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Sample records for fluorescence spectroscopy trlfs

  1. Incorporation of trivalent actinides into calcite: A time resolved laser fluorescence spectroscopy (TRLFS) study

    NASA Astrophysics Data System (ADS)

    Fernandes, M. Marques; Stumpf, T.; Rabung, T.; Bosbach, D.; Fanghänel, Th.

    2008-01-01

    In order to characterize and quantify the substitution of Ca(II) by Cm(III) (coordination, charge compensation), homogeneous Cm(III) coprecipitated calcite was synthesized in a mixed-flow-through experiment. Two sets of experiments were conducted at pH 8.1 and at pH 12.5. At pH 8.1 two calcites, a calcite with a low Cm3+ concentration (LCMpH8.1) and a calcite with a high M3+ (Gd3+ and Cm3+) concentration (HCMpH8.1) were grown and investigated by time resolved laser fluorescence spectroscopy. The Cm(III) emission spectra of LCMpH8.1 and HCMpH8.1 show the same Cm(III) fluorescence signals for two Cm(III) species; Cm(III) species (1) with a peak maximum at 606.2 nm and Cm(III) species (2) with a peak maximum at 620.3 nm. Cm(III) species (1) has a mean lifetime of τ = 386 ± 40 μs and Cm(III) species (2) has a mean lifetime of τ = 1874 ± 200 μs. A lifetime of 386 μs correlates with 1.3 water molecule in the first coordination sphere of the Cm ion whereas a lifetime of 1874 μs indicates the total loss of the Cm(III) hydration sphere. According to the fluorescence emission peak position and the fluorescence emission lifetime, Cm(III) species (1) is identified as a surface sorbed species whereas Cm(III) species (2) is identified as a Cm(III) incorporated into the calcite lattice. Cm(III) fluorescence emission spectra of Cm(III) doped calcite grown at pH 12.5 (LCMpH12.5) show the same peak maxima which are found for LCMpH8.1 and HCMpH8.1 grown at pH 8.1 but an additional emission band at 608.2 nm (3) is found, which can be assigned to a further Cm(III) species. Fluorescence emission lifetime measurements show that this Cm(III) species (3) has a lifetime of τ = 477 ± 25 μs, which correlates with 0.9 water molecules in the first coordination sphere. Cm(III) species (3) is suggested to be a CmOH2+ incorporated species.

  2. Complexation of Cm(III) with the recombinant N-lobe of human serum transferrin studied by time-resolved laser fluorescence spectroscopy (TRLFS).

    PubMed

    Bauer, N; Smith, V C; MacGillivray, R T A; Panak, P J

    2015-01-28

    The complexation of Cm(III) with the recombinant N-lobe of human serum transferrin (hTf/2N) is investigated in the pH range from 4.0 to 11.0 using TRLFS. At pH ≥ 7.4 a Cm(III) hTf/2N species is formed with Cm(III) bound at the Fe(III) binding site. The results are compared with Cm(III) transferrin interaction at the C-lobe and indicate the similarity of the coordination environment of the C- and N-terminal binding sites with four amino acid residues of the protein, two H2O molecules and three additional ligands (e.g. synergistic anions such as carbonate) in the first coordination sphere. Measurements at c(carbonate)tot = 0.23 mM (ambient carbonate concentration) and c(carbonate)tot = 25 mM (physiological carbonate concentration) show that an increase of the total carbonate concentration suppresses the formation of the Cm(III) hTf/2N species significantly. Additionally, the three Cm(III) carbonate species Cm(CO3)(+), Cm(CO3)2(-) and Cm(CO3)3(3-) are formed successively with increasing pH. In general, carbonate complexation is a competing reaction for both Cm(III) complexation with transferrin and hTf/2N but the effect is significantly higher for the half molecule. At c(carbonate)tot = 0.23 mM the complexation of Cm(III) with transferrin and hTf/2N starts at pH ≥ 7.4. At physiological carbonate concentration the Cm(III) transferrin species II forms at pH ≥ 7.0 whereas the Cm(III) hTf/2N species is not formed until pH > 10.0. Hence, our results reveal significant differences in the complexation behavior of the C-terminal site of transferrin and the recombinant N-lobe (hTf/2N) towards trivalent actinides.

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

    PubMed

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

    2011-01-01

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

  4. Sorption speciation of lanthanides/actinides on minerals by TRLFS, EXAFS and DFT studies: a review.

    PubMed

    Tan, Xiaoli; Fang, Ming; Wang, Xiangke

    2010-11-17

    Lanthanides/actinides sorption speciation on minerals and oxides by means of time resolved laser fluorescence spectroscopy (TRLFS), extended X-ray absorption fine structure spectroscopy (EXAFS) and density functional theory (DFT) is reviewed in the field of nuclear disposal safety research. The theoretical aspects of the methods are concisely presented. Examples of recent research results of lanthanide/actinide speciation and local atomic structures using TRLFS, EXAFS and DFT are discussed. The interaction of lanthanides/actinides with oxides and minerals as well as their uptake are also of common interest in radionuclide chemistry. Especially the sorption and inclusion of radionuclides into several minerals lead to an improvement in knowledge of minor components in solids. In the solid-liquid interface, the speciation and local atomic structures of Eu(III), Cm(III), U(VI), and Np(IV/VI) in several natural and synthetic minerals and oxides are also reviewed and discussed. The review is important to understand the physicochemical behavior of lanthanides/actinides at a molecular level in the natural environment.

  5. An EXAFS and TRLFS investigation on uranium(VI) sorption to pristine and leached albite surfaces.

    PubMed

    Walter, Marcus; Arnold, Thuro; Geipel, Gerhard; Scheinost, Andreas; Bernhard, Gert

    2005-02-15

    Uranium(VI) was sorbed to freshly ground and leached albite in batch and flow-through systems in the pH range 5.0-6.4. The uranium(VI) surface complexes were studied by extended X-ray absorption fine structure (EXAFS) spectroscopy and time-resolved laser-induced fluorescence spectroscopy (TRLFS). The EXAFS analysis of uranium(VI) sorbed on albite at pH 5.8 and 5 x 10(-6) M U(VI) showed one silicon atom at a USi distance of 3.09 A, which is indicative of the formation of an inner-sphere, mononuclear, bidentate uranium(VI) surface complex, Si(O)2UO2, on the silicate tetrahedra of albite. Two additional uranium(VI) sorption complexes were detected by TRLFS at higher initial aqueous U(VI) concentrations. However, the structure of these surface complexes could not be derived from EXAFS, since the measured EXAFS spectra represent the average of two surface complex structures. In order to simulate U(VI) sorption onto weathered feldspar surfaces, albite was leached with 0.01 M HClO4, resulting in surface material similar to amorphous silica gel. EXAFS showed that the equatorial oxygen shell of uranium(VI) sorbed on this material at pH 5.0 and 5.8 was split in two distances of 2.23 and 2.44 A. This indicates the formation of an inner-sphere surface complex.

  6. U(VI)-kaolinite surface complexation in absence and presence of humic acid studied by TRLFS.

    PubMed

    Krepelova, Adela; Brendler, Vinzenz; Sachs, Susanne; Baumann, Nils; Bernhard, Gert

    2007-09-01

    Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was applied to study the U(VI) surface complexes on kaolinite in the presence and absence of humic acid (HA). Two uranyl surface species with fluorescence lifetimes of 5.9 +/- 1.4 and 42.5 +/- 3.4 micros and 4.4 +/- 1.2 and 30.9 +/- 7.2 micros were identified in the binary (U(VI)-kaolinite) and ternary system (U(VI)-HA-kaolinite), respectively. The fluorescence spectra of adsorbed uranyl surface species are described with six and five fluorescence emission bands in the binary and ternary system, respectively. The positions of peak maxima are shifted significantly to higher wavelengths compared to the free uranyl ion in perchlorate medium. HA has no influence on positions of the fluorescence emission bands. In the binary system, both surface species can be attributed to adsorbed bidentate mononuclear surface complexes, which differ in the number of water molecules in their coordination environment. In the ternary system, U(VI) prefers direct binding on kaolinite rather than via HA, but it is sorbed as a uranyl-humate complex. Consequently, the hydration shell of the U(VI) surface complexes is displaced with complexed HA, which is simultaneously distributed between kaolinite particles. Aluminol binding sites are assumed to control the sorption of U(VI) onto kaolinite.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Multiphoton, optical fiber-based fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Bereś-Pawlik, ElŻbieta; Stawska, Hanna; Popenda, Maciej; Pajewski, Łukasz; Malinowska, Natalia; Hossa, Robert

    2016-12-01

    This paper presents investigation of normal and cancerous tissue by the means of one and two photon fluorescence spectroscopy. A comparison those methods has been conducted, allowing for eventual determination of granting the best possible diagnostic results.

  13. Fluorescence fluctuation spectroscopy in reduced detection volumes.

    PubMed

    Blom, H; Kastrup, L; Eggeling, C

    2006-02-01

    Fluorescence fluctuation spectroscopy is a versatile technique applied to in vitro and in vivo investigations of biochemical processes such as interactions, mobilities or densities with high specifity and sensitivity. The prerequisite of this dynamical fluorescence technique is to have, at a time, only few fluorescent molecules in the detection volume in order to generate significant fluorescence fluctuations. For usual confocal fluorescence microscopy this amounts to a useful concentration in the nanomolar range. The concentration of many biomolecules in living cell or on cell membranes is, however, often quite high, usually in the micro- to the millimolar range. To allow fluctuation spectroscopy and track intracellular interaction or localization of single fluorescently labeled biomolecules in such crowded environments, development of detection volumes with nanoscale resolution is necessary. As diffraction prevents this in the case of light microscopy, new (non-invasive) optical concepts have been developed. In this mini-review article we present recent advancements, implemented to decrease the detection volume below that of normal fluorescence microscopy. Especially, their combination with fluorescence fluctuation spectroscopy is emphasized.

  14. 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

  15. Complexation of europium(III) with the zwitterionic form of amino acids studied with ultraviolet-visible and time-resolved laser-induced fluorescence spectroscopy.

    PubMed

    Heller, Anne; Rönitz, Olivia; Barkleit, Astrid; Bernhard, Gert; Ackermann, Jörg-Uwe

    2010-08-01

    The complex formation of europium(III) with the zwitterionic form of amino acids (alanine, phenylalanine, and threonine) has been studied in aqueous solution. Measurements were performed at I = 0.1 M (NaCl/NaClO(4)), room temperature, and trace metal concentrations in the range of pH 2 to 8 using ultraviolet-visible (UV-Vis) and time-resolved laser-induced fluorescence spectroscopy (TRLFS). While complexation leads to a significant luminescence enhancement in the emission spectrum of the metal ion, absorption in the UV-Vis spectrum of the amino acid (AA) decreases. As zwitterionic species (AAH), all three ligands form weak complexes with 1:1 stoichiometry and a general formula of EuAAH(3+) with the metal. The complex stability constants were determined to be log K approximately 1 for all complexes, indicating the negligible contribution of the amino acid side chain to the complex formation reaction.

  16. 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.

  17. Fluorescence Spectroscopy with Surface Plasmon Excitation

    NASA Astrophysics Data System (ADS)

    Neumann, T.; Kreiter, M.; Knoll, W.

    In recent years, much effort has been directed towards the development of optical biosensors. While direct sensors are capable of monitoring the presence of an analyte without the use of labelling groups, the class of indirect sensors exploits the signal enhancement caused by bound marker molecules. Surface plasmon spectroscopy (SPS) as a direct detection method [1] is known to lack sensitivity for monitoring of low molecular mass analytes. In order to enhance the sensitivity and to improve the detection limit the technique was combined with fluorescence detection schemes in surface plasmon fluorescence spectroscopy (SPFS), as described recently [2]. Here, we briefly review the theory of plasmon excitation and the experimental realization of SPFS.

  18. 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

  19. Xanthines Studied via Femtosecond Fluorescence Spectroscopy.

    PubMed

    Changenet-Barret, Pascale; Kovács, Lajos; Markovitsi, Dimitra; Gustavsson, Thomas

    2016-12-03

    Xanthines represent a wide class of compounds closely related to the DNA bases adenine and guanine. Ubiquitous in the human body, they are capable of replacing natural bases in double helices and give rise to four-stranded structures. Although the use of their fluorescence for analytical purposes was proposed, their fluorescence properties have not been properly characterized so far. The present paper reports the first fluorescence study of xanthine solutions relying on femtosecond spectroscopy. Initially, we focus on 3-methylxanthine, showing that this compound exhibits non-exponential fluorescence decays with no significant dependence on the emission wavelength. The fluorescence quantum yield (3 × 10(-4)) and average decay time (0.9 ps) are slightly larger than those found for the DNA bases. Subsequently, we compare the dynamical fluorescence properties of seven mono-, di- and tri-methylated derivatives. Both the fluorescence decays and fluorescence anisotropies vary only weakly with the site and the degree of methylation. These findings are in line with theoretical predictions suggesting the involvement of several conical intersections in the relaxation of the lowest singlet excited state.

  20. 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.

  1. 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.

  2. 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

  3. Review of applications of fluorescence excitation spectroscopy to dermatology.

    PubMed

    Franco, W; Gutierrez-Herrera, E; Kollias, N; Doukas, A

    2016-03-01

    Endogenous molecules that exhibit fluorescence hold the potential to serve as reporters of tissue structure, activity and physiology. Fluorescence excitation spectroscopy is one means to measure and express tissue's innate fluorescence. This review focuses on the application of endogenous fluorescence ultraviolet excitation spectroscopy to dermatology.

  4. 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

  5. Long Range Surface Plasmon Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Kasry, Amal; Knoll, Wolfgang

    2007-03-01

    Surface plasmon modes, excited at the two sides of a thin metal layer surrounded by two (nearly) identical dielectric media interact via the overlap of their electromagnetic fields. This overlap results in two new-coupled modes, a short and a long-range surface plasmon (LRSP). We demonstrate that combining the LRSP optics with fluorescence spectroscopy can result in a huge enhancement of the fluorescence signal due to the enhanced optical field of the LRSP at the metal dielectric interface, and to its increased evanescent depth into the analyte. This was demonstrated for the detection of the fluorescence intensity of chromophore labeled protein bound to the surface sensor. Beside that, some fundamentals were studied leading to some interesting difference between SPFS and LRSPFS.

  6. Dual-focus fluorescence correlation spectroscopy.

    PubMed

    Pieper, Christoph; Weiß, Kerstin; Gregor, Ingo; Enderlein, Jörg

    2013-01-01

    This chapter introduces into the technique of dual-focus fluorescence correlation spectroscopy or 2fFCS. In 2fFCS, the fluorescence signals generated in two laterally shifted but overlapping focal regions are auto- and crosscorrelated. The resulting correlation curves are then used to determine diffusion coefficients of fluorescent molecules or particles in solutions or membranes. Moreover, the technique can also be used for noninvasively measuring flow-velocity profiles in three dimensions. Because the distance between the focal regions is precisely known and not changed by most optical aberrations, this provides an accurate and immutable external length scale for determining diffusivities and velocities, making 2fFCS the method of choice for accurately measuring absolute values of these quantities at pico- to nanomolar concentration.

  7. Fluorescence spectroscopy for diagnosis of esophageal cancer

    NASA Astrophysics Data System (ADS)

    Panjehpour, Masoud; Overholt, Bergein F.; Vo-Dinh, Tuan; Farris, Christie; Schmidhammer, James L.; Sneed, Rick E.; Buckley, Paul F., III

    1994-07-01

    Laser-induced fluorescence spectroscopy was employed to measure fluorescence emission of normal and malignant tissue during endoscopy in patients with esophageal adenocarcinoma. A nitrogen/dye laser tuned at 410 nm was used for excitation source. The fluorescence lineshape of each spectrum was determined and sampled at 15 nm intervals from 430 nm to 716 nm. A calibration set from normal and malignant spectra were selected. Using stepwise discriminate analysis, significant wavelengths that separated normal and malignant spectra were selected. The intensities at these wavelengths were used to formulate a classification model using linear discriminate analysis. The model was used to classify additional tissue spectra from 26 malignant and 108 normal sites into either normal or malignant spectra with a sensitivity of 100 percent and specificity of 98 percent.

  8. Fluorescence spectroscopy of protein oligomerization in membranes.

    PubMed

    Gorbenko, Galyna P

    2011-05-01

    Fluorescence spectroscopy is one of the most powerful tools for characterization of a multitude of biological processes. Of these, the phenomenon of protein oligomerization attracts especial interest due to its crucial role in the formation of fibrillar protein aggregates (amyloid fibrils) involved in ethiology of so-called protein misfolding diseases. It is becoming increasingly substantiated that protein fibrillization in vivo can be initiated and modulated at membrane-water interface. All steps of membrane-assisted fibrillogenesis, viz., protein adsorption onto lipid bilayer, structural transition of polypeptide chain into a highly aggregation-prone partially folded conformation, assembly of oligomeric nucleus from membrane-bound monomeric species and fiber elongation can be monitored with a mighty family of fluorescence-based techniques. Furthermore, the mechanisms behind cytotoxicity of prefibrillar protein oligomers are highly amenable to fluorescence analysis. The applications of fluorescence spectroscopy to monitoring protein oligomerization in a membrane environment are exemplified and some problems encountered in such kinds of studies are highlighted.

  9. Synchronous fluorescence spectroscopy of colon neoplasia

    NASA Astrophysics Data System (ADS)

    Borisova, Ekaterina; Semyachkina-Glushkovskaya, Oxana; Genova, Tsanislava; Penkov, Nikolay; Terziev, Ivan; Vladimirov, Borislav; Avramov, Latchezar

    2017-03-01

    Synchronous fluorescence spectroscopy (SFS) is a steady-state approach that we used for evaluation of specific fluorescence characteristics of cancerous colorectal tissues. SFS allow narrowing of the fluorescence spectra received, which increase the spectral resolution and improve the analysis of the fluorescence origin in such complex objects, such as biological tissues. In our study we investigate the characteristic differences, with diagnostic meaning, in the synchronous fluorescence spectra (SFS) of cancerous and healthy colorectal tissues ex vivo using a spectrofluorimeter FluoroLog3 (HORIBA, JobinYvon, France) for obtaining of the SFS data in a broad spectral range (300-800 nm) using excitation in the range of 280-440 nm with a delta lambda between 0 and 200 nm with a 10 nm step between scanning excitation and emission data. The procedure of obtaining the investigated samples ex vivo includes their excision during surgery for removal of neoplasia lesions. After the surgical removal biological samples are transported in isothermal conditions and safekeeping solution from the hospital to the spectral laboratory, where their spectral properties were investigated. All patients received and signed written informed consent and this research is approved by Ethics committee of University Hospital "Tsaritsa Yoanna", Sofia. Histological analysis was used as "gold standard" for evaluation of tissue samples and comparison of the spectral data received.

  10. Diagnosis of Breast Cancer Using Fluorescence and Reflectance Spectroscopy

    DTIC Science & Technology

    2004-09-01

    breast cancer based on fluorescence and diffuse reflectance spectroscopy . Our first objective include was to characterize the fluorescence properties of...device based on fluorescence and diffuse reflectance spectroscopy has the advantage of being fast, quantitative, and minimally invasive, and has the...Fluorescence and diffuse reflectance spectroscopy in the ultraviolet-visible wavelength range were made with a multi-separation probe at three illumination

  11. 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

  12. Position-sensitive scanning fluorescence correlation spectroscopy.

    PubMed

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

    2005-08-01

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

  13. 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.

  14. 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.

  15. Fluorescence correlation spectroscopy in semiadhesive wall proximity.

    PubMed

    Sanguigno, Luigi; De Santo, Ilaria; Causa, Filippo; Netti, Paolo A

    2011-11-01

    With examination of diffusion in heterogeneous media through fluorescence correlation spectroscopy, the temporal correlation of the intensity signal shows a long correlation tail and the characteristic diffusion time results are no longer easy to determine. Excluded volume and sticking effects have been proposed to justify such deviations from the standard behavior since all contribute and lead to anomalous diffusion mechanisms . Usually, the anomalous coefficient embodies all the effects of environmental heterogeneity providing too general explanations for the exotic diffusion recorded. Here, we investigated whether the reason of anomalies could be related to a lack of an adequate interpretative model for heterogeneous systems and how the presence of obstacles on the detection volume length scale could affect fluorescence correlation spectroscopy experiments. We report an original modeling of the autocorrelation function where fluorophores experience reflection or adsorption at a wall placed at distances comparable with the detection volume size. We successfully discriminate between steric and adhesion effects through the analysis of long time correlations and evaluate the adhesion strength through the evaluation of probability of being adsorbed and persistence time at the wall on reference data. The proposed model can be readily adopted to gain a better understanding of intracellular and nanoconfined diffusion opening the way for a more rational analysis of the diffusion mechanism in heterogeneous systems and further developing biological and biomedical applications.

  16. 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.

  17. Intraluminal fluorescence spectroscopy catheter with ultrasound guidance

    NASA Astrophysics Data System (ADS)

    Stephens, Douglas N.; Park, Jesung; Sun, Yang; Papaioannou, Thanassis; Marcu, Laura

    2009-05-01

    We demonstrate the feasibility of a time-resolved fluorescence spectroscopy (TRFS) technique for intraluminal investigation of arterial vessel composition under intravascular ultrasound (IVUS) guidance. A prototype 1.8-mm (5.4 Fr) catheter combining a side-viewing optical fiber (SVOF) and an IVUS catheter was constructed and tested with in vitro vessel phantoms. The prototype catheter can locate a fluorophore in the phantom vessel wall, steer the SVOF in place, perform blood flushing under flow conditions, and acquire high-quality TRFS data using 337-nm wavelength excitation. The catheter steering capability used for the coregistration of the IVUS image plane and the SVOF beam produce a guiding precision to an arterial phantom wall site location of 0.53+/-0.16 mm. This new intravascular multimodal catheter enables the potential for in vivo arterial plaque composition identification using TRFS.

  18. 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.

  19. Laser Induced Fluorescence Spectroscopy of Boron Carbide

    NASA Astrophysics Data System (ADS)

    Cheung, A. S.-C.; Ng, Y. W.; Pang, H. F.

    2011-06-01

    Laser induced fluorescence spectrum of boron carbide (BC) between 490 and 560 nm has been recorded and analyzed. Gas-phase BC molecule was produced by the reaction of B2H6 and CH4 in the presence of magnesium atom from laser ablation process. The (0, 0), (1, 0), and (2, 0) bands of the B4 Σ- - X4 Σ- transition were recorded and rotationally analyzed. Spectra of both isotopes: 10BC and 11BC were observed. Equilibrium molecular constants for the B4 Σ- and the X4 Σ- states for both isotopes were determined. A comparison of the determined gas-phase molecular constants with those obtained using matrix isolation spectroscopy and the theoretical calculations will be presented. Financial support from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKU 701008P) is gratefully acknowledged.

  20. 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.

  1. 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

  2. Theoretical methods in fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Torres, Tedman Anthony

    2009-12-01

    Fluorescence correlation spectroscopy (FCS) is a valuable tool in the study of reaction kinetics, diffusion, and photo-physics of bio-molecules. FCS utilizes correlation-function analysis of fluorescence intensity fluctuations from a small number of laser-excited molecules in a confocal optical system. The theoretical foundation of FCS was established in 1974 by Madge and Elson (ME). From this foundation, equations necessary to fit experimental correlations and extract parameters of interest are obtained. It can be shown that ME theory does not yield steady-state solutions, contradicting observed continuous intensity fluctuations from solutions in thermodynamic equilibrium. In this work, the contradiction is corrected through the application of stochastic process theory (SPT). To accomplish this, the master equation for a chemical reaction can be written; manipulations permit derivation of all equations necessary for FCS analysis and solve the contradiction. This new approach dispenses with the assumptions required in ME theory to write the reaction/diffusion equations and conditions on correlations at zero lag-time. These can be derived through SPT whereas ME methodology requires their assumption. It can be shown for non-linear reactions (at least for the types of non-linear, non-reversible reactions presented in this work) that the zero-time correlation conditions and correlation-functions from ME theory and SPT yield divergent results, converging for vanishing non-linearity. The SPT technique furnishes other possibilities not available in the ME technique. First, the SPT approach yields an efficient means of calculating spatial correlation-functions. While such functions have been derived previously, the approach in this work provides a means to obtain them in a direct and logical manner. Second, a power spectrum can be written in terms of the white noise driving the system. This allows one to easily derive the integrals for the FCS power spectrum for use in

  3. 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.

  4. 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.

  5. 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.

  6. Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens

    USDA-ARS?s Scientific Manuscript database

    This study deals with the rapid detection and classification of three bacteria, Escherichia coli, Salmonella, and Campylobacter, using fluorescence spectroscopy and multivariative analysis. Each bacterial sample was diluted in physiologic saline for analysis. Fluoroscence spectra were collected ...

  7. Determination of nitrite in waters by microplate fluorescence spectroscopy and HPLC with fluorescence detection.

    PubMed

    Büldt, A; Karst, U

    1999-08-01

    A selective and versatile fluorescence spectroscopic method for the determination of nitrite in waters has been developed. Nitrite reacts in the presence of mineral acids with the nonfluorescent N-methyl-4-hydrazino-7-nitrobenzofurazan forming N-methyl-4-amino-7-nitrobenzofurazan, which can be detected by fluorescence spectroscopy with an excitation maximum at lambda = 468 nm and an emission maximum at lambda = 537 nm in acetonitrile. Three new methods based on this reaction have been developed: Direct fluorescence spectroscopy, HPLC/fluorescence, or HPLC with UV/vis detector may be selected as detection techniques. On microplates, high-throughput fluorescence spectroscopy is achieved, while HPLC/fluorescence provides lower limits of detection, and HPLC with UV/vis detection enables evaluation of the reaction with standard instrumentation. Different water samples were investigated using all detection modes, and a photometric standard procedure was successfully employed to validate the new methods with an independent technique.

  8. Trivalent f-elements in human saliva: a comprehensive speciation study by time-resolved laser-induced fluorescence spectroscopy and thermodynamic calculations.

    PubMed

    Barkleit, Astrid; Wilke, Claudia; Heller, Anne; Stumpf, Thorsten; Ikeda-Ohno, Atsushi

    2017-01-31

    In the case of oral ingestion of radioactive contaminants, the first contact medium is saliva in the mouth. To gain a first insight into the interaction of radioactive contaminants in human saliva, the speciation of curium (Cm(iii)) and europium (Eu(iii)), i.e., trivalent f-elements, was investigated in different salivary media with time-resolved laser-induced fluorescence spectroscopy (TRLFS). The results indicate that these metal cations are primarily complexed with carbonates and phosphates, forming ternary complexes with a possible stoichiometry of 1 : 1 : 2 (M(iii) : carbonate : phosphate). For charge compensation, calcium is also involved in these ternary complexes. In addition to these inorganic components, organic substances, namely α-amylase, show a significant contribution to the speciation of the trivalent f-elements in saliva. This protein is the major enzyme in saliva and catalyzes the hydrolysis of polysaccharides. In this context, the effect of Eu(iii) on the activity of α-amylase was investigated to reveal the potential implication of these metal cations for the in vivo functions of saliva. The results indicate that the enzyme activity is strongly inhibited by the presence of Eu(iii), which is suppressed by an excess of calcium.

  9. Multiphoton excited fluorescence spectroscopy of biomolecular systems

    NASA Astrophysics Data System (ADS)

    Birch, David J. S.

    2001-09-01

    Recent work on the emerging application of multiphoton excitation to fluorescence studies of biomolecular dynamics and structure is reviewed. The fundamental principles and experimental techniques of multiphoton excitation are outlined, fluorescence lifetimes, anisotropy and spectra in membranes, proteins, hydrocarbons, skin, tissue and metabolites are featured, and future opportunities are highlighted.

  10. Speckle spectroscopy of fluorescent randomly inhomogeneous media

    NASA Astrophysics Data System (ADS)

    Zimnyakov, D. A.; Asharchuk, I. A.; Yuvchenko, S. A.; Sviridov, A. P.

    2016-11-01

    We propose a coherence optical method for probing fluorescent randomly inhomogeneous media based on the statistical analysis of spatial fluctuations of spectrally selected fluorescence radiation. We develop a phenomenological model that interrelates the flicker index of the spatial distribution of the fluorescence intensity at a fixed wavelength and the mean path difference of partial components of the fluorescence radiation field in the probed medium. The results of experimental approbation of the developed method using the layers of densely packed silicon dioxide particles saturated with the aqueous rhodamine 6G solution with a high concentration of the dye are presented. The experimentally observed significant decrease in the flicker index under the wavelength tuning from the edges of the fluorescence spectrum towards it central part is presumably a manifestation of spectrally dependent negative absorption in the medium.

  11. Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities.

    PubMed

    Heuff, Romey F; Swift, Jody L; Cramb, David T

    2007-04-28

    Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.

  12. Fluorescence Correlation Spectroscopy: A Review of Biochemical and Microfluidic Applications

    PubMed Central

    Tian, Yu; Martinez, Michelle M.

    2011-01-01

    Over the years fluorescence correlation spectroscopy (FCS) has proven to be a useful technique that has been utilized in several fields of study. Although FCS initially suffered from poor signal to noise ratios, the incorporation of confocal microscopy has overcome this drawback and transformed FCS into a sensitive technique with high figures of merit. In addition, tandem methods have evolved to include dual-color cross-correlation, total internal reflection fluorescence correlation, and fluorescence lifetime correlation spectroscopy combined with time-correlated single photon counting. In this review, we discuss several applications of FSC for biochemical, microfluidic, and cellular investigations. PMID:21396180

  13. Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

    PubMed Central

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

    2012-01-01

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

  14. Laser-induced fluorescence spectroscopy at endoscopy

    NASA Astrophysics Data System (ADS)

    Qu, Jianan Y.; MacAulay, Calum E.; Lam, Stephen; Palcic, Branko

    1994-07-01

    A spectrofluorometry system has been developed for the collection of laser induced fluorescense spectra of tissue during endoscopy. In this system, a catheter with seven optical fibers was used to deliver the excitation light and collect the emitted fluorescence. The system enables one to switch from regular endoscopy into fluorescence measurement in 50 ms using a computerized shutter system. The fluorescence spectra can be recorded in 100 ms. This spectrofluorometry system has been used to obtain spectra from bronchial, larynx and nasopharyngeal tissues when employed with the appropriate endoscopes. The results demonstrate that laser induced fluorescence can be used to differentiate abnormal tissue from normal tissue. The illumination and fluorescence collection patterns of this system have been modeled using a Monte Carlo simulation. The Monte Carlo simulation data shows that the spectra recorded by our collection pattern is very close to the intrinsic spectra of tissue. The experimental results and the Monte Carlo simulation suggest that changes in fluorescence intensity are more robust for the detection of early cancers than the differences in spectral characteristics.

  15. Assessment of skin flap viability using visible diffuse reflectance spectroscopy and auto-fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhu, Caigang; Chen, Shuo; Chui, Christopher Hoe-Kong; Liu, Quan

    2012-12-01

    The accurate assessment of skin flap viability is vitally important in reconstructive surgery. Early identification of vascular compromise increases the change of successful flap salvage. The ability to determine tissue viability intraoperatively is also extremely useful when the reconstructive surgeon must decide how to inset the flap and whether any tissue must be discarded. Visible diffuse reflectance and auto-fluorescence spectroscopy, which yield different sets of biochemical information, have not been used in the characterization of skin flap viability simultaneously to our best knowledge. We performed both diffuse reflectance and fluorescence measurements on a reverse MacFarlane rat dorsal skin flap model to identify the additional value of auto-fluorescence spectroscopy to the assessment of flap viability. Our result suggests that auto-fluorescence spectroscopy appears to be more sensitive to early biochemical changes in a failed flap than diffuse reflectance spectroscopy, which could be a valuable complement to diffuse reflectance spectroscopy for the assessment of flap viability.

  16. 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.

  17. Hydrosomes: femtoliter containers for fluorescence spectroscopy studies

    NASA Astrophysics Data System (ADS)

    Jofre, Ana M.; Tang, Jianyong; Greene, Mark E.; Lowman, Geoffrey M.; Hodas, Nathan; Kishore, Rani; Helmerson, Kristian; Goldner, Lori S.

    2007-09-01

    We report on improvements and innovations in the use of hydrosomes to encapsulate and study single molecules. Hydrosomes are optically-trappable aqueous nanodroplets. The droplets are suspended in a fluorocarbon medium that is immiscible with water and has an index of refraction lower than water, so hydrosomes are stable and optically trapped by a focused laser beam (optical tweezers). Using optical tweezers, we hold the hydrosomes within a confocal observation volume and interrogate the encapsulated molecule by fluorescence excitation. This method allows for long observation times of a molecule without the need for surface immobilization or liposome encapsulation. We have developed a new way for creating hydrosomes on demand by inertially launching them into the fluorocarbon matrix using a piezo-activated micropipette. Time-resolved fluorescence anisotropy studies are carried out to characterize the effects of the hydrosome interface boundary on biological molecules and to determine whether molecules encapsulated within hydrosomes diffuse freely throughout the available volume. We measured the fluorescence anisotropy decay of 20mer DNA duplexes, and enhanced green fluorescent protein (GFP). We conclude that the molecules rotate freely inside the nanodroplets and do not stick or aggregate at the boundary.

  18. Time-resolved spectroscopy of the fluorescence quenching of a donor — acceptor pair by halothane

    NASA Astrophysics Data System (ADS)

    Sharma, A.; Draxler, S.; Lippitsch, M. E.

    1992-04-01

    Donor (anthracene) sensitized acceptor (perylene) fluorescence is quenched more efficiently by halothane than is intrinsic perylene fluorescence. The underlying process of dynamic fluorescence quenching is investigated by time-resolved fluorescence spectroscopy.

  19. 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.

  20. 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.

  1. Fluorescent Lifetime Spectroscopy in Random Media

    NASA Astrophysics Data System (ADS)

    Hutchinson, Christina Laura

    Recently, an abundance of near-infrared phosphorescent and fluorescent probes have been developed whose lifetime is sensitive to changes in the local environment. The lifetime of these probes can be readily determined in a dilute, non-scattering media using conventional time- and frequency-domain techniques. From the lifetime, the concentration of metabolites can be found using the Stern-Volmer relationship. However, in highly scattering media such as tissues and particulate process streams, measurement of lifetime is complicated by the time delay associated with light scatter. In this dissertation, frequency-domain measurements of photon migration are developed for measuring fluorescent lifetimes independent of absorption and scattering properties of tissues and other random media. The measurement consists of launching, onto the surface of the medium, excitation light whose intensity is sinusoidally modulated at megahertz frequencies. The fluorescent light generated within the medium is intensity modulated at the same frequency, but phase-shifted and amplitude demodulated relative to the incident excitation source. In addition, the excitation light is also phase-shifted and amplitude demodulated relative to the incident excitation source. From Green's function analysis, finite element computations, and experimental measurements of fluorescent phase-shift and amplitude demodulation, we show it is possible to determine fluorophore lifetime of common laser dyes in a tissue mimicking phantom. Furthermore, the finite element computations of excitation and fluorescent light fluence show that when the fluorophore is uniformly distributed within a medium, signals re-emitted at the surface do not originate from significant depths if its lifetime is greater than the photon migration time associated with scatter. Consequently, this research points to the development of short-lived fluorescent compounds for biodiagnostics using properly referenced frequency

  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. 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.

  4. "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.

  5. "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.

  6. EMCCD-based spectrally resolved fluorescence correlation spectroscopy.

    PubMed

    Bestvater, Felix; Seghiri, Zahir; Kang, Moon Sik; Gröner, Nadine; Lee, Ji Young; Im, Kang-Bin; Wachsmuth, Malte

    2010-11-08

    We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.

  7. Combined fiber probe for fluorescence lifetime and Raman spectroscopy.

    PubMed

    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

    2015-11-01

    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. Graphical Abstract An image comparison between FLIm and Raman spectroscopy acquired with the bimodal probe onseveral tissue samples.

  8. 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.

  9. Laser Induced Fluorescence Spectroscopy of Cobalt Monoboride

    NASA Astrophysics Data System (ADS)

    Pang, H. F.; Ng, Y. W.; Cheung, A. S.-C.

    2011-06-01

    Laser induced fluorescence spectrum of cobalt monoboride (CoB) in the visible region between 465 and 560 nm has been observed. CoB molecule was produced by the reaction of laser ablated cobalt atom and diborane (B_2H_6) seeded in argon. Over twenty five vibronic bands have been recorded, and both Co10B and Co11B isotopic species have been observed and analyzed. Preliminary analysis of the rotational lines showed that the observed vibronic bands belong to two categories namely: the Ω' = 2 - Ω'' = 2 and the Ω' = 3 - Ω'' = 3 transitions, which indicated the ground state of CoB is consistent with an assignment of a ^3Δ_i state predicted from ab initio calculations. Unresolved hyperfine structure arising from the Co nucleus (I = 7/2) causes a broadening of spectral lines. This work represents the first experimental investigation of the spectrum of the CoB molecule. Financial support from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKU 701008P) is gratefully acknowledged.

  10. Americium(iii) and europium(iii) complex formation with lactate at elevated temperatures studied by spectroscopy and quantum chemical calculations.

    PubMed

    Barkleit, Astrid; Kretzschmar, Jerome; Tsushima, Satoru; Acker, Margret

    2014-08-07

    Thermodynamic parameters for the complex formation of Am(iii) and Eu(iii) with lactate were determined with UV-vis and time-resolved laser-induced fluorescence spectroscopy (TRLFS) in a temperature range between 25 and 70 °C. The reaction enthalpy decreased with increasing ionic strength. ATR FT-IR and NMR spectroscopy in combination with density functional theory (DFT) calculations revealed structural details of the Eu(iii) lactate 1 : 1 complex: a chelating coordination mode of the lactate with a monodentate binding carboxylate group and the hydroxyl group being deprotonated.

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

    PubMed

    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

    2007-07-31

    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.

  12. Sucrose Monoester Micelles Size Determined by Fluorescence Correlation Spectroscopy (FCS)

    PubMed Central

    Sanchez, Susana A.; Gratton, Enrico; Zanocco, Antonio L.; Lemp, Else; Gunther, German

    2011-01-01

    One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, Rh. As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured. PMID:22216230

  13. Chapter 1: In vivo applications of fluorescence correlation spectroscopy.

    PubMed

    Chen, Huimin; Farkas, Elaine R; Webb, Watt W

    2008-01-01

    Fluorescence correlation spectroscopy provides a sensitive optical probe of the molecular dynamics of life in vivo and in vitro. The kinetics of chemical binding, transport, and changes in molecular conformations are detected by measurement of fluctuations of fluorescence emission by sensitive marker fluorophores. The fluorophores within a defined volume are illuminated by laser light that excites their fluorescence. While conventional confocal illumination by short-wavelength laser light is sufficient for two-dimensional targets, multiphoton fluorescence excitation by simultaneous quantum absorption of two or more long-wavelength photons of approximately 100 fs laser pulses provides the more precise submicron three-dimensional spatial resolution required in cells and tissues. Chemical kinetics, molecular aggregation, molecular diffusion, fluid flows, photophysical interactions, conformational fluctuations, concentration fluctuations, and other dynamics of biological processes can be measured and monitored in volumes approximately 1 mum(3) at timescales from <1 mus and upward for many orders of magnitude. Theory, motivations, methods, in vivo applications, and future directions for improvement and new applications for fluorescence correlation spectroscopy are summarized in this chapter.

  14. Optical-fiber-microsphere for remote fluorescence correlation spectroscopy.

    PubMed

    Aouani, Heykel; Deiss, Frédérique; Wenger, Jérôme; Ferrand, Patrick; Sojic, Neso; Rigneault, Hervé

    2009-10-12

    Fluorescence correlation spectroscopy (FCS) is a versatile method that would greatly benefit to remote optical-fiber fluorescence sensors. However, the current state-of-the-art struggles with high background and low detection sensitivities that prevent the extension of fiber-based FCS down to the single-molecule level. Here we report the use of an optical fiber combined with a latex microsphere to perform FCS analysis. The sensitivity of the technique is demonstrated at the single molecule level thanks to a photonic nanojet effect. This offers new opportunities for reducing the bulky microscope setup and extending FCS to remote or in vivo applications.

  15. Femtosecond broadband fluorescence upconversion spectroscopy: Improved setup and photometric correction

    SciTech Connect

    Zhang, X.-X.; Wuerth, C.; Resch-Genger, U.; Zhao, L.; Ernsting, N. P.; Sajadi, M.

    2011-06-15

    A setup for fluorescence upconversion spectroscopy (FLUPS) is described which has 80 fs temporal response (fwhm) for emission in the spectral range 425-750 nm. Broadband phase matching is achieved with tilted gate pulses at 1340 nm. Background from harmonics of the gate pulse is removed and sensitivity increased compared to previous designs. Photometric calibration of the upconversion process is performed with a set of fluorescent dyes. For Coumarin 153 in methanol the peak position, bandwidth, and asymmetry depending on delay time are reported.

  16. 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

  17. Femtosecond broadband fluorescence upconversion spectroscopy: Improved setup and photometric correction

    NASA Astrophysics Data System (ADS)

    Zhang, X.-X.; Würth, C.; Zhao, L.; Resch-Genger, U.; Ernsting, N. P.; Sajadi, M.

    2011-06-01

    A setup for fluorescence upconversion spectroscopy (FLUPS) is described which has 80 fs temporal response (fwhm) for emission in the spectral range 425-750 nm. Broadband phase matching is achieved with tilted gate pulses at 1340 nm. Background from harmonics of the gate pulse is removed and sensitivity increased compared to previous designs. Photometric calibration of the upconversion process is performed with a set of fluorescent dyes. For Coumarin 153 in methanol the peak position, bandwidth, and asymmetry depending on delay time are reported.

  18. Diffusivity of asphaltene molecules by fluorescence correlation spectroscopy.

    PubMed

    Andrews, A Ballard; Guerra, Rodrigo E; Mullins, Oliver C; Sen, Pabitra N

    2006-07-06

    Using fluorescence correlation spectroscopy (FCS) we measure the translational diffusion coefficient of asphaltene molecules in toluene at extremely low concentrations (0.03-3.0 mg/L): where aggregation does not occur. We find that the translational diffusion coefficient of asphaltene molecules in toluene is about 0.35 x 10(-5) cm(2)/s at room temperature. This diffusion coefficient corresponds to a hydrodynamic radius of approximately 1 nm. These data confirm previously estimated size from rotational diffusion studied using fluorescence depolarization. The implication of this concurrence is that asphaltene molecular structures are monomeric, not polymeric.

  19. Interactions between natural organic ligands and trace metals studied by fluorescence lifetime and fluorescence quenching

    NASA Astrophysics Data System (ADS)

    Nouhi, Ayoub; Hajjoul, Houssam; Redon, Roland; Gagné, Jean-Pierre; Mounier, Stéphane

    2017-04-01

    Improved insight on the interactions between natural organic ligands and trace metals is of paramount importance for better understanding transport and toxicity pathways of metal ions in the environment. Fluorescence spectroscopy allows introspecting ligands-metals interactions. Time-resolved laser fluorescence spectroscopy (TRLFS) measures fluorophore lifetime probing the local molecular environment. Excitation Emission Fluorescence Matrices (EEFMs) and their statistical treatment : parallel factor analysis (PARAFAC) using PROGMEEF Matlab homemade program, can give insight on the number or nature of organic fluorophores involved in the interactions. Quenching of fluorescence by metals can occur following two processes: dynamic and static quenching (Lakowicz, 2013). In the first case, quenching is caused by physical collisions among molecules and in the second case fluorophores can form nonfluorescent complexes with quenchers. It is possible to identify the different mechanisms because each type of quenching corresponds to a different mathematical model (Lakowicz, 2013; Valeur and Berberan-Santos, 2012). In TRLFS, the study of fluorescence decay's laws induced by nanosecond pulsed laser will allow to exactly qualify the type of interaction. The crucial point of the temporal deconvolution will be the evaluation of the best fitting between the different physical models and the decays measured. From the most suitable time decay model, it will be possible to deduce the quenching which modifies the fluorescence. The aim of this study was to characterize interactions between natural organic ligands and trace metals using fluorescence tools to evaluate the fluorescence lifetime of the fluorophore, the occurrence of quenching in presence of metal, discuss its mechanism and estimate conditional stability constants if a complex organic ligand-metal is formed. This study has been done in two steps. First, we have examined the interactions between salicylic acid and copper in

  20. 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.

  1. 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.

  2. 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.

  3. Emerging applications of fluorescence spectroscopy in medical microbiology field.

    PubMed

    Shahzad, Aamir; Köhler, Gottfried; Knapp, Martin; Gaubitzer, Erwin; Puchinger, Martin; Edetsberger, Michael

    2009-11-26

    There are many diagnostic techniques and methods available for diagnosis of medically important microorganisms like bacteria, viruses, fungi and parasites. But, almost all these techniques and methods have some limitations or inconvenience. Most of these techniques are laborious, time consuming and with chances of false positive or false negative results. It warrants the need of a diagnostic technique which can overcome these limitations and problems. At present, there is emerging trend to use Fluorescence spectroscopy as a diagnostic as well as research tool in many fields of medical sciences. Here, we will critically discuss research studies which propose that Fluorescence spectroscopy may be an excellent diagnostic as well as excellent research tool in medical microbiology field with high sensitivity and specificity.

  4. Emerging applications of fluorescence spectroscopy in medical microbiology field

    PubMed Central

    2009-01-01

    There are many diagnostic techniques and methods available for diagnosis of medically important microorganisms like bacteria, viruses, fungi and parasites. But, almost all these techniques and methods have some limitations or inconvenience. Most of these techniques are laborious, time consuming and with chances of false positive or false negative results. It warrants the need of a diagnostic technique which can overcome these limitations and problems. At present, there is emerging trend to use Fluorescence spectroscopy as a diagnostic as well as research tool in many fields of medical sciences. Here, we will critically discuss research studies which propose that Fluorescence spectroscopy may be an excellent diagnostic as well as excellent research tool in medical microbiology field with high sensitivity and specificity. PMID:19941643

  5. 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.

  6. Two-dimensional fluorescence spectroscopy for application in biotechnology

    NASA Astrophysics Data System (ADS)

    Lindemann, Carsten; Marose, S.; Scheper, Thomas-Helmut; Nielsen, Hans O.; Hitzmann, Bernd; Belgardt, K.-H.

    1999-02-01

    A wide range of excitation and emission wavelengths is measured using the technique of two-dimensional (2D-) fluorescence spectroscopy. In a single, so called, two- dimensional fluorescence spectrum several biogenic fluorophors like proteins, vitamins and coenzymes can be detected simultaneously. This can give important information for bioprocess monitoring and control. An optical sensor (BioViewR) for on line fluorescence measurements at industrial (bio)-processes was used to get the results presented in this paper. This BioViewR-sensor is optimized to work in the harsh environment of production sites in biotechnological industry and -- using an optical light guide system with open-end detection -- it is very well suited for in vivo measurements, because it is non-invasive and the on line data can be performed in-situ.

  7. 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.

  8. Fast and Reversible Photoswitching of the Fluorescent Protein Dronpa as Evidenced by Fluorescence Correlation Spectroscopy

    PubMed Central

    Dedecker, Peter; Hotta, Jun-ichi; Ando, Ryoko; Miyawaki, Atsushi; Engelborghs, Yves; Hofkens, Johan

    2006-01-01

    Controlling molecular properties through photoirradiation holds great promise for its potential for noninvasive and selective manipulation of matter. Photochromism has been observed for several different molecules, including green fluorescent proteins, and recently the discovery of a novel photoswitchable green fluorescent protein called Dronpa was reported. Dronpa displays reversible and highly efficient on/off photoswitching of its fluorescence emission, and reversible switching of immobilized single molecules of Dronpa with response times faster than 20 ms was demonstrated. In this Letter, we expand these observations to freely diffusing molecules by using fluorescence correlation spectroscopy with simultaneous excitation at 488 and 405 nm. By varying the intensity of irradiation at 405 nm, we demonstrate the reversible photoswitching of Dronpa under these conditions, and from the obtained autocorrelation functions we conclude that this photoswitching can occur within tens of microseconds. PMID:16798811

  9. 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.

  10. Polarized fluorescence correlation spectroscopy of DNA-DAPI complexes.

    PubMed

    Barcellona, Maria Luisa; Gammon, Seth; Hazlett, Theodore; Digman, Michelle A; Gratton, Enrico

    2004-11-01

    We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods.

  11. Interaction between carbamazepine and humic substances: a fluorescence spectroscopy study.

    PubMed

    Bai, Yingchen; Wu, Fengchang; Liu, Congqiang; Guo, Jianyang; Fu, Pingqing; Li, Wen; Xing, Baoshan

    2008-01-01

    Carbamazepine is a popular drug that has been detected in natural environments, but little is known about its biogeochemical cycling, influencing factors, and eco-environmental effects in aquatic ecosystems. Interaction between carbamazepine and humic substances, including fulvic and humic acids, was studied using three-dimensional excitation-emission matrix fluorescence spectroscopy and synchronous-scan fluorescence spectroscopy. The intrinsic fluorescence of humic substances was quenched on the addition of carbamazepine, and static quenching was the primary mechanism. The binding parameters on their interaction, including the conditional binding constants (log K) and binding capacities (C(L)), were estimated by the Ryan-Weber nonlinear theory equation. Log K ranged from 3.41 to 5.04 L/mol at 25 degrees C and pH 7.0. The influence of pH on the complexation and the competition between carbamazepine and Cu(II) for fluorescence-binding sites also were discussed. The present results would be helpful in understanding the fate and biogeochemical cycling of other pharmaceuticals and personal care products in aquatic ecosystems.

  12. The spectroscopic basis of Fluorescence Triple Correlation Spectroscopy

    PubMed Central

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

    2012-01-01

    We have developed Fluorescence Triple Correlation Spectroscopy (F3CS) as an extension of the widely-used fluorescence microscopy technique Fluorescence Correlation Spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope. Experimental correlations were calculated from raw fluorescence data using triple correlation integrals that extend multiple-tau correlation theory to delay times in two dimensions. The quality of experimental data was improved by tuning specific spectroscopic parameters and employing multiple independent detectors to minimize optoelectronic artifacts. Experiments with the reversible system of freely-diffusing 16S rRNA revealed that triple correlation functions contain symmetries predicted from time-reversal arguments. Irreversible systems are shown to break these symmetries and correlation strategies were developed to detect time-reversal asymmetries in a comprehensive way with respect to two delay times, each spanning many orders of magnitude in time. The correlation strategies, experimental approaches and theory developed here enable studies of the composition and dynamics of complex systems using F3CS. PMID:22229664

  13. 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.

  14. 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.

  15. 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.

  16. In ovo sexing of chicken eggs by fluorescence spectroscopy.

    PubMed

    Galli, Roberta; Preusse, Grit; Uckermann, Ortrud; Bartels, Thomas; Krautwald-Junghanns, Maria-Elisabeth; Koch, Edmund; Steiner, Gerald

    2017-02-01

    Culling of day-old male chicks in production of laying hen strains involves several millions of animals every year worldwide and is ethically controversial. In an attempt to provide an alternative, optical spectroscopy was investigated to determine nondestructively in ovo the sex of early embryos of the domestic chicken. The extraembryonic blood circulation system was accessed by producing a window in the egg shell and the flowing blood was illuminated with a near-infrared laser. The strong fluorescence and the weak Raman signals were acquired and spectroscopically analyzed between 800 and 1000 nm. The increase of fluorescence intensity between 3.5 and 11.5 days of incubation was found to be in agreement with the erythropoietic stages, thus enabling to identify hemoglobin as fluorescence source. Sex-related differences in the fluorescence spectrum were found at day 3.5, and principal component (PC) analysis showed that the blood of males was characterized by a specific fluorescence band located at ∼910 nm. Supervised classification of the PC scores enabled the determination of the sex of 380 eggs at day 3.5 of incubation with a correct rate up to 93% by combining the information derived from both fluorescence and Raman scattering. Graphical abstract The fluorescence of blood obtained in ovo by illumination of embryonic vessels with a IR laser displays spectral differences that can be employed for sexing of eggs in early stage of incubation, before onset of embryo sensitivity and without hindering its development into a healthy chick.

  17. Fluorescence Fluctuation Spectroscopy of mCherry in Living Cells

    PubMed Central

    Wu, Bin; Chen, Yan; Müller, Joachim D.

    2009-01-01

    The red fluorescent protein mCherry is of considerable interest for fluorescence fluctuation spectroscopy (FFS), because the wide separation in color between mCherry and green fluorescent protein provides excellent conditions for identifying protein interactions inside cells. This two-photon study reveals that mCherry exists in more than a single brightness state. Unbiased analysis of the data needs to account for the presence of multiple states. We introduce a two-state model that successfully describes the brightness and fluctuation amplitude of mCherry. The properties of the two states are characterized by FFS and fluorescence lifetime experiments. No interconversion between the two states was observed over the experimentally probed timescales. The effect of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP) and mCherry is incorporated into the two-state model to describe protein hetero-oligomerization. The model is verified by comparing the predicted and measured brightness and fluctuation amplitude of several fusion proteins that contain mCherry and EGFP. In addition, hetero-fluorescence resonance energy transfer between mCherry molecules in different states is detected, but its influence on FFS parameters is small enough to be negligible. Finally, the two-state model is applied to study protein oligomerization in living cells. We demonstrate that the model successfully describes the homodimerization of nuclear receptors. In addition, we resolved a mixture of interacting and noninteracting proteins labeled with EGFP and mCherry. These results provide the foundation for quantitative applications of mCherry in FFS studies. PMID:19289064

  18. Applications of dynamic light scattering, fluorescence microscopy and fluorescence spectroscopy in DB-67 liposomal formulation studies

    NASA Astrophysics Data System (ADS)

    Kruszewski, Stefan; Ziomkowska, Blanka; Cyrankiewicz, Michał; Latus, Lori; Bom, David

    2005-08-01

    Campthothecin (CPT) and its analogues as prominent anticancer agents are currently the subject of the intensive studies. One of the most promising camptothecin analogues is 7-tert-butyldimethylsil- 1 0-hydroxycampthothecin called DB-67. It is characterized by high affinity to SUV (small unilamellar lipids vesicles) and relatively high stability in human blood. The studies of liposomal formulation as a delivery systems for DB-67 are the subject of this paper. The methods of dynamic light scattering (DLS), fluorescence microscopy (FM) and fluorescence spectroscopy (FS) are used to determine the physical properties of DB-67 liposomal formulation.

  19. Fluorescent rare earth solutions as intrinsic wavelength standards for protein fluorescence spectroscopy.

    PubMed

    Anderle, Heinz; Weber, Alfred

    2017-02-01

    Trivalent Gd, Tm, and Dy solutions can be used as intrinsic excitation and emission standards to validate the UV and violet-blue wavelength accuracy of a spectrofluorimeter. Europium extends the range into the red. To attain sufficient sensitivity, these luminescent rare earth ions require deuterated reagents or carbonate complexation, which allow the use of ordinary water and thus preparation in virtually any laboratory. Such solutions are particularly valuable as system suitability standards (SST) for protein fluorescence spectroscopy to detect red shifts of the intrinsic fluorescence maximum in stability and storage studies.

  20. 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.

  1. Time-Resolved Laser Fluorescence Spectroscopy Study of the Coordination Chemistry of a Hydrophilic CHON [1,2,3-Triazol-4-yl]pyridine Ligand with Cm(III) and Eu(III).

    PubMed

    Wagner, Christoph; Mossini, Eros; Macerata, Elena; Mariani, Mario; Arduini, Arturo; Casnati, Alessandro; Geist, Andreas; Panak, Petra J

    2017-02-20

    The complexation of Cm(III) and Eu(III) with the novel i-SANEX complexing agent 2,6-bis[1-(propan-1-ol)-1,2,3-triazol-4-yl]pyridine (PTD) was studied by time-resolved laser fluorescence spectroscopy (TRLFS). The formation of 1:3, 1:2, and 1:1 metal/ligand complexes was identified upon increasing PTD concentration in 10(-3) mol/L HClO4 and in 0.44 mol/L HNO3 solutions. For all these complexes, stability constants were determined at different acid concentrations. Though under the extraction conditions proposed for an An/Ln separation process, that is, for 0.08 mol/L PTD in 0.44 mol/L HNO3, 1:3 complexes represent the major species, a significant fraction of 1:2 complexes was found. This is caused by ligand protonation, and results in lower Eu(III)/Am(III) separation factors compared to SO3-Ph-BTP, until now considered the i-SANEX reference ligand. Focused extraction studies performed at lower proton concentration, where the 1:3 complex is formed exclusively, confirm this assumption.

  2. Optimized Time-Gated Fluorescence Spectroscopy for the Classification and Recycling of Fluorescently Labeled Plastics.

    PubMed

    Fomin, Petr; Zhelondz, Dmitry; Kargel, Christian

    2016-08-29

    For the production of high-quality parts from recycled plastics, a very high purity of the plastic waste to be recycled is mandatory. The incorporation of fluorescent tracers ("markers") into plastics during the manufacturing process helps overcome typical problems of non-tracer based optical classification methods. Despite the unique emission spectra of fluorescent markers, the classification becomes difficult when the host plastics exhibit (strong) autofluorescence that spectrally overlaps the marker fluorescence. Increasing the marker concentration is not an option from an economic perspective and might also adversely affect the properties of the plastics. A measurement approach that suppresses the autofluorescence in the acquired signal is time-gated fluorescence spectroscopy (TGFS). Unfortunately, TGFS is associated with a lower signal-to-noise (S/N) ratio, which results in larger classification errors. In order to optimize the S/N ratio we investigate and validate the best TGFS parameters-derived from a model for the fluorescence signal-for plastics labeled with four specifically designed fluorescent markers. In this study we also demonstrate the implementation of TGFS on a measurement and classification prototype system and determine its performance. Mean values for a sensitivity of [Formula: see text] = 99.93% and precision [Formula: see text] = 99.80% were achieved, proving that a highly reliable classification of plastics can be achieved in practice.

  3. 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.

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

    SciTech Connect

    Harilal, Sivanandan S.; LaHaye, Nicole L.; Phillips, Mark 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 solid aluminum targets 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, 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 continuous-wave laser at one transition. Time-delayed and gated detection of the emission spectrum is used to isolate the resonantly-excited fluorescence emission from the thermally-excited emission from the plasma. In addition, the tunable continuous-wave laser measures the absorption spectrum of the Al transition with ultra-high resolution after the plasma has cooled, resulting in narrower spectral linewidths than observed in emission spectra. Our results highlight that fluorescence spectroscopy employing continuous-wave laser re-excitation after pulsed laser ablation combines benefits of both traditional emission and absorption spectroscopic methods.

  5. Cross Talk Free Fluorescence Cross Correlation Spectroscopy in Live Cells

    PubMed Central

    Thews, Elmar; Gerken, Margarita; Eckert, Reiner; Zäpfel, Johannes; Tietz, Carsten; Wrachtrup, Jörg

    2005-01-01

    Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin. PMID:15951373

  6. Investigation on the effect of fluorescence quenching of bovine serum albumin by cefoxitin sodium using fluorescence spectroscopy and synchronous fluorescence spectroscopy.

    PubMed

    Li, Gaixia; Liu, Bao-Sheng; Zhang, Qiuju; Han, Rong

    2016-08-01

    The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of the synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanisms of drugs with protein and was consistent with one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between drug and protein and energy-transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role in the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. The primary binding site for cefoxitin sodium was sub-hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill's coefficients (nH ) was approximately equal to 1, which suggested no cooperativity in the bovine serum albumin-cefoxitin sodium system. The donor-to-acceptor distance r < 7 nm indicated that static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non-radiation energy-transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy.

    PubMed

    Widengren, Jerker; Chmyrov, Andriy; Eggeling, Christian; Löfdahl, Per-Ake; Seidel, Claus A M

    2007-01-25

    Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes

  8. An analog filter approach to frequency domain fluorescence spectroscopy

    DOE PAGES

    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

  9. 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

  10. Brain cancer probed by native fluorescence and stokes shift spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Yan; Liu, Cheng-hui; He, Yong; Pu, Yang; Li, Qingbo; Wang, Wei; Alfano, Robert R.

    2012-12-01

    Optical biopsy spectroscopy was applied to diagnosis human brain cancer in vitro. The spectra of native fluorescence, Stokes shift and excitation spectra were obtained from malignant meningioma, benign, normal meningeal tissues and acoustic neuroma benign tissues. The wide excitation wavelength ranges were used to establish the criterion for distinguishing brain diseases. The alteration of fluorescence spectra between normal and abnormal brain tissues were identified by the characteristic fluorophores under the excitation with UV to visible wavelength range. It was found that the ratios of the peak intensities and peak position in both spectra of fluorescence and Stokes shift may be used to diagnose human brain meninges diseases. The preliminary analysis of fluorescence spectral data from cancer and normal meningeal tissues by basic biochemical component analysis model (BBCA) and Bayes classification model based on statistical methods revealed the changes of components, and classified the difference between cancer and normal human brain meningeal tissues in a predictions accuracy rate is 0.93 in comparison with histopathology and immunohistochemistry reports (gold standard).

  11. Biosensors based on surface plasmon-enhanced fluorescence spectroscopy.

    PubMed

    Dostálek, Jakub; Knoll, Wolfgang

    2008-09-01

    The implementation of surface plasmon-enhanced fluorescence spectroscopy (SPFS) to surface plasmon resonance (SPR) biosensors enables increasing their sensitivity by several orders of magnitude. In SPR-based biosensors, surface plasmons probe the binding of target molecules contained in a liquid sample by their affinity partners attached to a metallic sensor surface. SPR biosensors relying on the detection of refractive index changes allow for direct observation of the binding of large and medium size molecules that produces sufficiently large refractive index changes. In SPR biosensors exploiting SPFS, the capture of fluorophore-labeled molecules to the sensor surface is observed by the detection of fluorescence light emitted from the surface. This technique takes advantage of the enhanced intensity of electromagnetic field accompanied with the resonant excitation of surface plasmons. The interaction with surface plasmons can greatly increase the measured fluorescence signal through enhancing the excitation rate of fluorophores and by more efficient collecting of fluorescence light. SPFS-based biosensors were shown to enable the analysis of samples with extremely low analyte concentrations and the detection of small molecules. In this review, we describe the fundamental principles, implementations, and current state of the art applications of SPFS biosensors. This review focuses on SPFS-based biosensors employing the excitation of surface plasmons on continuous metal-dielectric interfaces.

  12. Optimal algorithm for fluorescence suppression of modulated Raman spectroscopy.

    PubMed

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

    2010-05-24

    Raman spectroscopy permits probing of the molecular and chemical properties of the analyzed sample. However, its applicability has been seriously limited to specific applications by the presence of a strong fluorescence background. In our recent paper [Anal. Chem. 82, 738 (2010)], we reported a new modulation method for separating Raman scattering from fluorescence. By continuously changing the excitation wavelength, we demonstrated that it is possible to continuously shift the Raman peaks while the fluorescence background remains essentially constant. In this way, our method allows separation of the modulated Raman peaks from the static fluorescence background with important advantages when compared to previous work using only two [Appl. Spectrosc. 46, 707 (1992)] or a few shifted excitation wavelengths [Opt. Express 16, 10975 (2008)]. The purpose of the present work is to demonstrate a significant improvement of the efficacy of the modulated method by using different processing algorithms. The merits of each algorithm (Standard Deviation analysis, Fourier Filtering, Least-Squares fitting and Principal Component Analysis) are discussed and the dependence of the modulated Raman signal on several parameters, such as the amplitude and the modulation rate of the Raman excitation wavelength, is analyzed. The results of both simulation and experimental data demonstrate that Principal Component Analysis is the best processing algorithm. It improves the signal-to-noise ratio in the treated Raman spectra, reducing required acquisition times. Additionally, this approach does not require any synchronization procedure, reduces user intervention and renders it suitable for real-time applications.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Influence of the surface hydrophobicity on fluorescence correlation spectroscopy measurements

    NASA Astrophysics Data System (ADS)

    Boutin, Céline; Jaffiol, Rodolphe; Plain, Jérome; Royer, Pascal

    2007-02-01

    Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique used to analyze the diffusion at the single molecule level in solution. FCS is based on the temporal autocorrelation of fluorescent signal generated by dye molecules diffusing through a small confocal volume. These measurements are mostly carried out in a chambered coverglass, close to the glass substrate. In this report, we discuss how the chemical nature of the glass-water interface may interact with the free diffusion of molecules. Our results reveal a strong influence, up to a few μm from the interface, of the surface hydrophobicity degree. This influence is assessed through the relative weight of the two dimension diffusion process observed at the vicinity of the surface.

  18. Two-Photon-Excited Fluorescence-Encoded Infrared Spectroscopy.

    PubMed

    Mastron, Joseph N; Tokmakoff, Andrei

    2016-11-23

    We report on a method for performing ultrafast infrared (IR) vibrational spectroscopy using fluorescence detection. Vibrational dynamics on the ground electronic state driven by femtosecond mid-infrared pulses are detected by changes in fluorescence amplitude resulting from modulation of a two-photon visible transition by nuclear motion. We examine a series of coumarin dyes and study the signals as a function of solvent and excitation pulse parameters. The measured signal characterizes the relaxation of vibrational populations and coherences but yields different information than conventional IR transient absorption measurements. These differences result from the manner in which the ground-state dynamics are projected by the two-photon detection step. Extensions of this method can be adapted for a variety of increased-sensitivity IR measurements.

  19. Rapid-scan coherent 2D fluorescence spectroscopy.

    PubMed

    Draeger, Simon; Roeding, Sebastian; Brixner, Tobias

    2017-02-20

    We developed pulse-shaper-assisted coherent two-dimensional (2D) electronic spectroscopy in liquids using fluorescence detection. A customized pulse shaper facilitates shot-to-shot modulation at 1 kHz and is employed for rapid scanning over all time delays. A full 2D spectrum with 15 × 15 pixels is obtained in approximately 6 s of measurement time (plus further averaging if needed). Coherent information is extracted from the incoherent fluorescence signal via 27-step phase cycling. We exemplify the technique on cresyl violet in ethanol and recover literature-known oscillations as a function of population time. Signal-to-noise behavior is analyzed as a function of the amount of averaging. Rapid scanning provides a 2D spectrum with a root-mean-square error of < 0.05 after 1 min of measurement time.

  20. 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.

  1. Optical fiber fluorescence spectroscopy for detecting AFM1 in milk

    NASA Astrophysics Data System (ADS)

    Mignani, A. G.; Cucci, C.; Ciaccheri, L.; Dall'Asta, C.; Galaverna, G.; Dossena, A.; Marchelli, R.

    2008-04-01

    Fluorescence spectroscopy carried out by means of optical fibers was used for the rapid screening of M1 aflatoxin in milk, enabling the detection of concentrations up to the legal limit, which is 50 ppt. A compact fluorometric device equipped with a LED source, a miniaturized spectrometer, and optical fibers for illumination/detection of the measuring micro-cell was tested for measuring threshold values of AFM1 in pre-treated milk samples. Multivariate processing of the spectral data made it possible to obtain a preliminary screening at the earlier stages of the industrial process, as well as to discard contaminated milk stocks before their inclusion in the production chain.

  2. 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

  3. 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.

  4. 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

  5. The photon counting histogram in fluorescence fluctuation spectroscopy.

    PubMed Central

    Chen, Y; Müller, J D; So, P T; Gratton, E

    1999-01-01

    Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given by Mandel's formula. Based on this equation and considering the fluorescence intensity distribution in the two-photon excitation volume, a theoretical expression for the PCH as a function of the number of molecules in the excitation volume is derived. For a single molecular species two parameters are sufficient to characterize the histogram completely, namely the average number of molecules within the observation volume and the detected photon counts per molecule per sampling time epsilon. The PCH for multiple molecular species, on the other hand, is generated by successively convoluting the photon counting distribution of each species with the others. The influence of the excitation profile upon the photon counting statistics for two relevant point spread functions (PSFs), the three-dimensional Gaussian PSF conventionally employed in confocal detection and the square of the Gaussian

  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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. A universal model of restricted diffusion for fluorescence correlation spectroscopy.

    PubMed

    Piskorz, Tomasz K; Ochab-Marcinek, Anna

    2014-05-08

    Fluorescence correlation spectroscopy (FCS) is frequently used to study the processes of restricted diffusion. The most important quantity to determine is the size of the structures that hinder the Brownian motion of the molecules. We study three qualitatively different models of restricted diffusion, widely applied in biophysics and material science: Diffusion constrained by elastic force (i), walking confined diffusion (ii), and hop diffusion (iii). They cover the diversity of statistical behaviors, from purely Gaussian (i) to sharply non-Gaussian on intermediate time scales (ii) and, additionally, discrete (iii). We test whether one can use the Gaussian approximation of the FCS autocorrelation function to interpret the non-Gaussian data. We show that (i-iii) have approximately the same mean square displacements. Using simulations, we show that the FCS data suspected of restricted diffusion can be reliably interpreted using one archetypal model (i). Even if the underlying mechanism of the restriction is different or unknown, the accuracy of fitting the confinement size is excellent, and diffusion coefficients are also estimated with a good accuracy. This study gives a physical insight into the statistical behavior of different types of restricted diffusion and into the ability of fluorescence correlation spectroscopy to distinguish between them.

  9. Fluorescence correlation spectroscopy of repulsive systems: theory, simulation, and experiment.

    PubMed

    Feng, Ligang; Yang, Jingfa; Zhao, Jiang; Wang, Dapeng; Koynov, Kaloian; Butt, Hans-Jürgen

    2013-06-07

    The theoretical basis of fluorescence correlation spectroscopy (FCS) for repulsive systems, such as charged colloids or macromolecules, has been further expanded and developed. It is established that the collective correlation function can no longer be fitted using the theoretical model of non-interacting systems. Also, it is discovered that the collective correlation function can be divided into two parts: a self-part and a distinct-part, named as the self-correlation and cross-correlation function, respectively. The former indicates the self-diffusion of objects, while the latter describes mutual interactions. Dual-color fluorescence cross-correlation spectroscopy provides the direct measurements of the two parts. The particle concentration and mean squared displacement of single particles can be deduced from the self-correlation function, while the correlation volume between particles can be approximated from the cross-correlation function. In the case of charged colloids, the Debye length of the solution and particle surface charge number can be fitted from the cross-correlation function. These theoretical results are successfully proven using Brownian dynamics simulations and preliminary FCS experiments for model charged colloidal systems.

  10. Rapid measurement of meat spoilage using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Dahlberg, Kevin; Gao, Xin; Smith, Jason; Bailin, Jacob

    2017-02-01

    Food spoilage is mainly caused by microorganisms, such as bacteria. In this study, we measure the autofluorescence in meat samples longitudinally over a week in an attempt to develop a method to rapidly detect meat spoilage using fluorescence spectroscopy. Meat food is a biological tissue, which contains intrinsic fluorophores, such as tryptophan, collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) etc. As meat spoils, it undergoes various morphological and chemical changes. The concentrations of the native fluorophores present in a sample may change. In particular, the changes in NADH and FAD are associated with microbial metabolism, which is the most important process of the bacteria in food spoilage. Such changes may be revealed by fluorescence spectroscopy and used to indicate the status of meat spoilage. Therefore, such native fluorophores may be unique, reliable and nonsubjective indicators for detection of spoiled meat. The results of the study show that the relative concentrations of all above fluorophores change as the meat samples kept in room temperature ( 19° C) spoil. The changes become more rapidly after about two days. For the meat samples kept in a freezer ( -12° C), the changes are much less or even unnoticeable over a-week-long storage.

  11. Fluorescence spectroscopy of polynuclear aromatic compounds in environmental monitoring.

    PubMed

    Kumke, M U; Löhmannsröben, H G; Roch, T

    1995-06-01

    The occurrence of polynuclear aromatic compounds (PAC) in the environment and experimental techniques suitable for the detection of PAC in environmental compartments are briefly reviewed. The specific requirements for on-site andin situ environmental analysis are outlined. Particular emphasis is given to fluorescence spectroscopic techniques for the investigation of humic acid- and soil-containing samples. Some examples of studies in the literature on Shpol'skii and jet spectroscopy and on laser-induced fluorescence (OF) measurements of PAC and mineral oils are highlighted. Contaminants in the environment are usually encountered as multicomponent mixtures in very complex matrices. Total fluorescence analysis in combination with the chemometrical technique of rank annihilation factor analysis (RAFA) was employed for the evaluation of a six-component PAC mixture in toluene. It was shown that even in the presence of strong spectral overlap the qualitative identification of all compounds and the reliable quantification of five substances was possible. Results are presented from our stationary and time-resolved fluorescence investigations of the interactions between pyrene and humic acid in water. The Stern-Volmer analysis showed a significant effect of pH on the static quenching efficiency which can be explained by the pH-dependent macromolecular structure of humic acids. Preliminary results from studies of the deactivation of triplet PAC and quenching of delayed fluorescence by humic acid are reported. LIF measurements of mineral oils directly from soil surfaces and of a model oil in a soil column were performed with a fiber-optic coupled multichannel spectrometer. The fluorescence intensity/ concentration relationships were established for a crude and a fuel oil; the corresponding lower limits of detection (LOD) were determined to be 0.025 and 0.125% m/m (mass/mass percentages). These detection limits are compared with realistic oil contaminations of soils. In a soil

  12. 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.

  13. Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy.

    PubMed

    Chen, Yan; Wei, Li-Na; Müller, Joachim D

    2003-12-23

    Fluorescence fluctuation spectroscopy provides information about protein interactions in the intercellular environment from naturally occurring equilibrium fluctuations. We determine the molecular brightness of fluorescent proteins from the fluctuations by analyzing the photon counting histogram (PCH) or its moments and demonstrate the use of molecular brightness in probing the oligomerization state of proteins. We report fluorescence fluctuation measurements of enhanced GFP (EGFP) in cells up to concentrations of 10 microM by using an improved PCH theory. The molecular brightness of EGFP is constant in the concentration range studied. The brightness of a tandem EGFP construct, which carries two fluorophores, increases by a factor of two compared with EGFP alone, demonstrating the sensitivity of molecular brightness as a probe for protein complex formation. Oligomerization of nuclear receptors plays a crucial role in the regulation of gene expression. We probe the oligomerization state of the testicular receptor 4 and the ligand-binding domains of retinoid X receptor and retinoic acid receptor by observing molecular brightness changes as a function of protein concentration. The large concentration range accessible by experiment allows us to perform titration experiments on EGFP fusion proteins. An increase in the molecular brightness with protein concentration indicates the formation of homocomplexes. We observe the formation of homodimers of retinoid X receptor ligand binding domain upon addition of ligand. Resolving protein interactions in a cell is an important step in understanding cellular function on a molecular level. Brightness analysis promises to develop into an important tool for determining protein complex formation in cells.

  14. Single-molecule fluorescence spectroscopy using phospholipid bilayer nanodiscs.

    PubMed

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

    2010-01-01

    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 peripheral membrane-binding proteins islet amyloid polypeptide (IAPP) and alpha-synuclein, respectively. The monodisperse size distribution of Nanodiscs enhances control over the oligomeric state of the membrane protein of interest, and facilitates accurate solution-based measurements as well. 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. Copyright 2010 Elsevier Inc. All rights reserved.

  15. 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

  16. Comparison of absorption, fluorescence, and polarization spectroscopy of atomic rubidium

    NASA Astrophysics Data System (ADS)

    Ashman, Seth; Stifler, Cayla; Romero, Joaquin

    2015-05-01

    An ongoing spectroscopic investigation of atomic rubidium utilizes a two-photon, single-laser excitation process. Transitions accessible with our tunable laser include 5P1 / 2F' <-- 5S1 / 2 F and 5P3 / 2F' <-- 5S1 / 2 F . The laser is split into a pump and probe beam to allow for Doppler-free measurements of transitions between hyperfine levels. The pump and probe beams are overlapped in a counter-propagating geometry and the laser frequency scans over a transition. Absorption, fluorescence and polarization spectroscopy techniques are applied to this basic experimental setup. The temperature of the vapor cell and the power of the pump and probe beams have been varied to explore line broadening effects and signal-to-noise of each technique. This humble setup will hopefully grow into a more robust experimental arrangement in which double resonance, two-laser excitations are used to explore hyperfine state changing collisions between rubidium atoms and noble gas atoms. Rb-noble gas collisions can transfer population between hyperfine levels, such as 5P3 / 2 (F' = 3) <-- Collision 5P3 / 2 (F ' = 2) , and the probe beam couples 7S1 / 2 (F'' = 2) <-- 5P3 / 2 (F' = 3) . Polarization spectroscopy signal depends on the rate of population transfer due to the collision as well as maintaining the orientation created by the pump laser. Fluorescence spectroscopy relies only on transfer of population due to the collision. Comparison of these techniques yields information regarding the change of the magnetic sublevels, mF, during hyperfine state changing collisions.

  17. 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

  18. Assessing Raw and Treated Water Quality Using Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Bridgeman, J.; Baker, A.

    2006-12-01

    To date, much fluorescence spectroscopy work has focused on the use of techniques to characterize pollution in river water and to fingerprint pollutants such as, inter alia, treated and raw sewage effluent. In the face of tightening water quality standards associated with disinfection byproducts, there exists the need for a surrogate THM parameter which can be measured accurately and quickly at the water treatment works and which will give a satisfactory indication of the THM concentration leaving the water treatment works. In addition, water treatment works and distribution system managers require tools which are simple and quick, yet robust, to monitor plant and unit process performance. We extend the use of fluorescence techniques from raw water quality monitoring to (1) the monitoring of water treatment works intakes and the assessment of water treatment works performance by (2) assessing the removal of dissolved organic matter (DOM) through the unit process stages of various water treatment works treating different raw waters and (3) examining the prevalence of microbiological activity found at service reservoirs in the downstream distribution system. 16 surface water treatment works were selected in the central region of the UK and samples taken at works' intakes, downstream of each unit process, and in the distribution systems. The intakes selected abstract water from a broad range of upland and lowland water sources with varying natural and anthropogenic pollutant inputs and significantly different flows. The treatment works selected offer a range of different, but relatively standard, unit processes. The results demonstrate that raw waters exhibit more fluorescence than (partially) treated waters. However, noticeable differences between each site are observed. Furthermore, differences in unit process performance between works are also identified and quantified. Across all sites, treatment with Granular Activated Carbon is found to yield a significant

  19. Study of the Interaction of Cefonicid Sodium with Bovine Serum Albumin by Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Duan, Sh.-T.; Liu, B.-Sh.; Li, T.-T.; Cui, M.-M.

    2017-07-01

    The reaction mechanism of cefonicid sodium with bovine serum albumin was investigated by traditional fluorescence spectroscopy and synchronous fluorescence spectroscopy. The results demonstrated that cefonicid sodium caused a strong fluorescence quenching of bovine serum albumin through a static quenching mechanism, during which the electrostatic force played the dominant role in this system, and the number of binding sites in the system was close to 1. It also showed that the primary binding site for cefonicid sodium was closer to tryptophan residues located in sub-hydrophobic domain IIA. Moreover, circular dichroism spectroscopy showed that the secondary structure of bovine serum albumin changed. The donor-to-acceptor distance r < 8 nm indicated that the static fluorescence quenching of bovine serum albumin was a nonradiation energy transfer process. The data obtained from Δλ = 60 nm and λex = 295 nm indicated that synchronous fluorescence spectroscopy had higher sensitivity and accuracy compared to traditional fluorescence spectroscopy.

  20. 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.

  1. Monitoring helicase-catalyzed DNA unwinding by fluorescence anisotropy and fluorescence cross-correlation spectroscopy.

    PubMed

    Xi, Xu Guang; Deprez, Eric

    2010-07-01

    In order to elucidate molecular mechanism of helicases, we have developed two new rapid and sensitive fluorescence assays to measure helicase-mediated DNA unwinding. The fluorescence anisotropy (FA) assay takes the advantage of the substantial change in fluorescence polarization upon helicase binding to DNA and DNA unwinding. The extent of depolarization depends on the rate of tumbling of the fluorescently labeled DNA molecule, which decreases with increasing size. This assay therefore can simultaneously monitor the DNA binding of helicase and the subsequent helicase-catalyzed DNA unwinding in real-time. For size limitation reasons, the FA approach is more suitable for single-turnover kinetic studies. A fluorescence cross-correlation spectroscopy method (FCCS) is also described for measuring DNA unwinding. This assay is based on the degree of concomitant diffusion of the two complementary DNA strands in a small excitation volume, each labeled by a different color. The decrease in the amplitude of the cross-correlation signal is then directly related to the unwinding activity. By contrast with FA, the FCCS-based assay can be used to measure the unwinding activity under both single- and multiple-turnover conditions, with no limitation related to the size of the DNA strands constituting the DNA substrate. These methods used together have proven to be useful for studying molecular mechanism underlying efficient motor function of helicases. Here, we describe the theoretical basis and framework of FA and FCCS and some practical implications for measuring DNA binding and unwinding. We discuss sample preparation and potential troubleshooting. Special attention is paid to instrumentation, data acquisition and analysis.

  2. 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.

  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. Electrically induced microflows probed by fluorescence correlation spectroscopy.

    PubMed

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

    2005-03-01

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

  5. Parameter estimation and analysis model selections in fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Dong, Shiqing; Zhou, Jie; Ding, Xuemei; Wang, Yuhua; Xie, Shusen; Yang, Hongqin

    2016-10-01

    Fluorescence correlation spectroscopy (FCS) is a powerful technique that could provide high temporal resolution and detection for the diffusions of biomolecules at extremely low concentrations. The accuracy of this approach primarily depends on experimental condition requirements and the data analysis model. In this study, we have set up a confocal-based FCS system. And then we used a Rhodamine6G solution to calibrate the system and get the related parameters. An experimental measurement was carried out on one-component solution to evaluate the relationship between a certain number of molecules and concentrations. The results showed FCS system we built was stable and valid. Finally, a two-component solution experiment was carried out to show the importance of analysis model selection. It is a promising method for single molecular diffusion study in living cells.

  6. Detecting Nanodomains in Living Cell Membrane by Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    He, Hai-Tao; Marguet, Didier

    2011-05-01

    Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.

  7. Quick tour of fluorescence correlation spectroscopy from its inception.

    PubMed

    Elson, Elliot L

    2004-01-01

    Fluorescence correlation spectroscopy (FCS) was originally developed in the early 1970s as a way to measure the kinetics of chemical reactions under zero perturbation conditions. At its inception, the measurement was difficult due to experimental limitations and was primarily used during the 1970s and 1980s to characterize diffusion. More recently, as a result of technological advances, FCS measurements have become easier and more versatile. In addition to measurements of diffusion both in solution and in cells, FCS is now also used to measure not only chemical reaction kinetics but also extents of molecular aggregation, the dynamics of photophysical processes, conformational fluctuations, molecular interactions in solution and in cells, and has even found application as a pharmaceutical screening method. From its inception to the present, the contributions of Webb and his coworkers have had a central and defining role in the development and applications of FCS.

  8. 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.

  9. Profiling the near field of nanoshells using surface enhanced Raman spectroscopy and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lal, Surbhi

    Plasmon resonances in metal nanoparticles control the far field and near field optical properties of these metallic structures. The enhanced electromagnetic near field is strongest at the surface of the nanoparticles and rapidly decays away from the surface. This enhanced near field is exploited in surface enhanced spectroscopies including Surface Enhanced Raman Spectroscopy (SERS) and Metal Enhanced Fluorescence Spectroscopy (MEFS). A measurement of the decay profile of the fringing field is important both for further development of surface enhanced spectroscopy for sensor device application, and for understanding from a fundamental physics point of view. Gold nanoshells are spherical colloidal nanoparticles with a silica core covered by a thin gold shell. The plasmon resonance of nanoshells can be controllably tuned in the visible and infrared parts of the spectrum. The near field profile of nanoshells can be theoretically calculated on the basis of Mie scattering theory. The thesis describes a series of experiments designed to experimentally verify the near field profile of nanoshells. A scaffold of ss-DNA is used to place a fluorescein dye molecule at varying distances from the nanoshell surface. The SERS intensity from both the scaffold molecules and the fluorescein placed at the end of the tether is measured simultaneously and self consistently. The fluorescein-ss-DNA nanoshell conjugate structures are also used to study the distance dependence of the fluorescence emission from fluorescein. The thesis discusses the results of the SERS intensity profile agreement with the intensity profile calculated using Mie scattering theory. The quenching and enhancement of the fluorescence emission at varying distances from the nanoshell surface are also discussed.

  10. Optical biopsy fiber-based fluorescence spectroscopy instrumentation

    NASA Astrophysics Data System (ADS)

    Katz, Alvin; Ganesan, Singaravelu; Yang, Yuanlong; Tang, Gui C.; Budansky, Yury; Celmer, Edward J.; Savage, Howard E.; Schantz, Stimson P.; Alfano, Robert R.

    1996-04-01

    Native fluorescence spectroscopy of biomolecules has emerged as a new modality to the medical community in characterizing the various physiological conditions of tissues. In the past several years, many groups have been working to introduce the spectroscopic methods to diagnose cancer. Researchers have successfully used native fluorescence to distinguish cancerous from normal tissue samples in rat and human tissue. We have developed three generations of instruments, called the CD-scan, CD-ratiometer and CD-map, to allow the medical community to use optics for diagnosing tissue. Using ultraviolet excitation and emission spectral measurements on both normal and cancerous tissue of the breast, gynecology, colon, and aerodigestive tract can be separated. For example, from emission intensities at 340 nm to 440 nm (300 nm excitation), a statistically consistent difference between malignant tissue and normal or benign tissue is observed. In order to utilize optical biopsy techniques in a clinical setting, the CD-scan instrument was developed, which allows for rapid and reliable in-vitro and in-vivo florescence measurements of the aerodigestive tract with high accuracy. The instrumentation employs high sensitivity detection techniques which allows for lamp excitation, small diameter optical fiber probes; the higher spatial resolution afforded by the small diameter probes can increase the ability to detect smaller tumors. The fiber optic probes allow for usage in the aerodigestive tract, cervix and colon. Needle based fiber probes have been developed for in-vivo detection of breast cancer.

  11. Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms.

    PubMed

    Krieger, Jan W; Singh, Anand P; Bag, Nirmalya; Garbe, Christoph S; Saunders, Timothy E; Langowski, Jörg; Wohland, Thorsten

    2015-12-01

    Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS. Here we provide guidelines for imaging FCS/FCCS measurements on commercial and custom-built microscopes (including sample preparation, setup calibration, data acquisition and evaluation), as well as anticipated results for a variety of in vitro and in vivo samples. For a skilled user of an available SPIM or TIRF setup, sample preparation, microscope alignment, data acquisition and data fitting, as described in this protocol, will take ∼1 d, depending on the sample and the mode of imaging.

  12. [Outlier Detection of Time Series Three-Dimensional Fluorescence Spectroscopy].

    PubMed

    Yu, Shao-hui; Zhang, Yu-jun; Zhao, Nan-jing

    2015-06-01

    The qualitative and quantitative analysis are often interfered by the outliers in time series three-dimensional fluorescence spectroscopy. In this work, an efficient outlier detection method is proposed by taking advantage of the characteristics in time dimension and the spectral dimension. Firstly, the wavelength points that are mostly the outliers are extracted by the variance in time dimension. Secondly, by the analysis of the existence styles of outliers and similarity score of any two samples, the cumulative similarity is introduced in spectral dimension. At last, fluorescence intensity at each wavelength of all samples is modified by the correction matrix in time dimension and the outlier detection is completed according the to cumulative similarity scores. The application of the correction matrix in time dimension not only improves the validity of the method but also reduces the computation by the choice of characteristics region in correction matrix. Numerical experiments show that the outliers can still be detected by the 50 percent of all points in spectral dimension.

  13. 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.

  14. 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.

  15. Continuous Fluorescence Microphotolysis and Correlation Spectroscopy Using 4Pi Microscopy

    PubMed Central

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

    2007-01-01

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

  16. [Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy].

    PubMed

    Liang, Li-Fang; Da, Xing; Chen, Tong-Sheng; Pei, Yi-Hui

    2009-02-01

    In order to non-invasively investigate nucleoplasmic viscosity in real time with good temporal resolution, the present study firstly introduced a new method based on fluorescence correlation spectroscopy (FCS). FCS is a kind of single-molecule technique with high temporal and spatial resolution to analyze the dynamics of fluorescent molecules in nanomolar concentration. Through a time correlation analysis of spontaneous intensity fluctuations, this technique in conjunction with EGFP as a probe is capable of determining nucleoplasmic viscosity in terms of Stokes-Einstein equation as well as its corresponding analysis of the diffusion coefficient for EGFP in the nucleus. The results showed that nucleoplasmic viscosity of ASTC-a-1 cells and HeLa cells were respectively (2.55 +/- 0.61) cP and (2.04 +/- 0.49) cP at pH 7.4 and 37 degrees C, consistent with the results by traditional methods, and nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity. Meanwhile, the real-time analysis of nucleoplasmic viscosity in living cells exposed to hypotonic media proved that FCS could be used to track the changing rheological characteristics of the nucleoplasm in living cells. Taken together, this study suggests that FCS provides an accurate and non-invasive method to investigate the microenvironment in living cells on the femtoliter scale and it can be used as a powerful tool in researches on the dynamical processes of intracellular molecules.

  17. 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.

  18. 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.

  19. Single-Molecule Fluorescence Spectroscopy of Photosynthetic Systems.

    PubMed

    Kondo, Toru; Chen, Wei Jia; Schlau-Cohen, Gabriela S

    2017-01-25

    Photosynthesis begins when a network of pigment-protein complexes captures solar energy and transports it to the reaction center, where charge separation occurs. When necessary (under low light conditions), photosynthetic organisms perform this energy transport and charge separation with near unity quantum efficiency. Remarkably, this high efficiency is maintained under physiological conditions, which include thermal fluctuations of the pigment-protein complexes and changing local environments. These conditions introduce multiple types of heterogeneity in the pigment-protein complexes, including structural heterogeneity, energetic heterogeneity, and functional heterogeneity. Understanding how photosynthetic light-harvesting functions in the face of these fluctuations requires understanding this heterogeneity, which, in turn, requires characterization of individual pigment-protein complexes. Single-molecule spectroscopy has the power to probe individual complexes. In this review, we present an overview of the common techniques for single-molecule fluorescence spectroscopy applied to photosynthetic systems and describe selected experiments on these systems. We discuss how these experiments provide a new understanding of the impact of heterogeneity on light harvesting and thus how these systems are optimized to capture sunlight under physiological conditions.

  20. Fluorescence and Diffuse Reflectance Spectroscopy for Breast Cancer Diagnosis During Core Needle Biopsy

    DTIC Science & Technology

    2007-09-01

    The goal of this project is to explore the potential of using tissue fluorescence and diffuse reflectance spectroscopy for breast cancer detection...sensor based on tissue fluorescence and diffuse reflectance spectroscopy as an adjunct diagnostic tool, which has the potential to provide guidance for core needle breast biopsy.

  1. Methods of single-molecule fluorescence spectroscopy and microscopy

    NASA Astrophysics Data System (ADS)

    Moerner, W. E.; Fromm, David P.

    2003-08-01

    Optical spectroscopy at the ultimate limit of a single molecule has grown over the past dozen years into a powerful technique for exploring the individual nanoscale behavior of molecules in complex local environments. Observing a single molecule removes the usual ensemble average, allowing the exploration of hidden heterogeneity in complex condensed phases as well as direct observation of dynamical state changes arising from photophysics and photochemistry, without synchronization. This article reviews the experimental techniques of single-molecule fluorescence spectroscopy and microscopy with emphasis on studies at room temperature where the same single molecule is studied for an extended period. Key to successful single-molecule detection is the need to optimize signal-to-noise ratio, and the physical parameters affecting both signal and noise are described in detail. Four successful microscopic methods including the wide-field techniques of epifluorescence and total internal reflection, as well as confocal and near-field optical scanning microscopies are described. In order to extract the maximum amount of information from an experiment, a wide array of properties of the emission can be recorded, such as polarization, spectrum, degree of energy transfer, and spatial position. Whatever variable is measured, the time dependence of the parameter can yield information about excited state lifetimes, photochemistry, local environmental fluctuations, enzymatic activity, quantum optics, and many other dynamical effects. Due to the breadth of applications now appearing, single-molecule spectroscopy and microscopy may be viewed as useful new tools for the study of dynamics in complex systems, especially where ensemble averaging or lack of synchronization may obscure the details of the process under study.

  2. Fluorescence spectroscopy of fulvic acids from fen peatlands

    NASA Astrophysics Data System (ADS)

    Maryganova, Victoria; Wojciech Szajdak, Lech

    2010-05-01

    Intensive cultivation and agricultural use of peatlands lead to the degradation and mineralization of peat. Fulvic acids (FA) as the most mobile part of peat organic matter can be considered as an early indicator of its changes. One of the most sensitive and simple methods for studying the structural chemistry of humic substances is fluorescence spectroscopy. The objective of this study was to analyze comparatively the fluorescence properties of FA from low-moor peats of different genesis and decomposition degree with respect to the peculiarities of their chemical structure. FA were isolated from 4 peat samples collected from different fen peatlands of Belarus. Fluorescence spectra were obtained on water solutions of FA at a concentration of 50 mg/L after adjustment to pH=2, 6 and 13 on a MSL-4800 spectrofluorimeter (Perkin Elmer, USA.) at 20 ± 2 oC. Emission spectra were obtained using an excitation wavelength of 365 nm. Excitation spectra were recorded by varying the excitation wavelength from 260 to 520 nm and measuring the fluorescence emission at a fixed wavelength of 520 nm. Elemental composition of FA and optical density at 465 nm (D465) of FA solutions in 0.1 N NaOH were determined. Emission spectra of FA are characterized by a broad featureless band of the maximum wavelengths at λ=460-475 nm. Excitation spectra of FA have three peaks localized in different wavelength regions. The maximum wavelengths and intensities of the excitation peaks depend on the pH values. The highest intensities are observed at pH=6. FA exhibit a main excitation peak at λ=355-370 nm, a minor peak at λ=395-400 nm, and a weak band at λ=430-440 nm. At pH=2, all the peaks decrease in intensity. With increasing the pH to 13, the excitation maximum at λ=355-370 nm shifts from 10 to 20 nm towards longer wavelengths compared to acidic solutions. A general decrease in fluorescence intensity is observed, the intensity decline of the peak at λ=355-370 nm being more marked than of the

  3. Eu(3+)-mediated polymerization of benzenetetracarboxylic acid studied by spectroscopy, temperature-dependent calorimetry, and density functional theory.

    PubMed

    Barkleit, Astrid; Tsushima, Satoru; Savchuk, Olesya; Philipp, Jenny; Heim, Karsten; Acker, Margret; Taut, Steffen; Fahmy, Karim

    2011-06-20

    Thermodynamic parameters for the complexation of Eu(3+) with pyromellitic acid (1,2,4,5-benzenetetracarboxylic acid, BTC) as a model system for polymerizable metal-complexing humic acids were determined using temperature-dependent time-resolved laser-induced fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC). At low metal and ligand concentrations (<50 μM Eu(3+), <1 mM BTC), a 1:1 monomeric Eu-BTC complex was identified in the range of 25-60 °C. At elevated concentrations (>500 μM Eu(3+) and BTC) a temperature-dependent polymerization was observed, where BTC monomers are linked via coordinating shared Eu(3+) ions. The two methods lead to comparable thermodynamic data (ΔH = 18.5 ± 1.5/16.5 ± 0.1 kJ mol(-1); ΔS = 152 ± 5/130 ± 5 J mol(-1) K(-1); TRLFS/ITC) in the absence of polymerization. With the onset of polymerization, TRLFS reveals the water coordination number of the lanthanide, whereas calorimetry is superior in determining the thermodynamic data in this regime. Evaluating the heat uptake kinetics, the monomer and polymer formation steps could be separated by "time-resolved" ITC, revealing almost identical binding enthalpies for the sequential reactions. Structural features of the complexes were studied by Fourier-transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations showing predominantly chelating coordination with two carboxylate groups in the monomeric complex and monodentate binding of a single carboxylate group in the polymeric complex of the polycarboxylate with Eu(3+). The data show that pyromellitic acid is a suitable model for the study of metal-mediated polymerization as a crucial factor in determining the effect of humic acids on the mobility of heavy metals in the environment.

  4. 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

  5. Fluorescence quantum yield measurement in nanoparticle-fluorophore systems by thermal lens spectroscopy

    NASA Astrophysics Data System (ADS)

    Ferreira, M.; Piscitelli, V.

    2016-04-01

    Metallic nanoparticles have been used as a way to tailor the fluorescence properties like quantum yield, but regular fluorescence quantum yield measurements have to counter the reflection and dispersion of a sample for an accurate result. Thermal lens spectroscopy is a good alternative to resolve this problem because doesn't measure the fluorescence intensity but the heat generated by absorption. We studied the changes induced by silver nanoparticles, generated by laser ablation, in the fluorescence peak and quantum yield of Rhodamine B. We fund that the silver nanoparticles lowered the fluorescence peak and quenched the fluorescence of the Rhodamine B and how much is quenched also depends on its concentration.

  6. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    DOE PAGES

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

    2014-08-14

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

  7. 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.

  8. 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.

  9. The Intermediate Scattering Function in Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Guerra, Rodrigo; Andrews, Ballard; Sen, Pabitra

    2006-03-01

    We formulate the autocorrelation function for Fluorescence Correlation Spectroscopy (FCS) GD(τ) in reciprocal space in terms of the of the Intermediate Scattering Function ISF(k,t) and the fourier transform of the Optical Response Function ORF(k). In this way we may extend the use of FCS to processes that have been studied using NMR, DLS, and neutron scattering. This formalism is useful for the complicated propagators involved in confined systems and in the study of diffusion in cells: where diffusion is either restricted or permeation through membrane is important. Calculations in k-space produce approximate expressions for the ORF using cumulant expansions that are accurate for small wavevectors. This provides descriptions for longer timescales better suited for studying time-dependent diffusion ISF(k,t)->exp[-tD(t)k^2] and provides a natural separation of contributions from system dynamics and from optical artifacts and aberrations. We will show an explicit derivation of a semi-analytical fit function for free diffusion based on standard electromagnetic analysis of a confocal optical apparatus. This fit function is then used to analyze a representative data set and has no free fit parameters other than the diffusion constant.

  10. Intraoperative metastases detection by laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Vari, Sandor G.; Papazoglou, Theodore G.; van der Veen, Maurits J.; Fishbein, Michael C.; Young, J. D.; Chandra, Mudjianto; Papaioannou, Thanassis; Beeder, Clain; Shi, Wei-Qiang; Grundfest, Warren S.

    1991-06-01

    The authors studied the ability of Laser Induced Fluorescence Spectroscopy (LIFS) for the intraoperative identification of metastases using a photosensitizing agent Photofrin IIr to enhance spectroscopic detection. A He-Cd laser source (442 nm) was used to produce low-power illumination of tissue via a hand-held 400 micrometers fiberoptic probe. Through the same fiber, reflected and emitted light was returned to an optical multi-channel analyzer (OMA III) for analysis. Spectroscopic signals were displayed on a screen for immediate examination. Lobund Wistar rats, inoculated with Pollard rat adenocarcinoma cells, were used as an animal model. Photofrin IIr was administered intraperitoneal 24 or 48 hours prior to surgical exploration in doses varying from 0.75-7.5 mg/kg. Metastases detection was performed during abdominal exploration directed to ipsilateral and contralateral inguinal, iliac, para-aortic and renal lymph nodes. Nineteen tissue samples, identified as abnormal by LIFS, were removed for histologic analysis; 11 of these samples were larger than 5mm and histologic examination revealed malignancy in all cases. While LIFS signals showed malignancy in 8 tissue samples with dimensions less than 5mm, histology confirmed this in only 3. However, serial histologic sections were not performed. From the initial results, it was concluded that LIFS detection of malignant tissue is feasible and enhanced by the addition of Photofrin IIr. LIFS may be a promising technique for the intraoperative detection of primary malignant and metastatic tissue.

  11. 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.

  12. Subdiffusive molecular motion in nanochannels observed by fluorescence correlation spectroscopy.

    PubMed

    De Santo, Ilaria; Causa, Filippo; Netti, Paolo A

    2010-02-01

    The influence of confinement on biomolecule motion in glass channels of nanometric height has been investigated with fluorescence correlation spectroscopy (FCS). We measured intrachannel molecule diffusion time and concentration based on a single-component diffusion model as a function of molecule size to channel height (r(g)/h). Poly(ethylene glycol) (PEG) of 20 kDa and dextran of 40 kDa showed a reduction of their diffusion coefficients of almost 1 order of magnitude when nanochannel height approached probe diameter, whereas rhodamine 6G (Rh6G) was shown to be almost unaffected from confinement. Subdiffusive motion has been proven for flexible molecules in nanochannels, and deviations toward a square root dependence of mobility with time for confinement up to molecule size r(g)/h approximately 0.5 were registered. Diffusion coefficient time dependence has been evaluated and described with a model that accounts for diffusion time increase due to molecule rearrangements related to molecule flexibility and surface interactions dynamics. The evaluation of the subdiffusive mode and the key parameters extracted at the single-molecule level of partitioning, intrachannel diffusion time, desorption time, and binding probability at surfaces can be exploited for the engineering of bioanalytic nanodevices.

  13. Noninvasive determination of cell nucleoplasmic viscosity by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Liang, Lifang; Wang, Xichao; Xing, Da; Chen, Tongsheng; Chen, Wei R.

    2009-03-01

    Noninvasive and reliable quantification of rheological characteristics in the nucleus is extremely useful for fundamental research and practical applications in medicine and biology. This study examines the use of fluorescence correlation spectroscopy (FCS) to noninvasively determine nucleoplasmic viscosity (ɛnu), an important parameter of nucleoplasmic rheology. Our FCS analyses show that ɛnu of lung adenocarcinoma (ASTC-a-1) and HeLa cells are 1.77+/-0.42 cP and 1.40+/-0.27 cP, respectively, about three to four times larger than the water viscosity at 37 °C. ɛnu was reduced by 31 to 36% upon hypotonic exposure and increased by 28 to 52% from 37 to 24 °C. In addition, we found that ɛnu of HeLa cells reached the lowest value in the S phase and that there was no significant difference of ɛnu between in the G1 and G2 phases. Last, nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity in both HeLa and ASTC-a-1 cells. These results indicate that FCS can be used as a noninvasive tool to investigate the microenvironment of living cells. This is the first report on the measurement of ɛnu in living cells synchronized in the G1, S, and G2 phases.

  14. Structured illumination fluorescence correlation spectroscopy for velocimetry in Zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pozzi, Paolo; Rossetti, Leone; Sironi, Laura; Freddi, Stefano; D'Alfonso, Laura; Caccia, Michele; Bouzin, Margaux; Collini, Maddalena; Chirico, Giuseppe

    2013-02-01

    The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones.

  15. 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

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

    PubMed

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

    2009-09-01

    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.

  17. Time-resolved fluorescence spectroscopy of oil spill detected by ocean lidar

    NASA Astrophysics Data System (ADS)

    Li, Xiao-long; Chen, Yong-hua; Li, Jie; Jiang, Jingbo; Ni, Zuotao; Liu, Zhi-shen

    2016-10-01

    Based on time-resolved fluorescence of oils, an oceanographic fluorescence Lidar was designed to identify oil pollutions. A third harmonic (at 355nm) of Nd:YAG laser is used as the excitation source, and the fluorescence intensities and lifetimes of oil fluorescence at wavelength from 380 nm to 580 nm are measured by an intensified CCD (ICCD). In the experiments, time-resolved fluorescence spectra of 20 oil samples, including crude oils, fuel oils, lubricating oil, diesel oils and gasoline, are analyzed to discuss fluorescence spectral characteristics of samples for oil classification. The spectral characteristics of oil fluorescence obtained by ICCD with delay time of 2 ns, 4 ns, and 6 ns were studied by using the principal component analysis (PCA) method. Moreover, an efficient method is used to improve the recognition rate of the oil spill types, through enlarging spectral differences of oil fluorescence at different delay times. Experimental analysis shows that the optimization method can discriminate between crude oil and fuel oil, and a more accurate classification of oils is obtained by time-resolved fluorescence spectroscopy. As the result, comparing to traditional fluorescence spectroscopy, a higher recognition rate of oil spill types is achieved by time-resolved fluorescence spectroscopy which is also a feasibility technology for Ocean Lidar.

  18. Investigation of efflorescence of inorganic aerosols using fluorescence spectroscopy.

    PubMed

    Choi, Man Yee; Chan, Chak K

    2005-02-17

    The phase transition is one of the most fundamental phenomena affecting the physical and chemical properties of atmospheric aerosols. Efflorescence, in particular, is not well understood, partly because the molecular interactions between the solute and water molecules of saturated or supersaturated solution droplets have not been well characterized. Recently, we developed a technique that combines the use of an electrodynamic balance and a fluorescence dye, 8-hydroxyl-1,3,6-pyrenetrisulfonate (pyranine), to study the distributions of solvated and free water in aqueous droplets (Choi, M. Y.; Chan, C. K.; Zhang, Y. H. J. Phys. Chem. A 2004, 108, 1133). We found that the equality of the amounts of solvated and free water is a necessary but not sufficient condition for efflorescence. For efflorescing compounds such as Na2SO4, (NH4)2SO4, and a mixture of NaCl and Na2SO4, the amount of free water decreases, while that of solvated water is roughly constant in bulk measurements and decreases less dramatically than that of free water in single-particle measurements as the relative humidity (RH) decreases. Efflorescence of the supersaturated droplets of these solutions occurs when the amounts of free and solvated water are equal, which is consistent with our previous observation for NaCl. For nonefflorescing compounds in single-particle levitation experiments such as MgSO4 and Mg(NO3)2, the amounts of free and solvated water are equal at a water-to-solute molar ratio of about 6, at which spectral changes due to the formation of contact ion pairs between magnesium and the anions occur as shown by Raman spectroscopy. Fluorescence imaging shows that the droplets of diluted Mg(NO3)2 (at 80% RH) and MgSO4 are homogeneous but those of NaCl, Na2SO4, (NH4)2SO4, and supersaturated Mg(NO3)2 (at 10% RH) are heterogeneous in terms of the solvated-to-free water distribution. The solvated-to-free water ratios in NaCl, Na2SO4, and (NH4)2SO4 droplets are higher in the outer regions by about

  19. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Multiple stimulated emission fluorescence photoacoustic sensing and spectroscopy

    SciTech Connect

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

    2016-07-04

    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.

  2. 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.

  3. 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.

  4. The Application Of Picosecond-Resolved Fluorescence Spectroscopy In The Study Of Flavins And Flavoproteins

    NASA Astrophysics Data System (ADS)

    Visser, Antonie J.; van Hoek, Arie

    1988-06-01

    Picosecond relaxation processes of flavins and flavoproteins were investigated with mode-locked and synchronously pumped lasers as source of excitation and time-correlated single photon counting in detection. Free flavin rotational correlation times of 80-150 ps (values depending on the flavin derivative used) could be precisely determined. Picosecond-resolved fluorescence of the flavin bound in the electron-carrier protein flavodoxin from Desulfovibrio vulgaris yields a fluorescence lifetime component of 30 ps in the fluorescence decay. Time-resolved tryptophan fluorescence in flavodoxin exhibits a short lifetime component, which is attributed in part to energy transfer from tryptophan to flavin. Three-dimensional fluorescence spectroscopy and fluorescence anisotropy decay analysis of the two tryptophan residues in flavodoxin provide new evidence for specific flavin-tryptophan interaction. Finally, picosecond-resolved spectroscopy enables the direct measurement of energy transfer between two different chromophores in a protein, from which topographical details can be inferred.

  5. Application of fluorescence spectroscopy and chemometrics in the evaluation of processed cheese during storage.

    PubMed

    Christensen, J; Povlsen, V T; Sørensen, J

    2003-04-01

    Front face fluorescence spectroscopy is applied for an evaluation of the stability of processed cheese during storage. Fluorescence landscapes with excitation from 240 to 360 nm and emission in the range of 275 to 475 nm were obtained from cheese samples stored in darkness and light in up to 259 d, at 5, 20 and 37 degrees C, respectively. Parallel factor (PARAFAC) analysis of the fluorescence landscapes exhibits four fluorophores present in the cheese, all related to the storage conditions. The chemometric analysis resolves the fluorescence signal into excitation and emission profiles of the pure fluorescent compounds, which are suggested to be tryptophan, vitamin A and a compound derived from oxidation. Thus, it is concluded that fluorescence spectroscopy in combination with chemometrics has a potential as a fast method for monitoring the stability of processed cheese.

  6. Analytical Applications Of High-Resolution Molecular Fluorescence Spectroscopy In Low Temperature Solid Matrices

    NASA Astrophysics Data System (ADS)

    Hofstraat, Johannes W.; van Zeijl, W. J.; Smedes, F.; Ariese, Freek; Gooijer, Cees; Velthorst, Nel H.; Locher, R.; Renn, Alois; Wild, Urs P.

    1989-05-01

    High-resolution fluorescence spectroscopy may be used to obtain highly specific, vibrationally resolved spectral signatures of molecules. Two techniques are presented that both make use of low temperature, solid matrices. In Shpol'skii spectroscopy highly resolved spectra are obtained by employing n-alkanes as solvents that form neat crystalline matrices at low temperatures in which the guest molecules occupy well defined substitutional sites. Fluorescence line-narrowing spectroscopy is based on the application of selective (mostly laser-) excitation of the guest molecules. Principles and analytical applications of both techniques will be discussed. Specific attention will be paid to the determination of pyrene in bird meat by means of Shpol'skii spectroscopy and to the possibilities of applying two-dimensional fluorescence line-narrowing spectroscopy.

  7. Front face fluorescence spectroscopy and visible spectroscopy coupled with chemometrics have the potential to characterise ripening of Cabernet Franc grapes.

    PubMed

    Le Moigne, Marine; Dufour, Eric; Bertrand, Dominique; Maury, Chantal; Seraphin, Denis; Jourjon, Frédérique

    2008-07-21

    The potential of front-face spectroscopy for grape ripening dates discrimination was investigated on Cabernet Franc grapes from three parcels located on the Loire Valley and for six ripening dates. The 18 batches were analysed by front-face fluorescence spectroscopy and visible spectroscopy. The excitation spectra (250-310nm, emission wavelength=350nm) were characterised by a shoulder at 280nm. Grapes spectra were classified by factorial discriminant analysis (FDA). Ripening dates were well predicted by fluorescence spectra: grapes before veraison were separated from grapes after veraison and almost every ripening date was identified. The common spectroscopic space obtained by CCSWA showed that wavelengths corresponding to anthocyanin absorption in the visible were correlated to fluorescence wavelengths around the starting and ending points of the shoulder (263 and at 292nm). Then, regression models were investigated to predict total soluble solids (TSS), total acidity, malvidin-3G, total anthocyanins and total phenolics content from visible and fluorescence spectra. To predict technological indicators (TSS and total acidity), the PLS model with visible spectra (RMSECV=0.82 degrees Brix or 0.96gL(-1) H(2)SO(4)) was better than those with fluorescence one (RMSECV=1.39 degrees Brix or 2.06gL(-1) H(2)SO(4)). For malvidin-3G and total anthocyanins, all R(c)(2) and R(cv)(2) were superior to 0.90 and RMSECV were low. Visible and fluorescence spectroscopies succeeded in predicting anthocyanin content. Concerning total phenolic, the best prediction was provided by fluorescence spectroscopy.

  8. 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.

  9. Trace Chemical Detection through Vegetation Sentinels and Fluorescence Spectroscopy

    Treesearch

    John E. Anderson; Robert L. Fischer; Jean D. Nelson

    2006-01-01

    Detection of environmental contaminants through vegetation sentinels has long been a goal of remote sensing scientists. A promising technique that should be scalable to wide-area applications is the combined use of genetically modified vascular plants and fluorescence imaging. The ultimate goal of our research is to produce a bioreporter that will express fluorescence...

  10. 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.

  11. Multi-Photon Fluorescence Spectroscopy of Fluorescent Bio-Probes and Bio-Molecules

    DTIC Science & Technology

    2000-07-01

    the set-up of a multi-photon fluorescence microscope. The information can also be useful in the detection of multi-photon fluorescence in bio -chip...technology. In addition, we have investigated a few highly fluorescent bio -molecules commonly found in plant cells.

  12. 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.

  13. [Rapid identification of potato cultivars using NIR-excited fluorescence and Raman spectroscopy].

    PubMed

    Dai, Fen; Bergholt, Mads Sylvest; Benjamin, Arnold Julian Vinoj; Hong, Tian-Sheng; Zhiwei, Huang

    2014-03-01

    Potato is one of the most important food in the world. Rapid and noninvasive identification of potato cultivars plays a important role in the better use of varieties. In this study, The identification ability of optical spectroscopy techniques, including near-infrared (NIR) Raman spectroscopy and NIR fluorescence spectroscopy, for invasive detection of potato cultivars was evaluated. A rapid NIR Raman spectroscopy system was applied to measure the composite Raman and NIR fluorescence spectroscopy of 3 different species of potatoes (98 samples in total) under 785 nm laser light excitation. Then pure Raman and NIR fluorescence spectroscopy were abstracted from the composite spectroscopy, respectively. At last, the partial least squares-discriminant analysis (PLS-DA) was utilized to analyze and classify Raman spectra of 3 different types of potatoes. All the samples were divided into two sets at random: the calibration set (74samples) and prediction set (24 samples), the model was validated using a leave-one-out, cross-validation method. The results showed that both the NIR-excited fluorescence spectra and pure Raman spectra could be used to identify three cultivars of potatoes. The fluorescence spectrum could distinguish the Favorita variety well (sensitivity: 1, specificity: 0.86 and accuracy: 0.92), but the result for Diamant (sensitivity: 0.75, specificity: 0.75 and accuracy: 0. 75) and Granola (sensitivity: 0.16, specificity: 0.89 and accuracy: 0.71) cultivars identification were a bit poorer. We demonstrated that Raman spectroscopy uncovered the main biochemical compositions contained in potato species, and provided a better classification sensitivity, specificity and accuracy (sensitivity: 1, specificity: 1 and accuracy: 1 for all 3 potato cultivars identification) among the three types of potatoes as compared to fluorescence spectroscopy.

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

    PubMed

    Bose, Sayantan; Schönenbrücher, Holger; Richt, Jürgen A; Casey, Thomas A; Rasmussen, Mark A; Kehrli, Marcus E; Petrich, Jacob W

    2013-01-01

    Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated with 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 system to study age-related accumulation of lipofuscin, which has been investigated by monitoring the increasing fluorescence with age covering its entire life span. The current work aims at developing mice retina as a convenient model system to diagnose scrapie and other fatal TSE diseases in animals such as sheep and cows. The objective of the research reported here was to determine whether the spectral features are conserved between two different species namely mice and sheep, and whether an appropriate small animal model system could be identified for diagnosis of scrapie based on the fluorescence intensity in retina. The results were consistent with the previous reports on fluorescence studies of healthy and scrapie-infected retina of sheep. The fluorescence from the retinas of scrapie-infected sheep was significantly more intense and showed more heterogeneity than that from the retinas of uninfected mice. Although the structural characteristics of fluorescence spectra of scrapie-infected sheep and mice eyes are slightly different, more importantly, murine retinas reflect the enhancement of fluorescence intensity upon infecting the mice with scrapie, which is consistent with the observations in sheep eyes.

  15. Direct solid surface fluorescence spectroscopy of standard chemicals and humic acid in ternary system.

    PubMed

    Mounier, S; Nicolodelli, G; Redon, R; Milori, D M B P

    2017-04-15

    The front face fluorescence spectroscopy is often used to quantify chemicals in well-known matrices as it is a rapid and powerful technique, with no sample preparation. However it was not used to investigate extracted organic matter like humic substances. This work aims to fully investigate for the first time front face fluorescence spectroscopy response of a ternary system including boric acid, tryptophan and humic substances, and two binaries system containing quinine sulfate or humic substance in boric acid. Pure chemicals, boric acid, tryptophan, quinine sulfate and humic acid were mixed together in solid pellet at different contents from 0 to 100% in mass. The measurement of excitation emission matrix of fluorescence (3D fluorescence) and laser induced fluorescence were then done in the front face mode. Fluorescence matrices were decomposed using the CP/PARAFAC tools after scattering treatments. Results show that for 3D fluorescence there is no specific component for tryptophan and quinine sulfate, and that humic substances lead to a strong extinction effect for mixture containing quinine sulfate. Laser induced fluorescence gives a very good but non-specific related response for both quinine sulfate and tryptophan. No humic substances fluorescence response was found, but extinction effect is observed as for 3D fluorescence. This effect is stronger for quinine sulfate than for tryptophan. These responses were modeled using a simple absorbance versus emission model.

  16. Direct solid surface fluorescence spectroscopy of standard chemicals and humic acid in ternary system

    NASA Astrophysics Data System (ADS)

    Mounier, S.; Nicolodelli, G.; Redon, R.; Milori, D. M. B. P.

    2017-04-01

    The front face fluorescence spectroscopy is often used to quantify chemicals in well-known matrices as it is a rapid and powerful technique, with no sample preparation. However it was not used to investigate extracted organic matter like humic substances. This work aims to fully investigate for the first time front face fluorescence spectroscopy response of a ternary system including boric acid, tryptophan and humic substances, and two binaries system containing quinine sulfate or humic substance in boric acid. Pure chemicals, boric acid, tryptophan, quinine sulfate and humic acid were mixed together in solid pellet at different contents from 0 to 100% in mass. The measurement of excitation emission matrix of fluorescence (3D fluorescence) and laser induced fluorescence were then done in the front face mode. Fluorescence matrices were decomposed using the CP/PARAFAC tools after scattering treatments. Results show that for 3D fluorescence there is no specific component for tryptophan and quinine sulfate, and that humic substances lead to a strong extinction effect for mixture containing quinine sulfate. Laser induced fluorescence gives a very good but non-specific related response for both quinine sulfate and tryptophan. No humic substances fluorescence response was found, but extinction effect is observed as for 3D fluorescence. This effect is stronger for quinine sulfate than for tryptophan. These responses were modeled using a simple absorbance versus emission model.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Two-dimensional fluorescence spectroscopy of uranium isotopes in femtosecond laser ablation plumes

    DOE PAGES

    Phillips, Mark C.; Brumfield, Brian E.; LaHaye, Nicole; ...

    2017-06-19

    Here, we demonstrate measurement of uranium isotopes in femtosecond laser ablation plumes using two-dimensional fluorescence spectroscopy (2DFS). The high-resolution, tunable CW-laser spectroscopy technique clearly distinguishes atomic absorption from 235U and 238U in natural and highly enriched uranium metal samples. We present analysis of spectral resolution and analytical performance of 2DFS as a function of ambient pressure. Simultaneous measurement using time-resolved absorption spectroscopy provides information on temporal dynamics of the laser ablation plume and saturation behavior of fluorescence signals. The rapid, non-contact measurement is promising for in-field, standoff measurements of uranium enrichment for nuclear safety and security.

  2. Two-dimensional fluorescence spectroscopy of uranium isotopes in femtosecond laser ablation plumes.

    PubMed

    Phillips, Mark C; Brumfield, Brian E; LaHaye, Nicole; Harilal, Sivanandan S; Hartig, Kyle C; Jovanovic, Igor

    2017-06-19

    We demonstrate measurement of uranium isotopes in femtosecond laser ablation plumes using two-dimensional fluorescence spectroscopy (2DFS). The high-resolution, tunable CW-laser spectroscopy technique clearly distinguishes atomic absorption from (235)U and (238)U in natural and highly enriched uranium metal samples. We present analysis of spectral resolution and analytical performance of 2DFS as a function of ambient pressure. Simultaneous measurement using time-resolved absorption spectroscopy provides information on temporal dynamics of the laser ablation plume and saturation behavior of fluorescence signals. The rapid, non-contact measurement is promising for in-field, standoff measurements of uranium enrichment for nuclear safety and security.

  3. Excitation emission and time-resolved fluorescence spectroscopy of selected varnishes used in historical musical instruments.

    PubMed

    Nevin, Austin; Echard, Jean-Philippe; Thoury, Mathieu; Comelli, Daniela; Valentini, Gianluca; Cubeddu, Rinaldo

    2009-11-15

    The analysis of various varnishes from different origins, which are commonly found on historical musical instruments was carried out for the first time with both fluorescence excitation emission spectroscopy and laser-induced time-resolved fluorescence spectroscopy. Samples studied include varnishes prepared using shellac, and selected diterpenoid and triterpenoid resins from plants, and mixtures of these materials. Fluorescence excitation emission spectra have been collected from films of naturally aged varnishes. In parallel, time-resolved fluorescence spectroscopy of varnishes provides means for discriminating between short- (less than 2.0 ns) and long-lived (greater than 7.5 ns) fluorescence emissions in each of these complex materials. Results suggest that complementary use of the two non destructive techniques allows a better understanding of the main fluorophores responsible for the emission in shellac, and further provides means for distinguishing the main classes of other varnishes based on differences in fluorescence lifetime behaviour. Spectrofluorimetric data and time resolved spectra presented here may form the basis for the interpretation of results from future in situ fluorescence examination and time resolved fluorescence imaging of varnished musical instruments.

  4. 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

  5. Fluorescence spectroscopy of gastrointestinal tumors using δ-ALA

    NASA Astrophysics Data System (ADS)

    Borisova, E. G.; Vladimirov, B. G.; Angelov, I. G.; Avramov, L. A.

    2007-03-01

    In the recent study delta-aminolevulinic acid/Protoporphyrin IX (δ-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus and stomach. The δ-ALA is administered per os six hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built to use the light guide of standard video-endoscopic system (Olimpus Corp.). Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer (USB4000, OceanOptics Inc.). The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Mucosa autofluorescence lies at 450-600 nm region. The fluorescence of PpIX is clearly pronounced at the 630-710 nm region. Deep minima in the tumor fluorescence signals are observed in the region 540-575 nm, related to hemoglobin re-absorption. Such high hemoglobin content is an indication of the tumors neovascularisation and it is clearly pronounced in all dysplastic and tumor sites investigated. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of δ-ALA/PpIX only in abnormal sites and gives high contrast when lesion borders are determined from clinicians during video observation in the process of diagnostic procedure. Very good correlation between fluorescence signals and histology examination results of the lesions investigated is achieved.

  6. 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

  7. 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.

  8. Femtosecond broadband fluorescence spectroscopy by down- and up-conversion in β-barium borate crystals

    NASA Astrophysics Data System (ADS)

    Sajadi, M.; Quick, M.; Ernsting, N. P.

    2013-10-01

    Broadband transient fluorescence spectroscopy is performed by difference- and sum-frequency generation in β-barium borate crystals at a fixed geometry. Phase matching is spectrally broadened by using (i) 1340 nm gate pulses, (ii) a fluorescence angle of ˜4° extended at the crystal, and (iii) angular dispersion of the fluorescence in a calcite prism. The latter provides 1° angular separation between the two spectral wings of the fluorescence. By combining down- and up-conversion processes, a 26 000 cm-1 wide multiplex window is realized with ˜100 fs time resolution.

  9. 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.

  10. 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

    2015-10-05

    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.

  11. Hybrid native phosphorescence and fluorescence spectroscopy for cancer detection

    NASA Astrophysics Data System (ADS)

    Alimova, Alexandra; Katz, A.; Sriramoju, Vidyasagar; Budansky, Yuri; Bykov, Alexei A.; Zeylikovich, Roman; Alfano, R. R.

    2006-02-01

    Native fluorescence of tissues in the UV and visible spectral regions has been investigated for over two decades. Native fluorescence has been demonstrated to be an accurate tools for distinguish normal tissue from malignant and pre-malignant. Prior investigations have demonstrated that there are several ratio-based algorithms, which can distinguish malignant tissue from normal with high sensitivity and specificity.1 The wavelength combinations used in these ratios isolate the contributions from pairs of tissue fluorophors, one of which is frequently tryptophan (trp), the predominant tissue fluorophore with excitation in the UV (250-300 nm). In this work, algorithms using a combination of native fluorescence and trp phosphorescence were developed which show promise for providing enhanced detection accuracy. Using optical fibers to collect the emission from the specimen allowed interrogation of small regions of tissue, providing precise spatial information. Using a specially designed setup, specimens were excited in the UV and spectra were collected in the range of 300 to 700 nm. Three main emission bands were selected for analysis: 340 nm (trp fluorescence); 420 - 460 nm band (fluorescence from the extra cellular matrix); and 500 - 520 nm (trp phosphorescence). Normal specimens consistently exhibited a low ratio (<10) of 345 to 500 nm emission intensity while this same ratio was consistently high (>15) for cancer specimens. Creating intensities ratio maps from the tissue allows one to localize the malignant regions with high spatial precision. The study was performed on ex vivo human breast tissues. The ratio analysis correlated well with histopathology.

  12. 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.

  13. 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.

  14. Non-destructive identification of varnishes by UV fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Thoury, Mathieu; Elias, Mady; Frigerio, Jean Marc; Barthou, Carlos

    2005-06-01

    Qualitative UV-fluorescence of varnishes is commonly used to locate repaints on paintings or to specify the homogeneousness of a varnish layer. Photographers can now use flash UV-lamps coupled with a CCD camera to obtain colour images of the fluorescence of paintings, unveiling thus both interest and difficulty to interpret these colours. Starting from this point of view, UV-fluorescence spectra appear to be a potential technique to characterize the nature of varnishes and, if possible, their state of degradation. This identification will be non-invasive, without contact, obtained in real time and workable in situ, as the identification of pigments or dyes by reflectance spectrometry which is already done in our group. The last goal will be to realize both identifications with the same device. Emission fluorescence spectra are implemented with the Jobin-Yvon Fluorolog-3, providing an incident wavelength laying between 200 and 850 nm. The emission spectra are implemented with an optical fiber linked to a Jobin-Yvon spectrometer HR460 and a multi-channel CCD detector. In a first step, popular, fresh, raw resins used between the XVI th and the XIX th century, as mastic, dammar and sandarac, have been used to prepare varnishes films with different solvents. The fluorescence spectra of these films have been carried out at different excitation wavelengths to build databases. After having tested the coherence, the limits and the accuracy of the method, we suggest different applications of our method. A synthesis of the results will be presented to characterize each varnish by their fluorescence spectra.

  15. 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.

  16. 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.

  17. Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery.

    PubMed

    Hsiang, Jung-Cheng; Fleischer, Blake C; Dickson, Robert M

    2016-07-07

    Excitation of few-atom Ag cluster fluorescence produces significant steady-state dark state populations that can be dynamically optically depopulated with long wavelength coillumination. Modulating this secondary illumination dynamically repopulates the ground state, thereby directly modulating nanodot fluorescence without modulating background. Both fast and slow modulation enable unmodulated background to be quantitatively removed in fluorescence correlation spectroscopy (FCS) through simple correlation-based averaging. Such modulated dual-laser FCS enables recovery of pure Ag nanodot fluorescence correlations even in the presence of strong, spectrally overlapping background emission. Fluorescence recovery is linear with Fourier amplitude of the modulated fluorescence, providing a complementary approach to background-free quantitation of modulatable emitter concentration in high background environments. Using the expanding range of modulatable fluorophores, such methodologies should facilitate biologically relevant studies in both complex autofluorescent environments and multiplexed assays.

  18. 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.

  19. The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors.

    PubMed

    Kilpatrick, Laura E; Hill, Stephen J

    2016-04-15

    The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand-receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

  20. Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine.

    PubMed

    Boreham, Alexander; Brodwolf, Robert; Walker, Karolina; Haag, Rainer; Alexiev, Ulrike

    2016-12-24

    The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

  1. The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

    PubMed Central

    Kilpatrick, Laura E.; Hill, Stephen J.

    2016-01-01

    The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties. PMID:27068980

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  3. Fluorescence Spectroscopy of Conformational Changes of Single LH2 Complexes

    PubMed Central

    Rutkauskas, Danielis; Novoderezhkin, Vladimir; Cogdell, Richard J.; van Grondelle, Rienk

    2005-01-01

    We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between long-lived quasi-stable levels differing by up to 30 nm. The frequency and size of these fluorescence peak movements were found to increase linearly with the excitation intensity. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The probability for a particle to undergo significant spectral shift in either direction was found to be roughly the same. Using the modified Redfield theory, the observed changes in spectral shape and intensity were accounted for by changes in the realization of the static disorder. Long lifetimes of the quasi-stable states suggest large energetic barriers between the states characterized by different emission spectra. PMID:15501944

  4. 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.

  5. 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.

  6. Two-dimensional fluorescence correlation spectroscopy IV: Resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme

    NASA Astrophysics Data System (ADS)

    Fukuma, Hiroaki; Nakashima, Kenichi; Ozaki, Yukihiro; Noda, Isao

    2006-11-01

    Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve the fluorescence spectra of two tryptophan (Trp) residues in alcohol dehydrogenase and lysozyme. In each protein, one Trp residue is buried in a hydrophobic domain of the protein matrix and the other Trp residue is located at a hydrophilic domain close to the protein-water interface. Fluorescence quenching by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce the intensity change in the spectra. The Trp residue which is located at the hydrophilic domain is effectively quenched by the quencher, while the Trp residue located at the hydrophobic domain is protected from the quenching. Therefore, the fluorescence of these two Trp residues have a different sensitivity to the quenching, showing a different response to the concentration of the quencher. Fluorescence spectra of the two Trp residues in alcohol dehydrogenase, which are heavily overlapped in conventional one-dimensional spectra, have been successfully resolved by the 2D correlation technique. From the asynchronous correlation map, it was revealed that the quenching of Trp located at the hydrophobic part was brought about after that of Trp located at the hydrophilic part. In contrast, the fluorescence spectra of the two Trp residues could not be resolved after the alcohol dehydrogenase was denatured with guanidine hydrochloride. These results are consistent with the well-known structure of alcohol dehydrogenase. Furthermore, it was elucidated that the present 2D analysis is not interfered by Raman bands of the solvent, which sometimes bring difficulty into the conventional fluorescence analysis. Fluorescence spectra of the Trp residues in lysozyme could not be resolved by the 2D correlation technique. The differences between the two proteins are attributed to the fact that the Trp residue in the hydrophobic site of lysozyme is not sufficiently protected from the quenching.

  7. Potential of fluorescence spectroscopy to predict fatty acid composition of beef.

    PubMed

    Aït-Kaddour, A; Thomas, A; Mardon, J; Jacquot, S; Ferlay, A; Gruffat, D

    2016-03-01

    The present study aimed to evaluate and compare the ability of front face (FFFS) and synchronous fluorescence spectroscopy (SFS) to predict total fat and FA composition of beef LT muscles coming from 36 animals of 3 breeds (Angus, Limousin and Blond d'Aquitaine). The regression models were performed by using Partial Least Square (PLS) method. In spite of the low number of samples used, the results of this preliminary study demonstrated the ability of fluorescence spectroscopy to predict meat lipids. Nonetheless, the results suggested that the fluorescence spectroscopy is more suited to measure SFA (R(2)p≥0.66; RPD≥2.29) and MUFA (R(2)p≥0.48; RPD≥1.49) than PUFA (R(2)p≤0.48; RPD≤1.63). Moreover, R(2) and RPD factors obtained with FFFS were greater compared to the ones obtained with SFS suggesting that FFFS is more adapted to measure lipid composition of beef meat.

  8. Fluorescence decay characteristics of indole compounds revealed by time-resolved area-normalized emission spectroscopy.

    PubMed

    Otosu, Takuhiro; Nishimoto, Etsuko; Yamashita, Shoji

    2009-03-26

    Time-resolved fluorescence spectroscopy of tryptophan residue has been extensively applied to the studies on structure-function relationships of protein. Regardless of this, the fluorescence decay mechanism and kinetics of tryptophan residue in many proteins still remains unclear. Previous studies have demonstrated that conformational heterogeneity and relaxation dynamics are both involved in the peculiar multiexponential decay kinetics in subnanosecond resolution. In the present study, we characterized the fluorescence decay property of six indole compounds in glycerol by resolving the contribution of conformational heterogeneity and relaxation dynamics. We applied the time-resolved area-normalized fluorescence emission spectrum (TRANES) method for the fluorescence decay analysis. The results of TRANES, time-dependent shift of fluorescence spectral center of gravity, and fluorescence decay simulation demonstrated that the dielectric relaxation process independent of intrinsic rotamer/conformer and the individual fluorescence lifetime gives the peculiarity to the fluorescence decay of indole compounds. These results confirmed that TRANES and time-dependent spectral shift analysis are potent methods to resolve the origin of multiexponential decay kinetics of tryptophyl fluorescence in protein.

  9. Graphene as a substrate to suppress fluorescence in resonance Raman spectroscopy.

    PubMed

    Xie, Liming; Ling, Xi; Fang, Yuan; Zhang, Jin; Liu, Zhongfan

    2009-07-29

    We have measured resonance Raman spectra with greatly suppressed fluorescence (FL) background from rhodamine 6G (R6G) and protoporphyrin IX (PPP) adsorbed on graphene. The FL suppression is estimated to be approximately 10(3) times for R6G. The successful observation of resonance Raman peaks demonstrates that graphene can be used as a substrate to suppress FL in resonance Raman spectroscopy (RRS), which has potential applications in low-concentration detection and RRS study of fluorescent molecules.

  10. Surface intrinsic fluorescence spectroscopy of proteins using a UV linearly polarized pulsed laser beam

    NASA Astrophysics Data System (ADS)

    Yapoudjian, S.; Ivanova, M.; Uteza, Olivier P.; Marine, Vladimir I.; Sentis, Marc L.

    2000-04-01

    The proposed new interfacial spectroscopic method allows to measure the fluorescence emission spectra of the two tryptophans of the Bovine Serum Albumin (BSA) during its adsorption at the air/water and egg-lecithin (egg-PC)/water interfaces, using an UV excimer laser. Surface fluorescence spectroscopy shows changes in the spectroscopic properties of adsorbed BSA, spread BSA and mixed egg-PC/BSA films. These results are related to the surface pressure measurements which characterize the different BSA surface organizations.

  11. 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.

  12. Fluorescent probes for shock compression spectroscopy of microstructured materials

    NASA Astrophysics Data System (ADS)

    Christensen, James M.; Banishev, Alexandr A.; Dlott, Dana D.

    2017-01-01

    We are developing fluorescent probes to obtain dynamic two-dimensional pressure maps of shocked microstructured materials. We have fabricated silica nano-or micro-spheres doped with rhodamine 6G dye (R6G) which fluoresce strongly, and which may be dispersed throughout a microstructured sample. Alternatively we can grow thin skin layers of dye-doped silica on the surface of particles. The emissive microspheres were embedded in poly-methyl methacrylate (PMMA) and were excited by a quasi-continuous laser. When the samples were shocked to 3-8.4 GPa using laser-driven flyer plates, the emission redshifted and lost intensity. When encapsulating the dye in silica, the emission became brighter and the intensity-loss response became fast enough to monitor nanosecond shock effects. Preliminary data are reported showing the intensity loss in a shocked microstructured medium, an artificial sand, consisting of dye-coated silica microspheres.

  13. Time-resolved fluorescence spectroscopy for chemical sensors.

    PubMed

    Draxler, S; Lippitsch, M E

    1996-07-20

    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.

  14. 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.

  15. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy.

    PubMed

    Gallagher, Sean R; Desjardins, Philippe R

    2006-11-01

    Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques, as well as three microvolume methods that use fiber optic technology in specialized cells or instrumentation. These procedures allow quantitation of DNA solutions ranging from 1 pg/l to 50 mg/ml.

  16. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy.

    PubMed

    Gallagher, Sean R; Desjardins, Philippe R

    2008-05-01

    Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques, as well as three microvolume methods that use fiber optic technology in specialized cells or instrumentation. These procedures allow quantitation of DNA solutions ranging from 1 pg/liter to 50 mg/ml. Copyright 2008 by John Wiley & Sons, Inc.

  17. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy.

    PubMed

    Gallagher, Sean R; Desjardins, Philippe R

    2007-04-01

    Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques, as well as three microvolume methods that use fiber optic technology in specialized cells or instrumentation. These procedures allow quantitation of DNA solutions ranging from 1 pg/microl to 50 mg/ml.

  18. 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

  19. Investigation of the interaction between five alkaloids and human hemoglobin by fluorescence spectroscopy and molecular modeling.

    PubMed

    He, Wu; Dou, Huanjing; Li, Zhigang; Wang, Xiaogai; Wang, Lvjing; Wang, Ruiyong; Chang, Junbiao

    2014-04-05

    This work studied the interaction of human hemoglobin (HHb) with aminophylline, acefylline, caffeine, theophylline and diprophylline systematically by UV-vis absorption spectroscopy and fluorescence spectroscopy in combination with molecular modeling. Five alkaloids caused the fluorescence quenching of HHb by the formation of alkaloids-HHb complex. The binding constants and thermodynamic parameters were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize these complexes. Results of thermodynamic analysis and molecular modeling showed that aminophylline was the strongest quencher and diprophylline was the weakest quencher.

  20. Hadamard-Transform Fluorescence Excitation-Emission-Matrix Spectroscopy.

    PubMed

    Andrews, N L P; Ferguson, T; Rangaswamy, A M M; Bernicky, A R; Henning, N; Dudelzak, A; Reich, O; Barnes, J A; Loock, H-P

    2017-08-15

    We present a fluorescence excitation-emission-matrix spectrometer with superior data acquisition rates over previous instruments. Light from a white light emitting diode (LED) source is dispersed onto a digital micromirror array (DMA) and encoded using binary n-size Walsh functions ("barcodes"). The encoded excitation light is used to irradiate the liquid sample and its fluorescence is dispersed and detected using a conventional array spectrometer. After exposure to excitation light encoded in n different ways, the 2-dimensional excitation-emission-matrix (EEM) spectrum is obtained by inverse Hadamard transformation. Using this technique we examined the kinetics of the fluorescence of rhodamine B as a function of temperature and the acid-driven demetalation of chlorophyll-a into pheophytin-a. For these experiments, EEM spectra with 31 excitation channels and 2048 emission channels were recorded every 15 s. In total, data from over 3000 EEM spectra were included in this report. It is shown that the increase in data acquisition rate can be as high as [{n(n + 1)}/2]-fold over conventional EEM spectrometers. Spectral acquisition rates of more than two spectra per second were demonstrated.

  1. Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

    PubMed Central

    Chan, Jacky C. K.; Diebold, Eric D.; Buckley, Brandon W.; Mao, Sien; Akbari, Najva; Jalali, Bahram

    2014-01-01

    Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat’s-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures. PMID:25574449

  2. 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.

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

    PubMed

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

    2014-06-01

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

  4. 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.

  5. Laser Induced Fluorescence Spectroscopy of Soft Tissues of the Oral Cavity

    NASA Astrophysics Data System (ADS)

    Patil, Ajeetkumar; Unnikrishnan, V. K.; Bernard, Rodney; Pai, Keerthilatha M.; Ongole, Ravikiran; Kartha, V. B.; Chidangil, Santhosh

    2011-07-01

    The present study deals with the in vivo measurement of auto-fluorescence from different anatomical sites of oral cavities of healthy volunteers, using a homebuilt Laser Induced Fluorescence (LIF) Spectroscopy setup. Excitation wave length of 325 nm from a He-Cd laser was used as the source. From the 7 anatomical sites (say buccal mucosa, tongue, palate etc) of each oral cavity of 113 subjects, 1266 fluorescence spectra were recorded. The spectra were analysed using Principal Component Analysis (PCA) to see the correlation between different sites.

  6. Statistical analysis of excitation-emission matrices for laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Maslov, N. A.; Papaeva, E. O.

    2016-07-01

    An algorithm for statistical processing of the set of multicomponent excitation-emission matrices for laser-induced fluorescence spectroscopy is proposed that is based on principal component analysis. It is shown for the first time that the fluorescence emission and excitation spectra of unknown fluorophores in optically thin samples can be calculated. Using the proposed algorithm, it is possible to pass from principal components with alternating signs to positive quantities corresponding to the spectra of real substances. The method is applied to a mixture of three fluorescent dyes, and it is demonstrated that the obtained spectra of principal components well reproduce the spectra of initial dyes.

  7. Comparison of fluorescence spectroscopy and FTIR in differentiation of plant pollens

    NASA Astrophysics Data System (ADS)

    Mularczyk-Oliwa, Monika; Bombalska, Aneta; Kaliszewski, Miron; Włodarski, Maksymilian; Kopczyński, Krzysztof; Kwaśny, Mirosław; Szpakowska, Małgorzata; Trafny, Elżbieta A.

    2012-11-01

    Spectroscopic techniques are under investigation on possibility of differentiation of airborne particles. This paper describes pollen discrimination among others bio-particles in laboratory conditions. Pollen samples were characterized with UV-Vis fluorescence, drift and KBr pellet techniques of infrared spectroscopy. Principal Component Analysis of UV-Vis fluorescence and FTIR spectra revealed that pollens can be distinguished from other bio-materials with use of these methods. Both methods resulted in similar classification capability. Combined FTIR and fluorescence data analysis did not improve the discrimination between pollen allergens and other airborne biological materials.

  8. Prediction of Ba, Co and Ni for tropical soils using diffuse reflectance spectroscopy and X-ray fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Arantes Camargo, Livia; Marques Júnior, José; Reynaldo Ferracciú Alleoni, Luís; Tadeu Pereira, Gener; De Bortoli Teixeira, Daniel; Santos Rabelo de Souza Bahia, Angélica

    2017-04-01

    Environmental impact assessments may be assisted by spatial characterization of potentially toxic elements (PTEs). Diffuse reflectance spectroscopy (DRS) and X-ray fluorescence spectroscopy (XRF) are rapid, non-destructive, low-cost, prediction tools for a simultaneous characterization of different soil attributes. Although low concentrations of PTEs might preclude the observation of spectral features, their contents can be predicted using spectroscopy by exploring the existing relationship between the PTEs and soil attributes with spectral features. This study aimed to evaluate, in three geomorphic surfaces of Oxisols, the capacity for predicting PTEs (Ba, Co, and Ni) and their spatial variability by means of diffuse reflectance spectroscopy (DRS) and X-ray fluorescence spectroscopy (XRF). For that, soil samples were collected from three geomorphic surfaces and analyzed for chemical, physical, and mineralogical properties, and then analyzed in DRS (visible + near infrared - VIS+NIR and medium infrared - MIR) and XRF equipment. PTE prediction models were calibrated using partial least squares regression (PLSR). PTE spatial distribution maps were built using the values calculated by the calibrated models that reached the best accuracy using geostatistics. PTE prediction models were satisfactorily calibrated using MIR DRS for Ba, and Co (residual prediction deviation - RPD > 3.0), Vis DRS for Ni (RPD > 2.0) and FRX for all the studied PTEs (RPD > 1.8). DRS- and XRF-predicted values allowed the characterization and the understanding of spatial variability of the studied PTEs.

  9. Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy

    PubMed Central

    LUCHOWSKI, RAFAL; SZABELSKI, MARIUSZ; SARKAR, PABAK; APICELLA, ELISA; MIDDE, KRISHNA; RAUT, SANGRAM; BOREJDO, JULIAN; GRYCZYNSKI, ZYGMUNT; GRYCZYNSKI, IGNACY

    2011-01-01

    We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti : sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM). PMID:20719056

  10. Automated suppression of sample-related artifacts in Fluorescence Correlation Spectroscopy.

    PubMed

    Ries, Jonas; Bayer, Mathias; Csúcs, Gábor; Dirkx, Ronald; Solimena, Michele; Ewers, Helge; Schwille, Petra

    2010-05-24

    Fluorescence Correlation Spectroscopy (FCS) in cells often suffers from artifacts caused by bright aggregates or vesicles, depletion of fluorophores or bleaching of a fluorescent background. The common practice of manually discarding distorted curves is time consuming and subjective. Here we demonstrate the feasibility of automated FCS data analysis with efficient rejection of corrupted parts of the signal. As test systems we use a solution of fluorescent molecules, contaminated with bright fluorescent beads, as well as cells expressing a fluorescent protein (ICA512-EGFP), which partitions into bright secretory granules. This approach improves the accuracy of FCS measurements in biological samples, extends its applicability to especially challenging systems and greatly simplifies and accelerates the data analysis.

  11. Very High Spectral Resolution Imaging Spectroscopy: the Fluorescence Explorer (FLEX) Mission

    NASA Technical Reports Server (NTRS)

    Moreno, Jose F.; Goulas, Yves; Huth, Andreas; Middleton, Elizabeth; Miglietta, Franco; Mohammed, Gina; Nedbal, Ladislav; Rascher, Uwe; Verhoef, Wouter; Drusch, Matthias

    2016-01-01

    The Fluorescence Explorer (FLEX) mission has been recently selected as the 8th Earth Explorer by the European Space Agency (ESA). It will be the first mission specifically designed to measure from space vegetation fluorescence emission, by making use of very high spectral resolution imaging spectroscopy techniques. Vegetation fluorescence is the best proxy to actual vegetation photosynthesis which can be measurable from space, allowing an improved quantification of vegetation carbon assimilation and vegetation stress conditions, thus having key relevance for global mapping of ecosystems dynamics and aspects related with agricultural production and food security. The FLEX mission carries the FLORIS spectrometer, with a spectral resolution in the range of 0.3 nm, and is designed to fly in tandem with Copernicus Sentinel-3, in order to provide all the necessary spectral / angular information to disentangle emitted fluorescence from reflected radiance, and to allow proper interpretation of the observed fluorescence spatial and temporal dynamics.

  12. Fluorescence spectroscopy of kerosene vapour at high temperatures and pressures: potential for gas turbines measurements

    NASA Astrophysics Data System (ADS)

    Orain, M.; Baranger, P.; Ledier, C.; Apeloig, J.; Grisch, F.

    2014-09-01

    Laser-induced fluorescence spectroscopy of kerosene vapour was performed in a heated test cell operating between 450 and 900 K, at pressure from 0.1 to 3.0 MPa, for oxygen molar fraction between 0 and 21 %, with different laser excitation wavelengths (248, 266, 282 and 308 nm). Results show that, depending on the laser excitation scheme, kerosene fluorescence spectrum exhibits one or two fluorescence bands in the UV-visible range (attributed to aromatics naturally present in kerosene fuel). Fluorescence intensity of these bands decreases with increasing temperature, pressure and oxygen molar fraction. Different imaging strategies were derived from spectroscopic findings to simultaneously measure temperature and equivalence ratio fields in kerosene/air sprays, or flame structure and fuel spatial distribution in kerosene/air aeronautical combustors, by means of planar laser-induced fluorescence on kerosene vapour (K-PLIF).

  13. 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.

  14. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy.

    PubMed

    Gallagher, Sean R

    2011-07-01

    Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml.

  15. 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

  16. Quantitative fluorescence correlation spectroscopy on DNA in living cells

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  17. Optical biopsy using fluorescence spectroscopy for prostate cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Gao, Xin; Smith, Jason; Bailin, Jacob

    2017-02-01

    Native fluorescence spectra are acquired from fresh normal and cancerous human prostate tissues. The fluorescence data are analyzed using a multivariate analysis algorithm such as non-negative matrix factorization. The nonnegative spectral components are retrieved and attributed to the native fluorophores such as collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD) in tissue. The retrieved weights of the components, e.g. NADH and FAD are used to estimate the relative concentrations of the native fluorophores and the redox ratio. A machine learning algorithm such as support vector machine (SVM) is used for classification to distinguish normal and cancerous tissue samples based on either the relative concentrations of NADH and FAD or the redox ratio alone. The classification performance is shown based on statistical measures such as sensitivity, specificity, and accuracy, along with the area under receiver operating characteristic (ROC) curve. A cross validation method such as leave-one-out is used to evaluate the predictive performance of the SVM classifier to avoid bias due to overfitting.

  18. 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

  19. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. En route to traceable reference standards for surface group quantifications by XPS, NMR and fluorescence spectroscopy.

    PubMed

    Hennig, Andreas; Dietrich, Paul M; Hemmann, Felix; Thiele, Thomas; Borcherding, Heike; Hoffmann, Angelika; Schedler, Uwe; Jäger, Christian; Resch-Genger, Ute; Unger, Wolfgang E S

    2015-03-21

    The fluorine content of polymer particles labelled with 2,2,2-trifluoroethylamine was reliably quantified with overlapping sensitivity ranges by XPS and solid-state NMR. This provides a first step towards reference materials for the metrological traceability of surface group quantifications. The extension of this concept to fluorescence spectroscopy is illustrated.

  1. Application of Fluorescence Spectroscopy for Rapid Detection of Pathogens in Food

    USDA-ARS?s Scientific Manuscript database

    The potential of fluorescence spectroscopy was investigated for the detection food bone pathogens. E coli, Salmonella and Campylobactor, the most commonly present in food, were selectively identified. Each pathogen, grown in agar plate, was diluted in saline and prepared in different concentrations....

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. 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.

  4. Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy.

    PubMed

    Gallagher, Sean R

    2017-02-02

    Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml. © 2017 by John Wiley & Sons, Inc.

  5. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy.

    PubMed

    Gallagher, Sean R

    2011-01-01

    Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml. © 2011 by John Wiley & Sons, Inc.

  6. Rapid multiexcitation fluorescence spectroscopy system for in vivo tissue diagnosis

    NASA Astrophysics Data System (ADS)

    Zângaro, Renato Amaro; Silveira, Landulfo, Jr.; Manoharan, Ramasamy; Zonios, George; Itzkan, Irving; Dasari, Ramachandra R.; van Dam, Jacques; Feld, Michael S.

    1996-09-01

    We have designed, fabricated, and tested a compact, transportable, excitation-emission spectrofluorimeter with optical-fiber light delivery and collection for use in rapid analysis of tissues in a clinical setting. This system provides up to eleven different excitation wavelengths, permitting collection of all the corresponding emission spectra in approximately 600 ms. It uses a N2 laser that pumps a sequence of dyes placed in cuvettes on a rotating wheel. A white-light excitation source permits acquisition of the tissue's diffuse reflectance spectrum on each cycle. Return fluorescence and reflected light are dispersed by a small spectrograph and detected by a photodiode-array detector. The system can collect a single-shot spectrum from biological tissue with a signal-to-noise ratio in excess of 50:1.

  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. Dynamics of Nanoconfined Fluids measured by combined Force Microscopy and Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Subba Rao, Venkatesh; Pantea, Mircea; Grabowski, Christopher; Mukhopadhyay, Ashis; Hoffmann, Peter

    2009-03-01

    We present work performed on a model liquid, tetrakis(2-ethylhexoxy)silane (TEHOS), using Atomic Force microscopy (AFM) and Fluorescence Correlation Spectroscopy (FCS) to study its dynamical structure at the nanoscale. A novel homebuilt interferometer-based small amplitude AFM was used to measure directly the stiffness and damping coefficient of TEHOS film. Oscillations in stiffness and damping coefficient with period ˜1 nm (TEHOS molecular size) were observed. Translational diffusion in spin-coated TEHOS films was measured using Fluorescence Correlation Spectroscopy (FCS). Diffusion was found to be heterogeneous. Finally we present the ongoing work on an integrated platform of AFM and FCS to perform simultaneous measurements of nanoconfined fluids. Recent results using this new setup on a fluorescently labelled nanoparticle solution in confinement will be discussed.

  9. Fluorescence-free biochemical characterization of cells using modulated Raman spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

    The use of Raman spectroscopy for biomedical applications requires overcoming the obstacle of the broad fluorescence background that is generally generated in biological samples. Recently, we have developed a new modulation method for separating the weak Raman peaks from the strong fluorescence background. The novel method is based on the periodical modulation of the excitation wavelength and uses the principle of multi-channel lock-in detection. By continuously modulating the excitation wavelength it is possible to shift the Raman peaks while the fluorescence background remains essentially constant. The powerful capabilities of this novel method are demonstrated by acquiring spectra from different location (nucleus, cytoplasm and membrane) inside a CHO cell. In fact, we show that our modulated Raman spectroscopy provides, with higher efficiency than the standard one, Raman spectra of different locations within a single cell, suggesting that this minimally invasive optical technology could be applied for bio-medical diagnosis and imaging.

  10. 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.

  11. Study of the interaction between N-confused porphyrin and bovine serum albumin by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Yu, Xianyong; Liu, Ronghua; Yi, Rongqiong; Yang, Fengxian; Huang, Haowen; Chen, Jian; Ji, Danhong; Yang, Ying; Li, Xiaofang; Yi, Pinggui

    2011-04-01

    The fluorescence and ultraviolet spectroscopy were explored to study the interaction between N-confused porphyrins (NCP) and bovine serum albumin (BSA) under imitated physiological condition. The experimental results indicated that the fluorescence quenching mechanism between BSA and NCP was static quenching procedure at low NCP concentration at 293 and 305 K or a combined quenching (static and dynamic) procedure at higher NCP concentration at 305 K. The binding constants, binding sites and the corresponding thermodynamic parameters Δ H, Δ S, and Δ G were calculated at different temperatures. The comparison of binding potency of the three NCP to BSA showed that the substituting groups in benzene ring could enhance the binding affinity. From the thermodynamic parameters, we concluded that the action force was mainly hydrophobic interaction. The binding distances between NCP and BSA were calculated using Förster non-radiation energy transfer theory. In addition, the effect of NCP on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy.

  12. 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.

  13. 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.

  14. 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.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    Weger, Lukas; Hoffmann-Jacobsen, Kerstin

    2017-09-01

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

  17. Validation of a time-resolved fluorescence spectroscopy apparatus in a rabbit atherosclerosis model

    NASA Astrophysics Data System (ADS)

    Fang, Qiyin; Jo, Javier A.; Papaioannou, Thanassis; Dorafshar, Amir; Reil, Todd; Qiao, Jian-Hua; Fishbein, Michael C.; Freischlag, Julie A.; Marcu, Laura

    2004-07-01

    Time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) has been studied as a potential tool for in vivo diagnosis of atherosclerotic lesions. This study is to evaluate the potential of a compact fiber-optics based tr-LIFS instrument developed in our laboratory for in vivo analysis of atherosclerotic plaque composition. Time-resolved fluorescence spectroscopy studies were performed in vivo on fifteen New Zealand White rabbits (atherosclerotic: N=8, control: N=7). Time-resolved fluorescence spectra were acquired (range: 360-600 nm, increment: 5 nm, total acquisition time: 65 s) from normal aorta wall and lesions in the abdominal aorta. Data were analyzed in terms of fluorescence emission spectra and wavelength specific lifetimes. Following trichrome staining, tissue specimens were analyzed histopathologically in terms of intima/media thickness and biochemical composition (collagen, elastin, foam cells, and etc). Based on intimal thickness, the lesions were divided into thin and thick lesions. Each group was further separated into two categories: collagen rich lesions and foam cell rich lesions based on their biochemical composition. The obtained spectral and time domain fluorescence signatures were subsequently correlated to the histopathological findings. The results have shown that time-domain fluorescence spectral features can be used in vivo to separate atherosclerotic lesions from normal aorta wall as well discrimination within certain types of lesions.

  18. Use of fluorescence spectroscopy to control ozone dosage in recirculating aquaculture systems.

    PubMed

    Spiliotopoulou, Aikaterini; Martin, Richard; Pedersen, Lars-Flemming; Andersen, Henrik R

    2017-03-15

    The aim of this study was to investigate the potential of fluorescence spectroscopy to be used as an ozone dosage determination tool in recirculating aquaculture systems (RASs), by studying the relationship between fluorescence intensities and dissolved organic matter (DOM) degradation by ozone, in order to optimise ozonation treatment. Water samples from six different Danish facilities (two rearing units from a commercial trout RAS, a commercial eel RAS, a pilot RAS and two marine water aquariums) were treated with different O3 dosages (1.0-20.0 mg/L ozone) in bench-scale experiments, following which fluorescence intensity degradation was eventually determined. Ozonation kinetic experiments showed that RAS water contains fluorescent organic matter, which is easily oxidised upon ozonation in relatively low concentrations (0-5 mg O3/L). Fluorescence spectroscopy has a high level of sensitivity and selectivity in relation to associated fluorophores, and it is able to determine accurately the ozone demand of each system. The findings can potentially be used to design offline or online sensors based on the reduction by ozone of natural fluorescent-dissolved organic matter in RAS. The suggested indirect determination of ozone delivered into water can potentially contribute to a safer and more adequate ozone-based treatment to improve water quality.

  19. 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.

  20. 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.

  1. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.

    PubMed Central

    Schwille, P; Meyer-Almes, F J; Rigler, R

    1997-01-01

    The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude. Images FIGURE 2 FIGURE 3 PMID:9083691

  2. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Fluorescence spectroscopy and birefringence of molecular changes in maturing rat tail tendon.

    PubMed

    Korol, Renee M; Finlay, Helen M; Josseau, Melanie J; Lucas, Alexandra R; Canham, Peter B

    2007-01-01

    Tissue remodeling during maturation, wound healing, and response to vascular stress involves molecular changes of collagen and elastin in the extracellular matrix (ECM). Two optical techniques are effective for investigating these changes--laser-induced fluorescence (LIF) spectroscopy and polarizing microscopy. LIF spectroscopy integrates the signal from both elastin and collagen cross-linked structure, whereas birefringence is a measure of only collagen. Our purpose is (1) to evaluate the rat tail tendon (RTT) spectroscopy against data from purified extracted protein standards and (2) to correlate the two optical techniques in the study of RTT and skin. Spectra from tissue samples from 27 male rats and from extracted elastin and collagen were obtained using LIF spectroscopy (357 nm). Birefringence was measured on 5-mum histological sections of the same tissue. Morphometric analysis reveals that elastin represents approximately 10% of tendon volume and contributes to RTT fluorescence. RTT maximum fluorescence emission intensity (FEI(max)), which includes collagen and elastin, increases with animal weight (R(2)=0.64). Birefringence, when plotted against weight, increases to a plateau (nonlinear correlation: R(2)=0.90), tendon having greater birefringence than skin. LIF spectroscopy and collagen fiber birefringence are shown to provide complementary measurements of molecular structure (tendon birefringence versus FEI(max) at R(2)=0.60).

  4. 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.

  5. 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.

  6. Fluorescent core-shell star polymers based bioassays for ultrasensitive DNA detection by surface plasmon fluorescence spectroscopy.

    PubMed

    Feng, Chuan Liang; Yin, Meizhen; Zhang, Di; Zhu, Shenmin; Caminade, Anne Marie; Majoral, Jean Pierre; Müllen, Klaus

    2011-04-19

    Multilayers containing a perylene diimide labelled star polymers (FSP) donor adjacent to phosphorus dendrimer layer on a silver substrate were constructed by layer by layer (LBL) approach. Using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) technique, a time-resolved ultrasensitive and selective detection of DNA targets relying on enhanced optical fields associated with energy transfer (ET) were achieved under the excitation at 543 nm. The detection limit is about 8 orders of magnitude better than the achieved one under the excitation at 632 nm, which is ascribed to no energy transfer from the donor to the acceptor under the excitation at 632 nm, resulting in much weak detection signal in turn.

  7. A dark-field scanning spectroscopy platform for localized scatter and fluorescence imaging of tissue

    NASA Astrophysics Data System (ADS)

    Krishnaswamy, Venkataramanan; Laughney, Ashley M.; Paulsen, Keith D.; Pogue, Brian W.

    2011-03-01

    Tissue ultra-structure and molecular composition provide native contrast mechanisms for discriminating across pathologically distinct tissue-types. Multi-modality optical probe designs combined with spatially confined sampling techniques have been shown to be sensitive to this type of contrast but their extension to imaging has only been realized recently. A modular scanning spectroscopy platform has been developed to allow imaging localized morphology and molecular contrast measures in breast cancer surgical specimens. A custom designed dark-field telecentric scanning spectroscopy system forms the core of this imaging platform. The system allows imaging localized elastic scatter and fluorescence measures over fields of up to 15 mm x 15 mm at 100 microns resolution in tissue. Results from intralipid and blood phantom measurements demonstrate the ability of the system to quantify localized scatter parameters despite significant changes in local absorption. A co-registered fluorescence spectroscopy mode is also demonstrated in a protophorphyrin-IX phantom.

  8. 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.

  9. 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.

  10. 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.

  11. Water in Biomolecular Fluorescence Spectroscopy and Imaging: Side Effects and Remedies.

    PubMed

    Fürstenberg, Alexandre

    2017-02-22

    Historically, many of the classical organic fluorescent dyes were developed as laser dyes and characterized and optimized in organic solvents. Since then, fluorescence has, however, found a vast range of applications in the life sciences in which the fluorophores are usually surrounded by water and not by organic solvents. The omnipresence of water in biomolecular fluorescence spectroscopy and imaging leads to some unwanted but nonetheless unavoidable consequences on the photophysical properties of the dyes, which may impact the quality and complicate quantitative interpretation of the experiments. This paper discusses and illustrates with examples two such water-induced phenomena, namely chromophore aggregation in water and fluorescence quenching by water, as well as some ways to overcome them.

  12. Fluorescence and absorption spectroscopy of the weakly fluorescent chlorophyll a in cytochrome b6f of Synechocystis PCC6803.

    PubMed Central

    Peterman, E J; Wenk, S O; Pullerits, T; Pâlsson, L O; van Grondelle, R; Dekker, J P; Rögner, M; van Amerongen, H

    1998-01-01

    A spectroscopic characterization of the chlorophyll a (Chl) molecule in the monomeric cytochrome b6f complex (Cytb6f) isolated from the cyanobacterium Synechocystis PCC6803 is presented. The fluorescence lifetime and quantum yield have been determined, and it is shown that Chl in Cytb6f has an excited-state lifetime that is 20 times smaller than that of Chl in methanol. This shortening of the Chl excited state lifetime is not caused by an increased rate of intersystem crossing. Most probably it is due to quenching by a nearby amino acid. It is suggested that this quenching is a mechanism for preventing the formation of Chl triplets, which can lead to the formation of harmful singlet oxygen. Using site-selected fluorescence spectroscopy, detailed information on vibrational frequencies in both the ground and Qy excited states has been obtained. The vibrational frequencies indicate that the Chl molecule has one axial ligand bound to its central magnesium and accepts a hydrogen bond to its 13(1)-keto carbonyl. The results show that the Chl binds to a well-defined pocket of the protein and experiences several close contacts with nearby amino acids. From the site-selected fluorescence spectra, it is further concluded that the electron-phonon coupling is moderately strong. Simulations of both the site-selected fluorescence spectra and the temperature dependence of absorption and fluorescence spectra are presented. These simulations indicate that the Huang-Rhys factor characterizing the electron-phonon coupling strength is between 0.6 and 0.9. The width of the Gaussian inhomogeneous distribution function is 210 +/- 10 cm-1. PMID:9649396

  13. Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation

    PubMed Central

    Kittle, David S.; Vasefi, Fartash; Patil, Chirag G.; Mamelak, Adam; Black, Keith L.; Butte, Pramod V.

    2016-01-01

    The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data. We also show that TR-FS is able to quantify the relative concentration of fluorescence dyes in a mixture by the unmixing of lifetime decays. We show that the TR-FS prototype is able to identify in near-real time the concentrations of dyes in a complex mixture based on previously trained data. As a result, we demonstrate that in complex mixtures of fluorophores, the relative concentration information is encoded in the fluorescence lifetime across multiple spectral bands. We show for the first time the temporal and spectral measurements of a mixture of fluorochromes and the ability to differentiate relative concentrations of each fluorochrome mixture in real time. PMID:27929039

  14. 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.

  15. Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation.

    PubMed

    Kittle, David S; Vasefi, Fartash; Patil, Chirag G; Mamelak, Adam; Black, Keith L; Butte, Pramod V

    2016-12-08

    The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data. We also show that TR-FS is able to quantify the relative concentration of fluorescence dyes in a mixture by the unmixing of lifetime decays. We show that the TR-FS prototype is able to identify in near-real time the concentrations of dyes in a complex mixture based on previously trained data. As a result, we demonstrate that in complex mixtures of fluorophores, the relative concentration information is encoded in the fluorescence lifetime across multiple spectral bands. We show for the first time the temporal and spectral measurements of a mixture of fluorochromes and the ability to differentiate relative concentrations of each fluorochrome mixture in real time.

  16. Evaluating Activated Carbon Adsorption of Dissolved Organic Matter and Micropollutants Using Fluorescence Spectroscopy.

    PubMed

    Shimabuku, Kyle K; Kennedy, Anthony M; Mulhern, Riley E; Summers, R Scott

    2017-02-14

    Dissolved organic matter (DOM) negatively impacts granular activated carbon (GAC) adsorption of micropollutants and is a disinfection byproduct precursor. DOM from surface waters, wastewater effluent, and 1 kDa size fractions were adsorbed by GAC and characterized using fluorescence spectroscopy, UV-absorption, and size exclusion chromatography (SEC). Fluorescing DOM was preferentially adsorbed relative to UV-absorbing DOM. Humic-like fluorescence (peaks A and C) was selectively adsorbed relative to polyphenol-like fluorescence (peaks T and B) potentially due to size exclusion effects. In the surface waters and size fractions, peak C was preferentially removed relative to peak A, whereas the reverse was found in wastewater effluent, indicating that humic-like fluorescence is associated with different compounds depending on DOM source. Based on specific UV-absorption (SUVA), aromatic DOM was preferentially adsorbed. The fluorescence index (FI), if interpreted as an indicator of aromaticity, indicated the opposite but exhibited a strong relationship with average molecular weight, suggesting that FI might be a better indicator of DOM size than aromaticity. The influence of DOM intermolecular interactions on adsorption were minimal based on SEC analysis. Fluorescence parameters captured the impact of DOM size on the fouling of 2-methylisoborneol and warfarin adsorption and correlated with direct competition and pore blockage indicators.

  17. Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation

    NASA Astrophysics Data System (ADS)

    Kittle, David S.; Vasefi, Fartash; Patil, Chirag G.; Mamelak, Adam; Black, Keith L.; Butte, Pramod V.

    2016-12-01

    The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data. We also show that TR-FS is able to quantify the relative concentration of fluorescence dyes in a mixture by the unmixing of lifetime decays. We show that the TR-FS prototype is able to identify in near-real time the concentrations of dyes in a complex mixture based on previously trained data. As a result, we demonstrate that in complex mixtures of fluorophores, the relative concentration information is encoded in the fluorescence lifetime across multiple spectral bands. We show for the first time the temporal and spectral measurements of a mixture of fluorochromes and the ability to differentiate relative concentrations of each fluorochrome mixture in real time.

  18. 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.

  19. Method for rapid multidiameter single-fiber reflectance and fluorescence spectroscopy through a fiber bundle

    NASA Astrophysics Data System (ADS)

    Amelink, A.; Hoy, C. L.; Gamm, U. A.; Sterenborg, H. J. C. M.; Robinson, D. J.

    2014-03-01

    We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 μm, and a fluorescence spectrum at an effective diameter of 1000 μm. From these spectra, we measure the absolute absorption coefficient, μa, reduced scattering coefficient, μ's, phase function parameter, γ, and intrinsic fluorescence, Qμfa, across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Qμfa.

  20. Method for rapid multidiameter single-fiber reflectance and fluorescence spectroscopy through a fiber bundle.

    PubMed

    Hoy, Christopher L; Gamm, Ute A; Sterenborg, Henricus J C M; Robinson, Dominic J; Amelink, Arjen

    2013-10-01

    We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 μm, and a fluorescence spectrum at an effective diameter of 1000 μm. From these spectra, we measure the absolute absorption coefficient, μ(a), reduced scattering coefficient, μ'(s'), phase function parameter, γ, and intrinsic fluorescence, Qμ(a,x)(f), across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Qμ(a,x)(f).

  1. Classification of Aroma Styles and Geographic Origins of Chinese Liquors Using Chemometrics Based on Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Ma, Y.; Huo, D.-Q.; Qin, H.; Shen, C.-H.; Yang, P.; Hou, C.-J.

    2017-05-01

    The purpose of this paper is to study the feasibility of fluorescence spectroscopy as a reliable method for discrimination of Chinese liquor according to different aroma styles and geographic origins. The 84 Chinese liquors were analyzed by fluorescence spectroscopy and chemometrics. The results showed that Chinese liquors exhibit characteristic fluorescence spectra recorded at special excitation wavelengths that may be considered as fingerprints. Both principal component analysis (PCA) and stepwise linear discriminant analysis (SLDA) were carried out on the emission spectra (330-435 nm) recorded at excitation wavelength 300 nm to classify different aroma styles of Chinese liquors. The first two principal components explained 98.87% of the total variance, and the SLDA classified correctly 100%. Both hierarchical cluster analysis (HCA) and principal component analysis (PCA) were carried out on the emission spectra (325-420 nm) recorded at excitation wavelength 300 nm to identify different geographic origins of Chinese liquors. HCA accurately identified all the samples and the first three PCA explained 98.25% of the total variance. This study indicates that fluorescence spectroscopy coupled with chemometrics offers a promising approach for identifying Chinese liquors according to different flavor types and geographic origins.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

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

    PubMed

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

    2014-12-01

    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 km(2) 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.

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

    PubMed

    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.

  8. 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.

  9. 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.

  10. 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

  11. Center for Fluorescence Spectroscopy: advanced studies of fluorescence dynamics, lifetime imaging, clinical sensing, two-photon excitation, and light quenching

    NASA Astrophysics Data System (ADS)

    Lakowicz, Joseph R.; Malak, Henryk M.; Gryczynski, Ignacy; Szmacinski, Henryk; Kusba, Jozef; Akkaya, Engin; Terpetschnig, Ewald A.; Johnson, Michael L.

    1994-08-01

    The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the art time-resolved fluorescence instrumentation and software for scientists, whose research can be enhanced by such experimental data. The CFS is a national center, supported by the National Center for Research Resources Division of the National Institutes of Health, and in part by the National Science Foundation. Both time-domain (TD) and frequency- domain (FD) measurements (10 MHz to 10 Ghz) are available, with a wide range of excitation and emission wavelengths (UV to NIR). The data can be used to recover distances and site-to-site diffusion in protein, interactions between macromolecules, accessibility of fluorophores to quenchers, and the dynamic properties of proteins, membranes and nucleic acids. Current software provides for analysis of multi-exponential intensity and anisotropy decays, lifetime distribution, distance distributions for independent observation of fluorescence donors and acceptors, transient effects in collisional quenching, phase-modulation spectra and time-resolved emission spectra. Most programs provide for global analysis of multiple data sets obtained under similar experimental conditions. Data can be analyzed on-site by connection with the CFS computers through the internet. During six years of operation we have established scientific collaborations with over 30 academic and industrial groups in the United States. These collaborations have resulted in 63 scientific papers.

  12. 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. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

    PubMed

    Ruan, Qiaoqiao; Chen, Yan; Gratton, Enrico; Glaser, Michael; Mantulin, William W

    2002-12-01

    Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the

  14. Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Widengren, Jerker; Mets, Ülo; Rigler, Rudolf

    1999-12-01

    GFPs are upon excitation influenced by many different photophysical and photochemical processes effective over a very broad time scale. Much effort has been spent to investigate these processes. However, in the microsecond to millisecond time-range many processes still remain to be further characterized. This time-range can be conveniently covered by FCS, and is used here to study the photodynamical behaviour of wild-type (WT) and a F64L S65T mutant (BioST) of GFP. In addition to intersystem crossing to the triplet state, additional photophysical processes are seen, showing identical fluctuations in fluorescence to those found for a reversible photo-induced isomerization process, as well as fluctuations, not influenced by the electronic state of the chromophore unit. In the nanosecond time-range a contribution to the fluorescence correlation function is observed which can be attributed to rotational diffusion, suggesting a convenient way to measure rotational diffusion of proteins expressed with GFP on a microscopic scale.

  15. Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy.

    PubMed Central

    Murakami, A; Nakaura, M; Nakatsuji, Y; Nagahara, S; Tran-Cong, Q; Makino, K

    1991-01-01

    Fluorescein-labeled oligonucleotides as DNA-probes were synthesized and used to monitor hybrid formation, namely to detect DNA or oligonucleotide sequence in solution. The introduction of fluorescein to oligonucleotides was carried out by oxidation of a hydrogen phosphonate linkage with ethylenediamine or hexamethylenediamine as a tether and by a subsequent labeling of the primary amine moiety by FITC. Fluorescence anisotropy, r, was adopted as an index to monitor the behavior of F-probe in solution. An increase in the anisotropy was observed upon an increase in the chain-length of F-probe. When F-Probe formed a hybrid with its complementary oligonucleotide in solution, the r value increased compared to that of F-Probe itself. These observations clearly indicate that measurements of r in solution will readily lead to the monitoring of the presence of a hybrid in solution. Consequently, it is promising to detect a certain nucleic acid sequence in solution using fluorescent-labeled oligonucleotides. PMID:1870966

  16. In-vitro bacterial identification using fluorescence spectroscopy with an optical fiber system

    NASA Astrophysics Data System (ADS)

    Spector, Brian C.; Werkhaven, Jay A.; Smith, Dana; Reinisch, Lou

    2000-05-01

    Acute otitis media (AOM) remains a source of significant morbidity in children. With the emergence of antibiotic resistant strains of bacteria, tympanocentesis has become an important method of bacterial identification in the setting of treatment failures. Previous studies described a prototype system for the non-invasive fluorescence identification of bacteria in vitro. We demonstrate the addition of an optical fiber to allow for the identification of a specimen distant to the spectrofluorometer. Emission spectra from three bacteria, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus were successfully obtained in vitro. This represents a necessary step prior to the study of in vivo identification of bacteria in AOM using fluorescence spectroscopy.

  17. 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.

  18. Picosecond fluorescence spectroscopy of purple membrane in Halobacterium halobium with a photon-counting streak camera

    NASA Astrophysics Data System (ADS)

    Ohtani, Hiroyuki; Ishikawa, Mitsuru; Itoh, Hiroyasu; Takiguchi, Yoshihiro; Urakami, Tsuneyuki; Tsuchiya, Yutaka

    1990-05-01

    Fluorescence lifetimes and spectra of native and deionized purple membranes of Halobacterium halobium at 22°C were measured to be <3 and 12±4 ps, respectively, with a photon-counting streak camera system. The results confirmed that the blue-shifted transient previously found by absorption spectroscopy is attributed to bacteriorhodopsin in the lowest excited-singlet state. Ultraweak fluorescence of the light-adapted purple membrane with 2.5 × 10 -4 quantum yield could be detected even though the excitation pulse energy at 570 nm was reduced to 0.88 pJ (72 μW average power).

  19. 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.

  20. 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.

  1. AZIDE-SPECIFIC LABELLING OF BIOMOLECULES BY STAUDINGER-BERTOZZI LIGATION: PHOSPHINE DERIVATIVES OF FLUORESCENT PROBES SUITABLE FOR SINGLE-MOLECULE FLUORESCENCE SPECTROSCOPY

    PubMed Central

    Chakraborty, Anirban; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

    2010-01-01

    We describe the synthesis of phosphine derivatives of three fluorescent probes that have brightness and photostability suitable for single-molecule fluorescence spectroscopy and microscopy: Alexa488, Cy3B, and Alexa647. In addition, we describe procedures for use of these reagents in azide-specific, bioorthogonal labelling through use of the Staudinger-Bertozzi ligation and procedures for quantitation of labelling specificity and labelling efficiency. The reagents and procedures of this report enable chemoselective, site-selective labelling of azide-containing biomolecules for single-molecule fluorescence spectroscopy and microscopy. PMID:20580957

  2. Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics

    NASA Astrophysics Data System (ADS)

    Davis, Caitlin M.; Reddish, Michael J.; Dyer, R. Brian

    2017-05-01

    Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of < 0.2 mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50 ns to 0.5 ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics.

  3. 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.

  4. Imaging fluorescence correlation spectroscopy: nonuniform IgE distributions on planar membranes.

    PubMed Central

    Huang, Z; Thompson, N L

    1996-01-01

    Fluorescence correlation spectroscopy is useful for detecting and characterizing molecular clusters that are smaller than or approximately equal to optical resolution in size. Here, we report the development of an approach in which the pixel-to-pixel fluorescence fluctuations from a single fluorescence image are spatially autocorrelated. In these measurements, tetramethylrhodamine-labeled, anti-trinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenyl-aminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The antibody-coated membranes were illuminated with the evanescent field from a totally internally reflected laser beam, and the fluorescence arising from the IgE-coated membranes was recorded with a cooled CCD camera. The image was corrected for the elliptical Gaussian shape of the evanescent illumination after background subtraction. The spatial autocorrelation functions of the resulting images generated two useful parameters: the extrapolated initial values, which were related to the average cluster intensity and density; and the correlation distances, which were related to the average cluster size. These parameters varied with the IgE density, and unlabeled polyclonal anti-IgE enhanced the nonuniform IgE distributions. The autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Fluorescent beads were used to demonstrate the principles and the methods. Images FIGURE 3 PMID:8785359

  5. In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS).

    PubMed

    Krupka, Simone S; Wiltschi, Birgit; Reuning, Ute; Hölscher, Kerstin; Hara, Masahiko; Sinner, Eva-Kathrin

    2006-08-15

    Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.

  6. 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.

  7. Complexation between Hg(II) and biofilm extracellular polymeric substances: an application of fluorescence spectroscopy.

    PubMed

    Zhang, Daoyong; Pan, Xiangliang; Mostofa, Khan M G; Chen, Xi; Mu, Guijin; Wu, Fengchang; Liu, Jing; Song, Wenjuan; Yang, Jianying; Liu, Yanli; Fu, Qinglong

    2010-03-15

    The three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy was employed to investigate the interaction of extracellular polymeric substances (EPS) from natural biofilm with Hg(II). The EEM spectra demonstrated that EPS with molecular weight over 14 kDa had two protein-like fluorescence peaks. The fluorescence intensity at both peaks was strongly dependent on the solution pH in the absence and presence of Hg(II), with the maximal fluorescence intensity at neutral pH. Fluorescence of both protein-like peaks was significantly quenched by Hg(II). The values of conditional stability constants (log K(a)=3.28-4.48) derived from modified Stern-Volmer equation are approximate to those for humic substances and dissolved organic matter (DOM), indicating that fluorescent components in EPS have strong binding capacity for Hg(II). Our findings suggest that EPS from biofilm is a class of important organic ligands for complexation with Hg(II) and may significantly affect the chemical forms, mobility, bioavailability and ecotoxicity of heavy metals in the aquatic environment. (c) 2009 Elsevier B.V. All rights reserved.

  8. 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.

  9. Fluorescence quantum yield of Yb3+-doped tellurite glasses determined by thermal lens spectroscopy

    NASA Astrophysics Data System (ADS)

    Lima, S. M.; Souza, A. K. R.; Langaro, A. P.; Silva, J. R.; Costa, F. B.; Moraes, J. C. S.; Figueiredo, M. S.; Santos, F. A.; Baesso, M. L.; Nunes, L. A. O.; Andrade, L. H. C.

    2017-01-01

    In this work, the combination of three different thermal lens spectroscopic methodologies was used to better determine the fluorescence quantum yield and to observe the concentration quenching in Yb3+-doped binary tellurite glasses (in mol%, 80TeO2 - 20Li2O and 80TeO2 - 20WO3). The samples were synthesized by the conventional melt-quenching method and then studied using optical spectroscopy and thermal lens spectroscopy (TLS). These characterizations enabled investigation of the radiative and nonradiative processes involved in the ytterbium doped systems. High fluorescence quantum yield was obtained for low Yb3+ doping (>90%), and in both glasses the Yb3+ presented concentration quenching mainly caused by impurities, host-ion interaction and OH- vibrations. The observations suggested that there is a possibility of doping the glasses with higher Yb concentration (>1.6 × 1021 ions/cm3) with low reduction of the quantum yield.

  10. 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.

  11. Classification and characterization of beef muscles using front-face fluorescence spectroscopy.

    PubMed

    Sahar, Amna; Dufour, Eric

    2015-02-01

    The objective of this study was to evaluate the potential of fluorescence spectroscopy to identify different muscles and to predict some physicochemical and rheological parameters. Samples were taken from three muscles (Semitendinosus, Rectus abdominis and Infraspinatus) of Charolais breed. Dry matter content, fat content, protein content, texture and collagen content were determined. Moreover emission spectra were recorded in the range of 305-400nm, 340-540nm and 410-700nm by fixing the excitation wavelength at 290, 322 and 382nm, respectively. The results obtained were evaluated by partial least square discriminant analysis and partial least square regression. Results of our research work show that front-face fluorescence spectroscopy and chemometrics offer significant potential for the development of rapid and non-destructive methods for the identification and characterization of muscles.

  12. A fast, flexible algorithm for calculating correlations in Fluorescence Correlation Spectroscopy

    SciTech Connect

    Laurence, T; Fore, S; Huser, T

    2005-10-13

    A new algorithm is introduced for computing correlations of photon arrival time data acquired in single-molecule fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS). The correlation is first rewritten as a counting operation on photon pairs. For each photon, the contribution to the correlation function for each subsequent photon is calculated for arbitrary bin spacings of the correlation time lag. By retaining the bin positions in the photon sequence after each photon, the correlation can be performed efficiently. Example correlations for simulations of FCS experiments are shown, with comparable execution speed to the commonly used multiple-tau correlation technique. Also, wide bin spacings are possible that allow for real-time software calculation of correlations even for high count rates ({approx}350 kHz). The flexibility and broad applicability of the algorithm is demonstrated using results from single molecule photon antibunching experiments.

  13. Differential laser-induced perturbation spectroscopy and fluorescence imaging for biological and materials sensing

    NASA Astrophysics Data System (ADS)

    Burton, Dallas Jonathan

    The field of laser-based diagnostics has been a topic of research in various fields, more specifically for applications in environmental studies, military defense technologies, and medicine, among many others. In this dissertation, a novel laser-based optical diagnostic method, differential laser-induced perturbation spectroscopy (DLIPS), has been implemented in a spectroscopy mode and expanded into an imaging mode in combination with fluorescence techniques. The DLIPS method takes advantage of deep ultraviolet (UV) laser perturbation at sub-ablative energy fluences to photochemically cleave bonds and alter fluorescence signal response before and after perturbation. The resulting difference spectrum or differential image adds more information about the target specimen, and can be used in combination with traditional fluorescence techniques for detection of certain materials, characterization of many materials and biological specimen, and diagnosis of various human skin conditions. The differential aspect allows for mitigation of patient or sample variation, and has the potential to develop into a powerful, noninvasive optical sensing tool. The studies in this dissertation encompass efforts to continue the fundamental research on DLIPS including expansion of the method to an imaging mode. Five primary studies have been carried out and presented. These include the use of DLIPS in a spectroscopy mode for analysis of nitrogen-based explosives on various substrates, classification of Caribbean fruit flies versus Caribbean fruit flies that have been irradiated with gamma rays, and diagnosis of human skin cancer lesions. The nitrogen-based explosives and Caribbean fruit flies have been analyzed with the DLIPS scheme using the imaging modality, providing complementary information to the spectroscopic scheme. In each study, a comparison between absolute fluorescence signals and DLIPS responses showed that DLIPS statistically outperformed traditional fluorescence techniques

  14. Studies of multifrequency phase-resolved fluorescence spectroscopy for spectral fingerprinting

    SciTech Connect

    McGown, L.B.

    1989-01-01

    During the first project period, we have explored several different aspects of phase-resolved fluorescence spectroscopy (PRFS) for the fingerprinting of complex samples. It should be noted that our goal is not only fingerprinting'' per se, but also includes the characterization of complex samples with respect to dynamic interactions of luminescent molecules with each other and with sample matrix constituents. Each area of investigation is discussed in the following sections.

  15. Fluorescent atom coincidence spectroscopy of extremely neutron-deficient barium isotopes

    NASA Astrophysics Data System (ADS)

    Wells, S. A.; Evans, D. E.; Griffith, J. A. R.; Eastham, D. A.; Groves, J.; Smith, J. R. H.; Tolfree, D. W. L.; Warner, D. D.; Billowes, J.; Grant, I. S.; Walker, P. M.

    1988-09-01

    Fluorescent atom coincidence spectroscopy (FACS) has been used to measure the nuclear mean square radii and moments of the extremely neutron-deficient isotopes 120-124Ba. At N=65 an abrupt change in nuclear mean square charge radii is observed which can be understood in terms of the occupation of the spin-orbit partner g7/25/2[413] neutron and g9/29/2[404] proton orbitals and the consequent enhancement of the n-p interaction.

  16. Analysis of some Nigerian solid mineral ores by energy-dispersive X-ray fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Obiajunwa, E. I.

    2001-11-01

    Determination of major, minor and trace elements in some Nigerian solid mineral ores by energy-dispersive X-ray fluorescence (EDXRF) spectroscopy is described. Concentration values of major, minor and trace elements for Z>18 are reported. The mineral ores studied include (i) tantalite-coloumbite minerals, (ii) bismuth minerals and (iii) lead minerals. The accuracy and precision of the technique for chemical analysis was assured by analysing the geological standards mica-Fe (biotite) and NBS 278 (obsidian).

  17. Optical phantoms with variable properties and geometries for diffuse and fluorescence optical spectroscopy

    NASA Astrophysics Data System (ADS)

    Leh, Barbara; Siebert, Rainer; Hamzeh, Hussein; Menard, Laurent; Duval, Marie-Alix; Charon, Yves; Abi Haidar, Darine

    2012-10-01

    Growing interest in optical instruments for biomedical applications has increased the use of optically calibrated phantoms. Often associated with tissue modeling, phantoms allow the characterization of optical devices for clinical purposes. Fluorescent gel phantoms have been developed, mimicking optical properties of healthy and tumorous brain tissues. Specific geometries of dedicated molds offer multiple-layer phantoms with variable thicknesses and monolayer phantoms with cylindrical inclusions at various depths and diameters. Organic chromophores are added to allow fluorescence spectroscopy. These phantoms are designed to be used with 405 nm as the excitation wavelength. This wavelength is then adapted to excite large endogenous molecules. The benefits of these phantoms in understanding fluorescence tissue analysis are then demonstrated. In particular, detectability aspects as a function of geometrical and optical parameters are presented and discussed.

  18. Site-specific interaction of thrombin and inhibitors observed by fluorescence correlation spectroscopy.

    PubMed Central

    Klingler, J; Friedrich, T

    1997-01-01

    We report on the application of fluorescence correlation spectroscopy (FCS) to observe the interaction between thrombin and thrombin inhibitors. Two site-specific fluorescent labels were used to distinguish between inhibitors directed to the active site, the exosite, or both binding sites of thrombin. For several well-known inhibitors of thrombin, the binding sites observed by FCS correspond to previous studies. The interaction of the recently discovered thrombin inhibitor ornithodorin from the tick Ornithodorus moubata with thrombin was investigated. It was found that this inhibitor, like hirudin and rhodniin, binds to both the active site and exosite of thrombin simultaneously. This study shows the feasibility of FCS as a sensitive and selective method for observing protein-ligand interactions. As an additional technique, simultaneous labeling with both fluorescent labels was successfully demonstrated. Images FIGURE 1 PMID:9336216

  19. Analysis of RNA Folding and Ribonucleoprotein Assembly by Single-Molecule Fluorescence Spectroscopy

    PubMed Central

    Pljevaljčić, Goran; Robertson-Anderson, Rae; van der Schans, Edwin; Millar, David

    2013-01-01

    Summary To execute their diverse range of biological functions, RNA molecules must fold into specific tertiary structures and/or associate with one or more proteins to form ribonucleoprotein (RNP) complexes. Single-molecule fluorescence spectroscopy is a powerful tool for the study of RNA folding and RNP assembly processes, directly revealing different conformational subpopulations that are hidden in conventional ensemble measurements. Moreover, kinetic processes can be observed without the need to synchronize a population of molecules. In this chapter, we describe the fluorescence spectroscopic methods used for single-molecule measurements of freely diffusing or immobilized RNA molecules or RNA-protein complexes. We also provide practical protocols to prepare the fluorescently labeled RNA and protein molecules required for such studies. Finally, we provide two examples of how these various preparative and spectroscopic methods are employed in the study of RNA folding and RNP assembly processes. PMID:22573447

  20. 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

  1. Investigation of pH-dependent photophysical properties of quantum nanocrystals by fluorescence correlation spectroscopy.

    PubMed

    Oura, Makoto; Yamamoto, Johtaro; Jin, Takashi; Kinjo, Masataka

    2017-01-23

    Quantum dot (QD) and quantum rod (QR) nanocrystals are widely used non-organic nanocrystals. Their strong fluorescence and photostability make them suitable for biomedical imaging applications. However, their pH-dependence and antibunching properties have not been studied much, especially in aqueous conditions. In this report, we used fluorescence correlation spectroscopy (FCS) with high temporal resolution to demonstrate that the fluorescent blinking and antibunching of QDs/QRs can be changed by varying the pH of their solutions. Furthermore, herein, we reported the relationship between the aggregation and antibunching relaxation time of QDs/QRs for the first time. The findings of this study suggest that FCS can be used to discover novel environmental indicators via observing nanosecond and microsecond phenomena.

  2. Applying Fluorescence Correlation Spectroscopy to Investigate Peptide-Induced Membrane Disruption.

    PubMed

    Kristensen, Kasper; Henriksen, Jonas R; Andresen, Thomas L

    2017-01-01

    There is considerable interest in understanding the interactions of antimicrobial peptides with phospholipid membranes. Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that can be used to gain insight into these interactions. Specifically, FCS can be used to quantify leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles, thereby providing a tool for estimating the size of peptide-induced membrane disruptions. If fluorescently labeled lipids are incorporated into the membranes of the vesicles, FCS can also be used to obtain information about whether leakage occurs due to localized membrane perturbations or global membrane destabilization. Here, we outline a detailed step-by-step protocol on how to optimally implement an FCS-based leakage assay. To make the protocol easily accessible to other researchers, it has been supplemented with a number of practical tips and tricks.

  3. Fluorescence spectroscopy and molecular simulation on the interaction of caffeic acid with human serum albumin.

    PubMed

    Xiang, Yuhong; Duan, Lili; Ma, Qiang; Lv, Zizheng; Ruohua, Zhu; Zhang, Zhuoyong

    2016-12-01

    Fluorescence spectroscopy and molecular simulation were explored to study the interaction between caffeic acid and human serum albumin (HSA). The experimental results indicated that the fluorescence quenching mechanism between caffeic acid and HSA is a static quenching, which was proved again by the analysis of fluorescence lifetime by time-correlated single photon counting. The binding process is spontaneous and the hydrophobic force is the main force between caffeic acid and HSA. In addition, the binding of caffeic acid to HSA was modeled by molecular dynamics simulations. The root mean square deviations, root mean square fluctuations, radius of gyration and the number of hydrogen bonds of the molecular dynamic (MD) simulation process were analyzed. Both experimental and modeling results demonstrated strong binding between HSA and caffeic acid. HSA had a slight conformational change when it binds with caffeic acid. The obtained information is useful for HSA drug design. Copyright © 2016 John Wiley & Sons, Ltd.

  4. 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.

  5. 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.

  6. Fluorescence coincidence spectroscopy for single-molecule fluorescence resonance energy-transfer measurements.

    PubMed

    Orte, Angel; Clarke, Richard W; Klenerman, David

    2008-11-15

    Single-molecule fluorescence resonance energy transfer (FRET) is commonly used to probe different conformations and conformational dynamics of single biomolecules. However, the analysis of raw burst traces is not always straightforward. The presence of a "zero peak" and the skewness of peaks at high and low FRET efficiencies in proximity ratio histograms make the accurate evaluation of the histogram a challenging task. This is further compounded by the difficulty associated with siting two fluorophores in optimal range of each other. Here we present an alternative method of analysis, based on handling coincident FRET photon bursts, that addresses these problems. In addition, we demonstrate methods to enhance coincidence levels and thus the accuracy of FRET determination: the use of dual-color excitation, including direct excitation of the acceptor fluorophore; the addition of a remote dye to the biomolecule, not involved in the FRET process; or a combination of the two. We show the advantages of dual excitation by studying several labeled double-stranded DNA samples as FRET models. This method extends the application of single-molecule FRET to more complicated biological systems where only a small fraction of complexes are fully assembled.

  7. Arterial fluorescent components involved in atherosclerotic plaque instability: differentiation by time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Marcu, Laura; Grundfest, Warren S.; Maarek, Jean-Michel I.

    2001-05-01

    As part of our ongoing research on spectroscopic differentiation between unstable and stable atherosclerotic lesions, we report data on time-resolved fluorescence of components of arterial intima matrix (different types of cholesterols, lipoproteins, and collagens). Certain compositional features of atherosclerotic plaque have been associated with plaque instability and rupture. We have characterized and compared the time-resolved spectra of structural proteins (Types I and III collagens, and elastin), lipoproteins (LDL, VLDL), and cholesterols (free cholesterol, and cholesteryl oleate and linoleate) induced with nitrogen laser excitation pulses (337 nm, 3 ns) and detected (360-510 nm range, 5 nm interval) with an MCP-PMT connected to a fast digitizer (2 Gsamples/s). Spectral intensities and time-dependent parameters (lifetime (tau) f; decay constants (tau) 1 (fast-term), (tau) 2 (slow-term), A1 (fast-term amplitude contribution)) derived from the time-resolved spectra were used for samples characterization and comparison. We observed that time- resolved data distinguish collagens from cholesterols and from lipoproteins, and additionally, distinguish different types of cholesterols, different types of lipoproteins and different types of collagen from each other. For instance, the collagen lifetime (390 nm: Type I 5.2 ns, Type III 2.95 ns) was significantly longer than that of cholesterols (free 1.5 ns, linoleate 0.9 ns, oleate 1.0 ns) and that of lipoproteins (LDL 0.95 ns, VLDL 0.85 ns).

  8. Evanescent field in surface plasmon resonance and surface plasmon field-enhanced fluorescence spectroscopies.

    PubMed

    Ekgasit, Sanong; Thammacharoen, Chuchaat; Yu, Fang; Knoll, Wolfgang

    2004-04-15

    The highly sensitive nature of surface plasmon resonance (SPR) spectroscopy and surface plasmon field-enhanced fluorescence spectroscopy (SPFS) are governed by the strong surface plasmon resonance-generated evanescent field at the metal/dielectric interface. The greatest evanescent field amplitude at the interface and the maximum attenuation of the reflectance are observed when a nonabsorbing dielectric is employed. An absorbing dielectric decreases the evanescent field enhancement at the interface. The SPR curve of an absorbing dielectric is characterized by a greater reflectance minimum and a broader curve, as compared to those of the nonabsorbing dielectric with the same refractive index. For a weakly absorbing dielectric, such as nanometer-thick surface-confined fluorophores, the absorption is too small to induce a significant change in the SPR curve. However, the presence of a minute amount of the fluorophore can be detected by the highly sensitive SPFS. The angle with the maximum fluorescence intensity of an SPFS curve is always smaller than the resonance angle of the corresponding SPR curve. This discrepancy is due to the differences of evanescent field distributions and their decay characteristics within the metal film and the dielectric medium. The fluorescence intensity in an SPFS curve can be expressed in terms of the evanescent field amplitude. Excellent correlations between the experimentally measured fluorescence intensities and the evanescent field amplitudes are observed.

  9. 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.

  10. Investigation of the interaction of pepsin with ionic liquids by using fluorescence spectroscopy.

    PubMed

    Fan, Yunchang; Zhang, Sheli; Wang, Qiang; Li, Junhai; Fan, Haotian; Shan, Dongkai

    2013-06-01

    The molecular mechanism of the interaction between pepsin and two typical ionic liquids (ILs), 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and 1-octyl-3-methylimidazolium chloride ([C8mim]Cl), was investigated with fluorescence spectroscopy, ultraviolet absorption, and circular dichroism spectroscopy at a pH value of 1.6. The results suggest that ILs could quench the intrinsic fluorescence of pepsin, probably via a dynamic quenching mechanism. The fluorescence quenching constants were determined by employing the classic Stern-Volmer equation. The constant values are very small, indicating that only a very weak interaction between ILs and pepsin exists. The Gibbs free-energy change, enthalpy change (ΔH), and entropy change (ΔS) during the interaction of pepsin and ILs were estimated. Positive values of ΔH and ΔS indicate that the interaction between ILs and pepsin is mainly driven by hydrophobic interaction. Synchronous and three-dimensional fluorescence spectra demonstrate that the addition of ILs (0-0.20 mol L(-1) for each IL) does not bring apparent changes to the microenvironments of tyrosine and tryptophan residues. Activity experiments show that the activity of pepsin is concentration dependent; higher concentrations of ILs (>0.22 mol L(-1) for [C8mim]Cl and >0.30 mol L(-1) for [C4mim]Cl) cause the remarkable reduction of enzyme activity. The presence of ILs also does not improve the thermal stability of pepsin.

  11. 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.

  12. Effects of nerolidol and limonene on stratum corneum membranes: A probe EPR and fluorescence spectroscopy study.

    PubMed

    Mendanha, Sebastião Antonio; Marquezin, Cássia Alessandra; Ito, Amando Siuiti; Alonso, Antonio

    2017-10-30

    The sesquiterpene nerolidol and the monoterpene limonene are potent skin-permeation enhancers that have also been shown to have antitumor, antibacterial, antifungal and antiparasitic activities. Because terpenes are membrane-active compounds, we used electron paramagnetic resonance (EPR) spectroscopy of three membrane spin labels combined with the fluorescence spectroscopy of three lipid probes to study the interactions of these terpenes with stratum corneum (SC) intercellular membranes. An experimental apparatus was developed to assess the lipid fluidity of hydrated SC membranes via the fluorescence anisotropy of extrinsic membrane probes. Both EPR and fluorescence probes indicated that the intercellular membranes of neonatal SC rats undergo a main phase transition at approximately 50°C. Taken together, the results indicated that treatment with 1% nerolidol (v/v) caused large fluidity increases in the more ordered phases of SC membranes and that these effects gradually decreased with increasing temperature. Additionally, compared with (+)-limonene, nerolidol was better able to change the SC membrane dynamics. EPR and fluorescence data suggest that these terpenes act as spacers in lipid packaging and create increased lipid disorder in the more ordered regions and phases of SC membranes, notably leading to a population of probes with less restricted motion. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. [Analysis of Three Polycyclic Aromatic Hydrocarbons in Solution Based on Two-Dimensional Fluorescence Correlation Spectroscopy].

    PubMed

    Zhou, Chang-hong; Zhao, Mei-rong; Yang, Ren-jie; Zhu, Wen-bi; Dong, Gui-mei

    2016-02-01

    Polycyclic aromatic hydrocarbons (PAHs) are listed as the priority pollutants. It is difficult to resolve effectively the peaks of PAHs by conventional one-dimensional fluorescence spectroscopy due to its low content and the overlapping fluorescence three mixed ystems and a total of 27 samples, are to be prepared with different concentrations of three PAHs. Concentrations of three PAHS are monotonically increasing or decreasing in each mixed system. Then the 2D fluorescence correlation spectrum of each mixed systems will be calculated under the perturbation of the concentration of anthracene, phenanthrene and pyrene in solution. There are seven strong autopeaks at 425, 402, 381, 373, 365, 393 and 347 nm in synchronous 2D correlation spectrum. The fluorescence peak of phenanthrene at 347 nm is uncovered in three mixed systems, so the band at 347 nm is to be used as clues for further assignment. According to positive or negative cross peaks at 347 nm in synchronous 2D correlation spectrum, we can know that the peaks at 402, 381, 425 and 452 nm are assigned to anthracene, the peaks at 373 and 393 nm are assigned to pyrene, and the peaks at 365, 356 and 347 nm are assigned to phenanthrene. The fluorescence peak of phenanthrene at 385 nm is shown in asynchronous 2D correlation spectrum; it means the spectral resolution of asynchronous spectrum is better than the synchronous spectrum. The results are that it is feasible to analyze serious overlapping multi-component PAHs using two-dimensional fluorescence correlation spectroscopy, which can be extended to the detection of other pollutants in the air.

  14. 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

  15. Analyzing pH-induced changes in a myofibril model system with vibrational and fluorescence spectroscopy.

    PubMed

    Andersen, Petter Vejle; Veiseth-Kent, Eva; Wold, Jens Petter

    2017-03-01

    The decline of pH and ultimate pH in meat postmortem greatly influences meat quality (e.g. water holding capacity). Four spectroscopic techniques, Raman, Fourier transform infrared (FT-IR), near infrared (NIR) and fluorescence spectroscopy, were used to study protein and amino acid modifications to determine pH-related changes in pork myofibril extracts at three different pH-levels, 5.3, 5.8 and 6.3. Protonation of side-chain carboxylic acids of aspartic and glutamic acid and changes in secondary structure, mainly the amide I-III peaks, were the most important features identified by Raman and FT-IR spectroscopy linked to changes in pH. Fluorescence spectroscopy identified tryptophan interaction with the molecular environment as the most important contributor to changes in the spectra. NIR spectroscopy gave no significant contributions to interpreting protein structure related to pH. Results from our study are useful for interpreting spectroscopic data from meat where pH is an important variable.

  16. A high-resolution large-acceptance analyzer for X-ray fluorescence and Raman spectroscopy

    SciTech Connect

    Bergmann, Uwe; Cramer, Stephen P.

    2001-08-02

    A newly designed multi-crystal X-ray spectrometer and its applications in the fields of X-ray fluorescence and X-ray Raman spectroscopy are described. The instrument is based on 8 spherically curved Si crystals, each with a 3.5 inch diameter form bent to a radius of 86 cm. The crystals are individually aligned in the Rowland geometry capturing a total solid angle of 0.07 sr. The array is arranged in a way that energy scans can be performed by moving the whole instrument, rather than scanning each crystal by itself. At angles close to back scattering the energy resolution is between 0.3 and 1 eV depending on the beam dimensions at the sample. The instrument is mainly designed for X-ray absorption and fluorescence spectroscopy of transition metals in dilute systems such as metalloproteins. First results of the Mn K{beta} (3p -> 1s) emission in photosystem II are shown. An independent application of the instrument is the technique of X-ray Raman spectroscopy which can address problems similar to those in traditional soft X-ray absorption spectroscopies, and initial results are presented.

  17. Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Gilbert, Leona; Michel, Patrik; White, Daniel; Vuento, Matti; Oker-Blom, Christian

    2005-12-01

    Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment

  18. 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.

  19. On-chip integrated lensless fluorescence microscopy/spectroscopy module for cell-based sensors

    NASA Astrophysics Data System (ADS)

    Li, Wei; Knoll, Thorsten; Sossalla, Adam; Bueth, Heiko; Thielecke, Hagen

    2011-03-01

    The integration of a fluorescence microscopy/spectroscopy module in cell-based lab-on-a-chip systems is of high interest for applications in cell-based diagnostics and substance evaluation in situ. We present an on-chip integrated lensless fluorescence imaging module applying the principle of contact/proximate optical lithography. The pixel resolution is comparable with a 4 x objective microscope. The module can be used for morphology and fluorescence imaging of mammalian cells (15 - 20 μm) as well as for testing the concentration of a fluorescent substance. The biological samples or solutions are sustained in disposable sterilized microfluidic chips with 1 μm thick silicon nitride (Si3N4) membranes. These chips are assembled on the surface of a 5 megapixel colored CMOS image sensor array with 1.75 μm pixel size, which is coated with an additional interference filter. Each culturing chip consists of a MEMS cavity chip and a PDMS microfluidic interface. The surface of the CMOS image sensor is smoothened using SU-8 photoresist spin-coating for a commercial grade interference filter (optical density >= 5) coating by Plasma-Ion Assisted Deposition thereafter. The function is demonstrated by primary imaging results of the non-/fluorescent mammalian cells/microspheres as well as by differentiating different concentrations of FITC solutions.

  20. Assessment of drinking water quality at the tap using fluorescence spectroscopy.

    PubMed

    Heibati, Masoumeh; Stedmon, Colin A; Stenroth, Karolina; Rauch, Sebastien; Toljander, Jonas; Säve-Söderbergh, Melle; Murphy, Kathleen R

    2017-08-08

    Treated drinking water may become contaminated while travelling in the distribution system on the way to consumers. Elevated dissolved organic matter (DOM) at the tap relative to the water leaving the treatment plant is a potential indicator of contamination, and can be measured sensitively, inexpensively and potentially on-line via fluorescence and absorbance spectroscopy. Detecting elevated DOM requires potential contamination events to be distinguished from natural fluctuations in the system, but how much natural variation to expect in a stable distribution system is unknown. In this study, relationships between DOM optical properties, microbial indicator organisms and trace elements were investigated for households connected to a biologically-stable drinking water distribution system. Across the network, humic-like fluorescence intensities showed limited variation (RSD = 3.5-4.4%), with half of measured variation explained by interactions with copper. After accounting for quenching by copper, fluorescence provided a very stable background signal (RSD < 2.2%) against which a ∼2% infiltration of soil water would be detectable. Smaller infiltrations would be detectable in the case of contamination by sewage with a strong tryptophan-like fluorescence signal. These findings indicate that DOM fluorescence is a sensitive indicator of water quality changes in drinking water networks, as long as potential interferents are taken into account. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Effect of dielectric spacer thickness on signal intensity of surface plasmon field-enhanced fluorescence spectroscopy.

    PubMed

    Murakami, Takashi; Arima, Yusuke; Toda, Mitsuaki; Takiguchi, Hiromi; Iwata, Hiroo

    2012-02-15

    Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) combines enhanced field platform and fluorescence detection. Its advantages are the strong intensity of the electromagnetic field and the high signal/noise (S/N) ratio due to the localized evanescent field at the water/metal interface. However, the energy transfer from the fluorophore to the metal surface diminishes the fluorescence intensity, and this reduces the sensitivity. In this study, we tested whether polystyrene (PSt) could act as a dielectric layer to suppress the energy transfer from the fluorophore to the metal surface. We hypothesized that this would improve the sensitivity of SPFS-based immunoassays. We used α-fetoprotein (AFP) as a model tumor biomarker in the sandwich-type immunoassay. We determined the relationship between fluorescent signal intensity and PSt layer thickness and compared this to theoretical predictions. We found that the fluorescence signal increased by optimally controlling the thickness of the PSt layer. Our results indicated that the SPFS-based immunoassay is a promising clinical diagnostic tool for quantitatively determining the concentrations of low-level biomarkers in blood samples.

  2. 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.

  3. Solution conformation of 2-aminopurine dinucleotide determined by ultraviolet two-dimensional fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Widom, Julia R.; Johnson, Neil P.; von Hippel, Peter H.; Marcus, Andrew H.

    2013-02-01

    We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analogue 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 ± 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Fluorescence imaging and spectroscopy of ALA-induced protoporphyrin IX preferentially accumulated in tumor tissue

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert G.; Baumgartner, Reinhold; Beyer, Wolfgang; Knuechel, Ruth; Koerner, T. O.; Kriegmair, M.; Rick, Kai; Steinbach, Pia; Hofstetter, Alfons G.

    1995-12-01

    In a clinical pilot study performed on 104 patients suffering from bladder cancer it could be shown that intravesical instillation of a solution of 5-aminolevulinic acid (5-ALA) induces a tumorselective accumulation of Protoporphyrin IX (PPIX). Malignant lesions could be detected with a sensitivity of 97% and a specificity of 67%. The Kr+-laser as excitation light source could successfully be replaced by a filtered short arc Xe-lamp. Its emission wavelength band (375 nm - 440 nm) leads to an efficiency of 58% for PPIX- excitation compared to the laser. Two-hundred-sixty mW of output power at the distal end of a slightly modified cystoscope could be obtained. This is sufficient for recording fluorescence images with a target integrating color CCD-camera. Red fluorescence and blue remitted light are displayed simultaneously. Standard white light observation is possible with the same instrumentation. Pharmacokinetic measurements were performed on 18 patients after different routes of 5-ALA application (oral, inhalation and intravesical instillation). PPIX-fluorescence measurements were made on the skin and on the blood plasma. Pharmacokinetic of 5-ALA could be performed on blood plasma. Endoscopical florescence spectroscopy showed the high fluorescence contrast between tumor and normal tissue with a mean value of 10.7. Forthcoming clinical multicenter studies require an objective measure of the fluorescence intensity. Monte Carlo computer simulations showed that artifacts due to observation geometry and varying absorption can largely be reduced by ratioing fluorescence (red channel of camera) to remission (blue channel). Real time image ratioing provides false color images with a reliable fluorescence information.

  10. Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy.

    PubMed Central

    Chirico, G; Bettati, S; Mozzarelli, A; Chen, Y; Müller, J D; Gratton, E

    2001-01-01

    O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity. At least two components were detected, with lifetimes of approximately 2.5 and 0.5 ns. The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state. PMID:11259310

  11. 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.

  12. 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.

  13. 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.

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

    PubMed

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

    2014-03-01

    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 lifetime

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

    PubMed Central

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

    2014-01-01

    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

  16. 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

  17. 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.

  18. A reusable sensor for the label-free detection of specific oligonucleotides by surface plasmon fluorescence spectroscopy.

    PubMed

    Nöll, Gilbert; Su, Qiang; Heidel, Björn; Yu, Yaming

    2014-01-01

    The development of a reusable molecular beacon (MB)-based sensor for the label-free detection of specific oligonucleotides using surface plasmon fluorescence spectroscopy (SPFS) as the readout method is described. The MBs are chemisorbed at planar gold surfaces serving as fluorescence quenching units. Target oligonucleotides of 24 bases can be detected within a few minutes at high single-mismatch discrimination rates.

  19. 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.

  20. Fluorescence spectroscopy of teeth and bones of rats to assess demineralization: In vitro, in vivo and ex vivo studies.

    PubMed

    Paolillo, Fernanda Rossi; Romano, Renan Arnon; de Matos, Luciana; Rodrigues, Phamilla Gracielli Sousa; Panhóca, Vitor Hugo; Martin, Airton Abrahão; Soares, Luis Eduardo Silva; de Castro Neto, Jarbas Caiado; Bagnato, Vanderlei Salvador

    2016-12-01

    This study investigated the effects of demineralization on teeth and bones evaluated by fluorescence spectroscopy and micro energy-dispersive X-ray fluorescence spectrometry (μ-EDXRF) in rats. For in vitro study, 20 teeth of Wistar rats were removed and decalcified to evaluate fluorescence. For in vivo study, 10 female Wistar rats aged 6months were randomized into 2 groups: Control Group (C): non-ovariectomized rats; Ovariectomy Group (OV): ovariectomized rats to induce osteoporosis. The fluorescence spectroscopy of the teeth was performed for long-term (until 180days). For ex vivo study, the tooth and femur bone of the Wistar rats were removed at 180days to perform fluorescence spectroscopy using excitation laser at 408 and 532nm and μ-EDXRF for calcium (Ca) and phosphorus (P) analysis. There were no intergroup differences in fluorescence spectra with laser at 408nm (p≥0.05), but there were changes in the fluorescence spectra using laser at 532nm which led to both the wavelength shift and changes in the band area (p<0.05). The concentrations of P and Ca for the dentine and cortical bone, respectively, were significantly reduced in OV (p<0.05). Demineralization leading to loss of tissue quality may be assessed by fluorescence spectroscopy using 532nm laser. These findings corroborate those obtained by μ-EDXRF.

  1. 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.

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

    PubMed

    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.

  3. 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

  4. Accounting for misalignments and thermal fluctuations in fluorescence correlation spectroscopy experiments on membranes.

    PubMed

    Sanguigno, Luigi; Cosenza, Chiara; Causa, Filippo; Netti, Paolo Antonio

    2013-03-21

    Several authors have exploited the ability of the fluorescence correlation spectroscopy to probe motion at the molecular level. In a couple of decades, all their efforts have allowed the application of this technique even to the diffusion measurement of cellular components. Nowadays, the fluorescence correlation spectroscopy is considered a standard tool to measure diffusion in cells both in vivo and in vitro. Unfortunately, while the interpretation and the set-up have been consolidated for 3D diffusion measurements (i.e. diffusion in an aqueous solution), the experiments carried out on flat elements, such as membranes, show unusually high relative errors. Furthermore, long tail correlations are generally detected and ascribed to diffusion anomalies. The 2D fluorescence correlation measurements have been interpreted under certain hypotheses, whereby the membrane is assumed to be perfectly flat, motionless and aligned with the optical axes. Here, we investigated the robustness of these hypotheses, trying to understand, in an elementary but not trivial way, how misalignments and thermal fluctuations affect the temporal correlation of the intensity fluctuation collected during measurements on membranes.

  5. Experimental and clinical evaluation of a spectroscopy system for fluorescence-guided excimer laser angioplasty

    NASA Astrophysics Data System (ADS)

    Morguet, Andreas J.; Gabriel, Ruth E.; Buchwald, Arnd B.

    1996-12-01

    This study evaluated a single-laser approach for simultaneous ablation and fluorescence excitation for spectroscopic guidance of laser angioplasty. A spectroscopy system was developed and coupled to a clinical XeCl excimer laser. Ablation of 162 human aortic samples in saline and blood with 45 mJ/mm2 per pulse yielded 676 fluorescence spectra validated histologically. Five types of spectra could be differentiated: atheroma, fibrous plaque, calcified lesion in saline, normal media and calcified lesion in blood. Discriminant analysis prospectively classified 576 validation spectra with a sensitivity between 83.5 and 100 percent and a specificity between 96.8 and 100 percent. Subsequently, the equipment was used in 16 patients for angioplasty of 18 coronary stenoses applying 500 to 1725 pulses with 45 to 60 mJ/mm2 under saline flushing. A total of 783 spectra were recorded and validated by intracoronary ultrasound. Except for the media spectrum, all types of spectra were observed in vivo, too. The predominant sonographic category also prevailed in spectroscopy. In conclusion, using an excimer laser for angioplasty allows combining ablation and fluorescence excitation without a diagnostic laser. Principal types of atherosclerotic lesions and the media can be differentiated spectroscopically with this approach.

  6. Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions.

    PubMed

    Stengel, Gudrun; Knoll, Wolfgang

    2005-04-22

    Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) utilizes the evanescent electromagnetic field of a surface plasmon to excite chromophors in close proximity to the surface. While conventional surface plasmon resonance spectroscopy allows the observation of surface reactions by means of refractive index changes, SPFS additionally provides a channel for the read-out of fluorescence changes. Thus, the detection limit for low mass compounds, whose adsorption is only accompanied by small refractive index changes, can be substantially improved by fluorescent labeling. In this study, we present the first example that utilizes SPFS to follow the dynamics of an enzymatic reaction. The elongation of surface-tethered DNA has been observed by the incorporation of Cy5-labeled nucleotides into the nascent strand by the action of DNA polymerase I (Klenow fragment). The technique offers a rapid way to determine the binding constant and the catalytic activity of a DNA processing enzyme, here exemplified by the Klenow fragment. Furthermore, the effect of mispaired bases in the primer/template duplex and the influence of different label densities have been studied. The resulting sensitivity for nucleotide incorporation, being in the femtomolar regime, combined with the specificity of the enzyme for fully complementary DNA duplexes suggest the application of this assay as a powerful tool for DNA detection.

  7. Biosensor platform based on surface plasmon-enhanced fluorescence spectroscopy and responsive hydrogel binding matrix

    NASA Astrophysics Data System (ADS)

    Huang, Chun-Jen; Jonas, Ulrich; Dostálek, Jakub; Knoll, Wolfgang

    2009-05-01

    We report a novel biosensor platform based on surface plasmon-enhanced fluorescence spectroscopy (SPFS) and a responsive N-isopropylacrylamide (NIPAAm) hydrogel binding matrix. This binding matrix highly swells in aqueous environment and it can be modified with receptor biomolecules by using active ester coupling chemistry. After the binding of target analyte molecules contained in a sample by receptor biomolecules immobilized in the hydrogel matrix, the captured analyte molecules can be compacted on the surface through the collapse of the gel triggered by an external stimulus. A thin hydrogel NIPAAm-based film was attached to a gold sensor surface and modified with mouse IgG receptor molecules. The affinity binding of antibodies against mouse IgG that were labeled with Alexa Fluor chromophores was observed by surface plasmon-enhanced fluorescence spectroscopy. We demonstrate that the collapse of the hydrogel matrix results in the enhancement of measured fluorescence intensity owing to the increase in the concentration of captured molecules within the evanescent field of surface plasmons.

  8. 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.

  9. Impact of oxygen chemistry on the emission and fluorescence spectroscopy of laser ablation plumes

    NASA Astrophysics Data System (ADS)

    Hartig, K. C.; Brumfield, B. E.; Phillips, M. C.; Harilal, S. S.

    2017-09-01

    Oxygen present in the ambient gas medium may affect both laser-induced breakdown spectroscopy (LIBS) and laser-induced fluorescence (LIF) emission through a reduction of emission intensity and persistence. In this study, an evaluation is made on the role of oxygen in the ambient environment under atmospheric pressure conditions in LIBS and laser ablation (LA)-LIF emission. To generate plasmas, 1064 nm, 10 ns pulses were focused on an aluminum alloy sample. LIF was performed by frequency scanning a CW laser over the 396.15 nm (3s24s 2S1/2 → 3s23p 2P°3/2) Al I transition. Time-resolved emission and fluorescence signals were recorded to evaluate the variation in emission intensity caused by the presence of oxygen. The oxygen partial pressure (po) in the atmospheric pressure environment using N2 as the makeup gas was varied from 0 to 400 Torr O2. 2D-fluorescence spectroscopy images were obtained for various oxygen concentrations for simultaneous evaluation of the emission and excitation spectral features. Results showed that the presence of oxygen in the ambient environment reduces the persistence of the LIBS and LIF emission through an oxidation process that depletes the density of atomic species within the resulting laser-produced plasma (LPP) plume.

  10. Structural changes of soy proteins at the oil-water interface studied by fluorescence spectroscopy.

    PubMed

    Keerati-u-rai, Maneephan; Miriani, Matteo; Iametti, Stefania; Bonomi, Francesco; Corredig, Milena

    2012-05-01

    Fluorescence spectroscopy was used to acquire information on the structural changes of proteins at the oil/water interface in emulsions prepared by using soy protein isolate, glycinin, and β-conglycinin rich fractions. Spectral changes occurring from differences in the exposure of tryptophan residues to the solvent were evaluated with respect to spectra of native, urea-denatured, and heat treated proteins. The fluorescence emission maxima of the emulsions showed a red shift with respect to those of native proteins, indicating that the tryptophan residues moved toward a more hydrophilic environment after adsorption at the interface. The heat-induced irreversible transitions were investigated using microcalorimetry. Fluorescence spectroscopy studies indicated that while the protein in solution underwent irreversible structural changes with heating at 75 and 95°C for 15 min, the interface-adsorbed proteins showed very little temperature-induced rearrangements. The smallest structural changes were observed in soy protein isolate, probably because of the higher extent of protein-protein interactions in this material, as compared to the β-conglycinin and to the glycinin fractions. This work brings new evidence of structural changes of soy proteins upon adsorption at the oil water interface, and provides some insights on the possible protein exchange events that may occur between adsorbed and unadsorbed proteins in the presence of oil droplets. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Laser Induced Fluorescence Spectroscopy of Neutral and Ionized Polycyclic Aromatic Hydrocarbons in the Cosmic Simulation Chamber

    NASA Technical Reports Server (NTRS)

    Bejaoui, Salma; Salama, Farid; Contreras, Cesar; Sciamma O'Brien, Ella; Foing, Bernard; Pascale, Ehrenfreund

    2015-01-01

    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. Absorption spectra of several cold, isolated gas-phase PAHs have also been measured under experimental conditions that mimic the interstellar conditions. 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 and are performed with the Pulsed Discharge Nozzle (PDN) of the COSmIC laboratory facility at NASA Ames laboratory. The PDN generates a 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 examined.

  12. 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.

  13. 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.

  14. Multimodal fiber probe spectroscopy for tissue diagnostics applications: a combined Raman-fluorescence approach

    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.

    2014-03-01

    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 diagnosing melanocytic lesions in a good agreement with common routine histology. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. Further investigations were performed on colon and brain tissue samples in order to have a benchmark for diagnosing a broader range of tissue lesions and malignancies. 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.

  15. Study of the interaction between N-confused porphyrin and bovine serum albumin by fluorescence spectroscopy.

    PubMed

    Yu, Xianyong; Liu, Ronghua; Yi, Rongqiong; Yang, Fengxian; Huang, Haowen; Chen, Jian; Ji, Danhong; Yang, Ying; Li, Xiaofang; Yi, Pinggui

    2011-04-01

    The fluorescence and ultraviolet spectroscopy were explored to study the interaction between N-confused porphyrins (NCP) and bovine serum albumin (BSA) under imitated physiological condition. The experimental results indicated that the fluorescence quenching mechanism between BSA and NCP was static quenching procedure at low NCP concentration at 293 and 305 K or a combined quenching (static and dynamic) procedure at higher NCP concentration at 305 K. The binding constants, binding sites and the corresponding thermodynamic parameters ΔH, ΔS, and ΔG were calculated at different temperatures. The comparison of binding potency of the three NCP to BSA showed that the substituting groups in benzene ring could enhance the binding affinity. From the thermodynamic parameters, we concluded that the action force was mainly hydrophobic interaction. The binding distances between NCP and BSA were calculated using Förster non-radiation energy transfer theory. In addition, the effect of NCP on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Authentication of the botanical origin of honey by front-face fluorescence spectroscopy. A preliminary study.

    PubMed

    Ruoff, Kaspar; Karoui, Romdhane; Dufour, Eric; Luginbühl, Werner; Bosset, Jacques-Olivier; Bogdanov, Stefan; Amado, Renato

    2005-03-09

    The potential of front-face fluorescence spectroscopy for the authentication of unifloral and polyfloral honey types (n = 57 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis was evaluated. Emission spectra were recorded between 280 and 480 nm (excit: 250 nm), 305 and 500 nm (excit: 290 nm), and 380 and 600 nm (excit: 373 nm) directly on honey samples. In addition, excitation spectra (290-440 nm) were recorded with the emission measured at 450 nm. A total of four different spectral data sets were considered for data analysis. After normalization of the spectra, chemometric evaluation of the spectral data was carried out using principal component analysis (PCA) and linear discriminant analysis (LDA). The rate of correct classification ranged from 36% to 100% by using single spectral data sets (250, 290, 373, 450 nm) and from 73% to 100% by combining these four data sets. For alpine polyfloral honey and the unifloral varieties investigated (acacia, alpine rose, honeydew, chestnut, and rape), correct classification ranged from 96% to 100%. This preliminary study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey. It is nondestructive, rapid, easy to use, and inexpensive. The use of additional excitation wavelengths between 320 and 440 nm could increase the correct classification of the less characteristic fluorescent varieties.

  17. Fluorescence spectroscopy for the detection of tongue carcinoma--validation in an animal model.

    PubMed

    Kurachi, Cristina; Fontana, Carla R; Rosa, Luiz E B; Bagnato, Vanderlei S

    2008-01-01

    The efficacy of fluorescence spectroscopy to detect squamous cell carcinoma is evaluated in an animal model following laser excitation at 442 and 532 nm. Lesions are chemically induced with a topical DMBA application at the left lateral tongue of Golden Syrian hamsters. The animals are investigated every 2 weeks after the 4th week of induction until a total of 26 weeks. The right lateral tongue of each animal is considered as a control site (normal contralateral tissue) and the induced lesions are analyzed as a set of points covering the entire clinically detectable area. Based on fluorescence spectral differences, four indices are determined to discriminate normal and carcinoma tissues, based on intraspectral analysis. The spectral data are also analyzed using a multivariate data analysis and the results are compared with histology as the diagnostic gold standard. The best result achieved is for blue excitation using the KNN (K-nearest neighbor, a interspectral analysis) algorithm with a sensitivity of 95.7% and a specificity of 91.6%. These high indices indicate that fluorescence spectroscopy may constitute a fast noninvasive auxiliary tool for diagnostic of cancer within the oral cavity.

  18. A Linear Ion Trap with an Expanded Inscribed Diameter to Improve Optical Access for Fluorescence Spectroscopy.

    PubMed

    Rajagopal, Vaishnavi; Stokes, Chris; Ferzoco, Alessandra

    2017-08-18

    We report a custom-geometry linear ion trap designed for fluorescence spectroscopy of gas-phase ions at ambient to cryogenic temperatures. Laser-induced fluorescence from trapped ions is collected from between the trapping rods, orthogonal to the excitation laser that runs along the axis of the linear ion trap. To increase optical access to the ion cloud, the diameter of the round trapping rods is 80% of the inscribed diameter, rather than the roughly 110% used to approximate purely quadrupolar electric fields. To encompass as much of the ion cloud as possible, the first collection optic has a 25.4 mm diameter and a numerical aperture of 0.6. The choice of geometry and collection optics yields 10(7) detected photons/s from trapped rhodamine 6G ions. The trap is coupled to a closed-cycle helium refrigerator, which in combination with two 50 Ohm heaters enables temperature control to below 25 K on the rod electrodes. The purpose of the instrument is to broaden the applicability of fluorescence spectroscopy of gas-phase ions to cases where photon emission is a minority relaxation pathway. Such studies are important to understand how the microenvironment of a chromophore influences excited state charge transfer processes. Graphical Abstract ᅟ.

  19. A Linear Ion Trap with an Expanded Inscribed Diameter to Improve Optical Access for Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Rajagopal, Vaishnavi; Stokes, Chris; Ferzoco, Alessandra

    2017-08-01

    We report a custom-geometry linear ion trap designed for fluorescence spectroscopy of gas-phase ions at ambient to cryogenic temperatures. Laser-induced fluorescence from trapped ions is collected from between the trapping rods, orthogonal to the excitation laser that runs along the axis of the linear ion trap. To increase optical access to the ion cloud, the diameter of the round trapping rods is 80% of the inscribed diameter, rather than the roughly 110% used to approximate purely quadrupolar electric fields. To encompass as much of the ion cloud as possible, the first collection optic has a 25.4 mm diameter and a numerical aperture of 0.6. The choice of geometry and collection optics yields 107 detected photons/s from trapped rhodamine 6G ions. The trap is coupled to a closed-cycle helium refrigerator, which in combination with two 50 Ohm heaters enables temperature control to below 25 K on the rod electrodes. The purpose of the instrument is to broaden the applicability of fluorescence spectroscopy of gas-phase ions to cases where photon emission is a minority relaxation pathway. Such studies are important to understand how the microenvironment of a chromophore influences excited state charge transfer processes. [Figure not available: see fulltext.

  20. Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy.

    PubMed

    Korlann, You; Dertinger, Thomas; Michalet, Xavier; Weiss, Shimon; Enderlein, Jörg

    2008-09-15

    We present a new technique, polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS), based on the recently intro-duced dual-focus fluorescence correlation spectroscopy (2fFCS) to measure the absolute value of diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations. Analogous to 2fFCS, the new technique is robust against optical saturation in yielding correct values of the diffusion coefficient. This is in stark contrast to conventional FCS where optical saturation leads to an apparent decrease in the determined diffusion coefficient with increasing excitation power. However, compared to 2fFCS, the new technique is simpler to implement into a conventional confocal microscope setup and is compatible with cw-excitation, only needing as add-ons an electro-optical modulator and a differential interference contrast prism. With pmFCS, the measured diffusion coefficient (D) for Atto655 maleimide in water at 25?C is determined to be equal to (4.09 +/- 0.07) x 10(-6)cm(2)/s, in good agreement with the value of 4.04 x 10-6cm2/s as measured by 2fFCS.

  1. High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar

    NASA Astrophysics Data System (ADS)

    Koo, Juny; Czeslik, Claus

    2012-08-01

    Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 108 Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.

  2. Identification of oil contaminants on polymer coated beverage cans using fluorescence spectroscopy.

    PubMed

    Malecha, Michael; Bessant, Conrad; Saini, Selwayan

    2003-08-01

    In a recent publication we introduced a novel method for detection of impurities on beverage can surfaces using Raman spectroscopy. While investigating the technique, limitations emerged due to the low sensitivity of Raman scatter. This is a particular problem with the largest contributor of impurities in beverage cans: lubricants employed in the manufacturing process. This paper presents an alternative approach, using the more sensitive technique of fluorescence spectroscopy to tackle the same problem. Measurements using fluorescence spectroscopy were conducted indirectly in a cuvette as well as directly on a can surface with the aid of fiber optics. The chemometrics methods of linear discriminant analysis (LDA) and principal components analysis (PCA) were used to classify acquired spectra as belonging to one of the 16 investigated lubricants. Fiberoptic scans revealed that a successful detection and recognition of lubricant is possible down to a volume of 0.5 microL deposited on the can surface. Contaminant detection was possible at even lower levels down to 0.01 microL, but reliable identification proved difficult at such low volumes. Indirect measurements of lubricants dissolved in cyclohexane yielded the lowest detection limits between 1-0.1 ppb.

  3. On the photophysicochemical properties of selected fluoroquinolones: solvatochromic and fluorescence spectroscopy study.

    PubMed

    Bani-Yaseen, Abdulilah Dawoud; Hammad, Fawaz; Ghanem, Bader S; Mohammad, Elham G

    2013-01-01

    The photophysicochemical properties of selected fluoroquinolones in different solvents of various physical properties, including polarity and hydrogen bonding ability, were investigated using steady state fluorescence spectroscopy. The solvent-dependant fluorescence emission spectra of selected fluoroquinolones, namely ciprofloxacin (CIPR) and enrofloxacin (ENRO), were employed to gain insights concerning its photophysicochemical properties of interests. Interestingly, fluorescence spectra of the selected drugs exhibited structured emission spectra in nonpolar solvents such as hexane, whereas unstructured spectra were observed in more polar solvents such as alcohols and water. Also, a notable bathochromic shift in λ(max)(em) was observed in fluorescence spectra of both drugs with increasing solvent polarity that resulted in biphasic behavior upon applying the Lippert-Mataga correlation that correspond to general and specific solvent effects. Applying the Lippert-Mataga correlation to the fluorescence spectra of CIPR and ENRO in various solvents was employed to estimate the dipole moment difference between the ground and excited states of them, Δμ(μ(e) - μ(g)), where obtained results revealed the values of 9.4 and 16.2 Debye for the LE and ICT states of ENRO, respectively, and 8.0 and 16.2 Debye for the LE and ICT states of CIPR, respectively. Multiple linear regression analysis (MLRA) based on Kamlet-Taft equating was applied against absorption frequency (ν(abs)), emission frequency (ν(em)), Stokes shift (∆ν), and fluorescence quantum yield (Φ(f)), where obtained results revealed excellent correlation (R: 0.916-0.966) that are consistent with other results considering the effect of solvent polarizability, hydrogen bonding ability, and viscosity on the photophysicochemical properties of the studied fluoroquinolones.

  4. 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.

  5. [Identifying the origin of chromophoric dissolved organic matter in Xiamen Bay using fluorescence spectroscopy and chemometrics].

    PubMed

    Lin, Hui; Guo, Wei-Dong; Xu, Jing; Hu, Ming-Hui

    2013-02-01

    The fluorescent components of chromophoric dissolved matter (CDOM) in water samples collected from Xiamen Bay in spring and autumn, 2009 were examined using excitation-emission matrix fluorescence spectroscopy combined with parallel factor analysis (EEMs-PARAFAC). PARAFAC decomposed the fluorescence matrices of CDOM into three humic-like (C1: 250, 345/454 nm; C2: 230, 310/374 nm; C5: 265, 424/478 nm) and two protein-like (C3: 230/342 nm; C4: 230, 275/322 nm) components. Good linear correlation occurred among three humic-like components and between two protein-like components, respectively. This demonstrated that the same types of components (humic-like or protein-like) have similar origin and geochemical behaviors. High abundances of humic-like components were found at the upstream zone of the Jiulong Estuary, while the high abundance of protein-like components occurred at the northern part of semi-enclosed Western Xiamen Harbor. The significant negative correlations were found between the abundances of all fluorescence components and salinity in the estuary area. However, the high contents of chlorophyll a were in line with the high abundances of C3 and C4 in non-estuarine area, which implies that phytoplankton activity could be another important source of protein-like components besides the river runoff. A principal component analysis(PCA) of fluorescent components revealed that terrestrial runoff was the dominant sources of CDOM fluorescence components in Xiamen Bay, while the contribution of the in situ biological processes was relatively lower. This study demonstrates that the combination use of PARAFAC modeling and chemometrics (i. e. PCA) is very useful in identifying the origin of CDOM and quantifying the primary factors influencing their distributions.

  6. Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

    NASA Astrophysics Data System (ADS)

    van den Berg, Petra A. W.; Widengren, Jerker; Hink, Mark A.; Rigler, Rudolf; Visser, Antonie J. W. G.

    2001-09-01

    Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity (<10 kW/cm 2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations (> 50 mM of I -) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the

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

    NASA Astrophysics Data System (ADS)

    Lewis, William; Williams, Maura; Franco, Walfre

    2017-02-01

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

  8. Substrate-supported phospholipid membranes studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy.

    PubMed

    Tawa, Keiko; Morigaki, Kenichi

    2005-10-01

    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.

  9. 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.

  10. Fluorescence and Absorbance Spectroscopy Methods to Study Membrane Perturbations by Antimicrobial Host Defense Peptides.

    PubMed

    Arias, Mauricio; Vogel, Hans J

    2017-01-01

    Antimicrobial peptides (AMPs) are currently intensely studied because of their potential as new bactericidal and bacteriostatic agents. The mechanism of action of numerous AMPs involves the permeabilization of bacterial membranes. Several methods have been developed to study peptide-membrane interactions; in particular optical spectroscopy methods are widely used. The intrinsic fluorescence properties of the Trp indole ring in Trp-containing AMPs can be exploited by measuring the fluorescence blue shift and acrylamide-induced fluorescence quenching. One important aspect of such studies is the use of distinct models of the bacterial membrane, in most cases large unilamellar vesicles (LUVs) with different, yet well-defined, phospholipid compositions. Deploying LUVs that are preloaded with fluorescent dyes, such as calcein, also allows for the study of vesicle permeabilization by AMPs. In addition, experiments using genetically engineered live Escherichia coli cells can be used to distinguish between the effects of AMPs on the outer and inner membranes of gram-negative bacteria. In combination, these methods can provide a detailed insight into the mode of action of AMPs.

  11. 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.

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

    PubMed

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

    2014-01-01

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

  13. 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

  14. 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

    2016-03-03

    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.

  15. Fluorescence spectroscopy for tongue carcinoma detection: study in an animal model

    NASA Astrophysics Data System (ADS)

    Kurachi, Cristina; Fontana, Carla R.; Melo, Claudia A. S.; Rosa, Luiz E. B.; Bagnato, Vanderlei S.

    2005-04-01

    Fluorescence diagnosis of malignant lesions has been showed as an attractive optical technique due especially to its real-time response and a more objective and quantitative evaluation. Even though the oral cavity allows a direct examination many lesions are diagnosed when it is already in advanced stage, compromising the patient prognosis. In this study, the fluorescence spectroscopy was used to the detection of chemically induced carcinoma at the lateral border of the tongue in a hamster model. Two excitations wavelengths in visible region were applied: 442 and 532 nm. All the spectra results were analyzed comparing with the histopathological diagnosis. The better results were achieved with the 442 nm laser excitation. The spectra from carcinoma showed new emission bands and these were used to determined different ratios for a quantitative analysis. Using the 625-645 nm fluorescence range under 442 nm excitation (A3 coefficient) the percentage of false negative was of 9.1%, however the false positive percentage was of 18.5%. The 532 nm excitation provided a better normal tissue detection compared to 442 nm excitation. The ideal clinical condition is probably the use of multiple wavelengths excitation for a broader tissue fluorescence investigation.

  16. 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.

  17. [Measurement and analysis of lead in soil using X-ray fluorescence spectroscopy].

    PubMed

    Zhang, Rong; Zhang, Yu-Jun; Zhang, Wei; Chen, Dong; Yu, Xiao-Ya; Gao, Yan-Wei

    2013-02-01

    The present paper analyzed the characteristics of X-ray fluorescence spectroscopy (XRF) of metal element lead in soil using the NITON XLt793 portable X-ray fluorescence spectra of heavy metal analyzer under laboratory conditions. The characteristic spectral lines of L(alpha) (energy: 10. 55 keV) and L(beta) (energy: 12. 61 keV) with different matrix elements were selected respectively for lead in the experiment. By measuring the intensities of the characteristic spectral line with different Pb concentration, the results demonstrate that the relation between concentration [mass fraction 10 x 10(-6) - 1 800 x 10(-6)] of Pb element and the intensity of the characteristic spectrum is well linear. The calibration curve of Pb was plotted based on the different concentration measurement results, and the limit of detection of 7.89 x 10(-6) was obtained for Pb in soil.

  18. Vesicle fusion studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy.

    PubMed

    Morigaki, Kenichi; Tawa, Keiko

    2006-08-15

    Substrate-supported planar lipid bilayers are generated most commonly by the adsorption and transformation of phospholipid vesicles (vesicle fusion). We have recently demonstrated that simultaneous measurements of surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) are highly informative for monitoring lipid membranes on solid substrates. SPR and SPFS provide information on the amount and topography of adsorbed lipid membranes, respectively. In this study, the vesicle fusion process was studied in detail by measuring SPR-SPFS at a higher rate and plotting the obtained fluorescence intensity versus film thickness. We could track the initial adsorption of vesicles, the onset of vesicle rupture occurring at certain vesicle coverage of the surface, and the autocatalytic transformation into planar bilayers. We also monitored vesicle fusion of the same vesicle suspensions by quartz crystal microbalance with dissipation monitoring (QCM-D). We compared the results obtained from SPR-SPFS and QCM-D to highlight the unique information provided by SPR-SPFS.

  19. Detection of the presence of refined hazelnut oil in refined olive oil by fluorescence spectroscopy.

    PubMed

    Sayago, A; García-Gonzalez, D L; Morales, M T; Aparicio, R

    2007-03-21

    The fluorescence spectroscopy technique has been tested as regards its ability to differentiate between refined hazelnut and olive oils. Classification of these oils based on their excitation-emission fluorescence spectra data (spectral range 300-500 nm of the excitation spectra at lambdaem=655 and spectral range 650-900 of the emission spectra at lambdaex=50 nm) was performed using principal component analysis and artificial neural networks. Both methods provided good discrimination between the refined hazelnut and olive oils. The results have also pointed out the possibilities of a spectrofluorimetric method joined to multivariate analysis, to differentiate refined oils, and even to detect the presence of refined hazelnut oils in refined olive oils at percentages higher than 9%.

  20. Time-resolved fluorescence spectroscopy of photosynthetic membranes: experiment and model simulations

    NASA Astrophysics Data System (ADS)

    Freiberg, Arvi; Pullerits, Tonu; Timpmann, Kou

    1990-05-01

    The singlet excitation transfer and trapping kinetics in photosynthetic membranes in case of low excitation intensities is studied by spectrally selective picosecond-time- domain fluorescence spectroscopy and by numerical integration of an appropriate system of equations. The essential features of our models are spectral heterogeneity of the light- harvesting antenna, inclusion of temperature effects, nonabsolute excitation traps, correlation between spectral and spatial parmeters. A reasonably good agreement between theoretical and experimental fluorescence decay kinetics for several purple photosynthetic bacteria has been achieved. This comparison gives several interesting numerical constants characterizing microscopic excitation transfer between different light-harvesting-antenna pigment-protein complexes towards the reaction centres. Some aspects of the experiment are also discussed.

  1. 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.

  2. 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.

  3. Detection of lead in water using laser-induced breakdown spectroscopy and laser-induced fluorescence.

    PubMed

    Lui, Siu L; Godwal, Yogesh; Taschuk, Michael T; Tsui, Ying Y; Fedosejevs, Robert

    2008-03-15

    Laser-induced breakdown spectroscopy (LIBS) is a well-known technique for fast, stand-off, and nondestructive analysis of the elemental composition of a sample. We have been investigating micro-LIBS for the past few years and demonstrating its application to microanalysis of surfaces. Recently, we have integrated micro-LIBS with laser-induced fluorescence (LIF), and this combination, laser ablation laser-induced fluorescence (LA-LIF), allows one to achieve much higher sensitivity than traditional LIBS. In this study, we use a 170 microJ laser pulse to ablate a liquid sample in order to measure the lead content. The plasma created was re-excited by a 10 microJ laser pulse tuned to one of the lead resonant lines. Upon optimization, the 3sigma limit of detection was found to be 35 +/- 7 ppb, which is close to the EPA standard for the level of lead allowed in drinking water.

  4. Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe.

    PubMed

    Dochow, Sebastian; Fatakdawala, Hussain; Phipps, Jennifer E; Ma, Dinglong; Bocklitz, Thomas; Schmitt, Michael; Bishop, John W; Margulies, Kenneth B; Marcu, Laura; Popp, Jürgen

    2016-09-01

    Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged.

  5. Structure and dynamics of a DNA: polymerase complex by time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Millar, David P.; Benkovic, Stephen J.

    1990-05-01

    The interaction of a fluorescent DNA primer:template with the Klenow fragment of DNA polymerase I has been studied in solution using time-resolved fluorescence spectroscopy. The excited-state decay behavior and internal reorientation dynamics of a dansyl sulfonamide probe connected by a propyl chain to a modified uridine base in the primer strand were very sensitive to the local probe environment and exhibited characteristic changes upon binding of Kienow fragment to the DNA and elongation of the primer strand. Between 5 and 7 bases of duplex DNA upstream of the 3' primer terminus were protected from the solvent by the Kienow fragment and the strength of DNA:protein contacts varied within this region, being strongest at the 3' primer terminus. About 5% of the substrates were bound in a second spatially distinct site on the enzyme. Site-directed mutagenesis of the Kienow fragment was consistent with this being the active site for 3'->5' exonuclease activity.

  6. Fluorescence spectroscopy measures yeast PAH1-encoded phosphatidate phosphatase interaction with liposome membranes

    PubMed Central

    Xu, Zhi; Su, Wen-Min; Carman, George M.

    2012-01-01

    Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides. Fluorescence spectroscopy was used to measure the interaction of yeast PAH1-encoded PA phosphatase with model liposome membranes. PA phosphatase contains five tryptophan residues and exhibited inherit fluorescence that increased upon interaction with phosphatidylcholine liposomes. The interaction was enhanced by inclusion of other phospholipids and especially the substrate PA. Interaction was dependent on both the concentration of phosphatidylcholine-PA liposomes as well as the surface concentration of PA in liposomes. Mg2+ ions, which were required for catalysis, did not affect PA phosphatase interaction with phosphatidylcholine-PA liposomes. PA phosphatase was a substrate for protein kinase A, protein kinase C, and casein kinase II, and these phosphorylations decreased PA phosphatase interaction with phosphatidylcholine-PA liposome membranes. PMID:22180632

  7. 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.

  8. 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.

  9. 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.

  10. Characterization of metabolites in different kiwifruit varieties by NMR and fluorescence spectroscopy.

    PubMed

    Abdul Hamid, Nur Ashikin; Mediani, Ahmed; Maulidiani, M; Abas, Faridah; Park, Yong Seo; Leontowicz, Hanna; Leontowicz, Maria; Namiesnik, Jacek; Gorinstein, S

    2017-05-10

    It is known from our previous studies that kiwifruits, which are used in common human diet, have preventive properties of coronary artery disease. This study describes a combination of (1)H NMR spectroscopy, multivariate data analyses and fluorescence measurements in differentiating of some kiwifruit varieties, their quenching and antioxidant properties. A total of 41 metabolites were identified by comparing with literature data Chenomx database and 2D NMR. The binding properties of the extracted polyphenols against HSA showed higher reactivity of studied two cultivars in comparison with the common Hayward. The results showed that the fluorescence of HSA was quenched by Bidan as much as twice than by other fruits. The correlation between the binding properties of polyphenols in the investigated fruits, their relative quantification and suggested metabolic pathway was established. These results can provide possible application of fruit extracts in pharmaceutical industry.

  11. Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy

    PubMed Central

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

    2012-01-01

    A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate. PMID:22219367

  12. 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%.

  13. Changes in chlorophyll a fluorescence of glyphosate-tolerant soybean plants induced by glyphosate: in vivo analysis by laser-induced fluorescence spectroscopy.

    PubMed

    Fernandes, Joelson; Falco, William Ferreira; Oliveira, Samuel Leite; Caires, Anderson Rodrigues Lima

    2013-05-01

    A significant increase in the use of the herbicide glyphosate has generated many questions about its residual accumulation in the environment and possible damage to crops. In this study, changes in chlorophyll a (chl-a) fluorescence induced by glyphosate in three varieties of glyphosate-resistant soybean plants were determined with an in vivo analysis based on a portable laser-induced fluorescence system. Strong suppression of chl-a fluorescence was observed for all plants treated with the herbicide. Moreover, the ratio of the emission bands in the red and far-red regions (685 nm/735 nm) indicates that the application of glyphosate led to chlorophyll degradation. The results also indicated that the use of glyphosate, even at concentrations recommended by the manufacturer, suppressed chl-a fluorescence. In summary, this study shows that fluorescence spectroscopy can detect, in vivo, very early changes in the photosynthetic status of transgenic soybeans treated with this herbicide.

  14. 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

  15. Fluorescence lifetime cross correlation spectroscopy resolves EGFR and antagonist interaction in live cells.

    PubMed

    Chen, Jiji; Irudayaraj, Joseph

    2010-08-01

    Fluorescence correlation or cross-correlation spectroscopy (FCS or FCCS), a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. However, the inherent drawback of FCS is that species with similar molecular weight could not be differentiated. Although FCCS could resolve this through cross-correlation, it suffers from nonideal confocal volume overlap and spectral cross-talk which limits its application. In this work, we demonstrate for the first time the applicability of fluorescence lifetime correlation spectroscopy (FLCS) to monitor the interaction of an antagonist antibody with the epidermal growth factor receptor (EGFR) in live cells. As a proof of concept, we demonstrate the interaction of Cy5 labeled IgG and Alexa633 labeled anti-IgG using a single laser source (636 nm excitation) in vitro. The autocorrelation functions were separated based on their respective lifetime with a single detector and their K(d) value was determined to be 11 +/- 3 nM. An in vivo application constituting the interaction of EGFR neutralizing antibody labeled with Alexa488 and EGFR-GFP in live HEK293 cells was successfully demonstrated. The binding specificity of EGFR neutralizing antibody was confirmed by fluorescence lifetime cross-correlation measurements and fluorescence lifetime imaging (FLIM). The dissociation constant of this complex was found to be 9.2 +/- 2.7 nM. A quantitative assessment of receptor density calculations show that the density of EGFR significantly decreased, from 540 +/- 64 receptors/microm(2) to 38 +/- 7 receptors/microm(2) upon addition of the neutralizing EGFR antibody, indicating that the antagonist could induce receptor internalization. The demonstrated work not only opens up new opportunities in studying protein-protein interactions in solutions and in live cells but also provide new insights in biology to understand how the antagonists influence EGFR

  16. Determination of Yttrium in High Density Silicon Nitride by Emission and X-Ray Fluorescence Spectroscopy.

    DTIC Science & Technology

    1981-08-01

    AD-AI07 596 ARMY MATERIALS AND MECHANICS RESEARCH CENTER WATERTOWN MA F/S 7/4 DETERMINATION OF YTTRIUM IN HIGH DENSITY SILICON NITRIDE BY EMI-ETCIU...AUG Al B H STRAUSS. UNCLASSIFIED AMMRC-TR-Al-39 N AMMRC TR 81-39 A ~LEVEL ’ t’- .- DETERMINATION OF YTTRIUM IN 1 HIGH DENSITY SILICON NITRIDE BY...DETERMINATION OF YTTRIUM IN HIGH DENSITY SILICON NITRIDE BY EMISSION AND X-RAY Final Report FLUORESCENCE SPECTROSCOPY 6 PERFORMING ORG. REPORT NUMBER 7. AUTHOR

  17. Interactions of hypericin with a model mutagen - Acridine orange analyzed by light absorption and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Pietrzak, Monika; Szabelski, Mariusz; Kasparek, Adam; Wieczorek, Zbigniew

    2017-02-01

    The present study was designed to estimate the ability of hypericin to interact with a model mutagen - acridine orange. The hetero-association of hypericin and acridine orange was investigated with absorption and fluorescence spectroscopy methods in aqueous solution of DMSO. The data indicate that hypericin forms complexes with acridine orange and that the association constants are relatively high and depend on DMSO concentration. The absorption spectra of the hypericin - acridine orange complexes were examined as well. Owing to its ability to interact with flat aromatic compounds, hypericin may potentially be used as an interceptor molecule.

  18. 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.

  19. Layer with reduced viscosity at water-oil interfaces probed by fluorescence correlation spectroscopy.

    PubMed

    Wang, Dapeng; Pevzner, Leonid; Li, Chen; Peneva, Kalina; Li, Christopher Y; Chan, Derek Y C; Müllen, Klaus; Mezger, Markus; Koynov, Kaloian; Butt, Hans-Jürgen

    2013-01-01

    The two-dimensional diffusion of isolated molecular tracers at the water-n-alkane interface was studied with fluorescence correlation spectroscopy. The interfacial diffusion coefficients of larger tracers with a hydrodynamic radius of 4.0 nm agreed well with the values calculated from the macroscopic viscosities of the two bulk phases. However, for small molecule tracers with hydrodynamic radii of only 1.0 and 0.6 nm, notable deviations were observed, indicating the existence of an interfacial region with reduced effective viscosity and increased mobility.

  20. Hierarchical organization of the plasma membrane: investigations by single-molecule tracking vs. fluorescence correlation spectroscopy.

    PubMed

    Kusumi, Akihiro; Shirai, Yuki M; Koyama-Honda, Ikuko; Suzuki, Kenichi G N; Fujiwara, Takahiro K

    2010-05-03

    Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.

  1. 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.

  2. Time-resolved fluorescence spectroscopy for clinical diagnosis of actinic cheilitis.

    PubMed

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

    2016-10-01

    Actinic cheilitis is a potentially malignant disorder of the lips. Its first cause is believed to be UV sun radiation. The lesion is highly heterogeneous, making the choice of area to be biopsied difficult. This study exploits the capabilities of time-resolved fluorescence spectroscopy for the identification of the most representative area to be biopsied. A preliminary study was performed on fourteen patients. A classification algorithm was used on data acquired on nine different biopsies. The algorithm discriminated between absent, mild, and moderate dysplasia with a sensitivity of 92.9%, 90.0%, and 80.0%, respectively. The false positive rate for healthy tissue (specificity) was 88.8%.

  3. 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

  4. Applications of fluorescence spectroscopy and dynamic light scattering in biotechnology of camptothecins: promising anticancer drugs

    NASA Astrophysics Data System (ADS)

    Kruszewski, Stefan; Burke, Thomas G.

    2003-04-01

    Methods of optical measurements are widely used in biomedical research. Applications of these methods in determining of properties of camptothecins -- S-phase anticancer agents are described in this paper. Under physiological conditions CPT hydrolyses, and converts into inactive carboxylate form. Decrease of active form of CPT occurs especially fast in plasma of human blood. One of camptothecin analogues -- DB-67, exhibits, in contrary to CPT, relatively high stability in human blood. Fluorescence spectroscopy methods were used to determine physical and to predict biological properties of camptothecins, including DB-67. The dynamic light scattering (DLS) methods were applied to control the sizes of liposomes used as DB-67 delivery system.

  5. 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

  6. 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.

  7. Steady-state tryptophan fluorescence spectroscopy study to probe tertiary structure of proteins in solid powders.

    PubMed

    Sharma, Vikas K; Kalonia, Devendra S

    2003-04-01

    The purpose of this work was to obtain information about protein tertiary structure in solid state by using steady state tryptophan (Trp) fluorescence emission spectroscopy on protein powders. Beta-lactoglobulin (betaLg) and interferon alpha-2a (IFN) powder samples were studied by fluorescence spectroscopy using a front surface sample holder. Two different sets of dried betaLg samples were prepared by vacuum drying of solutions: one containing betaLg, and the other containing a mixture of betaLg and guanidine hydrochloride. Dried IFN samples were prepared by vacuum drying of IFN solutions and by vacuum drying of polyethylene glycol precipitated IFN. The results obtained from solid samples were compared with the emission scans of these proteins in solutions. The emission scans obtained from protein powders were slightly blue-shifted compared to the solution spectra due to the absence of water. The emission scans were red-shifted for betaLg samples dried from solutions containing GuHCl. The magnitude of the shifts in lambda(max) depended on the extent of drying of the samples, which was attributed to the crystallization of GuHCl during the drying process. The shifts in the lambda(max) of the Trp emission spectrum are associated with the changes in the tertiary structure of betaLg. In the case of IFN, the emission scans obtained from PEG-precipitated and dried sample were different compared to the emission scans obtained from IFN in solution and from vacuum dried IFN. The double peaks observed in this sample were attributed to the unfolding of the protein. In the presence of trehalose, the two peaks converged to form a single peak, which was similar to solution emission spectra, whereas no change was observed in the presence of mannitol. We conclude that Trp fluorescence spectroscopy provides a simple and reliable means to characterize Trp microenvironment in protein powders that is related to the tertiary conformation of proteins in the solid state. This study shows

  8. 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

  9. Kinetic study of delta-Ala induced porphyrins in mice using photoacoustic and fluorescence spectroscopies.

    PubMed

    Stolik, Suren; Tomás, Sergio A; Ramón-Gallegos, Eva; Sánchez, Feliciano

    2002-11-01

    The production of delta-aminolevulinic acid (ALA)-induced porphyrins in mice skin and blood was studied by photoacoustic and fluorescence spectroscopies. Mice were intraperitoneally administered with 30 mg/kg of ALA. The abdominal skin was subsequently excised at specific times within an 8-h interval and its absorption spectrum obtained by photoacoustics. The highest porphyrins concentration in skin, determined from the optical absorption of the Soret band at 410 nm, was found to occur nearly 2 h after ALA administration, but a first peak was also observed at approximately 15 min. Our hypothesis that the first peak represents the porphyrins content in blood vessels within the skin, whereas the second peak corresponds to porphyrins production in skin tissue, was confirmed by analysing the evolution of protoporphyrin IX content in plasma extracted intracardiacally. By finally applying phase resolved photoacoustic spectroscopy, we were able to evaluate the mean depth at which porphyrins are generated.

  10. Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics.

    PubMed

    Davis, Caitlin M; Reddish, Michael J; Dyer, R Brian

    2017-05-05

    Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of <0.2mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50ns to 0.5ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. 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.

  12. Synthesis, spectroscopy and photochemistry of novel branched fluorescent nitro-stilbene derivatives with benzopheonone groups.

    PubMed

    Gao, Fang; Liu, Jian; Peng, Huayong; Hu, Nvdan; Li, Hongru; Zhang, Shengtao

    2010-05-01

    In this article, we presented novel nitro-stilbene derivatives with one or two benzophenone groups as photoinitiators via multi-steps synthesis. The ultraviolet/visible spectroscopy and the emission spectroscopy of the compounds were determined in various solvents. The results showed that the ultraviolet/visible absorption spectroscopy of the derivatives with benzophenone moiety displayed overlap effects of nitro-stilbene and benzophenone parts. In non-polar solvents, the derivatives exhibited strong emission, while they displayed weak emission in modest and strong polar solvents. Dyes-linked benzopheonone groups displayed stronger fluorescence emission than simple chromophore parent molecules. Visible-light photoinitiating effects of the derivatives were investigated extensively. Methyl methacrylate could be photoinitiated efficiently by the derivatives with benzophenone moieties at very low concentration, even at 1 x 10(-5) mol/L. While the photopolymerization efficiency of styrene initiated by the derivatives was lower than that of methyl methacrylate. Our results showed that the dye-linked photoinitators had more efficient photoinitiating than the simple mixture of dye and photoinitator. Furthermore, the derivative with two benzophenone groups displayed more excellent photoiniatiating effects than the derivative with one benzophenone group. Thermodynamics driving for the occurrence of visible-light photoinduced intramolecular electron transfer from chromophore part to benzophenone part was evaluated. Benzopinacol moiety produced in photoreaction was confirmed by nuclear magnetic resonant spectroscopy. Thermal stability of the derivatives was analyzed.

  13. Tracking Biological Organic Compounds In Atmospheric Deposition In Alpine Environments With Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Mladenov, N.; Oldani, K. M.; Williams, M. W.; Schmidt, S. K.; Darcy, J.; Lemons, S.; Reche, I.

    2013-12-01

    Alpine environments, such as those of the Colorado Rocky Mountains, USA and the Sierra Nevada Mountains, Spain, contain undeveloped, barren soils that are carbon-limited. Atmospheric wet and dry deposition of organic carbon (OC) represents a substantial fraction of the OC load available to alpine soils, and includes contributions from atmospheric pollutants, dust, and biological aerosols, such as bacteria, algae, fungi, and plant debris. To evaluate the seasonal variability and sources of atmospheric deposition at these alpine sites, we measured the chemical characteristics of weekly wet and dry deposition and snowpack samples, including characterization of dissolved organic matter (DOM) and water soluble organic matter (WSOM) with fluorescence spectroscopy. The excitation-emission matrix (EEM) spectra we acquired show the presence of recurring peaks at low excitation and emission wavelengths typically associated with highly biodegradable organic carbon, presumably derived from the aromatic amino acids, tyrosine and tryptophan. Solar simulation experiments demonstrated that amino acid-like fluorescent components were more resistant to photo-degradation than humic- and fulvic-like fluorescent components. Our results also reveal the presence of a unique fluorophore, not previously described, that is found in both Rocky Mountains and the Sierra Nevada snowpack, wet deposition, and dry deposition and may be attributed to fluorescent pigments in bacteria. Biological aerosols may represent a labile source of carbon for alpine soil microbes, and consequently their deposition has important consequences for biogeochemical processes occurring in barren, alpine soils. Excitation emission matrix image of 24 Aug 2010 wet deposition sample from the Soddie site at Niwot Ridge, Colorado showing a unique fluorescent component with dual excitation peaks (285 nm and 340 nm) at 410 nm emission.

  14. 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.

  15. Photodynamic tumor therapy and on-line fluorescence spectroscopy after ALA administration using 633-nm light as therapeutic and fluorescence excitation radiation

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Kienle, Alwin; Boehncke, Wolf-Henning; Kaufmann, Roland; Rueck, Angelika C.; Meier, Thomas H.; Steiner, Rudolf W.

    1994-09-01

    Photodynamic therapy (PDT) and on-line fluorescence spectroscopy were carried out on human tumors after 5-aminolevulinic acid (ALA) administration using 633-nm light of a dye laser as therapeutic radiation and as fluorescence excitation radiation. This has the advantages of (1) enabling use of one laser for PDT and fluorescence diagnosis only, (2) enabling the possibility of on-line fluorescence measurements, and (3) exciting protoporphyrin molecules in deep tissue layers. Monte Carlo calculations were carried out to determine excitation and fluorescence phonon distribution in case of red and violet excitation radiation. The results show the possibility of depth-resolved measurements on the fluorophore distribution by variation of excitation wavelength. The high penetration depth of 633-nm radiation results in a higher ratio of the 700-nm protoporphyrin fluorescence of the xenotransplanted tumor It to Is compared with 407-nm excitation. No values greater than 1 for the ratio I/Is were found, however, in case of intravenous ALA injection even for red excitation. Therefore, a large amount of ALA will be metabolized in the skin and can cause photosensitivity of the patient when applied systematically. In contrast, protoporphyrin fluorescence limited to the pretreated skin area was detected in case of topically applied ALA to patients with mycosis funcoides and erythroplasy of Queyrat. The influence of remitted excitation light and of the spontaneous radiation from the laser as well as the possible excitation of foodbased degradation products of chlorophyll has to be considered in high-sensitivity fluorescence measurements.

  16. 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

  17. 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.

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

    PubMed

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

    2016-12-21

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

  19. Z-scan fluorescence correlation spectroscopy as a tool for diffusion measurements in planar lipid membranes.

    PubMed

    Steinberger, Tomáš; Macháň, Radek; Hof, Martin

    2014-01-01

    Studies of lateral diffusion are used for the characterization of the dynamics of biological membranes. One of the techniques that can be used for this purpose is fluorescence correlation spectroscopy (FCS), which belongs to the single-molecule techniques. Unfortunately, FCS measurements, when performed in planar lipid systems, are associated with a few sources of inaccuracy in the determination of the lateral diffusion coefficient. The main problems are related to the imperfect positioning of the laser focus relative to the plane of the sample. Another source of inaccuracy is the requirement for external calibration of the detection volume size. This protocol introduces a calibration-free method called Z-scan fluorescence correlation spectroscopy (Z-scan FCS), which is based on the determination of the diffusion time and particle number in steps along the optical (z-) axis by sequential FCS measurements. Z-scan FCS could be employed for diffusion measurements in planar membrane model systems-supported phospholipid bilayers (SPBs) and giant unilamellar vesicles (GUVs) and also in biological membranes. A result from measurements in SPBs is also presented in the protocol as a principle example of the Z-scan technique.

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

    PubMed

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

    2013-04-01

    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.

  1. 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

  2. Potential benefit of surfactants in a hydrocarbon contaminated soil washing process: fluorescence spectroscopy based assessment.

    PubMed

    Uhmann, Amandine; Aspray, Thomas J

    2012-06-15

    Soil washing is an ex situ soil remediation treatment process. The purpose of soil washing is to clean the major gravel and sand fractions, concentrating contamination into the fine silt and clay fractions. The addition of surfactants can improve the efficiency of this method. Here we report the use of UV fluorescence spectroscopy to assess the hydrocarbon cleaning process as a rapid and cost effective alternative to gas chromatography. Three wash solutions were tested on a total petroleum hydrocarbon contaminated soil: water, Sea Power 101 (SP101) at 1% (v/v) and Tween80 at 0.5% (w/v). The most effective to wash the gravel and sand was SP101 (54 and 65% improvement over the water only wash, respectively) which moved contamination to the silt fraction (94% of contaminants). Tween80 appeared not to enhance TPH removal efficiency from the gravel and sand fractions but did concentrate TPH in the effluent (95% more than water wash). In addition to TPH removal from gravel and sand, SP101 also showed potential benefit in the soil washing sedimentation process, enhancing sludge/water volume separation by 10% over the water only wash. In summary, fluorescence spectroscopy proved an effective technique to compare TPH removal efficiencies as part of soil washing laboratory based treatability testing. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Fluorescence spectroscopy for the detection of potentially malignant disorders and squamous cell carcinoma of the oral cavity.

    PubMed

    Francisco, Ana Lucia Noronha; Correr, Wagner Rafael; Azevedo, Luciane Hiramatsu; Kern, Vivian Galletta; Pinto, Clóvis Antônio Lopes; Kowalski, Luiz Paulo; Kurachi, Cristina

    2014-06-01

    Oral cancer is a public health problem with relevant incidence in the world population. The affected patient usually presents advanced stage disease and the consequence of this delay is a reduction in survival rates. Given this, it is essential to detect oral cancer at early stages. Fluorescence spectroscopy is a non-invasive diagnostic tool that can improve cancer detection in real time. It is a fast and accurate technique, relatively simple, which evaluates the biochemical composition and structure using the tissue fluorescence spectrum as interrogation data. Several studies have positive data regarding the tools for differentiating between normal mucosa and cancer, but the difference between cancer and potentially malignant disorders is not clear. The aim of this study was to evaluate the efficacy of fluorescence spectroscopy in the discrimination of normal oral mucosa, oral cancer, and potentially malignant disorders. The fluorescence spectroscopy was evaluated in 115 individuals, of whom 55 patients presented oral squamous cell carcinoma, 30 volunteers showing normal oral mucosa, and 30 patients having potentially malignant disorders. The spectra were classified and compared to histopathology to evaluate the efficiency in diagnostic discrimination employing fluorescence. In order to classify the spectra, a decision tree algorithm (C4.5) was applied. Despite of the high variance observed in spectral data, the specificity and sensitivity obtained were 93.8% and 88.5%, respectively at 406 nm excitation. These results point to the potential use of fluorescence spectroscopy as an important tool for oral cancer diagnosis and potentially malignant disorders.

  4. 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.

  5. 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

  6. 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.

  7. 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.

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

    PubMed

    Hu, Hongyan; Huang, Xiangyi; Ren, Jicun

    2016-05-01

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

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

    PubMed Central

    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

  10. On-the-flow differentiation between cells based on native fluorescence spectroscopy on a chip

    NASA Astrophysics Data System (ADS)

    Beck, Markus; Bassler, Michael; Kiesel, Peter; Johnson, Noble M.; Schmidt, Oliver

    2008-03-01

    Native fluorescence spectroscopy is a promising approach for the detection of pathogens without specific binding or tagging of the analyte. The distinction between different species is possible with (multi-color) UV excitation together with the detection of several spectral bands. We have developed a compact platform that combines a microfluidic quartz channel with chip-size wavelength-selective detection of the fluorescence from particles traversing the channel. The interaction between the UV excitation light and the analyte is enhanced by anti-resonantly guiding the light within fluid. We have recorded the intrinsic fluorescence of single cells (e.g. yeast, e-coli, and BT) passing the detection area. Knowing the particle speed and the physical dimensions of the observation window, we are able to determine particle positions with microscopic (˜10 microns) resolution. A special modulation technique allows us to achieve a high signal to noise ratio even for high particle speeds. Combining our technique with a cell sorting mechanism would allow for on-the-chip characterization and sorting of untagged cells.

  11. Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging.

    PubMed

    Kisley, Lydia; Brunetti, Rachel; Tauzin, Lawrence J; Shuang, Bo; Yi, Xiyu; Kirkeminde, Alec W; Higgins, Daniel A; Weiss, Shimon; Landes, Christy F

    2015-09-22

    Porous materials such as cellular cytosol, hydrogels, and block copolymers have nanoscale features that determine macroscale properties. Characterizing the structure of nanopores is difficult with current techniques due to imaging, sample preparation, and computational challenges. We produce a super-resolution optical image that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures by correlating stochastic fluctuations from diffusing fluorescent probes in the pores of the sample, dubbed here as "fluorescence correlation spectroscopy super-resolution optical fluctuation imaging" or "fcsSOFI". Simulations demonstrate that structural features and diffusion properties can be accurately obtained at sub-diffraction-limited resolution. We apply our technique to image agarose hydrogels and aqueous lyotropic liquid crystal gels. The heterogeneous pore resolution is improved by up to a factor of 2, and diffusion coefficients are accurately obtained through our method compared to diffraction-limited fluorescence imaging and single-particle tracking. Moreover, fcsSOFI allows for rapid and high-throughput characterization of porous materials. fcsSOFI could be applied to soft porous environments such hydrogels, polymers, and membranes in addition to hard materials such as zeolites and mesoporous silica.

  12. Entangled photon-pair two-dimensional fluorescence spectroscopy (EPP-2DFS).

    PubMed

    Raymer, M G; Marcus, Andrew H; Widom, Julia R; Vitullo, Dashiell L P

    2013-12-12

    We introduce a new method, called entangled photon-pair two-dimensional fluorescence spectroscopy (EPP-2DFS), to sensitively probe the nonlinear electronic response of molecular systems. The method incorporates a separated two-photon ('Franson') interferometer, which generates time-frequency-entangled photon pairs, into the framework of a fluorescence-detected 2D optical spectroscopic experiment. The entangled photons are temporally shaped and phase-modulated in the interferometer, and are used to excite a two-photon-absorbing (TPA) sample, whose excited-state population is selectively detected by simultaneously monitoring the sample fluorescence and the exciting fields. In comparison to 'classical' 2DFS techniques, major advantages of this scheme are the suppression of uncorrelated background signals, the enhancement of simultaneous time-and-frequency resolution, the suppression of diagonal 2D spectral features, and the enhancement and narrowing of off-diagonal spectral cross-peaks that contain information about electronic couplings. These effects are a consequence of the pure-state field properties unique to a parametric down-conversion light source, which must be included in the quantum mechanical description of the composite field-molecule system. We numerically simulate the EPP-2DFS observable for the case of an electronically coupled molecular dimer. The EPP-2DFS spectrum is greatly simplified in comparison to its classical 2D counterpart. Our results indicate that EPP-2DFS can provide previously unattainable resolution to extract model Hamiltonian parameters from electronically coupled molecular dimers.

  13. 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.

  14. [Determination of terbium (III) with EHPG-Tb (III) system by fluorescence spectroscopy].

    PubMed

    Zhao, Chun-gui; Li, Xiao-li; Yang, Bin-sheng

    2007-12-01

    The fluorescence of terbium was sensitized after addition of terbium to the ethylene-N, N'-bis (o-hydioxyphenylglycine) (EHPG) solution. A novel and simple method used for the determination of Tb (III) was developed by means of fluorescence spectroscopy in the presence of EHPG. It was showed that the relative fluorescence intensity is proportional to the concentration of terbium ions, while the molar ratio of terbium to EHPG is less than 1.0 in the system. The maximum wavelengths of excitation and emission are 295 and 547 nm respectively. The optimal range of pH is 7-9. The linear range of detection of the concentration of terbium is from 1.0 x 10(-8) mol x L(-1) to 1.0 x 10(-5) mol x L(-1), with a detection limit of 1.18 x 10(-9) mol x L(-1). The relative standard deviation is still within +/-3% in the presence of other lanthanide ions. The method was applied to the determination of the recoveries of synthetic samples and a rare earth sample with satisfactory results.

  15. Multimodal analysis of pearls and pearl treatments by using optical coherence tomography and fluorescence spectroscopy.

    PubMed

    Ju, Myeong Jin; Lee, Sang Jin; Kim, Yuri; Shin, Jun Geun; Kim, Hae Yeon; Lim, Yiheng; Yasuno, Yoshiaki; Lee, Byeong Ha

    2011-03-28

    We present an integrated optical system that consists of optical coherence tomography (OCT) and laser-induced fluorescence (LIF) spectroscopy for multimodal analysis of pearls and pearl treatments. The OCT source and the LIF excitation beams were aligned together to illuminate the same spot of a pearl fixed on the sample stage that was under rotation. As a result, both OCT images and LIF spectra of the pearls were detected at the same time and also at the same place. For OCT, a 1310 nm-centered swept laser source was used. For LIF, a 405 nm laser diode was used and a lensed multimode fiber was utilized as a fluorescence probe. The tomographic investigation on the internal structure of a pearl allowed us to evaluate and categorize the pearl nondestructively as was previously reported. In addition, the measurements of fluorescence spectrum and its decaying rate helped to determine the species of mother oyster. The proposed multimodal analysis made it possible to classify the pearls and also to disclose the treatments made on the pearls.

  16. Fluorescence spectroscopy as a tool for determining microbial quality in potable water applications.

    PubMed

    Cumberland, Susan; Bridgeman, John; Baker, Andy; Sterling, Mark; Ward, David

    2012-01-01

    Building on previous work where fluorescence spectroscopy has been used to detect sewage in rivers, a portable LED spectrophotometer was used for the first time to establish bacterial numbers in a range of water samples. A mixed-method approach was used with standard bacteria enumeration techniques on diluted river water and sewage works final effluent using a number of diluents (Ringer's solution, tap water and potable spring water). Fluorescence from uncultured dilutions was detected at a 280 nm excitation/360 nm emission wavelength (corresponding to the region of tryptophan and indole fluorescence) and compared with bacteria numbers on the same cultured sample. Good correlations were obtained for total coliforms, E. coli and heterotrophic bacteria with the portable LED spectrophotometer (R2 = 0.78, 0.72 and 0.81 respectively). The results indicate that the portable spectrophotometer could be applied to establish the quality of drinking water in areas of poor sanitation that are subject to faecal contamination, where infrastructure failure has occurred in the supply of clean drinking water. This would be particularly useful where laboratory facilities are not at hand.

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

    PubMed

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

    2017-02-01

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

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

    PubMed

    Hurst, Miranda N; DeLong, Robert K

    2016-09-14

    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.

  19. Analytical form of the autocorrelation function for the fluorescence correlation spectroscopy.

    PubMed

    Hołyst, Robert; Poniewierski, Andrzej; Zhang, Xuzhu

    2017-02-08

    Fluorescence correlation spectroscopy (FCS) can provide information about diffusion coefficients and rate constants of chemical reactions in small systems of interacting molecules. However, the interpretation of FCS experiments depends crucially on the model of the autocorrelation function for the fluorescence intensity fluctuations. In this theoretical work, we consider a system of fluorescent molecules that diffuse and interact with massive particles, e.g. surfactant micelles. Using the general formalism of FCS, we derive a new analytical approximation of the autocorrelation function for systems in which both diffusion and a binary reaction occur. This approximation provides a smooth interpolation between the limit of fast reaction (much faster than diffusion), and the opposite limit of slow reaction. Our studies of noncovalent interactions of micelles with dyes by FCS provided an experimental case to which the approximate autocorrelation function was successfully applied [X. Zhang, A. Poniewierski, A. Jelińska, A. Zagożdżon, A. Wisniewska, S. Hou and R. Hołyst, Soft Matter, 2016, 12, 8186-8194].

  20. Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy.

    PubMed

    Wang, Ying; Schellenberg, Helene; Walhorn, Volker; Toensing, Katja; Anselmetti, Dario

    2017-03-01

    Fluorescent dyes are broadly used in many biotechnological applications to detect and visualize DNA molecules. However, their binding to DNA alters the structural and nanomechanical properties of DNA and, thus, interferes with associated biological processes. In this work we employed magnetic tweezers and fluorescence spectroscopy to investigate the binding of PicoGreen to DNA at room temperature in a concentration-dependent manner. PicoGreen is an ultrasensitive quinolinium nucleic acid stain exhibiting hardly any background signal from unbound dye molecules. By means of stretching and overwinding single, torsionally constrained, nick-free double-stranded DNA molecules, we acquired force-extension and supercoiling curves which allow quantifying DNA contour length, persistence length and other thermodynamical binding parameters, respectively. The results of our magnetic tweezers single-molecule binding study were well supported through analyzing the fluorescent spectra of stained DNA. On the basis of our work, we could identify a concentration-dependent bimodal binding behavior, where, apparently, PicoGreen associates to DNA as an intercalator and minor-groove binder simultaneously.

  1. Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Ojala, Kirsi; Michel, Patrik O; Vuento, Matti; Oker-Blom, Christian

    2002-12-01

    Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

  2. Correlation Spectroscopy of Minor Fluorescent Species: Signal Purification and Distribution Analysis

    PubMed Central

    Laurence, Ted A.; Kwon, Youngeun; Yin, Eric; Hollars, Christopher W.; Camarero, Julio A.; Barsky, Daniel

    2007-01-01

    We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce “purified FCS”, which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce “single-molecule FCS”, which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant. PMID:17189306

  3. Measuring precise diffusion coefficients with two-focus fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Dertinger, Thomas; Gregor, Ingo; von der Hocht, Iris; Erdmann, Rainer; Krämer, Benedikt; Koberling, Felix; Hartmann, Rudolf; Enderlein, Jörg

    2006-02-01

    We present a new method for precisely measuring diffusion coefficients of fluorescent molecules at nanomolar concentrations. The method is based on a modified Fluorescence Correlation Spectroscopy (FCS)-setup which is robust against many artifacts that are inherent to standard FCS 1, 2. The core idea of the new method is the introduction of an external ruler by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by ab initio calculations of resulting correlation curves and subsequent affine transformation of these curves to match the measured auto- and cross-correlation functions. The affine transformation coefficient along the time axis then directly yields the correct diffusion coefficient. This method is not relying on the rather inexact assumption of a 3D Gaussian shaped detection volume. We measured the diffusion coefficient of the red fluorescent dye Atto-655 (Atto-Tec GmbH) in water and compared the obtained value with results from Gradient Pulsed Field NMR (GPF-NMR).

  4. A closed form for fluorescence correlation spectroscopy experiments in submicrometer structures.

    PubMed

    Sanguigno, Luigi; De Santo, Ilaria; Causa, Filippo; Netti, Paolo

    2010-12-01

    Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion coefficients in an open detection volume. However, in several practical cases, when FCS measurements are carried out in small compartments like microchannels, neglecting boundary effects could lead to erroneous results. Here, a close form solution is proposed to explicitly account for the presence of walls located at a distance comparable with the characteristic detection volume lengths. We derive a one-dimensional diffusion constrained model and then generalize the solution to the two- and the three-dimensional constrained cases. We further indicate within which limits the standard autocorrelation function (ACF) model gives reliable results in microconfinement. Our model relies just on the assumption of elastic hits at the system walls and succeeds in describing the ACF of fluorescent probes confined along one direction. Through the analysis of FCS experimental data, we are able to predict the correct shape of the ACF in channels of micrometric and submicrometric width and measure the extent of lateral confinement. In addition, it permits the investigation of microstructured material features such as cages and cavities having dimensions on the micrometric range. On the basis of the proposed model, we also show in which conditions confinement could generate an apparent time dependent probe mobility, thus allowing a proper interpretation of the transport process taking place in submicrometric compartments.

  5. 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.

  6. 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.

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

    NASA Astrophysics Data System (ADS)

    Hurst, Miranda N.; Delong, Robert K.

    2016-09-01

    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.

  8. Local conformations and excited state dynamics of porphyrins and nucleic acids by 2-dimensional fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Widom, Julia R.

    Biological systems present many challenges to researchers attempting to study them using spectroscopy. Low specificity, low sensitivity, and broad and overlapping lineshapes limit the amount of information that can be obtained in experiments. Two-dimensional fluorescence spectroscopy (2D FS) is a highly sensitive and information-rich spectroscopic technique that was developed to study the conformations and excited state dynamics of systems exhibiting exciton coupling. In this dissertation, I describe a variety of extensions of 2D FS that further increase its utility for the study of biological systems. I describe experiments on a dimer of zinc tetraphenylporphyrin embedded in a membrane, in which the signals from two conformational subpopulations were separated in order to study the thermodynamics of their interconversion. I present proof-of-principle experiments on nucleic acids