (DPQD) and Campinarana (DPQT), with areas of edaphic contacts between these two phytophysiognomies, which ranged from Open field (FDE) to closed Depression (DPQ). Preliminary results showed that the area closed Depression (DPQ) there was a continuous gradient of humification with increasing soil depth. A similar behavior was verified for area Forest (DPQD), where the highest values of HLIFS were obtained between the four points analyzed, indicating the magnitude of the molecular recalcitrance this organic matter in this area. The results obtained for area Campinarana (DPQT) and Open field (FDE) showed an opposite behavior. These points there

  15. New Homogeneous Standards by Atomic Layer Deposition for Synchrotron X-ray Fluorescence and Absorption Spectroscopies.

    SciTech Connect

    Butterworth, A.L.; Becker, N.; Gainsforth, Z.; Lanzirotti, A.; Newville, M.; Proslier, T.; Stodolna, J.; Sutton, S.; Tyliszczak, T.; Westphal, A.J.; Zasadzinski, J.

    2012-03-13

    Quantification of synchrotron XRF analyses is typically done through comparisons with measurements on the NIST SRM 1832/1833 thin film standards. Unfortunately, these standards are inhomogeneous on small scales at the tens of percent level. We are synthesizing new homogeneous multilayer standards using the Atomic Layer Deposition technique and characterizing them using multiple analytical methods, including ellipsometry, Rutherford Back Scattering at Evans Analytical, Synchrotron X-ray Fluorescence (SXRF) at Advanced Photon Source (APS) Beamline 13-ID, Synchrotron X-ray Absorption Spectroscopy (XAS) at Advanced Light Source (ALS) Beamlines 11.0.2 and 5.3.2.1 and by electron microscopy techniques. Our motivation for developing much-needed cross-calibration of synchrotron techniques is borne from coordinated analyses of particles captured in the aerogel of the NASA Stardust Interstellar Dust Collector (SIDC). The Stardust Interstellar Dust Preliminary Examination (ISPE) team have characterized three sub-nanogram, {approx}1{micro}m-sized fragments considered as candidates to be the first contemporary interstellar dust ever collected, based on their chemistries and trajectories. The candidates were analyzed in small wedges of aerogel in which they were extracted from the larger collector, using high sensitivity, high spatial resolution >3 keV synchrotron x-ray fluorescence spectroscopy (SXRF) and <2 keV synchrotron x-ray transmission microscopy (STXM) during Stardust ISPE. The ISPE synchrotron techniques have complementary capabilities. Hard X-ray SXRF is sensitive to sub-fg mass of elements Z {ge} 20 (calcium) and has a spatial resolution as low as 90nm. X-ray Diffraction data were collected simultaneously with SXRF data. Soft X-ray STXM at ALS beamline 11.0.2 can detect fg-mass of most elements, including cosmochemically important oxygen, magnesium, aluminum and silicon, which are invisible to SXRF in this application. ALS beamline 11.0.2 has spatial resolution

  16. Ex vivo optical coherence tomography and laser induced fluorescence spectroscopy imaging of murine gastrointestinal tract

    NASA Astrophysics Data System (ADS)

    Hariri, Lida; Tumlinson, Alexandre R.; Wade, Norman; Besselsen, David; Utzinger, Urs; Gerner, Eugene; Barton, Jennifer

    2005-04-01

    Optical Coherence Tomography (OCT) and Laser Induced Fluorescence Spectroscopy (LIF) have separately been found to have clinical potential in identifying human gastrointestinal (GI) pathologies, yet their diagnostic capability in mouse models of human disease is unknown. We combine the two modalities to survey the GI tract of a variety of mouse strains and sample dysplasias and inflammatory bowel disease (IBD) of the small and large intestine. Segments of duodenum and lower colon 2.5 cm in length and the entire esophagus from 10 mice each of two colon cancer models (ApcMin and AOM treated A/J) and two IBD models (Il-2 and Il-10) and 5 mice each of their respective controls were excised. OCT images and LIF spectra were obtained simultaneously from each tissue sample within 1 hour of extraction. Histology was used to classify tissue regions as normal, Peyer"s patch, dysplasia, adenoma, or IBD. Features in corresponding regions of OCT images were analyzed. Spectra from each of these categories were averaged and compared via the student's t-test. Features in OCT images correlated to histology in both normal and diseased tissue samples. In the diseased samples, OCT was able to identify early stages of mild colitis and dysplasia. In the sample of IBD, the LIF spectra displayed unique peaks at 635nm and 670nm, which were attributed to increased porphyrin production in the proliferating bacteria of the disease. These peaks have the potential to act as a diagnostic for IBD. OCT and LIF appear to be useful and complementary modalities for imaging mouse models.

  17. Simulated 'On-Line' Wear Metal Analysis of Lubricating Oils by X-Ray Fluorescence Spectroscopy

    NASA Technical Reports Server (NTRS)

    Kelliher, Warren C.; Partos, Richard D.; Nelson, Irina

    1996-01-01

    The objective of this project was to assess the sensitivity of X-ray Fluorescence Spectroscopy (XFS) for quantitative evaluation of metal particle content in engine oil suspensions and the feasibility of real-time, dynamic wear metal analysis. The study was focused on iron as the majority wear metal component. Variable parameters were: particle size, particle concentration and oil velocity. A commercial XFS spectrometer equipped with interchangeable static/dynamic (flow cell) sample chambers was used. XFS spectra were recorded for solutions of Fe-organometallic standard and for a series of DTE oil suspensions of high purity spherical iron particles of 2g, 4g, and 8g diameter, at concentrations from 5 ppm to 5,000 ppm. Real contaminated oil samples from Langley Air Force Base aircraft engines and NASA Langley Research Center wind tunnels were also analyzed. The experimental data conform the reliability of XFS as the analytical method of choice for this project. Intrinsic inadequacies of the instrument for precise analytic work at low metal concentrations were identified as being related to the particular x-ray beam definition, system geometry, and flow-cell materials selection. This work supports a proposal for the design, construction and testing of a conceptually new, miniature XFS spectrometer with superior performance, dedicated to on-line, real-time monitoring of lubricating oils in operating engines. Innovative design solutions include focalization of the incident x-ray beam, non-metal sample chamber, and miniaturization of the overall assembly. The instrument would contribute to prevention of catastrophic engine failures. A proposal for two-year funding has been presented to NASA Langley Research Center Internal Operation Group (IOG) Management, to continue the effort begun by this summer's project.

  18. [Investigation on Inhibitory Capacities of Seventeen Herbal Extracts on Oxidative Stress using Ultraviolet and Fluorescence Spectroscopy].

    PubMed

    Hou, Guang-yue; Zheng, Zhong; Song, Feng-rui; Liu, Zhi-qiang; Zhao, Bing

    2015-03-01

    Diabetic patients usually suffer from complications and the long-term secondary complications are the main cause of morbidity and mortality. The hyperglycemia-induced oxidative stress is one of the important pathogenesis of diabetic complications, while the oxidative stress is associated with the lipid peroxidation reaction and the formation of advanced glycation end products (AGEs). Our study was focus on the pathogenesis of diabetic complications and based on the oxidative stress reaction. In this research, the oxidative stress inhibiting effects of seventeen herbal extracts were studied based on spectroscopic methodology. The capacities of herbal extracts against the lipid peroxidation reaction of rat liver in vitro were investigated using spectrophotometric method. It showed that the inhibitory activity of Radix Scutellariae and Flos Sophorae Immaturus were better than other herbal extracts. Additionally, the herbal extracts rich in flavonoids, alkaloids and lignanoids showed good inhibitory activities on the lipid peroxidation reaction. On the contrary, the saponin-rich herbal extracts possessed weak inhibitory effects. We applied the BSA/glucose (fructose) system combined with fluorescent spectroscopy to determine the inhibitory activities of herbal extracts in glycation model reactions. The results showed that the AGEs formation inhibitory activity of Flos Sophorae Immaturus, Radix Scutellariae and Rhizoma Anemarrhenae were better than others in the BSA/glucose (fructose) system by fluorescene analysis. The results demonstrated that the herbal extracts rich in flavonoids were found to be more effective than that of those herbal extracts as alkaloids and terpenoids class in inhibiting oxidative stress, while the saponin-rich herbal extracts showed weak inhibitory activities against oxidative stress. The Flos Sophorae Immaturus and Radix Scutellariae extracts had better inhibitory activity to the oxidative stress, so their pharmacological activity could be

  19. Measurement of OH reactivity by laser flash photolysis coupled with laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Stone, Daniel; Whalley, Lisa K.; Ingham, Trevor; Edwards, Peter M.; Cryer, Danny R.; Brumby, Charlotte A.; Seakins, Paul W.; Heard, Dwayne E.

    2016-07-01

    OH reactivity (k'OH) is the total pseudo-first-order loss rate coefficient describing the removal of OH radicals to all sinks in the atmosphere, and is the inverse of the chemical lifetime of OH. Measurements of ambient OH reactivity can be used to discover the extent to which measured OH sinks contribute to the total OH loss rate. Thus, OH reactivity measurements enable determination of the comprehensiveness of measurements used in models to predict air quality and ozone production, and, in conjunction with measurements of OH radical concentrations, to assess our understanding of OH production rates. In this work, we describe the design and characterisation of an instrument to measure OH reactivity using laser flash photolysis coupled to laser-induced fluorescence (LFP-LIF) spectroscopy. The LFP-LIF technique produces OH radicals in isolation, and thus minimises potential interferences in OH reactivity measurements owing to the reaction of HO2 with NO which can occur if HO2 is co-produced with OH in the instrument. Capabilities of the instrument for ambient OH reactivity measurements are illustrated by data collected during field campaigns in London, UK, and York, UK. The instrumental limit of detection for k'OH was determined to be 1.0 s-1 for the campaign in London and 0.4 s-1 for the campaign in York. The precision, determined by laboratory experiment, is typically < 1 s-1 for most ambient measurements of OH reactivity. Total uncertainty in ambient measurements of OH reactivity is ˜ 6 %. We also present the coupling and characterisation of the LFP-LIF instrument to an atmospheric chamber for measurements of OH reactivity during simulated experiments, and provide suggestions for future improvements to OH reactivity LFP-LIF instruments.

  20. High-speed low-cost correlator for single molecule fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Lee, Hsu-Yang; Lin, Hsin-Yu; White, Jonathon D.; Fann, Wunshain

    2009-02-01

    Fluorescence correlation spectroscopy (FCS) has been extensively applied to study the kinetics and photophysics of molecules as well as interactions between molecules by extracting information from the fluctuation of signals. In particular, single molecule applications of FCS promise the greatest amounts of information. Ideally, one would like to carry out FCS in real-time; however, due to the time-consuming nature of the correlation process, performing the correlation in real-time is totally nontrivial. Generally an expensive hardware correlator or a TCSPC board is required for this purpose. Recently highly-efficient algorithms based on multi-tau method have been proposed to build up a software correlator. In this work, we set forth an innovative algorithm capable of realizing the real-time correlation, without turning to the multi-tau method. This algorithm takes advantage of the low count rate generally existing in the FCS experiments, directly using the time interval between each photon its adjacent photon to efficiently update the correlation function. Based on this efficiency, it is possible to build a low-cost software correlator with just an ordinary counter board. We practically demonstrate the feasibility by setting up this correlator to measure the diffusion motion of rhodamine 6G in water using FCS. The algorithm was validated by duplicating the signal from the photon detector and sending it to both the ordinary counter board with our software correlator and a commercial correlator simultaneously. The perfect coincidence of the correlation curves from these two correlators and the real-time display of the correlation function indicate the validity and practicability of our approach.

  1. Fluorescence correlation spectroscopy diffusion laws in the presence of moving nanodomains

    NASA Astrophysics Data System (ADS)

    Šachl, Radek; Bergstrand, Jan; Widengren, Jerker; Hof, Martin

    2016-03-01

    It has been shown by means of simulations that spot variation fluorescence correlation spectroscopy (sv-FCS) can be used for the identification and, to some extent, also characterization of immobile lipid nanodomains in model as well as cellular plasma membranes. However, in these simulations, the nanodomains were assumed to be stationary, whereas they actually tend to move like the surrounding lipids. In the present study, we investigated how such domain movement influences the diffusion time/spot-size dependence observed in FCS experiments, usually referred to as ‘diffusion law’ analysis. We show that domain movement might mask the effects of the ‘anomalous’ diffusion characteristics of membrane lipids or proteins predicted for stationary domains, making it difficult to identify such moving nanodomains by sv-FCS. More specifically, our simulations indicate that (i) for domains moving up to a factor of 2.25 slower than the surrounding lipids, such impeded diffusion cannot be observed and the diffusion behaviour of the proteins or lipids is indistinguishable from that of freely diffusing molecules, i.e. nanodomains are not detected; (ii) impeded protein/lipid diffusion behaviour can be observed in experiments where the radii of the detection volume are similar in size to the domain radii, the domain diffusion is about 10 times slower than that of the lipids, and the probes show a high affinity to the domains; and (iii) presence of nanodomains can only be reliably detected by diffraction limited sv-FCS when the domains move very slowly (about 200 times slower than the lipid diffusion). As nanodomains are expected to be in the range of tens of nanometres and most probes show low affinities to such domains, sv-FCS is limited to stationary domains and/or STED-FCS. However, even for that latter technique, diffusing domains smaller than 50 nm in radius are hardly detectable by FCS diffusion time/spot-size dependencies.

  2. Resonant inelastic scattering in dilute magnetic semiconductors by x-ray fluorescence spectroscopy

    SciTech Connect

    Lawniczak-Jablonska, K. |; Jia, J.J.; Underwood, J.H.

    1997-04-01

    As modern, technologically important materials have become more complex, element specific techniques have become invaluable in studying the electronic structure of individual components from the system. Soft x-ray fluorescence (SXF) and absorption (SXA) spectroscopies provide a unique means of measuring element and angular momentum density of electron states, respectively, for the valence and conducting bands in complex materials. X-ray absorption and the decay through x-ray emission are generally assumed to be two independent one-photon processes. Recent studies, however have demonstrated that SXF excited near the absorption threshold generate an array of spectral features that depend on nature of materials, particularly on the localization of excited states in s and d-band solids and that these two processes can no be longer treated as independent. Resonant SXF offers thus the new way to study the dynamics of the distribution of electronic valence states in the presence of a hole which is bound to the electron low lying in the conduction band. This process can simulate the interaction between hole-electron pair in wide gap semiconductors. Therefore such studies can help in understanding of transport and optics phenomena in the wide gap semiconductors. The authors report the result of Mn and S L-resonant emission in Zn{sub 1{minus}x}Mn{sub x}S (with x=0.2 and 0.3) and MnS as the energy of exciting radiation is tuned across the Mn and S L{sub 3,2} absorption edge, along with the resonant excited spectra from elemental Mn as a reference.

  3. Fluorescence Correlation Spectroscopy Reveals Biophysical Structure of Lung Endothelial Glycocalyx: The Canopy Model

    PubMed Central

    Stevens, Andrew; Hlady, Vladimir

    2008-01-01

    The endothelial glycocalyx is believed to play a major role in capillary permeability by functioning as a macromolecular sieve overlying the intercellular junction. Little is known about the three-dimensional organization of the glycocalyx, nor which constituents contribute to its overall structure-function relationship. We applied fluorescence correlation spectroscopy (FCS) to evaluate albumin diffusion and concentration profiles directly within the glycocalyx overlying the intercellular junctions of lung capillary endothelial cells. FCS data were obtained before and after enzymatic digestion of the glycocalyx with pronase, heparinase, or hyaluronidase. FCS revealed a structure interacting with albumin located from 1.0 to 2.0 μm above the cell membrane; this structure was capable of reducing albumin diffusion by 30% and increasing local albumin concentration by 5-fold. Digestion of the glycocalyx with pronase or heparinase resulted in only modest changes of albumin diffusion and concentration. Hyaluronidase digestion completely eliminated albumin-glycocalyx interactions. Based on these data, the biophysical structure of lung capillary glycocalyx appears like a dense canopy approximately 1.0 μm in thickness located well above the cell surface. These data also suggest that hyaluronan is a major determinant for albumin interactions with the lung endothelial glycocalyx structure. Confocal images of heparan sulfate and hyaluronan confirm a cell-surface layer 2-3 μm in thickness, thus validating FCS-derived measurements. In summary, we have used FCS to probe the extra-cellular structure of the endothelial glycocalyx and further our understanding of the structure-function relationship. PMID:17483194

  4. Picosecond time-resolved fluorescence spectroscopy of K-590 in the bacteriorhodopsin photocycle.

    PubMed Central

    Atkinson, G H; Blanchard, D; Lemaire, H; Brack, T L; Hayashi, H

    1989-01-01

    The fluorescence spectrum of a distinct isometric and conformational intermediate formed on the 10(-11) s time scale during the bacteriorhodopsin (BR) photocycle is observed at room temperature using a two laser, pump-probe technique with picosecond time resolution. The BR photocycle is initiated by pulsed (8 ps) excitation at 565 nm, whereas the fluorescence is generated by 4-ps laser pulses at 590 nm. The unstructured fluorescence extends from 650 to 880 nm and appears in the same general spectral region as the fluorescence spectrum assigned to BR-570. The transient fluorescence spectrum can be distinguished from that assigned to BR-570 by a larger emission quantum yield (approximately twice that of BR-570) and by a maximum intensity near 731 nm (shifted 17 nm to higher energy from the maximum of the BR-570 fluorescence spectrum). The fluorescence spectrum of BR-570 only is measured with low energy, picosecond pulsed excitation at 590 nm and is in good agreement with recent data in the literature. The assignment of the transient fluorescence spectrum to the K-590 intermediate is based on its appearance at time delays longer than 40 ps. The K-590 fluorescence spectrum remains unchanged over the entire 40-100-ps interval. The relevance of these fluorescence data with respect to the molecular mechanism used to model the primary processes in the BR photocycle also is discussed. PMID:2713439

  5. LASER BIOLOGY AND MEDICINE: A laser-spectroscopy system for fluorescent diagnostics and photodynamic therapy of diseases of eye retina and choroid

    NASA Astrophysics Data System (ADS)

    Meerovich, G. A.; Shevchik, S. A.; Loshchenov, M. V.; Budzinskaya, M. V.; Ermakova, N. A.; Kharnas, S. S.

    2002-11-01

    A laser-spectroscopy system for the fluorescent diagnostics and photodynamic therapy of pathologic eye-fundus changes combined with the use of the Photosens compound is developed. The system is tested on experimental animals (mice and rabbits).

  6. Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy

    PubMed Central

    Voltz, Karine; Léonard, Jérémie; Touceda, Patricia Tourón; Conyard, Jamie; Chaker, Ziyad; Dejaegere, Annick; Godet, Julien; Mély, Yves; Haacke, Stefan; Stote, Roland H.

    2016-01-01

    Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies. PMID:26896800

  7. Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.

    PubMed

    Voltz, Karine; Léonard, Jérémie; Touceda, Patricia Tourón; Conyard, Jamie; Chaker, Ziyad; Dejaegere, Annick; Godet, Julien; Mély, Yves; Haacke, Stefan; Stote, Roland H

    2016-04-20

    Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies. PMID:26896800

  8. Fluorescence spectroscopy as a means of distinguishing fulvic and humic acids from dissolved and sedimentary aquatic sources and terrestrial sources

    NASA Astrophysics Data System (ADS)

    Senesi, Nicola; Miano, Teodoro M.; Provenzano, Maria Rosaria

    Thirteen fulvic acids (FA) and humic acids (HA) isolated from river waters and sediment, marine sediments, leonardite, soils, and paleosol, have been investigated by fluorescence spectroscopy in the emission, excitation and, partly, synchronous scan excitation modes. Emission spectra are generally characterized by a unique broad band, whereas excitation spectra exhibit a variable number of peaks or shoulders of various intensity; these peaks are particularly well-resolved for sedimentary HA samples. A decrease in the relative intensity of fluorescence, which is associated with a red-shift (longer wavelengths) of both the emission maximum and the main excitation peaks, is observed when passing from dissolved aquatic and soil FA to river and marine sedimentary HA, to leonardite and soil HA, and, finally, to paleosol HA. Evident differences are shown in the relative intensity and wavelength maxima, measured in any mode, between soil FA and HA from the same source. For FA and HA of various nature and origin, the fluorescence is suggested to be caused by chemically different structural units. These units fluoresce from the blue-violet to the green and consist of variously extended, condensed, aromatic and/or heterocyclic ring systems, with a high degree of electronic conjugation and bearing suitable hydroxyl, alkoxyl and carbonyl groups (e.g. salicyl, cinnamic and hydroxybenzoic derivatives, naphtols, naphtoquinones, coumarin), and quinoline-derivatives, flavonoids and Schiffbase derivatives. Fluorescence properties of humic substances may represent an additional diagnostic criterium useful in distinguishing between FA and HA from the same or various natural sources.

  9. Spectroscopy of 3, 4, 9, 10-perylenetetracarboxylic dianhydride (PTCDA) attached to rare gas samples: Clusters vs. bulk matrices. II. Fluorescence emission spectroscopy

    NASA Astrophysics Data System (ADS)

    Dvorak, Matthieu; Müller, Markus; Knoblauch, Tobias; Bünermann, Oliver; Rydlo, Alexandre; Minniberger, Stefan; Harbich, Wolfgang; Stienkemeier, Frank

    2012-10-01

    The interaction between 3, 4, 9, 10-perylenetetracarboxylic dianhydride (PTCDA) molecules and solid rare gas samples is studied by means of fluorescence emission spectroscopy. Laser-excited PTCDA-doped large argon, neon, and para-hydrogen clusters along with PTCDA embedded in helium nanodroplets are spectroscopically characterized with respect to line broadening and shifting. A fast non-radiative relaxation is observed before a radiative decay in the electronic ground state takes place. In comparison, fluorescence emission studies of PTCDA embedded in bulk neon and argon matrices result in much more complex spectral signatures characterized by a splitting of the different emission lines. These can be assigned to the appearance of site isomers of the surrounding matrix lattice structure.

  10. Spectroscopy of 3, 4, 9, 10-perylenetetracarboxylic dianhydride (PTCDA) attached to rare gas samples: clusters vs. bulk matrices. II. Fluorescence emission spectroscopy.

    PubMed

    Dvorak, Matthieu; Müller, Markus; Knoblauch, Tobias; Bünermann, Oliver; Rydlo, Alexandre; Minniberger, Stefan; Harbich, Wolfgang; Stienkemeier, Frank

    2012-10-28

    The interaction between 3, 4, 9, 10-perylenetetracarboxylic dianhydride (PTCDA) molecules and solid rare gas samples is studied by means of fluorescence emission spectroscopy. Laser-excited PTCDA-doped large argon, neon, and para-hydrogen clusters along with PTCDA embedded in helium nanodroplets are spectroscopically characterized with respect to line broadening and shifting. A fast non-radiative relaxation is observed before a radiative decay in the electronic ground state takes place. In comparison, fluorescence emission studies of PTCDA embedded in bulk neon and argon matrices result in much more complex spectral signatures characterized by a splitting of the different emission lines. These can be assigned to the appearance of site isomers of the surrounding matrix lattice structure. PMID:23126705

  11. Fluorescence studies of cyanobacterial phycobiliproteins: I. Spectroscopy of allophycocyanin core complexes. II. Spectroscopy of two phycobilisome core insertion mutants

    SciTech Connect

    Maxson, P.

    1988-10-01

    The work described here relates to the mechanisms governing energy transfer in the core polypeptides of the cyanobacterial phycobilisome. Two approaches are represented: measurements were made on isolated core components for which a great deal of structural information is available; and the fluorescence properties were characterized for the whole phycobilisome from two phycobilisome core insertion mutants. 130 refs.

  12. In vivo native fluorescence spectroscopy and nicotinamide adinine dinucleotide/flavin adenine dinucleotide reduction and oxidation states of oral submucous fibrosis for chemopreventive drug monitoring

    NASA Astrophysics Data System (ADS)

    Sivabalan, Shanmugam; Vedeswari, C. Ponranjini; Jayachandran, Sadaksharam; Koteeswaran, Dornadula; Pravda, Chidambaranathan; Aruna, Prakasa Rao; Ganesan, Singaravelu

    2010-01-01

    Native fluorescence spectroscopy has shown potential to characterize and diagnose oral malignancy. We aim at extending the native fluorescence spectroscopy technique to characterize normal and oral submucous fibrosis (OSF) patients under pre- and post-treated conditions, and verify whether this method could also be considered in the monitoring of therapeutic prognosis noninvasively. In this study, 28 normal subjects and 28 clinically proven cases of OSF in the age group of 20 to 40 years are diagnosed using native fluorescence spectroscopy. The OSF patients are given dexamethasone sodium phosphate and hyaluronidase twice a week for 6 weeks, and the therapeutic response is monitored using fluorescence spectroscopy. The fluorescence emission spectra of normal and OSF cases of both pre- and post-treated conditions are recorded in the wavelength region of 350 to 600 nm at an excitation wavelength of 330 nm. The statistical significance is verified using discriminant analysis. The oxidation-reduction ratio of the tissue is also calculated using the fluorescence emission intensities of flavin adenine dinucleotide and nicotinamide adinine dinucleotide at 530 and 440 nm, respectively, and they are compared with conventional physical clinical examinations. This study suggests that native fluorescence spectroscopy could also be extended to OSF diagnosis and therapeutic prognosis.

  13. Determination of the PSI/PSII ratio in living plant cells at room temperature by spectrally resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Elgass, Kirstin; Zell, Martina; Maurino, Veronica G.; Schleifenbaum, Frank

    2011-02-01

    Leaf cells of living plants exhibit strong fluorescence from chloroplasts, the reaction centers of photosynthesis. Mutations in the photosystems change their structure and can, thus, be monitored by recording the fluorescence spectra of the emitted chlorophyll light. These measurements have, up to now, mostly been carried out at low temperatures (77 K), as these conditions enable the differentiation between the fluorescence of Photosystem I (PSI) and Photosystem II (PSII). In contrast, at room temperature, energy transfer processes between the various photosynthetic complexes result in very similar fluorescence emissions, which mainly consist of fluorescence photons emitted by PSII hindering a discrimination based on spectral ROIs (regions of interest). However, by statistical analysis of high resolution fluorescence spectra recorded at room temperature, it is possible to draw conclusions about the relative PSI/PSII ratio. Here, the possibility of determining the relative PSI/PSII ratio by fluorescence spectroscopy is demonstrated in living maize plants. Bundle-sheath chloroplasts of mature maize plants have a special morphologic characteristic; they are agranal, or exhibit only rudimentary grana, respectively. These chloroplasts are depleted in PSII activity and it could be shown that PSII is progressively reduced during leaf differentiation. A direct comparison of PSII activity in isolated chloroplasts is nearly impossible, since the activity of PSII in both mesophyll- and bundle-sheath chloroplasts decays with time after isolation and it takes significantly longer to isolate bundle-sheath chloroplasts. Considering this fact the measurement of PSI/PSII ratios with the 77K method, which includes taking fluorescence spectra from a diluted suspension of isolated chloroplasts at 77K, is questionable. These spectra are then used to analyze the distribution of energy between PSI and PSII. After rapid cooling to 77K secondary biochemical influences, which attenuate the

  14. Fluorescence spectroscopy as an aid to imaging latent fingermarks in the ultraviolet.

    PubMed

    Bramble, S K

    1996-11-01

    Two- and three-dimensional fluorescence spectroscopic data have been recorded from sebum-rich latent fingermarks on quartz and white card. The fingermark residue was found to fluoresce between 310 to 380 nm and have an excitation range between 260 to 300 nm. The data are used to describe the results observed when imaging the inherent ultraviolet photoluminescence of latent fingermarks. PMID:8914294

  15. Spectroscopy and Fluorescence Lifetime Imaging Microscopy To Probe the Interaction of Bovine Serum Albumin with Graphene Oxide.

    PubMed

    Kuchlyan, Jagannath; Kundu, Niloy; Banik, Debasis; Roy, Arpita; Sarkar, Nilmoni

    2015-12-29

    The interaction of graphene oxide (GO) with bovine serum albumin (BSA) in aqueous buffer solution has been investigated with various spectroscopic and imaging techniques. At single molecular resolution this interaction has been performed using fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) techniques. The conformational dynamics of BSA on GO's influence have been explored by FCS and circular dichroism (CD) spectroscopy. For the FCS studies BSA was labeled covalently by a fluorophore, Alexa Fluor 488. On the addition of GO in phosphate buffer of 10 mM at pH 7.4 the diffusion time (τD) and the hydrodynamic radius (Rh) of BSA increase due to adsorption of BSA. Conformational relaxation time components of native BSA drastically vary with the addition of GO, signifying the change of conformational dynamics of BSA after addition of GO. The adsorption isotherm also indicates significant adsorption of BSA on the GO surface. Adsorption of BSA on the GO surface has shown in direct images of atomic force microscopy (AFM) and FLIM. Fluorescence quenching study of BSA with addition of GO also indicates that there is strong interaction between BSA and GO. PMID:26646418

  16. Application of photoacoustic, photothermal and fluorescence spectroscopies in signal enhancement and the kinetics, chemistry and photophysics of several dyes

    SciTech Connect

    Isak, S.J.

    1992-06-01

    Modified photoacoustic and photothermal spectroscopies are applied in analytical studies of liquid and solid systems. Quenching of benzophenone by potassium iodide is used to demonstrate application of time resolved photothermal spectroscopies in study of fast (submicrosecond) deexcitation processes. Inherently weak X-ray photoacoustic signals at a synchrotron are enhanced by the introduction of a volatile liquid into a gas-microphone photoacoustic cell. Traditionally, photoacoustic signals have been detected either by gas coupling with a microphone or with a piezoelectric detector. However, optically detected photoacoustic signals have been used in the determination of physical properties of a liquid sample system and are successfully applied to the study of deexcitation processes of a number of dye molecules. Photothermal beam deflection photoacoustic (PBDPA), fluorescence and absorbance measurements are utilized to study the chemistry and photophysics of cresyl violet in aqueous, aqueous micellar and methanolic solutions. A concentration dependence of the fluorescence quantum yield of cresyl violet is investigated. Aspects of chemistry and photophysics relating to potential use of several diazo dyes as photothermal sensitizing dyes in photodynamic therapy are explored experimentally and discussed. Photothermal beam deflection, fluorescence and absorbance measurements are again utilized. The dyes are found to have a number of interesting chemical and photophysical properties. They are also determined to be ideal photothermal sensitizing dye candidates.

  17. 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. PMID:19566223

  18. 3D-front-face fluorescence spectroscopy and independent components analysis: A new way to monitor bread dough development.

    PubMed

    Garcia, Rebeca; Boussard, Aline; Rakotozafy, Lalatiana; Nicolas, Jacques; Potus, Jacques; Rutledge, Douglas N; Cordella, Christophe B Y

    2016-01-15

    Following bread dough development can be a hard task as no reliable method exists to give the optimal mixing time. Dough development is linked to the evolution of gluten proteins, carbohydrates and lipids which can result in modifications in the spectral properties of the various fluorophores naturally present in the system. In this paper, we propose to use 3-D-front-face-fluorescence (3D-FFF) spectroscopy in the 250-550nm domain to follow the dough development as influenced by formulation (addition or not of glucose, glucose oxidase and ferulic acid in the dough recipe) and mixing time (2, 4, 6 and 8min). In all the 32 dough samples as well as in flour, three regions of maximum fluorescence intensities have been observed at 320nm after excitation at 295nm (Region 1), at 420nm after excitation at 360nm (Region 2) and 450nm after excitation at 390nm (Region 3). The principal components analysis (PCA) of the evolution of these maxima shows that the formulations with and without ferulic acid are clearly separated since the presence of ferulic acid induces a decrease of fluorescence in Region 1 and an increase in Regions 2 and 3. In addition, a kinetic effect of the mixing time can be observed (decrease of fluorescence in the Regions 1 and 2) mainly in the absence of ferulic acid. The analysis of variance (ANOVA) on these maximum values statistically confirms these observations. Independent components analysis (ICA) is also applied to the complete 3-D-FFF spectra in order to extract interpretable signals from spectral data which reflect the complex contribution of several fluorophores as influenced by their environment. In all cases, 3 signals can be clearly separated matching the 3 regions of maximal fluorescence. The signals corresponding to regions 1 and 2 can be ascribed to proteins and ferulic acid respectively, whereas the fluorophores associated with the 3rd signal (corresponding to region 3) remain unidentified. Good correlations are obtained between the IC

  19. Characterization of copper binding properties of extracellular polymeric substances using a fluorescence quenching approach combining two-dimensional correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Hur, Jin; Lee, Bo-Mi

    2014-07-01

    Heterogeneous distributions of copper-binding sites within extracellular polymeric substances (EPS) were examined by using a fluorescence quenching titration method combined with two-dimensional correlation spectroscopy (2D-COS). The binding properties were compared for two types of the EPS extracted from the sludge formed under aerobic and anaerobic conditions. The quenching behaviors of the synchronous fluorescence spectra upon the addition of copper were similar for the two EPS. Protein-like fluorescence was substantially quenched by the copper addition while the changes of fulvic- and humic-like fluorescence were not obvious, suggesting that protein molecules were largely involved in binding copper for both EPS types. The logarithmic stability constants calculated at the wavelengths corresponding to the highest peaks were 4.73 and 4.22 for the aerobic and the anaerobic EPS, respectively. However, the 2D-COS revealed the possibility of the presence of multiple sites for copper binding within the protein-like fluorescent structures of the anaerobic EPS. No such heterogeneous distribution in the binding sites was found for the aerobic EPS. For the anaerobic EPS, the spectral change preferentially occurred in the wavelength order of 297 nm → 290 nm → 268 nm, exhibiting a range of the logarithmic values from 4.43 to 4.13. The extent of the binding affinities exactly followed the sequential orders interpreted from the 2D-COS results. Our study clearly demonstrated that fluorescence quenching combined with 2D-COS could be successfully used to provide a better understanding of the chemical heterogeneity associated with metal-binding sites within EPS.

  20. Probing the mechanisms of an air amplifier using a LTQ-FT-ICR-MS and fluorescence spectroscopy.

    PubMed

    Dixon, R Brent; Muddiman, David C; Hawkridge, Adam M; Fedorov, A G

    2007-11-01

    We report the first quantitative assessment of electrosprayed droplet/ion focusing enabled by the use of a voltage-assisted air amplifier between an electrospray ionization emitter and a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (ESI-LTQ-FT-ICR-MS). A solution of fluorescent dye was electrosprayed with a stainless steel mesh screen placed in front of the MS inlet capillary acting as a gas-permeable imaging plate for fluorescence spectroscopy. Without use of the air amplifier, no detectable FT-ICR signal was observed, as well as no detectable fluorescence on the screen upon imaging using a fluorescence scanner. When the air amplifier was turned ON while electrospraying the fluorescent dye, FT-ICR mass spectra with high signal to noise ratio were obtained with an average ion injection time of 21 ms for an AGC target value of 5 x 10(5). Imaging of the screen using a fluorescence scanner produced a distinct spot of cross-sectional area approximately 33.5 mm(2) in front of the MS inlet capillary. These experimental results provide direct evidence of aerodynamic focusing of electrosprayed droplets/ions enabled by an air amplifier, resulting in improved electrospray droplet/ion capture efficiency and reduced ion injection time. A second set of experiments was carried out to explore whether the air amplifier assists in desolvation. By electrospraying a mix of quaternary amines, ratios of increasingly hydrophobic molecules were obtained. Observation of the solvophobic effect associated with electrospray ionization resulted in a higher abundance of the hydrophobic molecule. This bias was eliminated when the air amplifier was turned ON and a response indicative of the respective component concentrations of the molecules in the bulk solution was observed. PMID:17855111

  1. Time-resolved spectroscopy of the intrinsic fluorescence of nucleic acid species

    NASA Astrophysics Data System (ADS)

    Daniels, Malcolm; Hart, Lucas P.; Ho, Paul S.; Ballini, Jean-Pierre; Vigny, Paul

    1990-05-01

    Polarization and lifetime studies have shown that the fluorescence from nucleic acid species is complex, both at the individual chromophore level and because of the effect of stacking interactions on the electronic states. Recent work aimed at elucidating some aspects of this behavior by decay analysis and time-resolved spectroscopy is surveyed. Experimental work has been carried out using the ACO synchrotron at LURE, France) with time-correlated single photon counting, or a frequency-doubled N2-pumped dye laser, pulse width 700 ps, with fast-gated (100 ps width) analog detection and signal averaging. Decay curves are treated by global analysis using the Marquardt non-linear least-squares algorithm (synchrotron data) or the SPLMOD program (EMBO), which carries out a non-linear leastsquares minimization using cubic splines, for the laser data. Resolution of the decay data gives a model-based estimate of the number of components and their lifetimes. This information is then used to deconvolute timewindowed spectra (time-delayed spectra) into the time-resolved spectra. It is a particular feature of the combination of delayed photon counting with the continuous wavelength distribution of pulsed synchrotron radiation that excitation spectra correlating with emissions of different lifetimes can be obtained by uninterrupted repetitive scanning over a wide range of exciting wavelengths, in the present work from 230 nm to 354 urn. Such time-delayed excitation spectra can also be deconvoluted into components corresponding to the various time-resolved emission spectra. Examples of these three types of information viz resolved lifetimes, time-resolved emission spectra and their excitation spectra are presented and discussed for the following systems. I. adenosine; 6N, 6N-dimethyladenosine; protonated adenosine; this work shows the role of rotamers in the excited state behavior of this chromophore and demonstrates the forbidden nature of the lowest excited state. II. d(AT); d

  2. Optical and structural properties of plasma-treated Cordyceps bassiana spores as studied by circular dichroism, absorption, and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lee, Geon Joon; Sim, Geon Bo; Choi, Eun Ha; Kwon, Young-Wan; Kim, Jun Young; Jang, Siun; Kim, Seong Hwan

    2015-01-01

    To understand the killing mechanism of fungal spores by plasma treatment, the optical, structural, and biological properties of the insect pathogenic fungus Cordyceps bassiana spores were studied. A nonthermal atmospheric-pressure plasma jet (APPJ) was used to treat the spores in aqueous solution. Optical emission spectra of the APPJ acquired in air indicated emission peaks corresponding to hydroxyl radicals and atomic oxygen. When the APPJ entered the aqueous solution, additional reactive species were derived from the interaction of plasma radicals with the aqueous solution. Fluorescence and absorption spectroscopy confirmed the generation of hydroxyl radicals and hydrogen peroxide in the plasma-activated water (PAW). Spore counting showed that plasma treatment significantly reduced spore viability. Absorption spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis of the DNA extracted from plasma-treated spores showed a reduction in spore DNA content. The magnitude of the dip in the CD spectrum was lower in the plasma-treated spores than in the control, indicating that plasma treatment causes structural modifications and/or damage to cellular components. Tryptophan fluorescence intensity was lower in the plasma-treated spores than in the control, suggesting that plasma treatment modified cell wall proteins. Changes in spore viability and DNA content were attributed to structural modification of the cell wall by reactive species coming from the APPJ and the PAW. Our results provided evidence that the plasma radicals and the derived reactive species play critical roles in fungal spore inactivation.

  3. Quarry identification of historical building materials by means of laser induced breakdown spectroscopy, X-ray fluorescence and chemometric analysis

    NASA Astrophysics Data System (ADS)

    Colao, F.; Fantoni, R.; Ortiz, P.; Vazquez, M. A.; Martin, J. M.; Ortiz, R.; Idris, N.

    2010-08-01

    To characterize historical building materials according to the geographic origin of the quarries from which they have been mined, the relative content of major and trace elements were determined by means of Laser Induced Breakdown Spectroscopy (LIBS) and X-ray Fluorescence (XRF) techniques. 48 different specimens were studied and the entire samples' set was divided in two different groups: the first, used as reference set, was composed by samples mined from eight different quarries located in Seville province; the second group was composed by specimens of unknown provenance collected in several historical buildings and churches in the city of Seville. Data reduction and analysis on laser induced breakdown spectroscopy and X-ray fluorescence measurements was performed using multivariate statistical approach, namely the Linear Discriminant Analysis (LDA), Principal Component Analysis (PCA) and Soft Independent Modeling of Class Analogy (SIMCA). A clear separation among reference sample materials mined from different quarries was observed in Principal Components (PC) score plots, then a supervised soft independent modeling of class analogy classification was trained and run, aiming to assess the provenance of unknown samples according to their elemental content. The obtained results were compared with the provenance assignments made on the basis of petrographical description. This work gives experimental evidence that laser induced breakdown spectroscopy measurements on a relatively small set of elements is a fast and effective method for the purpose of origin identification.

  4. Optical and structural properties of plasma-treated Cordyceps bassiana spores as studied by circular dichroism, absorption, and fluorescence spectroscopy

    SciTech Connect

    Lee, Geon Joon Sim, Geon Bo; Choi, Eun Ha; Kim, Jun Young; Jang, Siun; Kim, Seong Hwan

    2015-01-14

    To understand the killing mechanism of fungal spores by plasma treatment, the optical, structural, and biological properties of the insect pathogenic fungus Cordyceps bassiana spores were studied. A nonthermal atmospheric-pressure plasma jet (APPJ) was used to treat the spores in aqueous solution. Optical emission spectra of the APPJ acquired in air indicated emission peaks corresponding to hydroxyl radicals and atomic oxygen. When the APPJ entered the aqueous solution, additional reactive species were derived from the interaction of plasma radicals with the aqueous solution. Fluorescence and absorption spectroscopy confirmed the generation of hydroxyl radicals and hydrogen peroxide in the plasma-activated water (PAW). Spore counting showed that plasma treatment significantly reduced spore viability. Absorption spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis of the DNA extracted from plasma-treated spores showed a reduction in spore DNA content. The magnitude of the dip in the CD spectrum was lower in the plasma-treated spores than in the control, indicating that plasma treatment causes structural modifications and/or damage to cellular components. Tryptophan fluorescence intensity was lower in the plasma-treated spores than in the control, suggesting that plasma treatment modified cell wall proteins. Changes in spore viability and DNA content were attributed to structural modification of the cell wall by reactive species coming from the APPJ and the PAW. Our results provided evidence that the plasma radicals and the derived reactive species play critical roles in fungal spore inactivation.

  5. Utilization of fluorescence spectroscopy as a novel approach to evaluate the oxidative stability in beef retail displayed.

    PubMed

    Pouzo, L B; Zaritzky, N E; Pavan, E; Rossetti, L; Descalzo, A M

    2016-09-01

    Beef samples from grazing steers finished with different seed-supplemented diets were vacuum packaged for 3, 14 and 56days (VC) and subsequently exposed to aerobic conditions (AE) for 0 and 5days. Different fluorescent compounds of interest in the oxidation process were detected in meat, namely tryptophan residues, Schiff bases and porphyrins. Tryptophan intensity fluorescence increased with 14days of VC; while Schiff bases intensity increased (P<0.05) in beef samples stored under VC-56 and in all samples after AE-5days. Porphyrins increased (P<0.05) gradually with the extension of vacuum storage time, but were degraded in beef with long vacuum storage and 5days of AE. Higher levels of porphyrins in beef under VC were correlated (P<0.05) with lower redness and higher TBARS after AE-5. This study revealed the potential of fluorescence signals to detect oxidative changes in beef under different storage conditions using a fast and nondestructive method such as fluorescence spectroscopy. PMID:27111476

  6. Lipid Diffusion in Supported Lipid Bilayers: A Comparison between Line-Scanning Fluorescence Correlation Spectroscopy and Single-Particle Tracking

    PubMed Central

    Rose, Markus; Hirmiz, Nehad; Moran-Mirabal, Jose M.; Fradin, Cécile

    2015-01-01

    Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 μm2 · s−1 and DSPT ~ 2 μm2 · s−1, respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D ≳ 1 μm2 · s−1 for FCS and D ≲ 5 μm2 · s−1 for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusion of loosely-bound proteins hovering above the membrane. SPT, on the other hand, is ideal to study the motions of membrane-inserted proteins, especially those presenting different conformations, but only allows studying lipid diffusion in relatively viscous membranes, such as supported lipid bilayers and cell membranes. PMID:26610279

  7. Simultaneous determination of 2-naphthoxyacetic acid and indole-3-acetic acid by first derivation synchronous fluorescence spectroscopy.

    PubMed

    Liu, Xiangxiang; Wan, Yiqun

    2013-07-01

    A simple, rapid, sensitive and selective method for simultaneously determining 2-naphthoxyacetic acid (BNOA) and Indole-3-Acetic Acid (IAA) in mixtures has been developed using derivation synchronous fluorescence spectroscopy based on their synchronous fluorescence. The synchronous fluorescence spectra were obtained with Δλ=100 nm in a pH 8.5 NaH2PO4-NaOH buffer solution, and the detected wavelengths of quantitative analysis were set at 239 nm for BNOA and 293 nm for IAA respectively. The over lapped fluorescence spectra were well separated by the synchronous derivative method. Under optimized conditions, the limits of detection (LOD) were 0.003 μg/mL for BNOA and 0.012 μg/mL for IAA. This method is simple and expeditious, and it has been successfully applied to the determination of 2-naphthoxyacetic acid and indole-3-acetic acid in fruit juice samples with satisfactory results. The samples were only filtrated through a 0.45 μm membrane filter, which was free from the tedious separation procedures. The obtaining recoveries were in the range of 83.88-87.43% for BNOA and 80.76-86.68% for IAA, and the relative standard deviations were all less than 5.0%. Statistical comparison of the results with high performance liquid chromatography Mass Spectrometry (HPLC-MS) method revealed good agreement and proved that there were no significant difference in the accuracy and precision between these two methods. PMID:23651742

  8. Fluorescence spectroscopy approaches for the development of a real-time organophosphate detection system using an enzymatic sensor.

    PubMed

    Carullo, Paola; Cetrangolo, Giovanni Paolo; Mandrich, Luigi; Manco, Giuseppe; Febbraio, Ferdinando

    2015-01-01

    Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor. PMID:25671511

  9. Accurate Diffusion Coefficients of Organosoluble Reference Dyes in Organic Media Measured by Dual-Focus Fluorescence Correlation Spectroscopy.

    PubMed

    Goossens, Karel; Prior, Mira; Pacheco, Victor; Willbold, Dieter; Müllen, Klaus; Enderlein, Jörg; Hofkens, Johan; Gregor, Ingo

    2015-07-28

    Dual-focus fluorescence correlation spectroscopy (2fFCS) is a versatile method to determine accurate diffusion coefficients of fluorescent species in an absolute, reference-free manner. Whereas (either classical or dual-focus) FCS has been employed primarily in the life sciences and thus in aqueous environments, it is increasingly being used in materials chemistry, as well. These measurements are often performed in nonaqueous media such as organic solvents. However, the diffusion coefficients of reference dyes in organic solvents are not readily available. For this reason we determined the translational diffusion coefficients of several commercially available organosoluble fluorescent dyes by means of 2fFCS. The selected dyes and organic solvents span the visible spectrum and a broad range of refractive indices, respectively. The diffusion coefficients can be used as absolute reference values for the calibration of experimental FCS setups, allowing quantitative measurements to be performed. We show that reliable information about the hydrodynamic dimensions of the fluorescent species (including noncommercial compounds) within organic media can be extracted from the 2fFCS data. PMID:26144863

  10. Simultaneous determination of 2-naphthoxyacetic acid and indole-3-acetic acid by first derivation synchronous fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Xiangxiang; Wan, Yiqun

    2013-07-01

    A simple, rapid, sensitive and selective method for simultaneously determining 2-naphthoxyacetic acid (BNOA) and Indole-3-Acetic Acid (IAA) in mixtures has been developed using derivation synchronous fluorescence spectroscopy based on their synchronous fluorescence. The synchronous fluorescence spectra were obtained with Δλ = 100 nm in a pH 8.5 NaH2PO4-NaOH buffer solution, and the detected wavelengths of quantitative analysis were set at 239 nm for BNOA and 293 nm for IAA respectively. The over lapped fluorescence spectra were well separated by the synchronous derivative method. Under optimized conditions, the limits of detection (LOD) were 0.003 μg/mL for BNOA and 0.012 μg/mL for IAA. This method is simple and expeditious, and it has been successfully applied to the determination of 2-naphthoxyacetic acid and indole-3-acetic acid in fruit juice samples with satisfactory results. The samples were only filtrated through a 0.45 μm membrane filter, which was free from the tedious separation procedures. The obtaining recoveries were in the range of 83.88-87.43% for BNOA and 80.76-86.68% for IAA, and the relative standard deviations were all less than 5.0%. Statistical comparison of the results with high performance liquid chromatography Mass Spectrometry (HPLC-MS) method revealed good agreement and proved that there were no significant difference in the accuracy and precision between these two methods.

  11. Fluorescence Spectroscopy Approaches for the Development of a Real-Time Organophosphate Detection System Using an Enzymatic Sensor

    PubMed Central

    Carullo, Paola; Cetrangolo, Giovanni Paolo; Mandrich, Luigi; Manco, Giuseppe; Febbraio, Ferdinando

    2015-01-01

    Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor. PMID:25671511

  12. Laser induced fluorescence and Raman spectroscopy in capillary electrophoresis as an possible instrument for extraterrestrial life signs detection.

    NASA Astrophysics Data System (ADS)

    Mikhail, Gorlenko; Cheptcov, Vladimir; Anton, Maydykovskiy; Eugeniy, Vasilev

    The one of a significant aims in extraterrestrial exploration is a seeking for a life traces in a open space and planetary objects. Complex composition and unknown origin of suspected signs of life required у new analytical approaches and technical solutions. The promising assai here can be Laser induced fluorescence and Raman spectroscopy methods. The combined instrument developed by our team reveal the advantage of capillary electrophoresis assays in a junction with laser induced fluorescence detection technology. We optimized excitation configuration of fluorescence in capillary electrophoresis to reduce pumping laser power up to 1 mW and decrease background scattering. The improvement of the device sensitivity at poor sample concentration we achieved by incorporating fluorescence flow-through cuvette into spectrometer. That allows to simplify setup, to minimize weight and increase reproducibility of measurements. The device has been tasted in complex organic chemical mixes and microbial strains differentiation tasks. 3d multinational spectra allow us to increase the spectra information loads in comparison with ordinary capillary electrophoresis approaches. Possible updating the device with Raman approach can even furthermore multiple the differentiation power of the instrument. The analytical module developed using this approach can be potentially effectively used in extraterrestrial researches as a payload of the future spacecraft.

  13. Simultaneous fluorescence and breakdown spectroscopy of fresh and aging transformer oil immersed in paper using ArF excimer laser

    NASA Astrophysics Data System (ADS)

    Parvin, P.; Shoursheini, S. Z.; Khalilinejad, F.; Bavali, A.; Moshgel Gosha, M.; Mansouri, B.

    2012-11-01

    HV transformers are taken into account as the heart of the power distribution system. The on-line monitoring based on the oil analysis offers a rapid diagnostic technique to detect the probable faults. In fact, the transformer malfunctions can be detected using UV laser spectroscopic methods. Here, a novel technique is presented based on simultaneous laser induced fluorescence (LIF) and laser induced breakdown (LIB) spectroscopy for hyper sensitive identification of the oil degradation. Oil is mainly degraded due to the internal transformer faults such as overheating and partial discharge. The spectroscopic characteristics of oil in paper substrate were obtained due to ArF laser irradiation. It was shown that the amplitude of fluorescence signal increases when the oil suffers aging and degradation. A couple of additional characteristic carbon and Hα emissions appear in the corresponding breakdown spectra too.

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

    SciTech Connect

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

    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. 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.

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

    PubMed Central

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

    2014-01-01

    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. 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. PMID:25362365

  16. Greenish-blue fluorescence of 6-(4-aminophenyl)-4-(4-methoxyphenyl)-2-methoxynicotinonitrile: Synthesis, spectroscopy, crystal structure, and fluorescence property

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

    The title compound, C20H17N3O2, was synthesized by the cyclization of a chalcone derivative with malononitrile, and was characterized by 1H NMR and FT-IR spectroscopy techniques. The crystal structure was determined by single crystal X-ray structure determination. In the crystal packing, the molecules are linked by N-H···N hydrogen bonds forming a two dimensional network parallel to the (1 0-2) plane. The crystal structure is further stabilized by weak C-H···π interactions. The compound exhibits greenish-blue fluorescence in acetonitrile with an emission wavelength at 476 nm when was excited at 365 nm.

  17. Gadolinium Deposition in Nephrogenic Systemic Fibrosis: An Examination of Tissue using Synchrotron X-ray Fluorescence Spectroscopy

    SciTech Connect

    High, W.; Ranville, J; Brown, M; Punshon, T; Lanzirotti, A; Jackson, B

    2010-01-01

    Nephrogenic systemic fibrosis is a fibrosing disorder associated with gadolinium (Gd)-based contrast agents dosed during renal insufficiency. In two patients, Gd deposition in tissue affected by nephrogenic systemic fibrosis was quantified using inductively coupled plasma mass spectrometry. The presence of Gd was confirmed and mapped using synchrotron x-ray fluorescence spectroscopy. Affected skin and soft tissue from the lower extremity demonstrated 89 and 209 ppm ({micro}g/g, dry weight, formalin fixed) in cases 1 and 2, respectively. In case 2, the same skin and soft tissue was retested after paraffin embedding, with the fat content removed by xylene washes, and this resulted in a measured value of 189 ppm ({micro}g/g, dry weight, paraffin embedded). Synchrotron x-ray fluorescence spectroscopy confirmed Gd in the affected tissue of both cases, and provided high-sensitivity and high-resolution spatial mapping of Gd deposition. A gradient of Gd deposition in tissue correlated with fibrosis and cellularity. Gd deposited in periadnexal locations within the skin, including hair and eccrine ducts, where it colocalized to areas of high calcium and zinc content. Because of the difficulty in obtaining synchrotron x-ray fluorescence spectroscopy scans, tissue from only two patients were mapped. A single control with kidney disease and gadolinium-based contrast agent exposure did not contain Gd. Gd content on a gravimetric basis was impacted by processing that removed fat and altered the dry weight of the specimens. Gradients of Gd deposition in tissue corresponded to fibrosis and cellularity. Adnexal deposition of Gd correlated with areas of high calcium and zinc content.

  18. Scanning Single-Molecule Fluorescence Correlation Spectroscopy Enables Kinetics Study of DNA Hairpin Folding with a Time Window from Microseconds to Seconds.

    PubMed

    Bi, Huimin; Yin, Yandong; Pan, Bailong; Li, Geng; Zhao, Xin Sheng

    2016-05-19

    Single-molecule fluorescence measurements have been widely used to explore kinetics and dynamics of biological systems. Among them, single-molecule imaging (SMI) is good at tracking processes slower than tens of milliseconds, whereas fluorescence correlation spectroscopy (FCS) is good at probing processes faster than submilliseconds. However, there is still shortage of simple yet effective single-molecule fluorescence method to cover the time-scale between submilliseconds and tens of milliseconds. To effectively bridge this millisecond gap, we developed a single-molecule fluorescence correlation spectroscopy (smFCS) method that works on surface-immobilized single molecules through surface scanning. We validated it by monitoring the classical DNA hairpin folding process. With a wide time window from microseconds to seconds, the experimental data are well fitted to the two-state folding model. All relevant molecular parameters, including the relative fluorescence brightness, equilibrium constant, and reaction rate constants, were uniquely determined. PMID:27140004

  19. Fluorescence suppression in Raman spectroscopy using a time-gated CMOS SPAD.

    PubMed

    Kostamovaara, Juha; Tenhunen, Jussi; Kögler, Martin; Nissinen, Ilkka; Nissinen, Jan; Keränen, Pekka

    2013-12-16

    A Raman spectrometer technique is described that aims at suppressing the fluorescence background typical of Raman spectra. The sample is excited with a high power (65W), short (300ps) laser pulse and the time position of each of the Raman scattered photons with respect to the excitation is measured with a CMOS SPAD detector and an accurate time-to-digital converter at each spectral point. It is shown by means of measurements performed on an olive oil sample that the fluorescence background can be greatly suppressed if the sample response is recorded only for photons coinciding with the laser pulse. A further correction in the residual fluorescence baseline can be achieved using the measured fluorescence tails at each of the spectral points. PMID:24514736

  20. Quantitative photoabsorption and fluorescence spectroscopy of benzene, naphthalene, and some derivatives at 106-295 nm

    NASA Technical Reports Server (NTRS)

    Suto, Masako; Wang, Xiuyan; Shan, Jun; Lee, L. C.

    1992-01-01

    Photoabsorption and fluorescence cross sections of benzene, (o-, m-, p-) xylenes, naphthalene, 1-methylnaphthalene, and 2-ethylnaphthalene in the gas phase are measured at 106-295 nm using synchrotron radiation as a light source. Fluorescences are observed from the photoexcitation of benzene and xylenes at 230-280 nm and from naphthalene and its derivatives at 190-295 nm. The absolute fluorescence cross section is determined by calibration with respect to the emission intensity of the NO(A-X) system, for which the fluorescence quantum yield is equal to 1. To cross-check the current calibration method, the quantum yield of the SO2(C-X) system at 220-230 nm was measured since it is about equal to 1. The current quantum-yield data are compared with previously published values measured by different methods.

  1. Using Fluorescence Spectroscopy to Evaluate Hill Parameters and Heterogeneity of Ligand Binding to Cytochromes P450

    NASA Astrophysics Data System (ADS)

    Marsch, Glenn A.; Carlson, Benjamin; Hansen, Jennifer; Mihelc, Elaine; Martin, Martha V.; Guengerich, F. Peter

    2009-03-01

    The cytochromes P450 (CYPs) are hemoproteins that oxidize many drugs and carcinogens. Binding interactions of two CYPs with Nile Red, pyrene, and alpha-naphthoflavone were studied using fluorescence quenching. Upon interaction with CYPs, fluorescence from pyrene excited-state dimers was quenched more efficiently than fluorescence from pyrene monomers. Quenching data was fit to the Hill equation to determine binding affinities and the Hill parameter n for the interaction of substrates with CYPs. All ligands showed strong binding to the CYPs, especially alpha-naphthoflavone, but exhibited little or no cooperativity in the interaction. Modified Stern-Volmer plots were used to confirm binding affinities, and suggested heterogeneous populations of amino acid fluorophores. Fluorescence anisotropy experiments suggest that CYP molecules tumble more rapidly when alpha-naphthoflavone is added.

  2. Far wing depolarization of light - Generalized absorption profiles. [in laser fluorescence spectroscopy of Sr vapor

    NASA Technical Reports Server (NTRS)

    Thomann, P.; Burnett, K.; Cooper, J.

    1981-01-01

    An absorption (and/or emission) event which takes place during a strong collision is called a 'correlated event'. It is discussed how correlated events affect the far red wing depolarization of fluorescence. Attention is given to an atomic vapor which is irradiated by linearly polarized light of a frequency on the red side of the resonance line. Two limiting cases are considered, corresponding to excitation in the impact region and in the quasi-static wing. In the quasi-static wing, absorption of a photon followed by fluorescence (rather than Rayleigh scattering), occurs mostly during a collision. Correlated events dominate the scattering process. Expressions derived for the polarization of the fluorescent light are applied to far red wing depolarization. It is found that the polarization of the fluorescent light does not go to zero in the far wing, but depends crucially on the detailed nature of the anisotropy in the long-range part of the interatomic potential.

  3. 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.

  4. A direct experimental evidence for an aromatic C-H⋯O hydrogen bond by fluorescence-detected infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Venkatesan, V.; Fujii, A.; Ebata, T.; Mikami, N.

    2004-08-01

    Formation of a weak aromatic C-H⋯O hydrogen bond has been discerned both experimentally and computationally in the 1,2,4,5-tetrafluorobenzene (TFB)-water system. The intermolecular structure of the isolated TFB-water cluster in a supersonic jet was characterized using fluorescence-detected infrared spectroscopy. The formation of a weak hydrogen bond in the cluster was directly evidenced by a low-frequency shift and intensity enhancement of the hydrogen-bonded aromatic C-H stretch in the TFB moiety. This is the first direct observation of an aromatic C-H⋯O hydrogen bond in isolated gas phase clusters.

  5. A laser-spectroscopy complex for fluorescent diagnostics and photodynamic therapy of age-related macula degeneration

    NASA Astrophysics Data System (ADS)

    Shevchik, S. A.; Meerovich, Gennadii A.; Budzinskaya, M. V.; Ermakova, N. A.; Kharnas, Sergey S.; Loschenov, Victor B.

    2004-06-01

    A laser-spectroscopy complex was developed for fluorescent diagnostics and photodynamic therapy of age related macula degeneration using the Russian photosensitizer Photosense. The complex is based on slit lamp which was additionally equipped with an optical adapter, and the video adapter allows to combine the procedure of photodynamic therapy and the control of its carrying in the frame work of one procedure. The sensitivity and spatial resolution of the complex were investigated using a special test object. The availability of the developed complex and Photosense itself was examined on experimental animals.

  6. Anomalous Diffusion in Polymer Solution as Probed by Fluorescence Correlation Spectroscopy and Its Universal Importance in Biological Systems

    NASA Astrophysics Data System (ADS)

    Ushida, Kiminori

    2008-02-01

    Experimental evidence of anomalous diffusion occurring in an inhomogeneous media (hyaluronan aquous solution) was obtained by use of fluorescence correlation spectroscopy (FCS) combined with other techniques (PFG-NMR and Photochemical reactions). The diffusion coefficient was obtained as a function of diffusion time or diffusion distance. Since this polymer solution can be regarded as a model system of extracellular matrices (ECMs), intercellular communication, which takes part in ECM, is greatly influenced by this anomalous diffusion mode. Therefore universal importance of anomalous diffusion in biological activity is identified in this series of independent experiments to measure diffusion coefficients.

  7. Internal standards in fluorescent X-ray spectroscopy1 1 Publication authorized by the Director, U.S. Geological Survey.

    USGS Publications Warehouse

    Adler, I.; Axelrod, J.M.

    1955-01-01

    The use of internal standards in the analysis of ores and minerals of widely-varying matrix by means of fluorescent X-ray spectroscopy is frequently the most practical approach. Internal standards correct for absorption and enhancement effects except when an absorption edge falls between the comparison lines or a very strong emission line falls between the absorption edges responsible for the comparison lines. Particle size variations may introduce substantial errors. One method of coping with the particle size problem is grinding the sample with an added abrasive. ?? 1955.

  8. Ultralow detection limits for an organic dye determined by fluorescence spectroscopy with laser diode excitation

    SciTech Connect

    Johnson, P.A.; Barber, T.E.; Smith, B.W.; Winefordner, J.D. )

    1989-04-15

    Fluorescence of IR-140, a laser dye in methanol solution, is excited by a semiconductor laser diode. Analytical figures of merit are compared for three different instrumental configurations, with the dye measured in a cuvette, a liquid jet, and a compact instrument. The best limit of detection, 46,000 molecules, was achieved with a liquid jet. Linear dynamic range was 6 orders of magnitude. The laser diode operates in the near-infrared region, resulting in low background fluorescence.

  9. Time-resolved fluorescence spectroscopy for intraoperative assistance of thyroid surgery

    NASA Astrophysics Data System (ADS)

    Bachmann, L.; Brandao, M. P.; Iwakura, R.; Basilio, F. S.; Haleplian, K.; Ito, A. S.; Conti de Freitas, L. C.

    2016-03-01

    Searching for new methods to provide information of biochemical composition and structure is critical to improve the prognosis of thyroid diseases. The use of time-resolved fluorescence techniques to detect biochemical composition and tissue structure alterations could help develop a portable, minimally invasive, and non-destructive method to assist during surgical procedures. This research looks for employ a fluorescence technique based on lifetime measurements to differentiate healthy and benign lesions from malignant thyroid tissue. We employ a wide range of excitation and chose a more appropriate region for this work: 298-300 nm; and the fluorescence decay was measured at 340-450 nm. We observed fluorescence lifetimes at 340 nm emission of 0.80+/-0.26 and 3.94+/-0.47 ns for healthy tissue; 0.90+/-0.24 and 4.05+/-0.46 ns for benign lesions; and 1.21+/-0.14 and 4.63+/-0.25 ns for malignant lesions. For 450 nm emissions, we obtain lifetimes of 0.25+/-0.18 and 3.99+/-0.39 ns for healthy tissue, 0.24+/-0.17 and 4.20+/-0.48 ns for benign lesions, 0.33+/-0.32 and 4.55+/-0.55 ns for malignant lesions. We successfully demonstrated that fluorescence lifetimes at 340 nm emission can differentiate between thyroid malignant and healthy/benign tissues.

  10. Time-resolved and steady-state fluorescence spectroscopy from bacteria subjected to bactericidal agents

    NASA Astrophysics Data System (ADS)

    Katz, Alvin; Alimova, Alexandra; Siddique, Masood; Savage, Howard E.; Shah, Mahendra; Rosen, Richard; Alfano, Robert

    2004-03-01

    The time-resolved and steady-state changes in fluorescence were investigated from one spore-forming (Bacillus subtilis) and four non-spore forming (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) bacteria subjected to different bactericidal agents. The bactericidal agents were sodium hypochlorite (bleach) hydrogen peroxide, formaldehyde, and UV light exposure. Application of sodium hypochlorite resulted in an almost total lose of fluorescence signal and large decrease in the optical density of the bacterial suspension. Addition of hydrogen peroxide resulted in a 35% decrease in emission intensity fom the Sa and an 85-95% decrease for the other bacteria. Ultraviolet light exposure resulted in a 5-35% decrease in the emission intensity of the tryptophan band. The addition of formaldehyde to the bacteria did not result in significant changes in the steady-state emission intensity, but did shift the tryptophan emission peak position to shorter wavelengths by 3 to 5 nm. Time-resolved fluorescence measurements showed that the fluorescence lifetime of tryptophan in the bacteria could not be described by a single exponential decay, and was similar to that of tryptophan in neutral aqueous solution. Upon addition of formaldehyde to the Gram positive bacteria (Bs and Sa) the strength of the short lifetime component increased dramatically, while for the Gram negative bacteria, a smaller increase was observed. These fluorescence changes reflect the different mechanisms of the bactericidal agents and may provide a useful tool to monitor the effectiveness of disinfectants.

  11. Monitoring ALA-induced PpIX photodynamic therapy in the rat esophagus using fluorescence and reflectance spectroscopy.

    PubMed

    Kruijt, Bastiaan; de Bruijn, Henriette S; van der Ploeg-van den Heuvel, Angelique; de Bruin, Ron W F; Sterenborg, Henricus J C M; Amelink, Arjen; Robinson, Dominic J

    2008-01-01

    The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination. ALA at a dose of 200 mg kg(-1) was orally administered to 15 rats, five rats served as control animals. PDT was performed at an in situ measured fluence rate of 75 mW cm(-2) using a total fluence o