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Sample records for fragment fluorescence spectroscopy

  1. Detection of lead in soil with excimer laser fragmentation fluorescence spectroscopy (ELFFS)

    SciTech Connect

    Choi, J.H.; Damm, C.J.; O'Donovan, N.J.; Sawyer, R.F.; Koshland, C.P.; Lucas, D.

    2004-03-01

    Excimer laser fragmentation fluorescence spectroscopy (ELFFS) is used to monitor lead in soil sample and investigate laser-solid interactions. Pure lead nitrate salt and soil doped with lead nitrate are photolyzed with 193 nm light from an ArF excimer at fluences from 0.4 to 4 J/cm{sup 2}. Lead emission is observed at 357.2, 364.0, 368.3, 373.9 and 405.8 nm. Time-resolved data show the decay time of the lead emission at 405.8 nm grows with increasing fluence, and a plasma is formed above fluences of 2 J/cm{sup 2}, where a strong continuum emission interferes with the analyte signal. Fluences below this threshold allow us to achieve a detection limit of approximately 200 ppm in soil.

  2. Excimer laser fragmentation fluorescence spectroscopy for real-time monitoring of combustion generated pollutants

    NASA Astrophysics Data System (ADS)

    Damm, Christopher John

    Toxic pollutant emissions from combustion pose a hazard to public and environmental health. Better diagnostic techniques would benefit emissions monitoring programs and aid research aimed at understanding toxic pollutant formation and behavior. Excimer Laser Fragmentation Fluorescence Spectroscopy (ELFFS) provides sensitive, real-time, in situ measurements of several important combustion related pollutants. This thesis demonstrates the capabilities of ELFFS for detecting amines in combustion exhausts and carbonaceous particulate matter from engines. ELFFS photofragments target species using a 193 nm excimer laser to form fluorescent signature species. The NH (A--X) band at 336 nm is used to monitor ammonia, ammonium nitrate and ammonium sulfate. There are no major interferences in this spectral region. The sensitivity is approximately 100 ppb (1 second measurement) for ammonia in post flame gases and 100 ppb (mole fraction) for ammonium nitrate/sulfate in ambient air. Quenching of NH by the major combustion products does not limit the applicability of the detection method. Fluorescence from excited carbon atoms at 248 nm (1P 0 → 1S0) following photofragmentation measures particulate matter in a two-stroke gasoline engine and a four-stroke diesel engine. Fluorescence from CH (A2Delta → X 2pi, 431 nm) C2 (d3pig → a3piu, 468 nm) fragments is also observed. The atomic carbon fluorescence signal is proportional to the mass concentration of particles in the laser interrogation region. The 100-shot (1 second) detection limit for particles in the two-stroke gasoline engine exhaust is 0.5 ppb (volume fraction). The 100-shot detection limit for four-stroke diesel particulate matter is 0.2 ppb. Interferences from carbon monoxide and carbon dioxide are negligible. The ratios of atomic carbon, C2, and CH peaks provide information on the molecular forms of compounds condensed on or contained within the particles measured. The C/C2 signal ratio can be used to distinguish

  3. Preparatory study for detection of nickel in industrial flue gas by excimer laser-induced fragmentation fluorescence spectroscopy.

    PubMed

    Gottwald, U; Monkhouse, P

    2003-02-01

    The purpose of this work is to survey possibilities for detecting molecular nickel species in industrial flue gas using excimer laser-induced fragmentation fluorescence (ELIF), in particular to establish suitable detection schemes and to obtain a sensitivity estimate for Ni detection. Investigations were conducted in a heated laboratory cell under defined conditions of temperature and pressure, using NiCl2 as the precursor molecule. An ArF excimer laser (193 nm) was used for excitation and Ni atomic emission spectra were recorded in the range 300 to 550 nm. The dependence of ELIF signal on laser fluence was quadratic in the range of laser intensities investigated, as expected for a two-photon excitation process. The temporal behavior of the ELIF signals gave lifetimes significantly longer than the known natural lifetimes. This result and the energetics of the system suggest a Ni* production mechanism involving the formation of Ni+ and subsequent ion-electron recombination. The temperature dependence of the ELIF signal, determined in the range 773 to 1223 K, was found to follow the vapor-pressure curve (Antoine equation) known from the literature. Finally, quenching effects were investigated by measuring ELIF signals and lifetimes in nitrogen or air up to 1 atm. On the basis of the results so far, detection limits for Ni in practical combustion applications in the range of tens of ppb should be achievable, which will be sufficient for regulatory measurements in incinerators and power plants.

  4. Probing the binding of Cu(2+) ions to a fragment of the Aβ(1-42) polypeptide using fluorescence spectroscopy, isothermal titration calorimetry and molecular dynamics simulations.

    PubMed

    Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Żmudzińska, Wioletta; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

    2016-09-01

    Steady-state and time-resolved fluorescence quenching measurements supported by isothermal titration calorimetry (ITC) and molecular dynamics simulations (MD), with the NMR-derived restraints, were used to investigate the interactions of Cu(2+) ions with a fragment of the Aβ(1-42) polypeptide, Aβ(5-16) with the following sequence: Ac-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-NH2, denoted as HZ1. The studies presented in this paper, when compared with our previous results (Makowska et al., Spectrochim. Acta A 153: 451-456), show that the affinity of the peptide to metal ions is conformation-dependent. All the measurements were carried out in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, pH6.0. The Stern-Volmer equations, along with spectroscopic observations, were used to determine the quenching and binding parameters. The obtained results unequivocally suggest that Cu(2+) ions quench the fluorescence of HZ1 only through a static quenching mechanism, in contrast to the fragment from the N-terminal part of the FPB28 protein, with sequence Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr- NH2 (D9) and its derivative with a single point mutation: Ac-Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr- NH2 (D9_M), where dynamic quenching occurred. The thermodynamic parameters (ΔITCH, ΔITCS) for the interactions between Cu(2+) ions and the HZ1 peptide were determined from the calorimetric data. The conditional thermodynamic parameters suggest that, under the experimental conditions, the formation of the Cu(2+)-HZ1 complex is both an enthalpy and entropy driven process.

  5. 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.

  6. DNA fragment sizing and sorting by laser-induced fluorescence

    DOEpatents

    Hammond, Mark L.; Jett, James H.; Keller, Richard A.; Marrone, Babetta L.; Martin, John C.

    1996-01-01

    A method is provided for sizing DNA fragments using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA piece or the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is linearly related to the fragment length. The distribution of DNA fragment sizes forms a characterization of the DNA piece for use in forensic and research applications.

  7. DNA fragment sizing and sorting by laser-induced fluorescence

    SciTech Connect

    Jett, J.H.; Hammond, M.L.; Keller, R.A.; Marrone, B.L.; Martin, J.C.

    1992-12-31

    A method is provided for obtaining DNA fingerprints using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a selected sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is directly proportional to the fragment length. Additional dyes can be bound to the DNA piece and DNA fragments to provide information additional to length information. Oligonucleotide specific dyes and/or hybridization probes can be bound to the DNA fragments to provide information on oligonucleotide distribution or probe hybridization to DNA fragments of different sizes.

  8. 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.

  9. 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.

  10. Sizing of single fluorescently stained DNA fragments by scanning microscopy

    PubMed Central

    Laib, Stephan; Rankl, Michael; Ruckstuhl, Thomas; Seeger, Stefan

    2003-01-01

    We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-l-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7–14%. The proposed method is straightforward and can be applied to standardized microtiter plates. PMID:14602931

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Fluorescence spectroscopy for wastewater monitoring: A review.

    PubMed

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

    2016-05-15

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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

    SciTech Connect

    Didier, P.; Weiss, E.; Sibler, A.-P.; Philibert, P.; Martineau, P.; Bigot, J.-Y.; Guidoni, L.

    2008-02-22

    Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Fluorescent amplified fragment length polymorphism (fAFLP) analysis of Listeria monocytogenes.

    PubMed

    Amar, Corinne

    2014-01-01

    Fluorescent amplified fragment length polymorphism (fAFLP) is based on the selective PCR amplification of restriction fragments from a digest of total genomic DNA. Genomic DNA extracted from a purified bacterial isolate is completely digested with two endonucleases generating fragments which are ligated to specific double-stranded adaptors. The ligated fragments are then amplified by PCR using fluorescently labelled primers. Fluorescent amplified fragments are separated by size on an automated sequencer with a size standard. fAFLP is a rapid, highly reproducible technique which can be used to discriminate and subtype Listeria monocytogenes strains.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Fluorescence Emission and Excitation Spectra of Photo-Fragmented Nitrobenzene.

    NASA Astrophysics Data System (ADS)

    Lue, Christopher J.; Tanjaroon, Chakree; Johnson, J. Bruce; Allen, Susan D.; Reeve, Scott W.

    2012-06-01

    Upon absorption of a UV photon, nitrobenzene readily dissociates into C_6H_5, NO_2, C_6H_5NO, O, C_6H_5O, and NO through three different channels. We have recorded high resolution emission and excitation spectra of the NO resulting from photo-fragmented nitrobenzene using a pulsed picosecond tunable laser and a nanosecond dye laser. Specifically, the lasers probed the A^2Σ^+→ X^2π(1/2,3/2) NO band system between 225-260 nm using an one or two color process. In a one color process, the same energy (wavelength) photon is used to dissociate nitrobenzene and excite NO. In a two color process, photons of a particular energy are used to dissociate the nitrobenzene while photons of a different energy are used to probe the resultant NO. We have determined the rotational and vibrational temperatures of the nascent NO. And, we have examined the effect of the relative timing of the two photons on the fluorescence spectra to extract information about the photodissociation dynamics. Lin, M.-F.; Lee, Y. T.; Ni, C.-K.; Xu, S. and Lin, M. C. J. Chem. Phys., AIP, 2007, 126.

  17. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  19. 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.

  20. BIMOLECULAR FLUORESCENCE COMPLEMENTATION ANALYSIS OF INDUCIBLE PROTEIN INTERACTIONS: EFFECTS OF FACTORS AFFECTING PROTEIN FOLDING ON FLUORESCENT PROTEIN FRAGMENT ASSOCIATION

    PubMed Central

    Robida, Aaron M; Kerppola, Tom K

    2009-01-01

    Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. We investigated the temporal resolution and the quantitative accuracy of BiFC analysis using fragments of different fluorescent proteins. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of YFP fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 minutes after rapamycin addition and a ten-fold increase in the mean fluorescence intensity in 8 hours. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and YFP produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment of the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized prior to rapamycin addition formed BiFC complexes with the same efficiency as newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggest that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Jet Cooled Laser Induced Fluorescence Spectroscopy of FCH2CH2O and Other Photo-Fragments of XCH2CH2ONO (X=F, Cl, Br, Oh)

    NASA Astrophysics Data System (ADS)

    Chhantyal-Pun, Rabi; Chen, Ming-Wei; Miller, Terry A.

    2011-06-01

    HOCH2CH2O is one of the prototypical hydroxyalkoxy intermediates formed in the OH mediated oxidation of olefins in the atmosphere. Specifically, HOCH2CH2O is produced from NO assisted reduction of HOCH2CH2OO which in turn is formed by the OH mediated oxidation of ethene in the atmosphere. Halogen substituted ethoxy can be a model to study the hydroxy substituted ethoxy. Our group has successfully studied different primary, secondary and unsaturated alkoxy radicals using Laser Induced Fluorescence technique (LIF) coupled with supersonic free jet expansion in the past. In this talk we will present the jet cooled LIF spectrum of FCH2CH2O. FCH2CH2O was produced in the jet by 351nm photodissociation of FCH2CH2ONO. Aided by ab inito calculations and past experiments, we were able to assign our spectrum to different conformers of FCH2CH2O based on the G(±120°) and T(O°) orientations of the OCCF dihedral angle. Besides FCH2CH2O, we also found transitions belonging to HCHO and CH2CHO from FCH2CH2ONO photodissociation. HCHO and CH2CHO were also found in Cl, Br and OH substituted precursors.

  6. 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

  7. 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.

  8. 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.

  9. DSP Algorithms for Fission Fragment and Prompt Fission Neutron Spectroscopy

    SciTech Connect

    Zeynalova, O.; Zeynalov, Sh.; Hambsch, F.-J.; Oberstedt, S.; Fabry, I.

    2009-10-29

    Digital signal processing (DSP) algorithms are in high demand for modern nuclear fission investigation due to importance of increase the accuracy of fissile nuclear data for new generation of nuclear power stations. DSP algorithms for fission fragment (FF) and prompt fission neutron (PFN) spectroscopy are described in the present work. The twin Frisch-grid ionization chamber (GTIC) is used to measure the kinetic energy-, mass- and angular distributions of the FF in the {sup 252}Cf(SF) reaction. Along with the neutron time-of-flight (TOF) measurement the correlation between neutron emission and FF mass and energy is investigated. The TOF is measured between common cathode of the GTIC and the neutron detector (ND) pulses. Waveform digitizers (WFD) having 12 bit amplitude resolution and 100 MHz sampling frequency are used for the detector pulse sampling. DSP algorithms are developed as recursive procedures to perform the signal processing, similar to those available in various nuclear electronics modules, such as constant fraction discriminator (CFD), pulse shape discriminator (PSD), peak-sensitive analogue-to-digital converter (pADC) and pulse shaping amplifier (PSA). To measure the angle between FF and the cathode plane normal to the GTIC a new algorithm is developed having advantage over the traditional analogue pulse processing schemes. Algorithms are tested by comparing the numerical simulation of the data analysis of the {sup 252}Cf(SF) reaction with data available from literature.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. "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.

  15. 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.

  16. 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.

  17. Population inversion in fluorescing fragments of super-excited molecules inside an air filament

    NASA Astrophysics Data System (ADS)

    See-Leang, Chin; Huai-Liang, Xu

    2015-01-01

    An original idea is reviewed. When a molecule is pumped into a super-excited state, one of its decay channels is neutral dissociation. One or more of the neutral fragments will fluoresce. Hence, if a lower state of such fluorescing fragments was populated through other channels but with a lower probability, population inversion of the fluorescing fragments would be naturally realized. This idea seems to be validated, so far, by comparing published work on three hydrocarbon molecules, CH4, C2H2, C2H4, and water vapor, H2O. After super-excitation in a femtosecond laser filament in air mixed with these molecules, the fluorescence from the CH or OH fragments exhibits population inversion, i.e., amplified spontaneous emission was observed in the backscattering direction of the filament. Project supported by the Canada Research Chairs, the Natural Science and Engineering Research Council of Canada (NSERC), the FRQNT, the Canada Foundation for Innovation (CFI), the National Basic Research Program of China (Grant No. 2014CB921300), the National Natural Science Foundation of China (Grant No. 61235003), the Research Fund for the Doctoral Program of Higher Education of China, and the Scientific Research Foundation for Returned Scholars, Ministry of Education of China.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Cost-effective fluorescent amplified fragment length polymorphism (AFLP) analyses using a three primer system.

    PubMed

    Stölting, Kai N; Clarke, Andrew C; Meudt, Heidi M; Blankenhorn, Wolf U; Wilson, Anthony B

    2011-05-01

    The amplified fragment length polymorphism (AFLP) technique is a widely used multi-purpose DNA fingerprinting tool. The ability to size-separate fluorescently labelled AFLP fragments on a capillary electrophoresis instrument has provided a means for high-throughput genome screening, an approach particularly useful in studying the molecular ecology of nonmodel organisms. While the 'per-marker-generated' costs for AFLP are low, fluorescently labelled oligonucleotides remain costly. We present a cost-effective method for fluorescently end-labelling AFLPs that should make this tool more readily accessible for laboratories with limited budgets. Both standard fluorescent AFLPs and the end-labelled alternatives presented here are repeatable and produce similar numbers of fragments when scored using both manual and automated scoring methods. While it is not recommended to combine data using the two approaches, the results of the methods are qualitatively comparable, indicating that AFLP end-labelling is a robust alternative to standard methods of AFLP genotyping. For researchers commencing a new AFLP project, the AFLP end-labelling method outlined here is easily implemented, as it does not require major changes to PCR protocols and can significantly reduce the costs of AFLP studies.

  10. 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.

  11. 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

  12. Prompt {gamma}-ray spectroscopy of isotopically identified fission fragments

    SciTech Connect

    Shrivastava, A.; Caamano, M.; Rejmund, M.; Navin, A.; Rejmund, F.; Lemasson, A.; Schmitt, C.; Derkx, X.; Fernandez-Dominguez, B.; Golabek, C.; Roger, T.; Sieja, K.; Audouin, L.; Bacri, C. O.; Barreau, G.; Jurado, B.

    2009-11-15

    Measurements of prompt Doppler-corrected deexcitation {gamma} rays from uniquely identified fragments formed in fusion-fission reactions of the type {sup 12}C({sup 238}U,{sup 134}Xe)Ru are reported. The fragments were identified in both A and Z using the variable-mode, high-acceptance magnetic spectrometer VAMOS. States built on the characteristic neutron configurations forming high-spin isomers (7{sup -} and 10{sup +}) in {sup 134}Xe are presented and compared with the predictions of shell-model calculations using a new effective interaction in the region of Z{>=}50 and N{<=}82.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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).

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay.

    PubMed

    Sakamoto, Seiichi; Tanizaki, Yusuke; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-05-01

    A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv.

  9. 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.

  10. Assessment of fluorescent amplified fragment length polymorphism analysis for epidemiological genotyping of Legionella pneumophila serogroup 1.

    PubMed

    Fry, N K; Afshar, B; Visca, P; Jonas, D; Duncan, J; Nebuloso, E; Underwood, A; Harrison, T G

    2005-09-01

    This study assessed the reproducibility and epidemiological concordance of double-enzyme fluorescent amplified fragment length polymorphism (fAFLP) analysis for genotyping of Legionella pneumophila serogroup (sg) 1. fAFLP fragment analysis was performed on three different sequencing platforms (one gel- and two capillary-based) in different laboratories with a well-characterised set of 50 strains of L. pneumophila sg 1. fAFLP data were analysed with the Pearson correlation similarity coefficient, using a range of parameters, and dendrogram outputs were converted to arbitrary types after selection of a specified percentage similarity threshold. The results obtained were compared with those obtained by the standard non-fluorescent AFLP method and were found to be broadly concordant. Using optimised settings for each fAFLP method to analyse the panel of 50 strains, epidemiological concordance (E) and reproducibility (R) values of 1.00 were obtained, and the number of types ranged from nine to 15, compared with E=1.00 and R=1.00, with 16 types, for the non-fluorescent AFLP protocol. The study demonstrated the potential of fAFLP for typing strains of L. pneumophila sg 1 on all three platforms; however, inter-platform comparison of fAFLP data was not achieved. fAFLP analysis may have a role in the fingerprinting of multiple isolates during Legionella outbreak investigations, but further work is required before type designations and identification libraries can be developed.

  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. 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.

  13. 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.

  14. 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.

  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. 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.

  17. Generation of fluorescent CdSe nanocrystals by short-pulse laser fragmentation

    NASA Astrophysics Data System (ADS)

    Zholudov, Yu. T.; Sajti, C. L.; Slipchenko, N. N.; Chichkov, B. N.

    2015-12-01

    A simple liquid-phase laser fragmentation approach, resulting in the rapid transformation of CdSe microcrystals into colloidal quantum dots (QDs), is presented. Laser fragmentation is achieved by irradiating a CdSe suspension in dimethylformamide with intense infrared, picosecond laser pulses followed by surface passivation with oleylamine or different types of phosphines. The generated QDs reveal perfect colloidal stability preventing agglomeration and precipitation, and show characteristic QD absorption and fluorescence characteristics, whereas their emission properties strongly depend on the surface states and applied capping ligands. These QDs show distinct photoemission under 405-nm single-photon and 800-nm multi-photon excitations in the 560- to 610-nm spectral region corresponding to the QDs size of about 1.5-2 nm in diameter which is confirmed by transmission electron microscopy.

  18. 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.

  19. 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

  20. Ultrasensitive detection of genetically modified plants by fluorescence cross-correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Junfeng; Xing, Da; Chen, Tongsheng; Liu, Jinfeng

    2006-09-01

    In this study, a novel method for the direct detection of GMP without amplified by the general method of PCR is firstly presented and proved by experiments. In our method, fluorescence correlation spectroscopy, cleaving nucleic acid by restriction endonuclease and two nucleic acid probe hybridization techniques are combined to distinguish the caulifiower mosaic virus (CaMV) 35S promoter and determine whether samples contain genetically modified components. The detection principle is as follows: firstly two restriction endonucleases FOKI and BsrDlare used to cleave the genomic DNA and the 169bp fragments of CaMV 35S promoter are retrieved; secondly, two nucleic acid probes labeled by Rhodamine Green and y5 dyes respectively hybridize with cleaved 169bp fragments of CaMV 35S promoter; thirdly, the hybridization products simultaneously with two dye-labeled probes are detected by fluorescence cross-correlation spectroscopy and GMP is distinguished. As the detection and analysis by FCS can be performed at the level of single molecule, there is no need for any type of amplification. Genetically modified tobaccos are measured by this method. The results indicate this method can detect CaMV 35S promoter of GMP exactly and the sensitivity can be down to 3.47X10 -10M. Because no any type of amplification is involved, this method can avoid the non-specffic amplification and false-positive problems of PCR, Due to its high-sensitivity, simplicity, reliability and little need for sample amounts, this method promises to be a highly effective detection method for GMP.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  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. 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 +}.

  7. 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.

  8. 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.

  9. [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.

  10. 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.

  11. 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.

  12. Characterization of Titratable Amphiphiles in Lipid Membranes by Fluorescence Spectroscopy.

    PubMed

    Pierrat, Philippe; Lebeau, Luc

    2015-11-17

    Understanding the ionization behavior of lipid membranes is a key parameter for successful development of lipid-based drug delivery systems. Accurate determination of the ionization state of a titratable species incorporated in a lipid bilayer however requires special care. Herein we investigated the behavior of titratable lipids in liposomes by fluorescence spectroscopy and determined which extrinsic parameters-i.e., besides those directly related to their molecular structure-determine their ionization state. Two fluorescent dyes, TNS and R18, have been used to investigate basic and acidic titratable lipids, respectively. Our results suggest that the titration behavior of the ionizable lipid in the membrane is more sensitive to the composition of the membrane and to its physical state than to the presence of solutes in the aqueous phase. Essentially overlooked in earlier studies on ionizable lipid assemblies, the concentration of the titratable lipid in the membrane was found to have a major effect on the ionization state of the lipid polar head. This may result in a shift in the apparent pKa value which may be as large as two pKa units and cannot be satisfactorily predicted.

  13. 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.

  14. [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.

  15. 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.

  16. 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.

  17. 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.

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

    PubMed Central

    Thompson, N L; Axelrod, D

    1983-01-01

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

  19. Membrane effects of N-terminal fragment of apolipoprotein A-I: a fluorescent probe study.

    PubMed

    Trusova, Valeriya; Gorbenko, Galyna; Girych, Mykhailo; Adachi, Emi; Mizuguchi, Chiharu; Sood, Rohit; Kinnunen, Paavo; Saito, Hiroyuki

    2015-03-01

    The binding of monomeric and aggregated variants of 1-83 N-terminal fragment of apolipoprotein A-I with substitution mutations G26R, G26R/W@8, G26R/W@50 and G26R/W@72 to the model lipid membranes composed of phosphatidylcholine and its mixture with cholesterol has been investigated using fluorescent probes pyrene and Laurdan. Examination of pyrene spectral behavior did not reveal any marked influence of apoA-I mutants on the hydrocarbon region of lipid bilayer. In contrast, probing the membrane effects by Laurdan revealed decrease in the probe generalized polarization in the presence of aggregated proteins. suggesting that oligomeric and fibrillar apoA-I species induce increase in hydration degree and reduction of lipid packing density in the membrane interfacial region. These findings may shed light on molecular details of amyloid cytotoxicity.

  20. Fluorescence Correlation Spectroscopy: A Tool to Study Protein Oligomerization and Aggregation In Vitro and In Vivo.

    PubMed

    Sahoo, Bankanidhi; Drombosky, Kenneth W; Wetzel, Ronald

    2016-01-01

    Fluorescence correlation spectroscopy (FCS) is a highly sensitive analytical technique used to measure dynamic molecular parameters, such as diffusion time (from which particle size can be calculated), conformation, and concentration of fluorescent molecules. It has been particularly powerful in characterizing size distributions in molecular associations (e.g., dimer/multimer formation) both in well-behaved thermodynamically equilibrated systems in vitro as well as in more complex environments in vivo. Protein aggregation reactions like amyloid formation, in contrast, are complex, often involving a series of uniquely structured aggregation intermediates appearing at different time scales. Nonetheless, FCS can be used in appropriate cases to characterize the early stages of some aggregation reactions. Here are described step-by-step protocols and experimental procedures for the study of molecular complex formation in aggregation systems as observed in simple buffer systems, cell extracts, and living cells. The methods described are illustrated with examples from studies of the self-assembly of huntingtin fragments, but in principle can be adapted for any aggregating system.

  1. Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes.

    PubMed

    Nowicka, Anna; Grzebelus, Ewa; Grzebelus, Dariusz

    2012-03-01

    Carrot (Daucus carota L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  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. 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.

  11. 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

  12. 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.

  13. 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.

  14. γ -ray spectroscopy of fission fragments produced in 208Pb(18O ,f )

    NASA Astrophysics Data System (ADS)

    Banerjee, P.; Ganguly, S.; Pradhan, M. K.; Moin Shaikh, Md.; Sharma, H. P.; Chakraborty, S.; Palit, R.; Pillay, R. G.; Nanal, V.; Saha, S.; Sethi, J.; Biswas, D. C.

    2015-08-01

    Prompt gamma-ray spectroscopy of fission fragments produced in the heavy-ion induced fusion-fission reaction 208Pb(18O,f ) at E =90 MeV has been performed. The relative isotopic yields of the fission fragments and the fragment mass distribution have been studied. Structures in the mass distribution have been discussed in the light of earlier results. Relative yields of several odd-A isotopes of Mo, Ru, Pd, and Cd and the odd-A isotones with N =62 and 64 have been studied along with the yields of the neighboring even-Z , even-N fragments and correlated to nuclear structural effects. The average total neutron multiplicity during fission has been measured to be 5.48 ±0.59 . The level schemes of the two neutron-rich nuclei 110Pd and 116Cd have been studied from γ -ray triple coincidence data. A large number of transitions, previously reported only from β -decay studies, have been observed in 110Pd for the first time. The yrast band in 116Cd has been extended up to spin (16+). In addition, a rotational sequence built upon an excited 5- state in 116Cd has been observed up to (13-). The level schemes have been discussed in the context of existing results, both experimental and theoretical, in the literature.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Differentiation of Salmonella enteritidis isolates by fluorescent amplified fragment length polymorphism.

    PubMed

    Kober, Márcia Vargas; Abreu, Marina Bystronski; Bogo, Maurício Reis; Ferreira, Carlos Alexandre Sanchez; Oliveira, Sílvia Dias

    2011-01-01

    Salmonella Enteritidis is responsible for human gastroenteritis outbreaks worldwide, and the molecular characterization of isolates is an important tool for epidemiological studies. Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed on 31 Salmonella Enteritidis strains from South Brazil isolated from human, foods, swine, broiler carcasses, and other poultry-related samples to subtype isolates in comparison to pulsed-field gel electrophoresis (PFGE) analysis. Five strains of Salmonella Enteritidis from different geographical regions, Salmonella Enteritidis ATCC 13076, and four isolates of different Salmonella serovars were also tested. Among the 41 isolates tested, 96 polymorphic AFs and 40 distinct profiles were obtained, displaying a Simpson's index of diversity of 0.99; whereas the PFGE analysis presented 13 patterns and the resulting Simpson's index was 0.55. Nine FAFLP and seven PFGE clusters could be inferred based in Dice similarity coefficient. FAFLP clustering readily identified different serotypes of Salmonella but did not distinguish isolates epidemiologically nonrelated or distinct phage types. Therefore, these results indicate that FAFLP is a rapid method for epidemiological investigations of Salmonella outbreaks, presenting a high discriminatory power for subtyping of Salmonella Enteritidis.

  1. Genomic diversity amongst Vibrio isolates from different sources determined by fluorescent amplified fragment length polymorphism.

    PubMed

    Thompson, F L; Hoste, B; Vandemeulebroecke, K; Swings, J

    2001-12-01

    The genomic diversity among 506 strains of the family Vibrionaceae was analysed using Fluorescent Amplified Fragments Length Polymorphisms (FAFLP). Isolates were from different sources (e.g. fish, mollusc, shrimp, rotifers, artemia, and their culture water) in different countries, mainly from the aquacultural environment. Clustering of the FAFLP band patterns resulted in 69 clusters. A majority of the actually known species of the family Vibrionaceae formed separate clusters. Certain species e.g. V. alginolyticus, V. cholerae, V. cincinnatiensis, V. diabolicus, V. diazotrophicus, V. harveyi, V. logei, V. natriegens, V. nereis, V. splendidus and V. tubiashii were found to be ubiquitous, whereas V. halioticoli, V. ichthyoenteri, V. pectenicida and V. wodanis appear to be exclusively associated with a particular host or geographical region. Three main categories of isolates could be distinguished: (1) isolates with genomes related (i.e. with > or =45% FAFLP pattern similarity) to one of the known type strains; (2) isolates clustering (> or =45% pattern similarity) with more than one type strain; (3) isolates with genomes unrelated (<45% pattern similarity) to any of the type strains. The latter group consisted of 236 isolates distributed in 31 clusters indicating that many culturable taxa of the Vibrionaceae remain as yet to be described.

  2. 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.

  3. 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.

  4. The Use of Fluorescent Fragment Length Analysis (PCR-FFL) in the Direct Diagnosis and Identification of Cutaneous Leishmania Species

    PubMed Central

    Tomás-Pérez, Míriam; Fisa, Roser; Riera, Cristina

    2013-01-01

    Leishmaniasis is a disease caused by different species belonging to the genus Leishmania. It presents different epidemiological and clinical features and requires the development of rapid, sensitive techniques to improve specific diagnosis. In this study, we compared the traditional technique of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with PCR-fluorescent fragment length analysis (PCR-FFL). Fluorescently tagged primers, designed in the rRNA fragment ITS-1 and 7SL region, were used to amplify fragments, which were later digested and whose sizes were accurately determined using an automated DNA sequencer. We validated the technique using 19 Leishmania strains from five cutaneous Leishmania species before testing 36 clinical samples: 23 skin biopsies and 13 skin scrapings/lesion exudates on filter paper. In real diagnostic, PCR-FFL has proved to be quick, accurate, and more sensitive (83.3% testing the ITS-1 fragment and 94.4% testing the 7SL) than PCR-RFLP analysis (75% and 80.6%). Filter papers improved the specific diagnosis in both techniques using non-invasive samples. PMID:23382161

  5. 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.

  6. 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.

  7. Fragmentation and conformation study of ephedrine by low- and high-resolution mass selective UV spectroscopy

    NASA Astrophysics Data System (ADS)

    Chervenkov, S.; Wang, P. Q.; Braun, J. E.; Neusser, H. J.

    2004-10-01

    The neurotransmitter molecule, ephedrine, has been studied by mass-selective low- and high-resolution UV resonance enhanced two-photon ionization spectroscopy. Under all experimental conditions we observed an efficient fragmentation upon ionization. The detected vibronic peaks in the spectrum are classified according to the efficiency of the fragmentation, which leads to the conclusion that there exist three different species in the molecular beam: ephedrine-water cluster and two distinct conformers. The two-color two-photon ionization experiment with a decreased energy of the second photon leads to an upper limit of 8.3 eV for the ionization energy of ephedrine. The high-resolution (70 MHz) spectrum of the strongest vibronic peak in the spectrum measured at the fragment (m/z=58) mass channel displays a pronounced and rich rotational structure. Its analysis by the use of a specially designed computer-aided rotational fit process yields accurate rotational constants for the S0 and S1 states and the transition moment ratio, providing information on the respective conformational structure.

  8. [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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding.

    PubMed

    Hamilton, P B; Lewis, M D; Cruickshank, C; Gaunt, M W; Yeo, M; Llewellyn, M S; Valente, S A; Maia da Silva, F; Stevens, J R; Miles, M A; Teixeira, M M G

    2011-01-01

    Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi, T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes.

  19. 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.

  20. 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.

  1. High-sensitivity capillary electrophoresis of double-stranded DNA fragments using monomeric and dimeric fluorescent intercalating dyes

    SciTech Connect

    Zhu, H.; Clark, S.M.; Benson, S.C.; Rye, H.S.; Glazer, A.N.; Mathies, R.A. )

    1994-07-01

    Fluorescence-detected capillary electrophoresis separations of [phi]X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium bromide, two propidium dye derivatives, oxazole yellow, thiazole orange, and a polycationic thiazole orange derivative) were determined. The thiazole orange dyes provide the most sensitive detection with limiting sensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from [phi]X174/HaeIII samples containing only 1-2 fg of this fragment per microliter. Separations of preformed DNA-dimeric dye complexes were also performed. The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophoresis with 9-aminoacridine (9AA) in the column and running buffers. 43 refs., 10 figs., 1 tab.

  2. 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.

  3. 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.

  4. 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.

  5. Application of confocal X-ray fluorescence micro-spectroscopy to the investigation of paint layers.

    PubMed

    Sun, Tianxi; Liu, Zhiguo; Wang, Guangfu; Ma, Yongzhong; Peng, Song; Sun, Weiyuan; Li, Fangzuo; Sun, Xuepeng; Ding, Xunliang

    2014-12-01

    A confocal micro X-ray fluorescence (MXRF) spectrometer based on polycapillary X-ray optics was used for the identification of paint layers. The performance of the confocal MXRF was studied. Multilayered paint fragments of a car were analyzed nondestructively to demonstrate that this confocal MXRF instrument could be used in the discrimination of the various layers in multilayer paint systems.

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

    SciTech Connect

    ,; Neill, M

    2012-07-01

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

  7. 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.

  8. Conformation-specific spectroscopy of peptide fragment ions in a low-temperature ion trap.

    PubMed

    Wassermann, Tobias N; Boyarkin, Oleg V; Paizs, Béla; Rizzo, Thomas R

    2012-06-01

    We have applied conformer-selective infrared-ultraviolet (IR-UV) double-resonance photofragment spectroscopy at low temperatures in an ion trap mass spectrometer for the spectroscopic characterization of peptide fragment ions. We investigate b- and a-type ions formed by collision-induced dissociation from protonated leucine-enkephalin. The vibrational analysis and assignment are supported by nitrogen-15 isotopic substitution of individual amino acid residues and assisted by density functional theory calculations. Under such conditions, b-type ions of different size are found to appear exclusively as linear oxazolone structures with protonation on the N-terminus, while a rearrangement reaction is confirmed for the a (4) ion in which the side chain of the C-terminal phenylalanine residue is transferred to the N-terminal side of the molecule. The vibrational spectra that we present here provide a particularly stringent test for theoretical approaches.

  9. 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.

  10. 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.

  11. 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.

  12. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. 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.

  15. 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

  16. 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.

  17. Fluorescent SSCP of overlapping fragments (FSSCP-OF): a highly sensitive method for the screening of mitochondrial DNA variation.

    PubMed

    Salas, A; Rasmussen, E M; Lareu, M V; Morling, N; Carracedo, A

    2001-12-27

    The mtDNA analysis (mtDNA) is increasingly being demanded for forensic purposes due to the fact that many times the use of standard nuclear marker fails to analyze degraded samples (such as bones) and specially for the analysis of hair shafts (a common sample in the crime scene). However, analysis of mtDNA sequencing implies a great lab effort when a high number of samples must be analyzed. The present work introduces a novel and reliable method for the screening of mtDNA variation in the first and second hypervariables (HV1 and HV2) regions which we have denominated fluorescent single strand conformation polymorphism (SSCP) of overlapping fragments (FSSCP-OF). FSSCP-OF is based on the basic theory of SSCP analysis and combines two complementary strategies: the use of PCR amplified overlapping fragments and fluorescent detection technology. The overlap region contains a high percentage (50%) of the d-loop mtDNA variation and for this reason, the probability to detect a polymorphic position by SSCP analysis is clearly increased in comparison to conventional SSCP methods due to the fact that the same polymorphic position is usually placed in a different "relative" position in the two overlapped fragments. The use of multicolor fluorescent technology allows also the multiplex amplification of overlapping fragment and its subsequent analysis in an automatic sequencer. We have analyzed 50 samples of unrelated individuals through the FSSCP-OF technique and we have found that using this methodology the probability to distinguish two samples with different sequences is close to 100%. FSSCP-OF has other important advantages with respect to previous screening methods, such as the automation and standardization of the protocols, which is of special interest for the forensic routine.

  18. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    PubMed

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

  19. 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.

  20. 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.

  1. Burst analysis spectroscopy: a versatile single-particle approach for studying distributions of protein aggregates and fluorescent assemblies.

    PubMed

    Puchalla, Jason; Krantz, Kelly; Austin, Robert; Rye, Hays

    2008-09-23

    Many essential cellular functions depend on the assembly and disassembly of macromolecular complexes. The size, form, and distribution of these assemblies can be heterogeneous and complex, rendering their detailed characterization difficult. Here we describe a simple non-correlation-based method capable of directly measuring population distributions at very low sample concentrations. Specifically, we exploit the highest signal-to-noise light bursts from single fluorescent particles transiting a confocal excitation spot to recursively determine the brightness and size distribution of complex mixtures of fluorescent objects. We refer to this method as burst analysis spectroscopy (BAS) and demonstrate the sensitivity of this technique by examining the free-solution, time-resolved distribution of assembled protein aggregates by using two fluorescently labeled proteins: the aggregation-prone, chaperonin-dependent, folding model protein ribulose-bisphosphate carboxylase/oxygenase (RuBisCO), and an amyloidogenic fragment of the yeast prion protein Sup35. We find that the assembly kinetics of both proteins display complex multimodal behavior not readily quantifiable with other methods.

  2. Burst analysis spectroscopy: A versatile single-particle approach for studying distributions of protein aggregates and fluorescent assemblies

    PubMed Central

    Puchalla, Jason; Krantz, Kelly; Austin, Robert; Rye, Hays

    2008-01-01

    Many essential cellular functions depend on the assembly and disassembly of macromolecular complexes. The size, form, and distribution of these assemblies can be heterogeneous and complex, rendering their detailed characterization difficult. Here we describe a simple non-correlation-based method capable of directly measuring population distributions at very low sample concentrations. Specifically, we exploit the highest signal-to-noise light bursts from single fluorescent particles transiting a confocal excitation spot to recursively determine the brightness and size distribution of complex mixtures of fluorescent objects. We refer to this method as burst analysis spectroscopy (BAS) and demonstrate the sensitivity of this technique by examining the free-solution, time-resolved distribution of assembled protein aggregates by using two fluorescently labeled proteins: the aggregation-prone, chaperonin-dependent, folding model protein ribulose-bisphosphate carboxylase/oxygenase (RuBisCO), and an amyloidogenic fragment of the yeast prion protein Sup35. We find that the assembly kinetics of both proteins display complex multimodal behavior not readily quantifiable with other methods. PMID:18780782

  3. Structural study of human growth hormone-releasing factor fragment (1?29) by vibrational spectroscopy

    NASA Astrophysics Data System (ADS)

    Carmona, P.; Molina, M.; Lasagabaster, A.

    1995-05-01

    The conformational structure of fragment 1-29 of human growth hormone releasing factor, hGHRF (1-29), in aqueous solution and in the solid state is investigated by infrared and Raman spectroscopy. The polypeptide backbone is found to be unordered in the solid state. However, the spectra of the peptide prepared as 5% (w/w) aqueous solutions show that approximately 28% of the peptide is involved in intermolecular β-sheet aggregation. The remainder of the peptide exists largely as disordered and β-sheet conformations with a small portion of α-helices. Tyrosine residues are found to be exposed to the solvent. The secondary structures are quantitatively examined through infrared spectroscopy, the conformational percentages being near those obtained by HONDAet al. [ Biopolymers31, 869 (1991)] using circular dichroism. The fast hydrogen/deuterium exchange in peptide groups and the absence of any NMR sign indicative of ordered structure [ G. M. CLOREet al., J. Molec. Biol.191, 553 (1986)] support that the solution conformations of the non-aggregated peptide interconvert in dynamic equilibrium. Some physiological advantages that may derive from this conformational flexibility are also discussed

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

    PubMed Central

    Zhou, Xiaoming; Xing, Da; Tang, Yonghong; Chen, Wei R.

    2009-01-01

    The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids. PMID:19956680

  13. PCR-free detection of genetically modified organisms using magnetic capture technology and fluorescence cross-correlation spectroscopy.

    PubMed

    Zhou, Xiaoming; Xing, Da; Tang, Yonghong; Chen, Wei R

    2009-11-26

    The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 microg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.

  14. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    SciTech Connect

    Al-Gubory, Kais H. . E-mail: kais.algubory@jouy.inra.fr

    2005-11-01

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. Picosecond time-resolved fluorescence spectroscopy of phytochrome and stentorin

    NASA Astrophysics Data System (ADS)

    Song, Pill-Soon

    1991-05-01

    Phytochrome is a tetrapyrrole chromoprotein. It serves as a sensitive photosensor for red lightmediated gene expression and other developmental/morphological responses in plants. In this paper photochemical dynamics of the phytochrome molecule have been described in terms of photoisomerization of the tetrapyrrole chromophore in its singlet excited state and subsequent thermal processes in the Pr Pfr phototransformation of phytochrome. Stentorin acts as the photosensor molecule in the ciliate Stentor coeruleus. This unicellular protozoan is most sensitive to red light (610-620 urn). Stentor also senses the direction of light propagation as evidenced by their light-avoiding and negative phototactic swimming behaviors. This aneural photosensory phenomenon is triggered by the photoreceptor stentorin. The possible involvement of a light-induced transient proton release from the photoreceptor as the primary mechanism of light-signal processing has been discussed on the basis of picosecond fluorescence decays and time-resolved fluorescence spectra of stentorin in solution. An initial sensory signal generated by the primary photoprocess of stentorin then triggers subsequent transduction steps that include calcium ion influx from the extracellular medium. Calcium ion influx from the extracellular medium to the cytosol causes the Stentor cell to reverse its ciliary beating and subsequently steer away from the light trap. II.

  2. 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.

  3. 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

  4. Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycocluster to lectin and tetanus toxin c-fragment

    NASA Astrophysics Data System (ADS)

    Okada, Tomoko; Minoura, Norihiko

    2010-02-01

    We have developed a fluorescent ruthenium metalloglycocluster as a powerful molecular probe for evaluating a binding event between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analysis. The fluorescent ruthenium metalloglycoclusters, [Ru(bpy-2Gal)3] and [Ru(bpy-2Glc)3], possess clustered galactose and glucose surrounding the ruthenium center. Changes in FE and FP of these metalloglycoclusters were measured by adding each lectin (Peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), Concanavalin A (ConA), or Wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA, the FE spectrum of [Ru(bpy- 2Gal)3] showed new emission peak and the FP value of [Ru(bpy-2Gal)3] increased. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] showed new emission peak and the FP value increased following the addition of ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were proved by the FE and FP measurement. From nonlinear least-squares fitting, dissociation constants (Kd) of [Ru(bpy-2Gal)3] to PNA was 6.1 μM, while the Kd values of [Ru(bpy)2(bpy-2Gal)] to PNA was ca. 10-4 M. Therefore, the clustered carbohydrates were proved to increase affinity to lectins. Furthermore, the FP measurements proved specific binding of [Ru(bpy-2Gal)3] to TCF.

  5. Measuring the Quenching of no Fluorescence Produced from the Excitation of Photo-Fragmented Nitrobenzene Using a Picosecond Laser.

    NASA Astrophysics Data System (ADS)

    Lue, Christopher J.; Tanjaroon, Chakree; Johnson, J. Bruce; Reeve, Scott W.; Allen, Susan D.

    2013-06-01

    The military is interested in using spectroscopic methods to detect nitroaromatic compounds related to explosives. Upon absorption of a UV photon, nitrobenzene can dissociate into C_6H_5O and NO. Wynn, et al. have shown that looking at NO fluorescence from the photodissociated nitrobenzene could be a possible detection method. However, the fluorescence can easily be quenched by molecular oxygen and other constituents in air. We have measured fluorescence lifetimes of the nascent NO resulting from photo-fragmented nitrobenzene using a pulsed picosecond tunable laser (pulse width ≈15 ps) by means of a two-color process. In the two-color process, photons of a particular energy dissociated the nitrobenzene while photons of a different energy probed the A^2Σ^+← X^2Π_{(1/2,3/2)} NO band system between 225-260 nm. We have performed the measurements with different background pressures of He, N_2, and air. We present the results of these measurements which indicate considerable quenching of the NO fluorescence due to oxygen. Wynn, C. M.; Palmacci, S.; Kunz, R. R.; and Rothschild, M.Opt. Express, OSA, 2010, 18, 5399-5406

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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....

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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).

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Detection of Energetic Materials by Laser Photofragmentation/Fragment Detection and Pyrolysis/Laser-Induced Fluorescence

    DTIC Science & Technology

    2001-02-01

    Analysis." Applied Spectroscopy Reviews, vol. 31, pp. 1-72, 1996. 4. Huang, S. D., L. Kolaitis, and D. M. Lubman. "Detection Of Explosives Using Laser...Desorption/Mass Spectrometry." Applied Spectroscopy , vol. 41, pp. 137 1-1376, 1987. 5. Riris, H., C. B. Carisle, D. F. McMillen, and D. E. Cooper...Photofragmentation/Ionization Spectrometry." Applied Spectroscopy , vol. 47, no. 11, pp. 1907-1912, 1993. 10. Wu, D., J. Singh, F. Yueh, and D. Monts. Ŗ,4,6

  5. 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.

  6. 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.

  7. Dual-color fluorescence cross-correlation spectroscopy on a planar optofluidic chip.

    PubMed

    Chen, A; Eberle, M M; Lunt, E J; Liu, S; Leake, K; Rudenko, M I; Hawkins, A R; Schmidt, H

    2011-04-21

    Fluorescence cross-correlation spectroscopy (FCCS) is a highly sensitive fluorescence technique with distinct advantages in many bioanalytical applications involving interaction and binding of multiple components. Due to the use of multiple beams, bulk optical FCCS setups require delicate and complex alignment procedures. We demonstrate the first implementation of dual-color FCCS on a planar, integrated optofluidic chip based on liquid-core waveguides that can guide liquid and light simultaneously. In this configuration, the excitation beams are delivered in predefined locations and automatically aligned within the excitation waveguides. We implement two canonical applications of FCCS in the optofluidic lab-on-chip environment: particle colocalization and binding/dissociation dynamics. Colocalization is demonstrated in the detection and discrimination of single-color and double-color fluorescently labeled nanobeads. FCCS in combination with fluorescence resonance energy transfer (FRET) is used to detect the denaturation process of double-stranded DNA at nanomolar concentration.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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).

  16. 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.

  17. 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.

  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. 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.

  20. New Homogeneous Standards by Atomic Layer Deposition for Synchrotron X-ray Fluorescence and Absorption Spectroscopies.

    SciTech Connect

    Butterworth, A.L.; Becker, N.; Gainsforth, Z.; Lanzirotti, A.; Newville, M.; Proslier, T.; Stodolna, J.; Sutton, S.; Tyliszczak, T.; Westphal, A.J.; Zasadzinski, J.

    2012-03-13

    Quantification of synchrotron XRF analyses is typically done through comparisons with measurements on the NIST SRM 1832/1833 thin film standards. Unfortunately, these standards are inhomogeneous on small scales at the tens of percent level. We are synthesizing new homogeneous multilayer standards using the Atomic Layer Deposition technique and characterizing them using multiple analytical methods, including ellipsometry, Rutherford Back Scattering at Evans Analytical, Synchrotron X-ray Fluorescence (SXRF) at Advanced Photon Source (APS) Beamline 13-ID, Synchrotron X-ray Absorption Spectroscopy (XAS) at Advanced Light Source (ALS) Beamlines 11.0.2 and 5.3.2.1 and by electron microscopy techniques. Our motivation for developing much-needed cross-calibration of synchrotron techniques is borne from coordinated analyses of particles captured in the aerogel of the NASA Stardust Interstellar Dust Collector (SIDC). The Stardust Interstellar Dust Preliminary Examination (ISPE) team have characterized three sub-nanogram, {approx}1{micro}m-sized fragments considered as candidates to be the first contemporary interstellar dust ever collected, based on their chemistries and trajectories. The candidates were analyzed in small wedges of aerogel in which they were extracted from the larger collector, using high sensitivity, high spatial resolution >3 keV synchrotron x-ray fluorescence spectroscopy (SXRF) and <2 keV synchrotron x-ray transmission microscopy (STXM) during Stardust ISPE. The ISPE synchrotron techniques have complementary capabilities. Hard X-ray SXRF is sensitive to sub-fg mass of elements Z {ge} 20 (calcium) and has a spatial resolution as low as 90nm. X-ray Diffraction data were collected simultaneously with SXRF data. Soft X-ray STXM at ALS beamline 11.0.2 can detect fg-mass of most elements, including cosmochemically important oxygen, magnesium, aluminum and silicon, which are invisible to SXRF in this application. ALS beamline 11.0.2 has spatial resolution

  1. Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycoclusters to lectins and tetanus toxin C-fragment

    NASA Astrophysics Data System (ADS)

    Okada, Tomoko; Minoura, Norihiko

    2011-03-01

    We develop a fluorescent ruthenium metalloglycocluster for use as a powerful molecular probe in evaluating the binding between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analyses. Changes in the FE and FP of these metalloglycoclusters are measured following the addition of lectin [peanut agglutinin (PNA), Ricinus communis agglutinin 120, Concanavalin A (ConA), or wheat germ agglutinin] or tetanus toxin c-fragment (TCF). After the addition of PNA, the FE spectrum of [Ru(bpy-2Gal)3] shows a new emission peak and the FP value of [Ru(bpy-2Gal)3] increases. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] shows a new emission peak and the FP value increases on addition of ConA. Because other combinations of metalloglycoclusters and lectins show little change, specific binding of galactose to PNA and that of glucose to ConA are confirmed by the FE and FP measurements. Resulting dissociation constants (Kd) prove that the metalloglycoclusters with highly clustered carbohydrates show higher affinity for the respective lectins than those with less clustered carbohydrates. Furthermore, specific binding of [Ru(bpy-2Gal)3] to TCF was confirmed by the FP measurement.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  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. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

    2017-02-02

    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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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).

  20. Tea quality and classification evaluation using multi-wavelength light-emitting diodes induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Feng, Chao; Liu, Xuan; Yan, Chunsheng; He, Sailing; Mei, Liang

    2013-03-01

    In this paper, we demonstrated a detective system to evaluate the quality and classification of different tea samples based on multi-wavelength LED-induced fluorescence spectroscopy. By utilizing multiple excitation wavelengths, we obtained much more physical and chemical information from the detected samples than single excitation wavelength. By utilizing principal component analysis (PCA), we extracted the dominant features of the samples to classify and characterize the tea samples.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  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. 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.

  12. 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.

  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. Determination of flue gas alkali concentrations in fluidized-bed coal combustion by excimer-laser-induced fragmentation fluorescence

    SciTech Connect

    Hartinger, K.T.; Monkhouse, P.B.; Wolfrum, J.; Baumann, H.; Bonn, B.

    1994-12-31

    Gas-phase sodium concentrations were measured for the first time in situ in the flue gas of a fluidized-bed reactor by the excimer-laser-induced fragmentation fluorescence (ELIF) technique. This method involves using ArF-excimer laser light at 193 nm to simultaneously photodissociate the alkali compounds of interest and excite electronically the alkali atoms formed. The resulting fluorescence from Na (3{sup 2}P) atoms can he readily detected at 589 nm. Measured signals were converted to absolute concentrations using a calibration system that monitors alkali compounds under known conditions of temperature, pressure, and composition and rising the same optical setup as at the reactor. Several different coals were investigated under a specific set of reactor conditions at total pressures close to 1 bar. Sodium concentrations ranging from the sub-ppb region to 20 ppb were obtained, and a detection limit for sodium of 0.1 ppb under the present conditions was estimated. Over the course of the reactor program, contrasting concentration histories were observed for the two lignites and the hard coal investigated. In particular, significantly higher sodium concentrations were found for the hard coal, consistent with both the higher chlorine and sodium contents determined in the corresponding coal analysis.

  16. 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

  17. 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.

  18. 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.

  19. A wavelength dependent investigation of the indole photophysics via ionization and fragmentation pump-probe spectroscopies.

    PubMed

    Godfrey, T J; Yu, Hui; Biddle, Michael S; Ullrich, Susanne

    2015-10-14

    A wavelength dependent study investigating the low-lying (1)La and (1)Lb states, both possessing (1)ππ* character, and the (1)πσ* state in the deactivation process of indole is presented here. Relaxation dynamics following excitation at 241, 250, 260, 270, 273, and 282 nm are examined using three gas-phase, pump-probe spectroscopic techniques: (1) hydrogen atom (H-atom) time-resolved kinetic energy release (TR-KER), (2) time-resolved photoelectron spectroscopy (TR-PES), and (3) time-resolved ion yield (TR-IY). Applied in combination, a more complete picture of the indole relaxation dynamics may be gleaned. For instance, TR-PES experiments directly observe all relaxation pathways by probing the evolution of the excited states following photoexcitation; whereas, TR-KER measurements indirectly, yet specifically, probe for (1)πσ*-state activity through the detection of H-atoms eliminated along the indole nitrogen-hydrogen (N-H) stretch coordinate-a possible outcome of (1)πσ*-state relaxation in indole. In addition, mass information obtained via TR-IY monitors fragmentation dynamics that may occur within the neutral electronically excited and/or cationic states. The work herein assesses the onset and importance of the (1)πσ* state at various pump wavelengths by systematically tuning across the ultraviolet absorption spectrum of indole with a particular focus on those pump wavelengths longer than 263 nm, where the involvement of the (1)πσ* state is under current debate. As far as this experimental work is concerned, there does not appear to be any significant involvement by the (1)πσ* state in the indole relaxation processes following excitation at 270, 273, or 282 nm. This investigation also evaluates the primary orbital promotions contributing to the (1)La, (1)Lb, and (1)πσ* transitions based on ionization preferences observed in TR-PES spectra. Relaxation time constants associated with dynamics along these states are also reported for excitation at all

  20. 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

  1. 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.

  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. 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.

  6. 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.

  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. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  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. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Factors affecting quantification of total DNA by UV spectroscopy and PicoGreen fluorescence.

    PubMed

    Holden, Marcia J; Haynes, Ross J; Rabb, Savelas A; Satija, Neena; Yang, Kristina; Blasic, Joseph R

    2009-08-26

    The total amount of DNA in a preparation extracted from tissues can be measured in several ways, each method offering advantages and disadvantages. For the sake of accuracy in quantitation, it is of interest to compare these methodologies and determine if good correlation can be achieved between them. Different answers can also be clues to the physical state of the DNA. In this study, we investigated the lack of correlation between ultraviolet (UV) absorbance and fluorescent (PicoGreen) measurements of the concentration of DNAs isolated from plant tissues. We found that quantitation based on the absorbance-based method correlated with quantitation based on phosphorus content, while the PicoGreen-based method did not. We also found evidence of the production of single-stranded DNA under conditions where the DNA was not fragmented into small pieces. The PicoGreen fluorescent signal was dependent on DNA fragment size but only if the DNA was in pure water, while DNA in buffer was much less sensitive. Finally, we document the high sensitivity of the PicoGreen assays to the detergent known as CTAB (cetyldimethylethylammonium bromide). The CTAB-based method is highly popular for low-cost DNA extraction with many published variations for plant and other tissues. The removal of residual CTAB is important for accurate quantitation of DNA using PicoGreen.

  13. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations

    PubMed Central

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I.; Kudla, Jörg

    2014-01-01

    Summary The analyses of protein-protein interactions is crucial for understanding cellular processes including signal transduction, protein trafficking and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms, and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  14. 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.

  15. [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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  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. 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.

  5. Spectroscopy of exotic 123,125Ag produced in fragmentation reactions

    NASA Astrophysics Data System (ADS)

    Stefanescu, I.; Walters, W. B.; Hoteling, N.; Tomlin, B.; Mantica, P. F.; Pereira, J.; Becerril, A.; Fleckenstein, T.; Hecht, A. A.; Lorusso, G.; Quinn, M.; Pinter, J. S.; Stoker, J. B.

    2008-04-01

    We extended the experimental knowledge in the mass-region around 132Sn by identifying the decay of high-spin isomers in the exotic odd-mass 123,125Ag. The two isotopes were produced at the NSCL laboratory by projectile fragmentation of a 136Xe beam at 120MeV/u directed onto a thick Be target. The NSCL Beta Counting System, was used identify secondary beam fragments. Prompt and delayed gamma-rays following the deexcitation of the fragments were detected with the SEGA array. Partial level schemes for 123,125Ag are proposed for the first time. The observed states show single-particle characteristics, indicating strong Z=50 and N=82 shell gaps and also support the proposed weakened nucleon-nucleon interaction in this mass-region.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  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. 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.

  12. 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.

  13. 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%.

  14. 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.

  15. [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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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%.

  2. 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.

  3. 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.

  4. 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.

  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. 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

  7. ImmunoPET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer with a Dual-Labeled Bispecific Antibody Fragment.

    PubMed

    Luo, Haiming; England, Christopher G; Goel, Shreya; Graves, Stephen A; Ai, Fanrong; Liu, Bai; Theuer, Charles P; Wong, Hing C; Nickles, Robert J; Cai, Weibo

    2017-03-24

    Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab' antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide ((64)Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF(+/+)) or PANC-1 (CD105/TF(-/-)) tumor xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of (64)Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4%ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0%ID/g; n = 4; p < 0.01) and ALT-836 (7.6 ± 3.7%ID/g; n = 4; p < 0.01) at 24 h postinjection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by 4-fold with TF blocking and 2-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1%ID/g; n = 4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated that the dual targeting with (64)Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like (64)Cu-NOTA-heterodimer-ZW800, may increase the overall tumor

  8. In vitro binding kinetics of DNA double strand break repair proteins Ku70/80 and DNA-PKcs quantified by fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Abdisalaam, Salim; Chen, David J.; Alexandrakis, George

    2012-02-01

    DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage that occurs in eukaryotic cells. There are two distinct pathways of repairing DSBs, homologous recombination (HR) and non-homologous end joining (NHEJ). In the NHEJ repairing pathway, DSB recognition and repair initiation is directed by the interaction of DNAbinding subunit Ku70/80 heterodimer with the DNA-PK protein catalytic subunit (DNA-PKcs). Mutations in these proteins result in repair stalling and eventual DNA misrepair that may lead to genomic instability. Studying the binding kinetics of these repair proteins is therefore important for understanding the conditions under which DSB repair stalls. Currently open questions are, what is the minimum DNA length that this complex needs to get a foothold onto a DSB and how tightly does DNA-PKcs bind onto the DNA-Ku70/80 complex. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) techniques have the potential to give information about the binding kinetics of DNA-protein and protein-protein interactions at the single-molecule level. In this work, FCS/FCCS measurements were performed to explore the minimum DNA base-pair (bp) length that Ku70/80 needed as a foothold to bind effectively onto the tips of different lengths of double-stranded DNA (dsDNA) fragments that mimic DSBs. 25 bp, 33 bp and 50 bp of dsDNA were used for these experiments and binding was studied as a function of salt concentration in solution. It was found that the 25 bp binding was weak even at physiological salt concentrations while the dissociation constant (Kd) remained constant for 33 and 50 bp dsDNA strand lengths. These studies indicated that the minimum binding length for the Ku70/8 is in the vicinity of 25 bp. The specificity of binding of Ku70/80 was proven by competitive binding FCCS experiments between Cy5-labeled DNA, GFP-Ku70/80 and titrations of unlabeled Ku70/80. Finally, using FCCS it was possible to estimate

  9. 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.

  10. Quantitative multiplex PCR of short fluorescent fragments for the detection of large intragenic POLG rearrangements in a large French cohort

    PubMed Central

    Rouzier, Cécile; Chaussenot, Annabelle; Serre, Valérie; Fragaki, Konstantina; Bannwarth, Sylvie; Ait-El-Mkadem, Samira; Attarian, Shahram; Kaphan, Elsa; Cano, Aline; Delmont, Emilien; Sacconi, Sabrina; de Camaret, Bénédicte Mousson; Rio, Marlène; Lebre, Anne-Sophie; Jardel, Claude; Deschamps, Romain; Richelme, Christian; Pouget, Jean; Chabrol, Brigitte; Paquis-Flucklinger, Véronique

    2014-01-01

    Polymerase gamma (POLG) is the gene most commonly involved in mitochondrial disorders with mitochondrial DNA instability and causes a wide range of diseases with recessive or dominant transmission. More than 170 mutations have been reported. Most of them are missense mutations, although nonsense mutations, splice-site mutations, small deletions and insertions have also been identified. However, to date, only one large-scale rearrangement has been described in a child with Alpers syndrome. Below, we report a large cohort of 160 patients with clinical, molecular and/or biochemical presentation suggestive of POLG deficiency. Using sequencing, we identified POLG variants in 22 patients (18 kindreds) including five novel pathogenic mutations. Two patients with novel mutations had unusual clinical presentation: the first exhibited an isolated ataxic neuropathy and the second was a child who presented with endocrine signs. We completed the sequencing step by quantitative multiplex PCR of short fluorescent fragments (QMPSF) analysis in 37 patients with either only one POLG heterozygous variant or a family history suggesting a dominant transmission. We identified a large intragenic deletion encompassing part of intron 21 and exon 22 of POLG in a child with refractory epilepsia partialis continua. In conclusion, we describe the first large French cohort of patients with POLG mutations, expanding the wide clinical and molecular spectrum observed in POLG disease. We confirm that large deletions in the POLG gene are rare events and we highlight the importance of QMPSF in patients with a single heterozygous POLG mutation, particularly in severe infantile phenotypes. PMID:23921535

  11. Quantitative multiplex PCR of short fluorescent fragments for the detection of large intragenic POLG rearrangements in a large French cohort.

    PubMed

    Rouzier, Cécile; Chaussenot, Annabelle; Serre, Valérie; Fragaki, Konstantina; Bannwarth, Sylvie; Ait-El-Mkadem, Samira; Attarian, Shahram; Kaphan, Elsa; Cano, Aline; Delmont, Emilien; Sacconi, Sabrina; Mousson de Camaret, Bénédicte; Rio, Marlène; Lebre, Anne-Sophie; Jardel, Claude; Deschamps, Romain; Richelme, Christian; Pouget, Jean; Chabrol, Brigitte; Paquis-Flucklinger, Véronique

    2014-04-01

    Polymerase gamma (POLG) is the gene most commonly involved in mitochondrial disorders with mitochondrial DNA instability and causes a wide range of diseases with recessive or dominant transmission. More than 170 mutations have been reported. Most of them are missense mutations, although nonsense mutations, splice-site mutations, small deletions and insertions have also been identified. However, to date, only one large-scale rearrangement has been described in a child with Alpers syndrome. Below, we report a large cohort of 160 patients with clinical, molecular and/or biochemical presentation suggestive of POLG deficiency. Using sequencing, we identified POLG variants in 22 patients (18 kindreds) including five novel pathogenic mutations. Two patients with novel mutations had unusual clinical presentation: the first exhibited an isolated ataxic neuropathy and the second was a child who presented with endocrine signs. We completed the sequencing step by quantitative multiplex PCR of short fluorescent fragments (QMPSF) analysis in 37 patients with either only one POLG heterozygous variant or a family history suggesting a dominant transmission. We identified a large intragenic deletion encompassing part of intron 21 and exon 22 of POLG in a child with refractory epilepsia partialis continua. In conclusion, we describe the first large French cohort of patients with POLG mutations, expanding the wide clinical and molecular spectrum observed in POLG disease. We confirm that large deletions in the POLG gene are rare events and we highlight the importance of QMPSF in patients with a single heterozygous POLG mutation, particularly in severe infantile phenotypes.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Branching out of single-molecule fluorescence spectroscopy: challenges for chemistry and influence on biology.

    PubMed

    Tinnefeld, Philip; Sauer, Markus

    2005-04-29

    In the last decade emerging single-molecule fluorescence-spectroscopy tools have been developed and adapted to analyze individual molecules under various conditions. Single-molecule-sensitive optical techniques are now well established and help to increase our understanding of complex problems in different disciplines ranging from materials science to cell biology. Previous dreams, such as the monitoring of the motility and structural changes of single motor proteins in living cells or the detection of single-copy genes and the determination of their distance from polymerase molecules in transcription factories in the nucleus of a living cell, no longer constitute unsolvable problems. In this Review we demonstrate that single-molecule fluorescence spectroscopy has become an independent discipline capable of solving problems in molecular biology. We outline the challenges and future prospects for optical single-molecule techniques which can be used in combination with smart labeling strategies to yield quantitative three-dimensional information about the dynamic organization of living cells.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  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. Formation and fragmentation of radical peptide anions: insights from vacuum ultra violet spectroscopy.

    PubMed

    Brunet, Claire; Antoine, Rodolphe; Dugourd, Philippe; Canon, Francis; Giuliani, Alexandre; Nahon, Laurent

    2012-02-01

    We have studied the photodissociation of gas-phase deprotonated caerulein anions by vacuum ultraviolet (VUV) photons in the 4.5 to 20 eV range, as provided by the DESIRS beamline at the synchrotron radiation facility SOLEIL (France). Caerulein is a sulphated peptide with three aromatic residues and nine amide bonds. Electron loss is found to be the major relaxation channel at every photon energy. However, an increase in the fragmentation efficiency (neutral losses and peptide backbone cleavages) as a function of the energy is also observed. The oxidized ions, generated by electron photodetachment were further isolated and activated by collision (CID) in a MS(3) scheme. The branching ratios of the different fragments observed by CID as a function of the initial VUV photon energy are found to be independent of the initial photon energy. Thus, there is no memory effect of the initial excitation energy on the fragmentation channels of the oxidized species on the time scale of our tandem MS experiment. We also report photofragment yields as a function of photon energy for doubly deprotonated caerulein ions, for both closed-shell ([M-2H](2-)) non-radical ions and open-shell ([M-3H](2-•)) radical ions. These latter ions are generated by electron photodetachment from [M-3H](3-) precursor ions. The detachment yield increases monotonically with the energy with the appearance of several absorption bands. Spectra for radical and non-radical ions are quite similar in terms of observed bands; however, the VUV fragmentation yield is enhanced by the presence of a radical in caerulein peptides.

  3. Formation and Fragmentation of Radical Peptide Anions: Insights from Vacuum Ultra Violet Spectroscopy

    NASA Astrophysics Data System (ADS)

    Brunet, Claire; Antoine, Rodolphe; Dugourd, Philippe; Canon, Francis; Giuliani, Alexandre; Nahon, Laurent

    2012-02-01

    We have studied the photodissociation of gas-phase deprotonated caerulein anions by vacuum ultraviolet (VUV) photons in the 4.5 to 20 eV range, as provided by the DESIRS beamline at the synchrotron radiation facility SOLEIL (France). Caerulein is a sulphated peptide with three aromatic residues and nine amide bonds. Electron loss is found to be the major relaxation channel at every photon energy. However, an increase in the fragmentation efficiency (neutral losses and peptide backbone cleavages) as a function of the energy is also observed. The oxidized ions, generated by electron photodetachment were further isolated and activated by collision (CID) in a MS3 scheme. The branching ratios of the different fragments observed by CID as a function of the initial VUV photon energy are found to be independent of the initial photon energy. Thus, there is no memory effect of the initial excitation energy on the fragmentation channels of the oxidized species on the time scale of our tandem MS experiment. We also report photofragment yields as a function of photon energy for doubly deprotonated caerulein ions, for both closed-shell ([M-2H]2-) non-radical ions and open-shell ([M-3H]2-•) radical ions. These latter ions are generated by electron photodetachment from [M-3H]3- precursor ions. The detachment yield increases monotonically with the energy with the appearance of several absorption bands. Spectra for radical and non-radical ions are quite similar in terms of observed bands; however, the VUV fragmentation yield is enhanced by the presence of a radical in caerulein peptides.

  4. Interaction of bombesin and its fragments with gold nanoparticles analyzed using surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Tąta, Agnieszka; Szkudlarek, Aleksandra; Kim, Younkyoo; Proniewicz, Edyta

    2017-02-01

    This work demonstrates the application of commercially available stable surface composed of gold nanograins with diameters ranging from 70 to 226 nm deposited onto silicon wafer for surface-enhanced Raman scattering investigations of biologically active compounds, such as bombesin (BN) and its fragments. BN is an important neurotransmitter involved in a complex signaling pathways and biological responses; for instance, hypertensive action, contractive on uterus, colon or ileum, locomotor activity, stimulation of gastric and insulin secretion as well as growth promotion of various tumor cell lines, including: lung, prostate, stomach, colon, and breast. It has also been shown that 8-14 BN C-terminal fragment partially retains the biological activity of BN. The SERS results for BN and its fragment demonstrated that (1) three amino acids from these peptides sequence; i.e., L-histidine, L-methionine, and L-tryptophan, are involved in the interaction with gold coated silicon wafer and (2) the strength of these interactions depends upon the aforementioned amino acids position in the peptide sequence.

  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. 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.

  7. 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.

  8. 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

  9. 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).

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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].

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. [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.

  2. 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.

  3. 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.

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

    NASA Astrophysics Data System (ADS)

    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.

  5. 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.

  6. 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.

  7. 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.

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

    PubMed

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

    2016-07-02

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

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

    PubMed Central

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

    2016-01-01

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

  10. 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 that utilize fluorescence resonance energy transfer to separate signals from different subpopulations. I also describe experiments in which 2D FS was performed using ultraviolet excitation to determine the conformation of a dinucleotide of a fluorescent analogue of the nucleic acid base adenine. I discuss experiments on porphyrin dimers in which 2D FS was used as a probe of excited state dynamics. Finally, I present model calculations for a proposed variation of 2D FS in which entangled photons would be used as the excitation source. These calculations suggest that this approach has the potential to yield significantly narrower spectral lineshapes than conventional 2D FS. These experiments and calculations yield new insight into the systems investigated and establish a `toolbox' of variations of 2D FS that can be used to gain as much information as possible from experiments on challenging systems such as protein-DNA complexes.

  11. Hemiparasite--host plant interactions in a fragmented landscape assessed via imaging spectroscopy and LiDAR.

    PubMed

    Barbosa, Jomar M; Sebástian-González, Esther; Asner, Gregory P; Knapp, David E; Anderson, Christopher; Martin, Roberta E; Dirzo, Rodolfo

    2016-01-01

    Species interactions are susceptible to anthropogenic changes in ecosystems, but this has been poorly investigated in a spatially explicit manner in the case of plant parasitism, such as the omnipresent hemiparasitic mistletoe-host plant interactions. Analyzing such interactions at a large spatial scale may advance our understanding of parasitism patterns over complex landscapes. Combining high-resolution airborne imaging spectroscopy and LiDAR, we studied hemiparasite incidence within and among tree host stands to examine the prevalence and spatial distribution of hemiparasite load in ecosystems. Specifically, we aimed to assess: (1) detection accuracy of mistletoes on their oak hosts; (2) hemiparasitism prevalence within host tree canopies depending on tree height, and (3) spatial variation in hemiparasitism across fragmented woodlands, in a low-diversity mediterranean oak woodland in California, USA. We identified mistletoe infestations with 55-96% accuracy, and detected significant differences in remote-sensed spectra between oak trees with and without mistletoe infestation. We also found that host canopy height had little influence on infestation degree, whereas landscape-level variation showed consistent; non-random patterns: isolated host trees had twice the infestation load than did trees located at the core of forest fragments. Overall, we found that canopy exposure (i.e., lower canopy density or proximity to forest edge) is more important than canopy height for mistletoe infestation, and that by changing landscape structure, parasitic prevalence increased with woodland fragmentation. We conclude that reducing fragmentation in oak woodlands will minimize anthropogenic impact on mistletoe infestation at the landscape level. We argue that advanced remote sensing technology can provide baselines to quantitatively analyze and monitor parasite-host trajectories in light of global environmental change, and that this is a promising approach to be further tested

  12. Fluorescence spectroscopy: a promising tool for gear-oil condition monitoring

    NASA Astrophysics Data System (ADS)

    Dorigo, Daniel D.; Wiesent, Benjamin R.; Simsek, Özlem; Pérez Grassi, A.; Koch, Alexander W.

    2012-04-01

    Wind power is one of the most promising green energy sources, especially when produced in offshore power plants. Corrective operations in wind turbines cause a considerable part of the maintenance costs of such plants. One preventive action for reducing such operations is the periodic off-line control of oil samples from the wind turbines. The time delay between sampling and availability of the results is a major disadvantage of this kind of controlling. In-situ condition monitoring is a solution to this problem. In-situ monitoring allows real time detection of random, time discrete events, thus enabling a better scheduling of preventive actions and reducing costs and downtime. Fluorescence spectroscopy is a complementary technique to absorption spectroscopy. Due to absorption of UV or visible light, the electrons of specific molecules are excited from a ground electronic state to a vibrational state of higher energy. By collision with other molecules, the excited electron looses a part of the acquired energy and relaxes to a lower vibrational state. The remaining acquired energy is emitted during the electron's transition to the ground state. The resulting frequency shift between excitation and emission energy, known as Stokes shift, is unique and characteristic for each active molecule. In this paper gear-oil condition monitoring based on fluorescence spectroscopy is proposed. Three typical commercial gear-oils for wind turbines were studied. The spectra gained by UV excitation of the samples were analyzed by means of partial least square (PLS) regression. Good prediction results were obtained for the total acid number (TAN). The latter is a measure for the oil acidity and is considered to be a proxy variable for oil age. Other parameters delivering information about gear-oil additive depletion and the related oil aging condition, like phosphor, sulfur and molybdenum concentration, were also analyzed.

  13. Characterization and matching of oil samples using fluorescence spectroscopy and parallel factor analysis.

    PubMed

    Christensen, Jan H; Hansen, Asger B; Mortensen, John; Andersen, Ole

    2005-04-01

    A novel approach for matching oil samples by fluorescence spectroscopy combined with three-way decomposition of spectra is presented. It offers an objective fingerprinting based on the relative composition of polycyclic aromatic compounds (PACs) in oils. The method is complementary to GC-FID for initial screening of oil samples but can also be used for prescreening in the field, onboard ships, using a portable fluorescence spectrometer. Parallel factor analysis (PARAFAC) was applied to fluorescence excitation-emission matrixes (EEMs) of heavy fuel oils (HFOs), light fuel oils, lubricating oils, crude oils, unknown oils, and a sample collected in the spill area two weeks after the Baltic Carrier oil spill (Denmark, 2001). A total of 112 EEMs were decomposed into a five-factor PARAFAC model using excitation wavelengths from 245 to 400 nm and emission wavelengths from 280 to 550 nm. The PARAFAC factors were compared to EEMs of PAC standards with two to five rings, and the comparisons indicate that each of the factors can be related to a mixture of PACs with similar fluorescence characteristics: a mixture of naphthalenes and dibenzothiophenes, fluorenes, phenanthrenes, chrysenes, and five-ring PACs, respectively. Oils were grouped in score plots according to oil type. Except for HFOs and crude oils, the method easily discriminated between the four oil types. Minor overlaps of HFOs and crude oils were observed along all five PARAFAC factors, and the variability of crude oils was large along factor 2 due to a varying content of five-ring PACs. The spill sample was correctly assigned as a HFO with similar PAC pattern as oil from the cargo tank of the Baltic Carrier by comparing the correlation coefficient of scores for the oil spill sample and possible source oils (i.e., oils in the database).

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

    PubMed

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

    2015-10-01

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

  15. Evaluation of the overall quality of olive oil using fluorescence spectroscopy.

    PubMed

    Guzmán, Elena; Baeten, Vincent; Pierna, Juan Antonio Fernández; García-Mesa, José A

    2015-04-15

    The fluorescence spectra of some olive oils were examined in their natural and oxidised state, with wavelength range emissions of 300-800 nm and 300-400 nm used as excitation radiation. The fluorescence emissions were measured and an assessment was made of the relationship between them and the main quality parameters of olive oils, such as peroxide value, K232, K270 and acidity. These quality parameters (peroxide value, K232, K270 and acidity) are determined by laboratory methods, which though not too sophisticated, they are required solvents and materials as well as time consuming and sample preparation; there is a need for rapid analytical techniques and a low-cost technology for olive oil quality control. The oxidised oils studied had a strong fluorescence band at 430-450 nm. Extra virgin olive oil gave a different but interesting fluorescence spectrum, composed of three bands: one low intensity doublet at 440 and 455 nm; one strong band at 525 nm; and one of medium intensity at 681 nm. The band at 681 nm was identified as the chlorophyll band. The band at 525 nm was derived, at least partially, from vitamin E. The results presented demonstrate the ability of the fluorescence technique, combined with multivariate analysis, to characterise olive oils on the basis of all the quality parameters studied. Prediction models were obtained using various methods, such as partial least squares (PLS), N-way PLS (N-PLS) and external validation, in order to obtain an overall evaluation of oil quality. The best results were obtained for predicting K270 with a root mean square (RMS) prediction error of 0.08 and a correlation coefficient obtained with the external validation of 0.924. Fluorescence spectroscopy facilitates the detection of virgin olive oils obtained from defective or poorly maintained fruits (high acidity), fruits that are highly degraded in the early stages (with a high peroxide value) and oils in advanced stages of oxidation, with secondary oxidation compounds

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

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

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

    PubMed

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

    2008-03-01

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

  18. The characterization of canvas painting by the Serbian artist Milo Milunović using X-ray fluorescence, micro-Raman and FTIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Damjanović, Lj.; Gajić-Kvaščev, M.; Đurđević, J.; Andrić, V.; Marić-Stojanović, M.; Lazić, T.; Nikolić, S.

    2015-10-01

    A canvas painting by Milo Milunović "The Inspiration of the poet" was studied by energy dispersive X-Ray fluorescence (EDXRF), micro-Raman and Fourier transform infrared (FTIR) spectroscopy in order to identify materials used by the artist and his painting technique. Study is perfomed combining in situ non-destructive method with the preparation and study of cross-section samples and raw fragments of the samples. Milo Milunović, an eminent painter from Balkan region, made a copy of the Nicolas Poussin's original painting in Louvre in 1926/27. Obtained results revealed following pigments on the investigated canvas painting: vermilion, minium, cobalt blue, ultramarine, lead white, zinc white, cadmium yellow, chrome-based green pigment and several earth pigments - red and yellow ocher, green earth and umber. Ground layer was made of lead white mixed with calcium carbonate.

  19. Comparative study of carbonaceous meteoritic fragments by micro-Raman spectroscopy and SEM/EDS.

    NASA Astrophysics Data System (ADS)

    Dall'Asén, AnalíA.; Mittelstaedt, Jacob; Kim, Jin-Sun; Baer, Brandon; Paul, Raka; Gerton, Jordan; Bromley, Benjamin; Kenyon, Scott

    2017-01-01

    Meteorites provide precious clues about the formation of planets in the solar system. In particular, carbonaceous chondritic meteorites, considered the most primitive surviving materials from the early Solar System, can contribute to understand how planetisimals (the precursors to planets, of 1-100 km in radius) formed from dust (micron-size grains). These relics are mainly composed of chondrules (micro/millimeter-sized inclusions) surrounded by a matrix of microparticles. Here we present a comparative study of the structure and composition of the chondrules and surrounding matrix of different carbonaceous chondritic meteorites using low- and high-resolution micro-Raman spectroscopy and SEM/EDS (Scanning Electron Microscopy/Energy Dispersive X-ray Spectroscopy). We examine how these properties vary in different regions of the chondrules and matrix, capturing details from micrometer to millimeter scales. We compare the structure and composition between different samples, looking for signatures of the physical processes that drove their formation.

  20. Spectral decomposition of NAD(P)H fluorescence components recorded by multi-wavelength fluorescence lifetime spectroscopy in living cardiac cells

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Mateasik, Anton; Chorvat, Dusan, Jr.

    2013-12-01

    We report a novel analytical approach to identify individual components of a cell’s endogenous fluorescence, recorded by spectrally-resolved time-correlated single photon counting (TCSPC). Time-resolved area-normalized emission spectroscopy (TRANES) and principal component analysis (PCA) were applied to estimate the number of spectral components after metabolic modulation of cardiac cells following excitation with a 375 nm picosecond laser. Linear unmixing of TCSPC data spectrally decomposed individual components in living cells, while using characteristics of endogenously fluorescing molecules in solvents as a reference spectral database. Our data demonstrate the presence of three individual components, corresponding to the nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in organic and inorganic solvents and to the residual flavoprotein fluorescence. The presented analytical approach offers a new alternative for the spectral separation of multi-wavelength fluorescence lifetime spectroscopy data to the conventional analysis, and opens a new possibility for the use of pattern recognition for fast resolution of components in 2D fluorescence lifetime microscopy images.

  1. Weighted PARAFAC and Non-linear Regression for Handling Intensity Changes in Fluorescence Spectroscopy caused by pH Fluctuations.

    PubMed

    Svendsen, Carina; Skov, Thomas; van den Berg, Frans W J

    2016-07-22

    Fluorescence spectroscopy is a sensitive and selective technique, which can be of great value in bioprocesses to provide online, real-time measures of chemical compounds. Although fluorescence spectroscopy is a widely studied method, not much attention has been given to issues concerning intensity variations in the fluorescence landscapes due to pH fluctuations. This study elucidates how pH fluctuations cause intensity changes in fluorescence measurements and thereby decreases the quality of the subsequent quantification. A photo-degradation process of riboflavin was investigated by fluorescence spectroscopy and used as a model system. A two-step modeling approach, combining weighted PARAllel FACtor analysis (PARAFAC) with weighted non-linear regression of the known reaction kinetics, is suggested as a way of handling the fluorescence intensity shifts caused by the pH changes. The suggested strategy makes it possible to compensate for uncertainties in the shifted data and thereby obtain more reliable concentration profiles for the chemical compounds and kinetic parameters of the reaction.

  2. Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments.

    PubMed

    Ohashi, Kazumasa; Kiuchi, Tai; Shoji, Kazuyasu; Sampei, Kaori; Mizuno, Kensaku

    2012-01-01

    The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca(2+)-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.

  3. Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy.

    PubMed

    Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

    2011-01-01

    p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.

  4. Detection of uranium in solids by using laser-induced breakdown spectroscopy combined with laser-induced fluorescence

    SciTech Connect

    Shen, X. K.; Lu, Y. F

    2008-04-10

    Detection of uranium in solids by using laser-induced breakdown spectroscopy has been investigated in combination with laser-induced fluorescence. An optical parametric oscillator wavelength-tunable laser was used to resonantly excite the uranium atoms and ions within the plasma plumes generated by a Q-switched Nd:YAG laser. Both atomic and ionic lines can be selected to detect their fluorescence lines. A uranium concentration of 462 ppm in a glass sample can be detected by using this technique at an excitation wavelength of 385.96 nm for resonant excitation of U II and a fluorescence line wavelength of 409.0 nm from U II.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  6. Parallel online multi-wavelength (2D) fluorescence spectroscopy in each well of a continuously shaken microtiter plate.

    PubMed

    Ladner, Tobias; Beckers, Mario; Hitzmann, Bernd; Büchs, Jochen

    2016-12-01

    Small-scale high-throughput screening devices are becoming increasingly important in bioprocess development. Conventional dipping probes for process monitoring are often too large to be used in these devices. Thus, optical measurements are often the method of choice. Even some parameters that cannot directly be measured by fluorescence become accessible via sensitive fluorescence dyes. However, not all compounds of interest are measurable by this technique. Recent studies applying multi-wavelength (2D) fluorescence spectroscopy in combination with chemometrics have shown that information on numerous analytes is obscured by the fluorescence data. Hitherto, this measurement technique has only been available on the scale of stirred tank fermenters. This work introduces a new device for multi-wavelength (2D) fluorescence spectroscopy in each well of a continuously shaken microtiter plate. Using a combination of spectrograph and CCD detector, the required time per measurement cycle in a 48-well microtiter plate was 0.5 h. Cultures of Hansenula polymorpha and Escherichia coli are monitored. The concentrations of glycerol, glucose and acetate as well as pH are determined using partial least square (PLS) models. Because a pH-sensitive fluorescence dye was not required, no dependency of the pKa of a fluorescence dye exists, and measurements in the low pH range can be obtained.

  7. The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

    PubMed

    Drabik, Dominik; Przybyło, Magda; Sikorski, Aleksander; Langner, Marek

    2016-03-01

    Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.

  8. Diffusion and segmental dynamics of rodlike molecules by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Winkler, Roland G.

    2007-08-01

    The dynamics of weakly bending polymers is analyzed on the basis of a Gaussian semiflexible chain model and the fluorescence correlation spectroscopy (FCS) correlation function is determined. Particular attention is paid to the influence of the rotational motion on the decay of the FCS correlation function. An analytical expression for the correlation function is derived, from which the averaged segmental mean square displacement can be determined independent of any specific model for the polymer dynamcis. The theoretical analysis exhibits a strong dependence of the correlation function on the rotational motion for semiflexible polymers with typical lengths and persistence lengths of actin filaments or fd viruses. Hence, FCS allows for a measurement of the rotational motion of such semiflexible polymers. The theoretical results agree well with experimental measurements on actin filaments and confirm the importance of large relaxation times.

  9. Lasing dynamics study by femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy

    NASA Astrophysics Data System (ADS)

    Wei, Dang; Qing, Liao; Peng-Cheng, Mao; Hong-Bing, Fu; Yu-Xiang, Weng

    2016-05-01

    Femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy (FNOPAS) is a versatile technique with advantages of high sensitivity, broad detection bandwidth, and intrinsic spectrum correction function. These advantages should benefit the study of coherent emission, such as measurement of lasing dynamics. In this letter, the FNOPAS was used to trace the lasing process in Rhodamine 6G (R6G) solution and organic semiconductor nano-wires. High-quality transient emission spectra and lasing dynamic traces were acquired, which demonstrates the applicability of FNOPAS in the study of lasing dynamics. Our work extends the application scope of the FNOPAS technique. Project supported by the National Natural Science Foundation of China (Grant Nos. 20925313 and 21503066), the Innovation Program of Chinese Academy of Sciences (Grant No. KJCX2-YW-W25), the Postdoctoral Project of Hebei University, China, and the Project of Science and Technology Bureau of Baoding City, China (Grant No. 15ZG029).

  10. Application of dual-focus fluorescence correlation spectroscopy to microfluidic flow-velocity measurement.

    PubMed

    Arbour, Tyler J; Enderlein, Jörg

    2010-05-21

    Several methods exist to measure and map fluid velocities in microfluidic devices, which are vital to understanding properties on the micro- and nano-scale. Fluorescence correlation spectroscopy (FCS) is a method traditionally exploited for its ability to measure molecular diffusion coefficients. However, several reports during the past decade have shown that FCS can also be successfully used to measure precise flow rates in microfluidics with very high spatial resolution, making it a competitive alternative to other common flow-measurement methods. In 2007 we introduced a modified version of conventional FCS that overcomes many of the artifacts troubling the standard technique. Here we show how the advantages of this method, called dual-focus FCS, extend to flow measurements. To do so, we have measured the velocity flow profile along the cross-section of a square-bore microfluidic channel and compared the result to the theoretical prediction.

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

    PubMed Central

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

    2016-01-01

    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%. PMID:27867726

  12. Line scan fluorescence correlation spectroscopy for three-dimensional microfluidic flow velocity measurements

    NASA Astrophysics Data System (ADS)

    Pan, Xiaotao; Shi, Xianke; Korzh, Vladimir; Yu, Hanry; Wohland, Thorsten

    2009-03-01

    The flow direction of microfluidics in biological applications is not limited to two dimensions, but often extends to three dimensions. Currently there are optical methods available for the measurement of 3-D microfluidic flow vectors, but with low spatial resolution. Line scan fluorescence correlation spectroscopy (FCS) was proposed to determine flow directions in 2-D within microchannels and small blood vessels in our previous work. Importantly, its spatial resolution was demonstrated to be as good as 0.5 μm. In this work, we extend line scan FCS to the third dimension for the characterization of 3-D flow velocity vectors. The spatial resolution is close to the diffraction limit using a scan length of 0.5 μm in all three dimensions. The feasibility of line scan FCS for 3-D microfluidic flow is verified by measurements in microchannels and small blood vessels of zebrafish embryos.

  13. Study of mechanical properties of DNA in E. coli cells by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Kafle, Rudra; Liebeskind, Molly; Meiners, Jens-Christian

    Mechanical quantities like the elasticity of cells are conventionally measured by directly probing them mechanically. Measurements of these quantities for subcellular structures in living cells are almost impossible this way. We use fluorescence correlation spectroscopy (FCS) to measure such mechanical quantities in chromosomal DNA in E. coli cells. We present methods to address complexities of live-cell FCS such as photobleaching, and calculate the viscoelastic moduli from the FCS data. We compare the measured viscoelastic moduli of live cells with those that are ATP-depleted to stop all molecular motor action and find substantial differences. Active processes are stopped in ATP-depleted cells and hence the bacterial DNA appears to become stiffer and the surrounding intracellular medium more viscous. We also compare our results with the FCS data obtained from the lambda DNA solution in various concentrations to mimic the cellular environment.

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

  15. Fluorescence spectroscopy coupled with PARAFAC and PLS DA for characterization and classification of honey.

    PubMed

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

    2015-05-15

    Fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC) and Partial least squares Discriminant Analysis (PLS DA) were used for characterization and classification of honey. Excitation emission spectra were obtained for 95 honey samples of different botanical origin (acacia, sunflower, linden, meadow, and fake honey) by recording emission from 270 to 640 nm with excitation in the range of 240-500 nm. The number of fluorophores present in honey, excitation and emission spectra of each fluorophore, and their relative concentration are determined using a six-component PARAFAC model. Emissions from phenolic compounds and Maillard reaction products exhibited the largest difference among classes of honey of different botanical origin. The PLS DA classification model, constructed from PARAFAC model scores, detected fake honey samples with 100% sensitivity and specificity. Honey samples were also classified using PLS DA with errors of 0.5% for linden, 10% for acacia, and about 20% for both sunflower and meadow mix.

  16. Probing the photoluminescence properties of gold nanoclusters by fluorescence lifetime correlation spectroscopy

    SciTech Connect

    Yuan, C. T. Lin, T. N.; Shen, J. L.; Lin, C. A.; Chang, W. H.; Cheng, H. W.; Tang, J.

    2013-12-21

    Gold nanoclusters (Au NCs) have attracted much attention for promising applications in biological imaging owing to their tiny sizes and biocompatibility. So far, most efforts have been focused on the strategies for fabricating high-quality Au NCs and then characterized by conventional ensemble measurement. Here, a fusion single-molecule technique combining fluorescence correlation spectroscopy and time-correlated single-photon counting can be successfully applied to probe the photoluminescence (PL) properties for sparse Au NCs. In this case, the triplet-state dynamics and diffusion process can be observed simultaneously and the relevant time constants can be derived. This work provides a complementary insight into the PL mechanism at the molecular levels for Au NCs in solution.

  17. Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy.

    PubMed

    Daniels, Charlisa R; Reznik, Carmen; Kilmer, Rachel; Felipe, Mary Jane; Tria, Maria Celeste R; Kourentzi, Katerina; Chen, Wen-Hsiang; Advincula, Rigoberto C; Willson, Richard C; Landes, Christy F

    2011-11-01

    The present work reports on in situ observations of the interaction of organic dye probe molecules and dye-labeled protein with different poly(ethylene glycol) (PEG) architectures (linear, dendron, and bottle brush). Fluorescence correlation spectroscopy (FCS) and single molecule event analysis were used to examine the nature and extent of probe-PEG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe-PEG interactions. Small probes are trapped by more dense PEG architectures and large probes interact more with less dense PEG surfaces. These results, and the tunable pore structure of the PEG dendrons employed in this work, suggest the viability of electrochemically-active materials for tunable surfaces.

  18. High resolution laser induced fluorescence Doppler velocimetry utilizing saturated absorption spectroscopy

    SciTech Connect

    Aramaki, Mitsutoshi; Ogiwara, Kohei; Etoh, Shuzo; Yoshimura, Shinji; Tanaka, Masayoshi Y.

    2009-05-15

    A high resolution laser induced fluorescence (LIF) system has been developed to measure the flow velocity field of neutral particles in an electron-cyclotron-resonance argon plasma. The flow velocity has been determined by the Doppler shift of the LIF spectrum, which is proportional to the velocity distribution function. Very high accuracy in velocity determination has been achieved by installing a saturated absorption spectroscopy unit into the LIF system, where the absolute value and scale of laser wavelength are determined by using the Lamb dip and the fringes of a Fabry-Perot interferometer. The minimum detectable flow velocity of a newly developed LIF system is {+-}2 m/s, and this performance remains unchanged in a long-time experiment. From the radial measurements of LIF spectra of argon metastable atoms, it is found that there exists an inward flow of neutral particles associated with neutral depletion.

  19. Temperature-dependent conformations of a membrane supported zinc porphyrin tweezer by 2D fluorescence spectroscopy.

    PubMed

    Widom, Julia R; Lee, Wonbae; Perdomo-Ortiz, Alejandro; Rappoport, Dmitrij; Molinski, Tadeusz F; Aspuru-Guzik, Alán; Marcus, Andrew H

    2013-07-25

    We studied the equilibrium conformations of a zinc porphyrin tweezer composed of two carboxylphenyl-functionalized zinc tetraphenyl porphyrin subunits connected by a 1,4-butyndiol spacer, which was suspended inside the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) liposomes. By combining phase-modulation two-dimensional fluorescence spectroscopy (2D FS) with linear absorbance and fluorimetry, we determined that the zinc porphyrin tweezer adopts a mixture of folded and extended conformations in the membrane. By fitting an exciton-coupling model to a series of data sets recorded over a range of temperatures (17-85 °C) and at different laser center wavelengths, we determined that the folded form of the tweezer is stabilized by a favorable change in the entropy of the local membrane environment. Our results provide insights toward understanding the balance of thermodynamic factors that govern molecular assembly in membranes.

  20. Monitoring laboratory-scale bioventing using synchronous scan fluorescence spectroscopy: analysis of the vapor phase.

    PubMed

    Bachman, J; Kanan, S M; Patterson, H H

    2001-01-01

    Bioventing is an improved method of soil remediation that is being used with increasing frequency. In this paper, we refine techniques to measure the progress of petroleum hydrocarbon decomposition by monitoring vapor phase composition with synchronous scan fluorescence spectroscopy (SSFS). Analysis of the vapor phase has advantages compared to standard extraction techniques that require extensive sample handling and clean up. For comparison, hydrocarbon contamination in the soil was measured by analysis of Soxhlet extractions with gas chromatography-mass spectrometry (GC-MS). Comparison of the GC-MS and SSFS data showed that changes in hydrocarbon composition measured in the vapor phase provide an accurate measure of decomposition reactions taking place in the soil.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

    The study of microbes from the environment is often facilitated by the fixation of samples prior to analyses in the laboratory. Samples not appropriately preserved can show dramatic changes e.g. unwanted growth, loss of biomass and sample degradation between collection and analysis. To move Deep-UV Raman analyses from model lab organisms to environmental samples the effect of preservation must be evaluated. Deep UV Raman and Fluorescence (i.e. excitation <250 nm) has been shown capable discriminating various types of microbes as well as giving some information on the growth stage of the culture. The fluorescence signal is typically 3-4 orders of magnitude more intense than the Raman signal and enables rapid location of bacteria on a surface and crudely split them into categories. However it suffers from broad spectral features making discrete classification of bacteria problematic. While a far weaker phenomenon, the chemical specificity of Raman spectroscopy has been shown capable of discriminating between different bacterial species and has even shown spectral variation in same species under differing growth conditions or growth stages and has even been used to measure microbial activity by measuring the incorporation of stable isotope labeled substrates. Typically these analyses are carried out on well-studied, lab-grown model organisms and while relatively easy, these analyses are performed on cells grow under non-environmentally relevant conditions using rich media types not often found in nature. Here we show the effect on the Raman and fluorescence signal (248 nm Deep-UV excitation) from E. coli and other bacteria, grown in more nutrient limited environments, and fixed/preserved in ethanol, PFA and formalin. These fixatives not only preserve the cells for spectroscopic analysis but are compatible with many common techniques that can be used for further characterization of environmental microbial samples. Ethanol appears to heavily degrade the signals from

  2. Photoelectron Spectroscopy and Ionic Fragmentation of OSeCl2 and Its Analogue OSCl2 under VUV Irradiation.

    PubMed

    Geronés, Mariana; Rodríguez Pirani, Lucas S; Erben, Mauricio F; Romano, Rosana M; Cavasso Filho, Reinaldo L; Tong, Sheng Rui; Ge, Maofa; Della Védova, Carlos O

    2015-07-23

    The electronic structure and the dissociative ionization of selenium oxychloride, OSeCl2, have been investigated in the valence region by using results from both photoelectron spectroscopy (PES) and synchrotron-based photoelectron photoion coincidence (PEPICO) spectra. The PES is assigned with the help of quantum chemical calculations at the outer-valence Green's function (OVGF) and symmetry adapted cluster/configuration interaction (SAC-CI) levels. The first energy ionization is observed at 11.47 eV assigned to the ionization of electrons formally delocalized over the Se, Cl, and O lone pair orbitals. Irradiation of OSeCl2 with photons in the valence region leads to the formation of OSeCl2(•+), OSeCl(+), SeCl2(•+), SeCl(+), and SeO(•+) ions. Furthermore, the inner shell Se 3p, Cl 2p, and Se 3s electronic regions of OSeCl2 together with S 2p, Cl 2p, and S 2s electronic regions of thionyl chloride, OSCl2, have been studied by using tunable synchrotron radiation. Thus, total ion yield spectra and the fragmentation patterns deduced from PEPICO spectra at the various excitation energies have been studied. Cl(+), O(•+), and Se(•+) ions appear as the most intense fragments in the OSeCl2 PEPICO spectra, like in the sulfur analogue OSCl2, whose photofragmentation is dominated by the Cl(+), O(•+), and S(•+) ions. Fragmentation processes in OSCl2 leading to the formation of the double coincidences involving atomic ions appear as the most intense in the PEPIPICO spectra.

  3. Fluorescence Spectroscopy: An Adjunct Diagnostic Tool to Image-Guided Core Needle Biopsy of the Breast

    PubMed Central

    Zhu, Changfang; Burnside, Elizabeth S.; Sisney, Gale A.; Salkowski, Lonie R.; Harter, Josephine M.; Yu, Bing

    2009-01-01

    We explored the use of a fiber-optic probe for in vivo fluorescence spectroscopy of breast tissues during percutaneous image-guided breast biopsy. A total of 121 biopsy samples with accompanying histological diagnosis were obtained clinically and investigated in this study. The tissue spectra were analyzed using partial least-squares analysis and represented using a set of principal components (PCs) with dramatically reduced data dimension. For nonmalignant tissue samples, a set of PCs that account for the largest amount of variance in the spectra displayed correlation with the percent tissue composition. For all tissue samples, a set of PCs was identified using a Wilcoxon rank-sum test as showing statistically significant differences between: 1) malignant and fibrous/benign; 2) malignant and adipose; and 3) malignant and nonmalignant breast samples. These PCs were used to distinguish malignant from other nonmalignant tissue types using a binary classification scheme based on both linear and nonlinear support vector machine (SVM) and logistic regression (LR). For the sample set investigated in this study, the SVM classifier provided a cross-validated sensitivity and specificity of up to 81% and 87%, respectively, for discrimination between malignant and fibrous/benign samples, and up to 81% and 81%, respectively, for discriminating between malignant and adipose samples. Classification based on LR was used to generate receiver operator curves with an area under the curve (AUC) of 0.87 for discriminating malignant versus fibrous/benign tissues, and an AUC of 0.84 for discriminating malignant from adipose tissue samples. This study demonstrates the feasibility of performing fluorescence spectroscopy during clinical core needle breast biopsy, and the potential of this technique for identifying breast malignancy in vivo. PMID:19272976

  4. A mixed alkanethiol based immunosensor for surface plasmon field-enhanced fluorescence spectroscopy in serum.

    PubMed

    Scholten, Andreas; Menges, Bernhard; Juebner, Martin; Rothschild, Markus A; Bender, Katja

    2013-03-21

    This paper describes a simple and sensitive immuno-based biosensor for interference-reduced detection of C-reactive protein (CRP) in serum. The detection was performed by using a non-competitive sandwich immunoassay in combination with surface plasmon field-enhanced fluorescence spectroscopy (SPFS). CRP is an important marker for the diagnosis of inflammatory processes and cardiovascular diseases (CVD). It is nowadays detected by high-sensitivity enzyme-linked immunosorbent assays (ELISA) in blood serum. CRP was used as a model analyte in this work because it is well-characterized. However, interfering effects of matrix components affect the limit of detection (LOD) and quantification (LOQ) in general. Therefore, the availability of fast, sensitive and robust analytical methods is of major interest. A number of biosensor approaches have been described already, but only a few have demonstrated their usefulness in authentic samples such as serum. Thus our aim was to develop a simple and sensitive immunoassay-based biosensor for an interference-reduced detection of CRP in serum with surface plasmon enhanced fluorescence spectroscopy (SPFS). LODs and LOQs were experimentally determined both for CRP spiked buffer and serum. SPFS in combination with our biosensor allows sensitive analysis of CRP, achieving in buffer a LOD of 0.016 μg mL(-1) and a LOQ of 0.049 μg mL(-1). In serum the accomplished LOD was 0.026 μg mL(-1) and the LOQ was found to be 0.08 μg mL(-1). These low LODs and LOQs demonstrate the applicability of the designed biosensor for qualitative and semi-quantitative analysis of trace amounts of substances in very small sample volumes of body fluids.

  5. Fluorescence spectroscopy of single molecules at room temperature and its applications

    SciTech Connect

    Ha, Taekjip

    1996-12-01

    We performed fluorescence spectroscopy of single and pairs of dye molecules on a surface at room temperature. Near field scanning optical microscope (NSOM) and far field scanning optical microscope with multi-color excitation/detection capability were built. The instrument is capable of optical imaging with 100nm resolution and has the sensitivity necessary for single molecule detection. A variety of dynamic events which cannot be observed from an ensemble of molecules is revealed when the molecules are probed one at a time. They include (1) spectral jumps correlated with dark states, (2) individually resolved quantum jumps to and from the meta-stable triplet state, (3) rotational jumps due to desorption/readsorption events of single molecules on the surface. For these studies, a computer controlled optical system which automatically and rapidly locates and performs spectroscopic measurements on single molecules was developed. We also studied the interaction between closely spaced pairs of molecules. In particular, fluorescence resonance energy transfer between a single resonant pair of donor and acceptor molecules was measured. Photodestruction dynamics of the donor or acceptor were used to determine the presence and efficiency of energy transfer Dual molecule spectroscopy was extended to a non-resonant pair of molecules to obtain high resolution differential distance information. By combining NSOM and dual color scheme, we studied the co-localization of parasite proteins and host proteins on a human red blood cell membrane infected with malaria. These dual-molecule techniques can be used to measure distances, relative orientations, and changes in distances/orientations of biological macromolecules with very good spatial, angular and temporal resolutions, hence opening new capabilities in the study of such systems.

  6. Different mechanisms of action of antimicrobial peptides: insights from fluorescence spectroscopy experiments and molecular dynamics simulations.

    PubMed

    Bocchinfuso, Gianfranco; Palleschi, Antonio; Orioni, Barbara; Grande, Giacinto; Formaggio, Fernando; Toniolo, Claudio; Park, Yoonkyung; Hahm, Kyung-Soo; Stella, Lorenzo

    2009-09-01

    Most antimicrobial peptides exert their activity by interacting with bacterial membranes, thus perturbing their permeability. They are investigated as a possible solution to the insurgence of bacteria resistant to the presently available antibiotic drugs. However, several different models have been proposed for their mechanism of membrane perturbation, and the molecular details of this process are still debated. Here, we compare fluorescence spectroscopy experiments and molecular dynamics (MD) simulations regarding the association with lipid bilayers and lipid perturbation for two different amphiphilic helical antimicrobial peptides, PMAP-23 and trichogin GA IV. PMAP-23, a cationic peptide member of the cathelicidin family, is considered to induce membrane permeability according to the Shai-Matsuzaki-Huang "carpet" model, while trichogin GA IV is a neutral peptide, member of the peptaibol family. Although several lines of evidence suggest a "barrel-stave" mechanism of pore formation for the latter peptide, its length is only half the normal thickness of a lipid bilayer. Both fluorescence spectroscopy experiments and MD simulations indicated that PMAP-23 associates with membranes close to their surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. By contrast, trichogin GA IV can undergo a transition from a surface-bound state to a transmembrane orientation. In the first arrangement, it does not cause any strong membrane perturbation, while in the second orientation it might be able to span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane.

  7. The use of one- and two- photon induced fluorescence spectroscopy for the optical characterization of carcinogenic aflatoxins

    NASA Astrophysics Data System (ADS)

    Smeesters, L.; Meulebroeck, W.; Raeymaekers, S.; Thienpont, H.

    2014-09-01

    Carcinogenic and toxic contaminants in food and feed products are nowadays mostly detected by destructive, time-consuming chemical analyses, like HPLC and LC-MS/MS methods. However, as a consequence of the severe and growing regulations on food products by the European Union, there arose an increased demand for the ultra-fast, high-sensitive and non-destructive detection of contaminants in food and feed products. Therefore, we have investigated fluorescence spectroscopy for the characterization of carcinogenic aflatoxins. With the use of a tunable titanium-sapphire laser in combination with second and third harmonic wavelength generation, both one- and two-photon induced fluorescence excitation wavelengths could be generated using the same setup. We characterized and compared the one- and two-photon induced fluorescence spectra of pure aflatoxin powder, after excitation with 365nm and 730nm respectively. Moreover, we investigated the absolute fluorescence intensity as function of the excitation power density. Afterwards, we applied our characterization setup to the detection of aflatoxins in maize grains. The fluorescence spectra of both healthy and contaminated maize samples were experimentally characterized. In addition to the fluorescence spectrum of the pure aflatoxin, we observed an unwanted influence of the intrinsic fluorescence of the maize. Depending on the excitation wavelength, a varying contrast between the fluorescence spectra of the healthy and contaminated samples was obtained. After a comparison of the measured fluorescence signals, a detection criterion for the optical identification of the contaminated maize samples could be defined. As a result, this illustrates the use of fluorescence spectroscopy as a valuable tool for the non-destructive, real-time and high-sensitive detection of aflatoxins in maize.

  8. Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

    PubMed

    Smith, Adam W

    2015-01-01

    Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These

  9. THE USE OF FLUORESCENCE CORRELATION SPECTROSCOPY TO PROBE CHROMATIN IN THE CELL NUCLEUS

    SciTech Connect

    Sorscher, Stanley M.; Bartholemew, James C.; Klein, Melvin P.

    1980-03-01

    All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small systems, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well characterized DNA preparations, including calf thymus DNA, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and decreases as the cells leave the resting state, and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.

  10. The Citrate Carrier CitS Probed by Single-Molecule Fluorescence Spectroscopy

    PubMed Central

    Kästner, Christopher N.; Prummer, Michael; Sick, Beate; Renn, Alois; Wild, Urs P.; Dimroth, Peter

    2003-01-01

    A prominent region of the Na+-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitS-sC398, CitS-sC414) were selectively labeled with the thiol-reactive fluorophores AlexaFluor 546/568 C5 maleimide (AF546, AF568). While both single-cysteine mutants were catalytically active citrate carriers, labeling with the fluorophore was only tolerated at C398. Upon citrate addition to the functional protein fluorophore conjugate CitS-sC398-AF546, complete fluorescence quenching of the majority of molecules was observed, indicating a citrate-induced conformational change of the fluorophore-containing domain of CitS. This quenching was specific for the physiological substrate citrate and therefore most likely reflecting a conformational change in the citrate transport mechanism. Single-molecule studies with dual-labeled CitS-sC398-AF546/568 and dual-color detection provided strong evidence for a homodimeric association of CitS. PMID:12609868

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

    PubMed

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

    2013-10-01

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

  12. Light adaptation of the unicellular red alga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.

    PubMed

    Ueno, Yoshifumi; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

    2015-08-01

    Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed.

  13. Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

    PubMed

    Kekic, M; Huang, W; Moens, P D; Hambly, B D; dos Remedios, C G

    1996-07-01

    We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region.

  14. Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy*S⃞

    PubMed Central

    Laurence, Ted A.; Kwon, Youngeun; Johnson, Aaron; Hollars, Christopher W.; O'Donnell, Mike; Camarero, Julio A.; Barsky, Daniel

    2008-01-01

    DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III β subunit or β clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the β clamp diffusing along DNA is on the order of 10–14 m2/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the β clamp remains at the 3′ end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA. PMID:18556658

  15. Trimodal detection of early childhood caries using laser light scanning and fluorescence spectroscopy: clinical prototype

    PubMed Central

    Kim, Amy S.; Ridge, Jeremy S.; Nelson, Leonard Y.; Berg, Joel H.; Seibel, Eric J.

    2013-01-01

    Abstract. There is currently a need for a safe and effective way to detect and diagnose early stages of childhood caries. A multimodal optical clinical prototype for diagnosing caries demineralization in vivo has been developed. The device can be used to quickly image and screen for any signs of demineralized enamel by obtaining high-resolution and high-contrast surface images using a 405-nm laser as the illumination source, as well as obtaining autofluorescence and bacterial fluorescence images. When a suspicious region of demineralization is located, the device also performs dual laser fluorescence spectroscopy using 405- and 532-nm laser excitation. An autofluorescence ratio of the two excitation lasers is computed and used to quantitatively diagnose enamel health. The device was tested on five patients in vivo as well as on 28 extracted teeth with clinically diagnosed carious lesions. The device was able to provide detailed images that highlighted the lesions identified by the clinicians. The autofluorescence spectroscopic ratios obtained from the extracted teeth successfully quantitatively discriminated between sound and demineralized enamel. PMID:23986369

  16. Fluorescence spectroscopy for monitoring reduction of natural organic matter and halogenated furanone precursors by biofiltration.

    PubMed

    Peleato, Nicolás M; McKie, Michael; Taylor-Edmonds, Lizbeth; Andrews, Susan A; Legge, Raymond L; Andrews, Robert C

    2016-06-01

    The application of fluorescence spectroscopy to monitor natural organic matter (NOM) reduction as a function of biofiltration performance was investigated. This study was conducted at pilot-scale where a conventional media filter was compared to six biofilters employing varying enhancement strategies. Overall reductions of NOM were identified by measuring dissolved organic carbon (DOC), and UV absorbance at 254 nm, as well as characterization of organic sub-fractions by liquid chromatography-organic carbon detection (LC-OCD) and parallel factors analysis (PARAFAC) of fluorescence excitation-emission matrices (FEEM). The biofilter using granular activated carbon media, with exhausted absorptive capacity, was found to provide the highest removal of all identified PARAFAC components. A microbial or processed humic-like component was found to be most amenable to biodegradation by biofilters and removal by conventional treatment. One refractory humic-like component, detectable only by FEEM-PARAFAC, was not well removed by biofiltration or conventional treatment. All biofilters removed protein-like material to a high degree relative to conventional treatment. The formation potential of two halogenated furanones, 3-chloro-4(dichloromethyl)-2(5H)-furanone (MX) and mucochloric acid (MCA), as well as overall treated water genotoxicity are also reported. Using the organic characterization results possible halogenated furanone and genotoxicity precursors are identified. Comparison of FEEM-PARAFAC and LC-OCD results revealed polysaccharides as potential MX/MCA precursors.

  17. Use of COD, TOC, and Fluorescence Spectroscopy to Estimate BOD in Wastewater.

    PubMed

    Christian, Evelyn; Batista, Jacimaria R; Gerrity, Daniel

    2017-02-01

      Common to all National Pollutant Discharge Elimination System (NPDES) permits in the United States is a limit on biochemical oxygen demand (BOD). Chemical oxygen demand (COD), total organic carbon (TOC), and fluorescence spectroscopy are also capable of quantifying organic content, although the mechanisms of quantification and the organic fractions targeted differ for each test. This study explores correlations between BOD5 and these alternate test procedures using facility influent, primary effluent, and facility effluent samples from a full-scale water resource recovery facility. Relative reductions of the water quality parameters proved to be strong indicators of their suitability as surrogates for BOD5. Suitable correlations were generally limited to the combined datasets for the three sampling locations or the facility effluent alone. COD exhibited relatively strong linear correlations with BOD5 when considering the three sample points (r = 0.985) and the facility effluent alone (r = 0.914), while TOC exhibited a suitable linear correlation with BOD5 in the facility effluent (r = 0.902). Exponential regressions proved to be useful for estimating BOD5 based on TOC or fluorescence (r > 0.95).

  18. Comparative Studies of Interactions between Fluorodihydroquinazolin Derivatives and Human Serum Albumin with Fluorescence Spectroscopy.

    PubMed

    Wang, Yi; Zhu, Meiqing; Liu, Feng; Wu, Xiangwei; Pan, Dandan; Liu, Jia; Fan, Shisuo; Wang, Zhen; Tang, Jun; Na, Risong; Li, Qing X; Hua, Rimao; Liu, Shangzhong

    2016-10-14

    In the present study, 3-(fluorobenzylideneamino)-6-chloro-1-(3,3-dimethylbutanoyl)-phenyl-2,3-dihydroquinazolin-4(1H)-one (FDQL) derivatives have been designed and synthesized to study the interaction between fluorine substituted dihydroquinazoline derivatives with human serum albumin (HSA) using fluorescence, circular dichroism and Fourier transform infrared spectroscopy. The results indicated that the FDQL could bind to HSA, induce conformation and the secondary structure changes of HSA, and quench the intrinsic fluorescence of HSA through a static quenching mechanism. The thermodynamic parameters, ΔH, ΔS, and ΔG, calculated at different temperatures, revealed that the binding was through spontaneous and hydrophobic forces and thus played major roles in the association. Based on the number of binding sites, it was considered that one molecule of FDQL could bind to a single site of HSA. Site marker competition experiments indicated that the reactive site of HSA to FDQL mainly located in site II (subdomain IIIA). The substitution by fluorine in the benzene ring could increase the interactions between FDQL and HSA to some extent in the proper temperature range through hydrophobic effect, and the substitution at meta-position enhanced the affinity greater than that at para- and ortho-positions.

  19. Fluorescence excitation-emission matrix spectroscopy analysis of landfill leachate DOM in coagulation-flocculation process.

    PubMed

    Zhu, Guocheng; Wang, Chuang; Dong, Xingwei

    2016-09-29

    Landfill leachate contains a variety of organic matters, some of which can be excited and emit fluorescence signal. In order to degrade these organic matters, the pretreatment of the leachate is needed, which can improve the degradation performance of post-treatment process. Coagulation-flocculation is one of the important pretreatment processes to treat landfill leachate. Assessing the chemical compositions of landfill leachate is helpful in the understanding of their sources and fates as well as the mechanistic behaviors in the water environment. The present work aimed to use fluorescence excitation-emission matrix spectroscopy (EEMs) to characterize the chemical fractions of landfill leachate dissolved organic matter (DOM) in conjunction with parallel factor analysis (PARAFAC). Results showed that the DOM of landfill leachate tested in this study was identified resulting from microbial input, which included five typical characteristic peaks and four kinds of PARAFAC fractions. These fractions were mainly composed of hydrophobic macromolecule humic acid-like (HM-HA), hydrophilic intermediate molecular fulvic acid-like (HIM-FA), and hydrophilic small molecule protein-like substances (HSM-PS). HM-HA and HIM-FA were found to be easier to remove than HSM-PS. Further research on HSM-PS removal by coagulation-flocculation still needs to be improved.

  20. Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

    PubMed Central

    Kekic, M; Huang, W; Moens, P D; Hambly, B D; dos Remedios, C G

    1996-01-01

    We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region. Images FIGURE 4 FIGURE 5 PMID:8804587

  1. Detection and characterization of stomach cancer and atrophic gastritis with fluorescence and Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Xiaozhou; Lin, Junxiu; Jia, Chunde; Wang, Rong

    2003-12-01

    In this paper, we attempt to find a valid method to distinguish gastric cancer and atrophic gastritis. Auto-fluorescence and Raman spectroscopy of laser induced (514.5 nm and 488.0 nm) was measured. The serum spectrum is different between normal and cancer. Average value of diagnosis parameter for normal serum, red shift is less than 12 nm and Raman relative intensity of peak C by 514.5 nm excited is stronger than that of 488.0 nm. To gastric cancer, its red shift of average is bigger than 12 nm and relative intensity of Raman peak C by 514.5 nm excited is weaker than that by 488.0 nm. To atrophic gastritis, the distribution state of Raman peaks is similar with normal serum and auto-fluorescence spectrum's shape is similar to that of gastric cancer. Its average Raman peak red shift is bigger than 12 nm and the relative intensity of peak C by 514.5 excited is stronger than that of by 488.0. We considered it as a criterion and got an accuracy of 85.6% for diagnosis of gastric cancer compared with the result of clinical diagnosis.

  2. Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

    NASA Astrophysics Data System (ADS)

    Swift, Kerry M.; Matayoshi, Edmund D.

    2008-02-01

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

  3. Information content in fluorescence correlation spectroscopy: binary mixtures and detection volume distortion.

    PubMed

    Lam, Jonathan D; Culbertson, Michael J; Skinner, Nathan P; Barton, Zachary J; Burden, Daniel L

    2011-07-01

    When properly implemented, fluorescence correlation spectroscopy (FCS) reveals numerous static and dynamic properties of molecules in solution. However, complications arise whenever the measurement scenario is complex. Specific limitations occur when the detection region does not match the ideal Gaussian geometry ubiquitously assumed by FCS theory, or when properties of multiple fluorescent species are assessed simultaneously. A simple binary solution of diffusers, where both mole fraction and diffusion constants are sought, can face interpretive difficulty. In order to better understand the limits of FCS, this study systematically explores the relationship between detection-volume distortion, diffusion constants, species mole fraction, and fitting methodology in analyses that utilize a two-component autocorrelation model. FCS measurements from solution mixtures of dye-labeled protein and free dye are compared to simulations, which predict the performance of FCS under a variety of experimental circumstances. The results reveal a range of conditions necessary for performing accurate measurements and describe experimental scenarios that should be avoided. The findings also provide guidelines for obtaining meaningful measurements when grossly distorted detection volumes are utilized and generally assess the latent information contained in FCS datasets.

  4. Fluorescent substituted amidines of benzanthrone: Synthesis, spectroscopy and quantum chemical calculations

    NASA Astrophysics Data System (ADS)

    Gonta, Svetlana; Utinans, Maris; Kirilov, Georgii; Belyakov, Sergey; Ivanova, Irena; Fleisher, Mendel; Savenkov, Valerij; Kirilova, Elena

    2013-01-01

    Several new substituted amidine derivatives of benzanthrone were synthesized by a condensation reaction from 3-aminobenzo[de]anthracen-7-one and appropriate aromatic and aliphatic amides. The obtained derivatives have a bright yellow or orange fluorescence in organic solvents and in solid state. The novel benzanthrone derivatives were characterized by TLC analysis, 1H NMR, IR, MS, UV/vis, and fluorescence spectroscopy. The solvent effect on photophysical behaviors of these dyes was investigated, and the results showed that the Stoke's shift increased, whereas quantum yield decreased with the growth of the solvent polarity. The structure of some dyes was confirmed by the X-ray single crystal structure analysis. AM1, ZINDO/S and ab initio calculations using Gaussian software were carried out to estimate the electron system of structures. The calculations show planar configurations for the aromatic core of these compounds and two possible orientations of amidine substituents. The calculation results correlate well with red-shifted absorption and emission spectra of compounds.

  5. Fluorescence and Circular Dichroism Spectroscopy of Cytochrome c in Alkylammonium Formate Ionic Liquids

    PubMed Central

    Wei, Wenjun; Danielson, Neil D.

    2012-01-01

    The structural stability of cytochrome c has been studied in alkylammonium formate (AAF) ionic liquids such as methylammonium formate (MAF) and ethylammonium formate (EAF) by fluorescence and circular dichroism (CD) spectroscopy. At room temperature, the native structure of cytochrome c is maintained in relatively high ionic liquid concentrations (50%–70% AAF/water or AAF/phosphate buffer pH 7.0) in contrast to denaturation of cytochrome c in similar solutions of methanol or acetonitrile, with water or buffer co-solvents. Fluorescence and CD spectra indicate the conformation of cytochrome c is maintained in 20% AAF-80% water from 30 – 50 °C. No such temperature stability is found in 80% AAF-20% water. About one third of the enzyme activity of cytochrome c in 80% AAF-20% water can be maintained as compared to phosphate buffer and this is greater than the activities measured in corresponding methanol and acetonitrile aqueous solutions. This biophysical study shows that AAFs have potential application as organic solvent replacements at moderate temperature in the mobile phase for the separation of proteins in their native form by reversed phase liquid chromatography. PMID:21210672

  6. Discrimination of Rhizoma Gastrodiae (Tianma) using 3D synchronous fluorescence spectroscopy coupled with principal component analysis

    NASA Astrophysics Data System (ADS)

    Fan, Qimeng; Chen, Chaoyin; Huang, Zaiqiang; Zhang, Chunmei; Liang, Pengjuan; Zhao, Shenglan

    2015-02-01

    Rhizoma Gastrodiae (Tianma) of different variants and different geographical origins has vital difference in quality and physiological efficacy. This paper focused on the classification and identification of Tianma of six types (two variants from three different geographical origins) using three dimensional synchronous fluorescence spectroscopy (3D-SFS) coupled with principal component analysis (PCA). 3D-SF spectra of aqueous extracts, which were obtained from Tianma of the six types, were measured by a LS-50B luminescence spectrofluorometer. The experimental results showed that the characteristic fluorescent spectral regions of the 3D-SF spectra were similar, while the intensities of characteristic regions are different significantly. Coupled these differences in peak intensities with PCA, Tianma of six types could be discriminated successfully. In conclusion, 3D-SFS coupled with PCA, which has such advantages as effective, specific, rapid, non-polluting, has an edge for discrimination of the similar Chinese herbal medicine. And the proposed methodology is a useful tool to classify and identify Tianma of different variants and different geographical origins.

  7. Ablation plume structure and dynamics in ambient gas observed by laser-induced fluorescence imaging spectroscopy

    NASA Astrophysics Data System (ADS)

    Miyabe, M.; Oba, M.; Iimura, H.; Akaoka, K.; Khumaeni, A.; Kato, M.; Wakaida, I.

    2015-08-01

    The dynamic behavior of an ablation plume in ambient gas has been investigated by laser-induced fluorescence imaging spectroscopy. The second harmonic beam from an Nd:YAG laser (0.5-6 J/cm2) was focused on a sintered oxide pellet or a metal chip of gadolinium. The produced plume was subsequently intersected with a sheet-shaped UV beam from a dye laser so that time-resolved fluorescence images were acquired with an intensified CCD camera at various delay times. The obtained cross-sectional images of the plume indicate that the ablated ground state atoms and ions of gadolinium accumulate in a hemispherical contact layer between the plume and the ambient gas, and a cavity containing a smaller density of ablated species is formed near the center of the plume. At earlier expansion stage, another luminous component also expands in the cavity so that it coalesces into the hemispherical layer. The splitting and coalescence for atomic plume occur later than those for ionic plume. Furthermore, the hemispherical layer of neutral atoms appears later than that of ions; however, the locations of the layers are nearly identical. This coincidence of the appearance locations of the layers strongly suggests that the neutral atoms in the hemispherical layer are produced as a consequence of three-body recombination of ions through collisions with gas atoms. The obtained knowledge regarding plume expansion dynamics and detailed plume structure is useful for optimizing the experimental conditions for ablation-based spectroscopic analysis.

  8. Heterogeneity in binary mixtures of dimethyl sulfoxide and glycerol: fluorescence correlation spectroscopy.

    PubMed

    Chattoraj, Shyamtanu; Chowdhury, Rajdeep; Ghosh, Shirsendu; Bhattacharyya, Kankan

    2013-06-07

    Diffusion of four coumarin dyes in a binary mixture of dimethyl sulfoxide (DMSO) and glycerol is studied using fluorescence correlation spectroscopy (FCS). The coumarin dyes are C151, C152, C480, and C481. In pure DMSO, all the four dyes exhibit a very narrow (almost uni-modal) distribution of diffusion coefficient (Dt). In contrast, in the binary mixtures all of them display a bimodal distribution of Dt with broadly two components. One of the components of D(t) corresponds to the bulk viscosity. The other one is similar to that in pure DMSO. This clearly indicates the presence of two distinctly different nano-domains inside the binary mixture. In the first, the micro-environment of the solute consists of both DMSO and glycerol approximately at the bulk composition. The other corresponds to a situation where the first layer of the solute consists of DMSO only. The burst integrated fluorescence lifetime (BIFL) analysis also indicates presence of two micro-environments one of which resembles DMSO. The relative contribution of the DMSO-like environment obtained from the BIFL analysis is much larger than that obtained from FCS measurements. It is proposed that BIFL corresponds to an instantaneous environment in a small region (a few nm) around the probe. FCS, on the contrary, describes the long time trajectory of the probes in a region of dimension ~200 nm. The results are explained in terms of the theory of binary mixtures and recent simulations of binary mixtures containing DMSO.

  9. Determination of liposome permeability of ionizable carbamates of zidovudine by steady state fluorescence spectroscopy.

    PubMed

    Raviolo, Mónica A; Sanchez, Julieta M; Briñón, Margarita C; Perillo, María A

    2008-02-15

    In the present paper the relative permeabilities of AZT-Pyp and AZT-Ethy across a phospholipid bilayer were estimated by the means of fluorescence spectroscopy. The center of spectral mass of both non-encapsulated AZT-derivatives (AZT-der) emission spectra increased as a function of the illumination time inside the spectrofluorimeter cell. This phenomenon was even more evident when drugs were incubated under an UV mercury lamp, suggesting its photolytic origin. AZT-der were protected from photolysis inside liposomes and decomposed upon irradiation when they were free in the aqueous phase. The time-dependent decrease in the fluorescence intensity at a constant wavelength was fitted to a two-exponential equation and the values of rate constants for permeability and photolysis were calculated. It was concluded that AZT-Pyp but not AZT-Ethy diffused across the bilayer. This behavior correlated with the molecular volumes of AZT-Pyp (379.6A(3)) and AZT-Ethy (450.5A(3)), determined from the minimum energy conformations but not with previously reported logP values. These results reinforce the concept that not only lipophilicity but also membrane structure and AZT-der molecular size had a critical influence in passive diffusion across bilayers and may help in future refinements of other AZT-der molecular design.

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

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  11. Effect of Exogenous Phytase Addition on Soil Phosphatase Activities: a Fluorescence Spectroscopy Study.

    PubMed

    Yang, Xiao-zhu; Chen, Zhen-hua; Zhang, Yu-lan; Chen, Li-jun

    2015-05-01

    The utilization of organic phosphorus (P) has directly or indirectly improved after exogenous phytase was added to soil. However, the mechanism by which exogenous phytase affected the soil phosphatases (phosphomonoesterase and phosphodiesterase) activities was not clear. The present work was aimed to study red soil, brown soil and cinnamon soil phosphomonoesterase (acid and alkaline) (AcP and AlP) and phosphodiesterase (PD) activities responding to the addition of exogenous phytase (1 g phytase/50 g air dry soil sample) based on the measurements performed via a fluorescence detection method combined with 96 microplates using a TECAN Infinite 200 Multi-Mode Microplate Reader. The results indicated that the acid phosphomonoesterase activity was significantly enhanced in red soil (p≤0. 01), while it was significantly reduced in cinnamon soil; alkaline phosphomonoesterase activity was significantly enhanced in cinnamon soil (p≤ 0. 01), while it was significantly reduced in red soil; phosphodiesterase activity was increased in three soils but it was significantly increased in brown soil (p≤0. 01) after the addition of exogenous phytase. The activities still remained strong after eight days in different soils, which indicated that exogenous phytase addition could be enhance soil phosphatases activities effectively. This effect was not only related to soil properties, such as pH and phosphorus forms, but might also be related to the excreted enzyme amount of the stimulating microorganism. Using fluorescence spectroscopy to study exogenous phytase addition influence on soil phosphatase activities was the first time at home and abroad. Compared with the conventional spectrophotometric method, the fluorescence microplate method is an accurate, fast and simple to use method to determine the relationships among the soil phosphatases activities.

  12. Kinetics of lactone hydrolysis in antitumor drugs of camptothecin series as studied by fluorescence spectroscopy.

    PubMed

    Chourpa, I; Millot, J M; Sockalingum, G D; Riou, J F; Manfait, M

    1998-03-02

    Potent antitumor activity exhibited by 20-S-camptothecin (CPT) and numerous derivatives is known to be lost upon opening of the alpha-hydroxy-lactone ring of these drugs, hydrolyzable at neutral and basic pH. To quantify in 'real time' the lactone hydrolysis reaction in CPTs under physiological conditions, we have applied a non-perturbing approach by fluorescence spectroscopy. CPT and a set of its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38, topotecan, tricyclic ketone-CPT) with antitumor activity varying from negligible to 10 times that of CPT have been studied. Prior to the kinetic measurements, the effects of substitutions, pH, polarity of molecular environment, lactone ring opening (lactone-carboxylate transition) have been investigated in terms of the UV-visible absorption and fluorescence emission spectra of CPTs. Then the determined parameters of the fluorescence emission spectra corresponding to the respective lactone and carboxylate forms have been used to estimate the residual lactone percentage as a function of time. The reproducibility of the obtained data demonstrates that the spectroscopic approach provides a satisfactory precision for this kind of measurements. For CPT at pH 7.3, the lactone half-life was 29.4 +/- 1.7 min and the lactone percentage at equilibrium was 20.9 +/- 0.3%. Within a series of derivatives with substitutions at quinoline rings, the lactone half-life varied from 29 to 32 min and the equilibrium lactone content varied from 15% to 23%. For each compound, even slight increase of pH from 7.1 to 7.3 or from 7.3 to 7.6 logically leads to a remarkable decrease of both lactone half-life and equilibrium lactone percentage.

  13. High resolution x-ray fluorescence spectroscopy - a new technique for site- and spin-selectivity

    SciTech Connect

    Wang, Xin

    1996-12-01

    X-ray spectroscopy has long been used to elucidate electronic and structural information of molecules. One of the weaknesses of x-ray absorption is its sensitivity to all of the atoms of a particular element in a sample. Through out this thesis, a new technique for enhancing the site- and spin-selectivity of the x-ray absorption has been developed. By high resolution fluorescence detection, the chemical sensitivity of K emission spectra can be used to identify oxidation and spin states; it can also be used to facilitate site-selective X-ray Absorption Near Edge Structure (XANES) and site-selective Extended X-ray Absorption Fine Structure (EXAFS). The spin polarization in K fluorescence could be used to generate spin selective XANES or spin-polarized EXAFS, which provides a new measure of the spin density, or the nature of magnetic neighboring atoms. Finally, dramatic line-sharpening effects by the combination of absorption and emission processes allow observation of structure that is normally unobservable. All these unique characters can enormously simplify a complex x-ray spectrum. Applications of this novel technique have generated information from various transition-metal model compounds to metalloproteins. The absorption and emission spectra by high resolution fluorescence detection are interdependent. The ligand field multiplet model has been used for the analysis of K{alpha} and K{beta} emission spectra. First demonstration on different chemical states of Fe compounds has shown the applicability of site selectivity and spin polarization. Different interatomic distances of the same element in different chemical forms have been detected using site-selective EXAFS.

  14. Recombinant phytochrome of the moss Ceratodon purpureus (CP2): fluorescence spectroscopy and photochemistry.

    PubMed

    Sineshchekov, V; Koppel, L; Hughes, J; Lamparter, T; Zeidler, M

    2000-07-01

    The recombinant phytochrome of the moss Ceratodon purpureus (CP2) expressed in Saccharomyces cerevisiae and reconstituted with phycocyanobilin (PCB) was investigated using fluorescence spectroscopy. The pigment had an emission maximum at 670 nm at low temperature (85 K) and at 667 nm at room temperature (RT) and an excitation maximum at 650-652 nm at 85 K (excitation spectra could not be measured at RT). Both spectra had a half-band width of approx. 30-35 nm at 85 K. The fluorescence intensity revealed a steep temperature dependence with an activation energy of fluorescence decay (Ea) of 5.9-6.4 and 12.6-14.7 kJ mol(-1) in the interval from 85 to 210 K and from 210 to 275 K, respectively. The photochemical properties of CP2/PCB were characterised by the extent of the red-induced (lambda(a) = 639 nm) Pr conversion into the first photoproduct lumi-R at 85 K (gamma1) of approximately 0.07 and into Pfr at RT (gamma2) of approximately 0.7. From these characteristics, CP2/PCB can be attributed to the Pr" photochemical type with gamma1 < or = 0.05, which comprises the minor phyA fraction (phyA"), phyB, Adiantum phy1 and Synechocystis Cph1 in contrast to the major phyA' fraction (Pr' type with gamma1 = 0.5). Within the Pr" type, it is closer to phyA" than to phyB and Cph1.

  15. Evaluating fluorescence spectroscopy as a tool to characterize cyanobacteria intracellular organic matter upon simulated release and oxidation in natural water.

    PubMed

    Korak, Julie A; Wert, Eric C; Rosario-Ortiz, Fernando L

    2015-01-01

    Intracellular organic matter (IOM) from cyanobacteria may be released into natural waters following cell death in aquatic ecosystems and during oxidation processes in drinking water treatment plants. Fluorescence spectroscopy was evaluated to identify the presence of IOM from three cyanobacteria species during simulated release into natural water and following oxidation processes (i.e. ozone, free chlorine, chloramine, chlorine dioxide). Peak picking and the fluorescence index (FI) were explored to determine which IOM components (e.g., pigments) provide unique and persistent fluorescence signatures with minimal interferences from the background dissolved organic matter (DOM) found in Colorado River water (CRW). When IOM was added to ultrapure water, the fluorescence signature of the three cyanobacteria species showed similarities to each other. Each IOM exhibited a strong protein-like fluorescence and fluorescence at Ex 370 nm and Em 460 nm (FDOM), where commercial fluorescence sensors monitor. All species also had strong phycobiliprotein fluorescence (i.e. phycocyanin or phycoerythrin) in the higher excitation range (500-650 nm). All three IOM isolates had FI values greater than 2. When IOM was added to CRW, phycobiliprotein fluorescence was quenched through interactions between IOM and CRW-DOM. Mixing IOM and CRW demonstrated that protein-like and FDOM intensity responses were not a simple superposition of the starting material intensities, indicating that interactions between IOM and CRW-DOM fluorescing moieties were important. Fluorescence intensity in all regions decreased with exposure to ozone, free chlorine, and chlorine dioxide, but the FI still indicated compositional differences compared to CRW-DOM. The phycobiliproteins in IOM are not promising as a surrogate for IOM release, because their fluorescence intensity is quenched by interactions with DOM and decreased during oxidation processes. Increases in both FDOM intensity and FI are viable qualitative

  16. Characterization of carbonaceous meteoritic fragments found in Antarctica by high-resolution Raman spectroscopy and SEM/EDS

    NASA Astrophysics Data System (ADS)

    Dall Asen, Analia; Baer, Brandon; Mittelstaedt, Jake; Gerton, Jordan; Bromley, Benjamin; Kenyon, Scott

    2016-03-01

    Carbonaceous chondritic meteorites are composed mainly of chondrules (micro/millimeter-sized inclusions) surrounding by a matrix of microparticles, and are considered the most primitive surviving materials from the early Solar System. Understanding their properties and history may provide clues to the formation of planets from micron-size dust grains in the Solar nebula. Our approach is to study the structure and composition of carbonaceous chondrites with high-resolution micro-Raman spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. These techniques enable us to capture details on a wide range of spatial scales, from micrometers to millimeters. Here we provide the first analysis of a set of meteorite fragments from Antarctica (MIL 07002 and ALH 84028), mapping elemental and molecular abundances, as well as large-scale morphological features. We present characterizations of individual chondrules and the surrounding matrix, and we consider on how our findings reflect physical processes believed to be operating during the early stages of planet formation.

  17. Interactions of gemini surfactants with two model proteins: NMR, CD, and fluorescence spectroscopies.

    PubMed

    Amiri, Razieh; Bordbar, Abdol-Khalegh; García-Mayoral, Ma Flor; Khosropour, Ahmad Reza; Mohammadpoor-Baltork, Iraj; Menéndez, Margarita; Laurents, Douglas V

    2012-03-01

    Gemini surfactants have two polar head groups and two hydrocarbon tails. Compared with conventional surfactants, geminis have much lower (μM vs. mM) critical micelle concentrations and possess slower (ms vs. μs) monomer <-- / --> micelle kinetics. The structure of the gemini surfactants studied is [HOCH(2)CH(2)-, CH(3)-, CH(3)(CH(2))(15)-N(+)-(CH(2))(s)-N(+)-(CH(2))(15)CH(3),-CH(3),-CH(2)CH(2)OH]·2Br(-) where s=4, 5, or 6. Our objective is to reveal the effect of these cationic gemini surfactants on the structure and stability of two model proteins: Ribonuclease A (RNase A) and Hen Egg White Lysozyme (HEWL). 2D (1)H NMR and Circular Dichroism (CD) spectroscopies show that the conformation of RNase A and HEWL is unaffected at low to neutral pH where these proteins are positively charged, although hydrogen exchange shows that RNase A's conformational stability is slightly lowered. At alkaline pH, where these proteins lose their net positive charge, fluorescence and CD spectroscopies and ITC experiments show that they do interact with gemini surfactants, and multiple protein•gemini complexes are observed. Based on the results, we conclude that these cationic gemini surfactants neither interact strongly with nor severely destabilize these well folded proteins in physiological conditions, and we advance that they can serve as useful membrane mimetics for studying the interactions between membrane components and positively charged proteins.

  18. Trace elemental analysis of titanium dioxide pigments and automotive white paint fragments for forensic examination using high-energy synchrotron radiation x-ray fluorescence spectrometry.

    PubMed

    Nishiwaki, Yoshinori; Watanabe, Seiya; Shimoda, Osamu; Saito, Yasuhiro; Nakanishi, Toshio; Terada, Yasuko; Ninomiya, Toshio; Nakai, Izumi

    2009-05-01

    High-energy synchrotron radiation x-ray fluorescence spectrometry (SR-XRF) utilizing 116 keV x-rays was used to characterize titanium dioxide pigments (rutile) and automotive white paint fragments for forensic examination. The technique allowed analysis of K lines of 9 trace elements in 18 titanium dioxide pigments (rutile), and 10 trace elements in finish coat layers of seven automotive white paint fragments. High-field strength elements (HFSE) were found to strongly reflect the origin of the titanium dioxide (TiO(2)) pigments, and could be used as effective parameters for discrimination and classification of the pigments and paint fragments. A pairwise comparison of the finish coat layers of seven automotive white paint fragments was performed. The trace elements in the finish coat layers detected by the high-energy SR-XRF were especially effective for identification. By introducing the trace element information of primer and electrocoat layers, all the automotive white paint fragments could be discriminated by this technique.

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

    NASA Astrophysics Data System (ADS)

    Bejaoui, Salma; Salama, Farid

    2015-08-01

    Polycyclic aromatic hydrocarbon (PAH) molecules are considered the best carriers to account for the ubiquitous infrared emission bands. PAHs have also been proposed as candidates to explain the diffuse interstellar bands (DIBs), a series of absorption features seen on the interstellar extinction curve and are plausible carriers for the extended red emission (ERE), a photoluminescent process associated with a wide variety of interstellar environments. Extensive efforts have been devoted over the past two decades to characterize the physical and chemical properties of PAH molecules and ions in space. Absorption spectra of PAH molecules and ions trapped in solid matrices have been compared to the DIBs [1, 2]. Absorption spectra of several cold, isolated gas-phase PAHs have also been measured under experimental conditions that mimic the interstellar conditions [see 3 for a review]. The purpose of this study is to provide a new dimension to the existing spectroscopic database of neutral and single ionized PAHs that is largely based on absorption spectra by adding emission spectroscopy data. The measurements are based on the laser-induced fluorescence (LIF) technique [4] and are performed with the Pulsed Discharge Nozzle (PDN) of the COSmIC laboratory facility at NASA Ames laboratory. The PDN generates plasma in a free supersonic jet expansion to simulate the physical and the chemical conditions in interstellar environments. We focus, here, on the fluorescence spectra of large neutral PAHs and their cations where there is a lack of fluorescence spectroscopy data. The astronomical implications of the data (e.g., ERE) are examinedReferences[1] F. Salama, E. Bakes, L.J. Allamandola, A.G.G.M. Tielens, Astrophys. J., 458 (1996) p.621[2] F. Salama, The ISO Revolution, EDP Sciences, Les Ulis, France (1999) p.65[3] Salama F., In Organic Matter in Space, IAU Symposium 251, Kwok & Sandford Eds.Cambridge University Press,4, S251,(2008), p. 357 (2008) and references therein.[4

  20. Investigation on laser induced salivary stone fragmentation

    NASA Astrophysics Data System (ADS)

    Sroka, Ronald; Pongratz, Thomas; Eder, Matthias; Domes, Mona; Vogeser, Michael; Johnson, Thorsten; Siedeck, Vanessa; Schroetzlmair, Florian; Zengel, Pamela

    2014-03-01

    Objective: It was the objective of this in-vitro study to investigate photon-based techniques for identifying the composition and fragmentation of salivary stones using a Ho:YAG laser. Materials and Method: Salivary stones (n=47) extracted from patients with clinical symptoms of sialolithiasis were examined in-vitro. After extraction, the stones were kept in Ringers solution until size and volume measurements could be performed. Thereafter, dual-energy CT scans (DECT) were performed to classify the composition of the stones. Subsequently, fluorescence measurements were performed by taking images under blue light excitation as well as by fluorescence spectroscopy, measuring excitation-emission-matrixes (EEM). Further investigation to identify the exact composition of the stone was performed by Raman spectroscopy and FTIR spectroscopy of stone fragments and debris. Fragmentation was performed in an aquarium set-up equipped with a mesh (hole: 1.5mm) using a Ho:YAG-laser to deliver laser pulses of 0.5, 1.0 and 1.5J/pulse at a frequency of 3Hz through a 200μm-fibre to the stone surface. The collected data were analyzed and fragmentation rates were calculated. Finally, correlation between stone composition and fragmentation was performed. Results: Blue light fluorescence excitation resulted in either fluorescence in the green spectral region or in a combination of green and red fluorescence emission. EEM-measurement showed the corresponding spectra. Raman spectroscopy showed a mixture of carbonate apatite and keratin. DECT results in evidence of calcium containing components. FTIR-spectroscopy results showed that carbonate apatite is the main component. Fragmentation experiment showed a dependency on the energy per pulse applied if the evaluation implies the ratio of fragmented weight to pulse, while the ratio fragmented weight to energy remains about constant for the three laser parameter used. Conclusion: The composition of salivary stones could be determined using

  1. Characteristics of dissolved organic carbon revealed by ultraviolet/visible absorbance and fluorescence spectroscopy: The current status and future exploration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dissolved organic carbon (DOC) is an important research subject for various disciplines. The objectives of this chapter are to review and summarize recent advancement in characterization of DOC by ultraviolet/visible (UV/Vis) and fluorescence spectroscopies and to identify the information gaps for ...

  2. Two-photon two-focus fluorescence correlation spectroscopy with a tunable distance between the excitation volumes.

    PubMed

    Didier, Pascal; Godet, Julien; Mély, Yves

    2009-05-01

    In the present work, a Michelson interferometer was combined with a two-photon excitation microscope to perform two-focus Fluorescence Correlation Spectroscopy. This simple and original approach allows us to tune the distance between the two excitation volumes and determine absolute diffusion constants. The technique was validated on different model systems that demonstrate the sensitivity of the approach.

  3. Fluorescence and Fourier-transform infrared spectroscopy for the analysis of iconic Italian design lamps made of polymeric materials.

    PubMed

    Toja, Francesca; Nevin, Austin; Comelli, Daniela; Levi, Marinella; Cubeddu, Rinaldo; Toniolo, Lucia

    2011-03-01

    The preservation of design object collections requires an understanding of their constituent materials which are often polymeric blends. Challenges associated with aging of complex polymers from objects with an unknown physical history may compromise the interpretation of data from analytical techniques, and therefore complicate the assessment of the condition of polymers in indoor museum environments. This study focuses on the analysis of polymeric materials from three well-known Italian design lamps from the 1960s. To assess the degree of chemical modifications in the polymers, non-destructive molecular spectroscopic techniques, Fourier-transform infrared (FTIR) and fluorescence spectroscopy, have been applied directly on the object surfaces using an optical fiber probe and through examination of micro samples. FTIR spectra of the different polymers, polyvinylacetate (PVAc) for the lamps Taraxacum and Fantasma, and both acrylonitrile-butadiene-styrene polymer (ABS) and cellulose acetate (CA) for the lamp Nesso, allowed the detection of ongoing deterioration processes. Fluorescence spectroscopy proved particularly sensitive for the detection of molecular changes in the polymeric objects, as the spectra obtained from the examined lamps differ significantly from those of the unaged reference materials. Differences in fluorescence spectra are also detected between different points on the same object further indicating the presence of different chemical species on the surfaces. With the aid of complementary data from FTIR spectroscopy, an interpretation of the emission spectra of the studied polymeric objects is here proposed, further suggesting that fluorescence spectroscopy may be useful for following the degradation of historical polymeric objects.

  4. Time-correlated Raman and fluorescence spectroscopy based on a silicon photomultiplier and time-correlated single photon counting technique.

    PubMed

    Zhang, Chunling; Zhang, Liying; Yang, Ru; Liang, Kun; Han, Dejun

    2013-02-01

    We report a time-correlated Raman spectroscopy technique based on a silicon photomultiplier (SiPM) and a time-correlated single photon counting (TCSPC) technique to exploit the natural temporal separation between Raman and fluorescence phenomena to alleviate the high fluorescence background with conventional Raman detection. The TCSPC technique employed can greatly reduce the effect of high dark count rate (DCR) and crosstalk of SiPM that seriously hinder its application in low light level detection. The operating principle and performance of the 400 ps time resolution system are discussed along with the improvement of the peak-to-background ratio (PBR) for bulk trinitrotoluene (TNT) Raman spectrum relative to a commercial Raman spectrometer with charge coupled device (CCD). The fluorescence lifetime for solid TNT and Surface Enhanced Raman Scattering (SERS) spectrum for 10(-6) mol/L trace TNT have also been obtained by this system, showing excellent versatility and convenience in spectroscopy measurement.

  5. Optical response of magnetic fluorescent microspheres used for force spectroscopy in the evanescent field.

    PubMed

    Bijamov, Alex; Shubitidze, Fridon; Oliver, Piercen M; Vezenov, Dmitri V

    2010-07-20

    Force spectroscopy based on magnetic tweezers is a powerful technique for manipulating single biomolecules and studying their interactions. The resolution in magnetic probe displacement, however, needs to be commensurate with molecular sizes. To achieve the desirable sensitivity in tracking displacements of the magnetic probe, some recent approaches have combined magnetic tweezers with total internal reflection fluorescence microscopy. In this situation, a typical force probe is a polymer microsphere containing two types of optically active components: a pure absorber (magnetic nanoparticles for providing the pulling force) and a luminophore (semiconducting nanoparticles or organic dyes for fluorescent imaging). To assess the system's capability fully with regard to tracking the position of the force probe with subnanometer accuracy, we developed a body-of-revolution formulation of the method of auxiliary sources (BOR-MAS) to simulate the absorption, scattering, and fluorescence of microscopic spheres in an evanescent electromagnetic field. The theoretical formulation uses the axial symmetry of the system to reduce the dimensionality of the modeling problem and produces excellent agreement with the reported experimental data on forward scattering intensity. Using the BOR-MAS numerical model, we investigated the probe detection sensitivity for a high numerical aperture objective. The analysis of both backscattering and fluorescence observation modes shows that the total intensity of the bead image decays exponentially with the distance from the surface (or the length of a biomolecule). Our investigations demonstrate that the decay lengths of observable optical power are smaller than the penetration depth of the unperturbed excitation evanescent wave. In addition, our numerical modeling results illustrate that the expected sensitivity for the decay length changes with the angle of incidence, tracking the theoretical penetration depth for a two-media model, and is

  6. Total Absorption Spectroscopy of Fission Fragments Relevant for Reactor Antineutrino Spectra and Decay Heat Calculations

    NASA Astrophysics Data System (ADS)

    Porta, A.; Zakari-Issoufou, A.-A.; Fallot, M.; Algora, A.; Tain, J. L.; Valencia, E.; Rice, S.; Bui, V. M.; Cormon, S.; Estienne, M.; Agramunt, J.; Äystö, J.; Bowry, M.; Briz, J. A.; Caballero-Folch, R.; Cano-Ott, D.; Cucouanes, A.; Elomaa, V.-V.; Eronen, T.; Estévez, E.; Farrelly, G. F.; Garcia, A. R.; Gelletly, W.; Gomez-Hornillos, M. B.; Gorlychev, V.; Hakala, J.; Jokinen, A.; Jordan, M. D.; Kankainen, A.; Karvonen, P.; Kolhinen, V. S.; Kondev, F. G.; Martinez, T.; Mendoza, E.; Molina, F.; Moore, I.; Perez-Cerdán, A. B.; Podolyák, Zs.; Penttilä, H.; Regan, P. H.; Reponen, M.; Rissanen, J.; Rubio, B.; Shiba, T.; Sonzogni, A. A.; Weber, C.

    2016-03-01

    Beta decay of fission products is at the origin of decay heat and antineutrino emission in nuclear reactors. Decay heat represents about 7% of the reactor power during operation and strongly impacts reactor safety. Reactor antineutrino detection is used in several fundamental neutrino physics experiments and it can also be used for reactor monitoring and non-proliferation purposes. 92,93Rb are two fission products of importance in reactor antineutrino spectra and decay heat, but their β-decay properties are not well known. New measurements of 92,93Rb β-decay properties have been performed at the IGISOL facility (Jyväskylä, Finland) using Total Absorption Spectroscopy (TAS). TAS is complementary to techniques based on Germanium detectors. It implies the use of a calorimeter to measure the total gamma intensity de-exciting each level in the daughter nucleus providing a direct measurement of the beta feeding. In these proceedings we present preliminary results for 93Rb, our measured beta feedings for 92Rb and we show the impact of these results on reactor antineutrino spectra and decay heat calculations.

  7. Diffusion and conformation of peptide-functionalized polyphenylene dendrimers studied by fluorescence correlation and 13C NMR spectroscopy.

    PubMed

    Koynov, K; Mihov, G; Mondeshki, M; Moon, C; Spiess, H W; Müllen, K; Butt, H-J; Floudas, G

    2007-05-01

    We report on the combined use of fluorescence correlation spectroscopy (FCS) and 1H and 13C NMR spectroscopy to detect the size and type of peptide secondary structures in a series of poly-Z-L-lysine functionalized polyphenylene dendrimers bearing the fluorescent perylenediimide core in solution. In dilute solution, the size of the molecule as detected from FCS and 1H NMR diffusion measurements matches nicely. We show that FCS is a sensitive probe of the core size as well as of the change in the peptide secondary structure. However, FCS is less sensitive to functionality. A change in the peptide secondary conformation from beta-sheets to alpha-helices detected by 13C NMR spectroscopy gives rise to a steep increase in the hydrodynamic radii for number of residues n > or = 16. Nevertheless, helices are objects of low persistence.

  8. Zeolite-Supported Organorhodium Fragments: Essentially Molecular Surface Chemistry Elucidated with Spectroscopy

    SciTech Connect

    Liang, A.J.; Craciun, R.; Chen, M.; Kelly, T.G.; Kletnieks, P.W.; Haw, J.F.; Dixon, D.A.; Gates, B.C.

    2009-07-21

    Structures of zeolite-anchored organorhodium complexes undergoing conversions with gas-phase reactants were characterized by infrared spectra bolstered by calculations with density functional theory and analysis of the gas-phase products. Structurally well-defined zeolite-supported rhodium diethylene complexes were synthesized by chemisorption of Rh(C{sub 2}H{sub 4}){sub 2}(acac) (acac = CH{sub 3}COCHCOCH{sub 3}) on dealuminated Y zeolite, being anchored by two Rh-O bonds, as shown by extended X-ray absorption fine structure (EXAFS) spectroscopy. In contrast to the nonuniformity of metal complexes anchored to metal oxides, the near uniformity of the zeolite-supported species allowed precise determination of their chemistry, including the role of the support as a ligand. The anchored rhodium diethylene complex underwent facile, reversible ligand exchange with deuterated ethylene at 298 K, and ethylene ligands were hydrogenated by reverse spillover of hydrogen from support hydroxyl groups. The supported complexes reacted with CO to form rhodium gem-dicarbonyls, which, in the presence of ethylene, gave rhodium monocarbonyls. The facile removal of ethylene ligands from the complex in H{sub 2}-N{sub 2} mixtures created coordinatively unsaturated rhodium complexes; the coordinative unsaturation was stabilized by the site isolation of the complexes, allowing reaction with N2 to form rhodium complexes with one and with two N{sub 2} ligands. The results also provide evidence of a new rhodium monohydride species incorporating a C{sub 2}H{sub 4} ligand.

  9. Investigation of electronically excited indole relaxation dynamics via photoionization and fragmentation pump-probe spectroscopy

    SciTech Connect

    Godfrey, T. J.; Yu, Hui; Ullrich, Susanne

    2014-07-28

    The studies herein investigate the involvement of the low-lying {sup 1}L{sub a} and {sup 1}L{sub b} states with {sup 1}ππ{sup *} character and the {sup 1}πσ{sup *} state in the deactivation process of indole following photoexcitation at 201 nm. Three gas-phase, pump-probe spectroscopic techniques are employed: (1) Time-resolved photoelectron spectroscopy (TR-PES), (2) hydrogen atom (H-atom) time-resolved kinetic energy release (TR-KER), and (3) time-resolved ion yield (TR-IY). Each technique provides complementary information specific to the photophysical processes in the indole molecule. In conjunction, a thorough examination of the electronically excited states in the relaxation process, with particular focus on the involvement of the {sup 1}πσ{sup *} state, is afforded. Through an extensive analysis of the TR-PES data presented here, it is deduced that the initial excitation of the {sup 1}B{sub b} state decays to the {sup 1}L{sub a} state on a timescale beyond the resolution of the current experimental setup. Relaxation proceeds on the {sup 1}L{sub a} state with an ultrafast decay constant (<100 femtoseconds (fs)) to the lower-lying {sup 1}L{sub b} state, which is found to possess a relatively long lifetime of 23 ± 5 picoseconds (ps) before regressing to the ground state. These studies also manifest an additional component with a relaxation time of 405 ± 76 fs, which is correlated with activity along the {sup 1}πσ{sup *} state. TR-KER and TR-IY experiments, both specifically probing {sup 1}πσ{sup *} dynamics, exhibit similar decay constants, further validating these observations.

  10. Tracking transport and transformation of dissolved organic matter using fluorescence spectroscopy at Rifle vadose zone, Colorado

    NASA Astrophysics Data System (ADS)

    Dong, W.; Wan, J.; Tokunaga, T. K.; Gilbert, B.; Kim, Y.; Williams, K. H.

    2015-12-01

    Dissolved organic matter (DOM) represents the most mobile and active form of natural organic matter. It plays important roles in terrestrial C transport and biogeochemical cycles. Its reactivity makes it sensitive to seasonal variations and climate change. The objective of this study is to investigate the transport and transformation of DOM by tracking the spatial and seasonal variations of DOM concentrations and characteristics throughout the vadose zone and groundwater within a semi-arid floodplain at Rifle, Colorado. Three sets of vertically stratified pore water samplers were installed along a groundwater flow transect, and allowed collection of temporally resolved pore water samples from different depths. Fluorescence excitation-emission matrix (EEM) spectroscopy was used to trace changes in DOM characteristics. The humification index (HIX) was applied to evaluate variations in humification extent of DOM. EEM analysis identified fulvic-like, humic-like, tryptophan-like and tyrosine-like substances as the major fluorescent components of DOM in pore waters. Tryptophan-like and tyrosine-like compounds are typically considered as the recent microbial by-products, and they showed higher concentrations in the deeper vadose zone in late spring, and decrease from spring to winter. HIX values are smaller within the deeper vadose zone (1.5 ̶ 3.5 m) than in the overlying 1.0 m soil water and underlying groundwater samples (≥ 3.5 m), suggesting that some non- or less-humified DOM (or "fresh" microbial-derived DOM) was transferred during late spring. HIX value at each depth increased continuously from late spring to winter, with rapid humification occurring in late spring to early summer. These results suggest an annual cycle in which less humified soil organic matter is transferred into the deeper vadose zone during snowmelt/rainfall events, and then humified further through microbial transformation.

  11. Studies of cytochrome c-551 unfolding using fluorescence correlation spectroscopy and other biophysical techniques.

    PubMed

    Sil, Pallabi; Paul, Simanta Sarani; Silvio, Eva Di; Travaglini-Allocatelli, Carlo; Chattopadhyay, Krishnananda

    2016-09-21

    In this paper, we have studied the equilibrium unfolding transitions of cytochrome c from Pseudomonas aeruginosa (cytc551), a small bacterial protein. Similar to eukaryotic cytochrome c, cytc551 folds sequentially, although significant differences exist in the order of folding units (foldons). There are two regions of cytc551 (N-terminal helix with residue number 3 to 10 and the loop 2 region containing residues 34 to 45), in which no foldon unit could be assigned. In addition, the helix containing the Cys-X-X-Cys-His motif, adjacent to the N-terminal helix (residue number 3 to 10), shows unexplained ultra-fast collapse. To obtain further insights, we have studied cytc551 site-directed mutants using fluorescence correlation spectroscopy (FCS) and molecular dynamics simulation. We have found out that cytc551 unfolds through the formation of a fluorescently dark intermediate state and the amplitude of the dark component depends on the position of labeling. We have utilized this position dependence to propose a shape change model during the unfolding of cytc551. The present results show that the N-terminal helix remains in a collapsed position even in the completely unfolded state and this helix may act as a rigid support to guide the folding of its adjacent helix. This rigid support may be responsible for the ultra-fast collapse of the adjacent helix region, which occurs during the initial events of folding. The present results also show that the C-terminal end of loop 2 traverses a large distance during unfolding compared to the N-terminal end, which justifies the observed flexibility of the loop 2 region.

  12. Noninvasive fluorescence excitation spectroscopy for the diagnosis of oral neoplasia in vivo

    NASA Astrophysics Data System (ADS)

    Ebenezar, Jeyasingh; Ganesan, Singaravelu; Aruna, Prakasarao; Muralinaidu, Radhakrishnan; Renganathan, Kannan; Saraswathy, Thillai Rajasekaran

    2012-09-01

    Fluorescence excitation spectroscopy (FES) is an emerging approach to cancer detection. The goal of this pilot study is to evaluate the diagnostic potential of FES technique for the detection and characterization of normal and cancerous oral lesions in vivo. Fluorescence excitation (FE) spectra from oral mucosa were recorded in the spectral range of 340 to 600 nm at 635 nm emission using a fiberoptic probe spectrofluorometer to obtain spectra from the buccal mucosa of 30 sites of 15 healthy volunteers and 15 sites of 10 cancerous patients. Significant FE spectral differences were observed between normal and well differentiated squamous cell carcinoma (WDSCC) oral lesions. The FE spectra of healthy volunteers consists of a broad emission band around 440 to 470 nm, whereas in WDSCC lesions, a new primary peak was seen at 410 nm with secondary peaks observed at 505, 540, and 580 nm due to the accumulation of porphyrins in oral lesions. The FE spectral bands of the WDSCC lesions resemble the typical absorption spectra of a porphyrin. Three potential ratios (I410/I505, I410/I540, and I410/I580) were calculated from the FE spectra and used as input variables for a stepwise linear discriminant analysis (SLDA) for normal and WDSCC groups. Leave-one-out (LOO) method of cross-validation was performed to check the reliability on spectral data for tissue characterization. The diagnostic sensitivity and specificity were determined for normal and WDSCC lesions from the scatter plot of the discriminant function scores. It was observed that diagnostic algorithm based on discriminant function scores obtained by SLDA-LOO method was able to distinguish WDSCC from normal lesions with a sensitivity of 100% and specificity of 100%. Results of the pilot study demonstrate that the FE spectral changes due to porphyrin have a good diagnostic potential; therefore, porphyrin can be used as a native tumor marker.

  13. Enzymatic surface modification and functionalization of PET: a water contact angle, FTIR, and fluorescence spectroscopy study.

    PubMed

    Donelli, Ilaria; Taddei, Paola; Smet, Philippe F; Poelman, Dirk; Nierstrasz, Vincent A; Freddi, Giuliano

    2009-08-01

    The purpose of this study was to investigate the changes induced by a lypolytic enzyme on the surface properties of polyethylene terephthalate (PET). Changes in surface hydrophilicity were monitored by means of water contact angle (WCA) measurements. Fourier Transform Infrared spectroscopy (FTIR) in the Attenuated Total Reflectance mode (ATR) was used to investigate the structural and conformational changes of the ethylene glycol and benzene moieties of PET. Amorphous and crystalline PET membranes were used as substrate. The lipolytic enzyme displayed higher hydrolytic activity towards the amorphous PET substrate, as demonstrated by the decrease of the WCA values. Minor changes were observed on the crystalline PET membrane. The effect of enzyme adhesion was addressed by applying a protease after-treatment which was able to remove the residual enzyme protein adhering to the surface of PET, as demonstrated by the behavior of WCA values. Significant spectral changes were observed by FTIR-ATR analysis in the spectral regions characteristic of the crystalline and amorphous PET domains. The intensity of the crystalline marker bands increased while that of the amorphous ones decreased. Accordingly, the crystallinity indexes calculated as band intensity ratios (1,341/1,410 cm(-1) and 1,120/1,100 cm(-1)) increased. Finally, the free carboxyl groups formed at the surface of PET by enzyme hydrolysis were esterified with a fluorescent alkyl bromide, 2-(bromomethyl)naphthalene (BrNP). WCA measurements confirmed that the reaction proceeded effectively. The fluorescence results indicate that the enzymatically treated PET films are more reactive towards BrNP. FTIR analysis showed that the surface of BrNP-modified PET acquired a more crystalline character.

  14. Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization.

    SciTech Connect

    Kelly, J. J.; Chernov, B. K.; Tovstanovsky, I.; Mirzabekov, A. D.; Bavykin, S. G.; Biochip Technology Center; Northwestern Univ.; Engelhardt Inst. of Molecular Biology

    2002-12-15

    DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific 'chemical nucleases' to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms.We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.

  15. Laser-induced Fluorescence Spectroscopy for applications in chemical sensing and optical refrigeration

    NASA Astrophysics Data System (ADS)

    Kumi Barimah, Eric

    Laser-induced breakdown spectroscopy (LIBS) is an innovative technique that has been used as a method for fast elemental analysis in real time. Conventional ultraviolet-visible (UV-VIS) LIBS has been applied to detect the elemental composition of different materials, including explosives, pharmaceutical drugs, and biological samples. The extension of conventional LIBS to the infrared region (˜1-12 mum) promises to provide additional information on molecular emission signatures due to rotational-vibrational transitions. In this research, a pulsed Nd: YAG laser operating at 1064 nm was focused onto several sodium compounds (NaCl, NaClO3, Na2CO3 and NaClO4) and potassium compounds (KCl, KClO3, K2CO3 and KClO4) to produce an intense plasma at the target surface. Several distinct infrared (IR) atomic emission signatures were observed from all sodium and potassium containing compounds. The atomic emission lines observed from the investigated samples matched assigned transitions of neutral sodium and potassium atoms published in the National Institute of Standards and Technology (NIST) atomic database. In addition to the intense atomic lines, the rst evidence of molecular LIBS emission structures were observed at ˜10.0 m in KClO3 and NaClO3 for the chlorate anion (ClO3 --1), at ˜6.7 to 8.0 mum in KNO3 and NaNO 3 for the nitrate anion (NO3--1 ), ˜8.0 to 10.0 mum in KClO4 and NaClO4 for perchlorate anion (ClO4--1 ), and ˜6.88 mum and 11.53 mum in Na2CO3 for the carbonate anion (CO3--1 ). The observed molecular emission showed strong correlation with the conventional Fourier Transform Infrared Spectrometry (FTIR) absorption spectra of the investigated samples. IR LIBS was also applied to determine the limit of detection (LOD) for the perchlorate anion in KClO4 using the 8.0 -11.0 mum IR-LIBS emission band. The calibration curve of ClO4 in KClO4 was constructed using peak and integrated emission intensities for known concentrations of mixed KClO4/NH4NO3 samples. The

  16. Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence.

    PubMed

    Pawlak, M; Grell, E; Schick, E; Anselmetti, D; Ehrat, M

    1998-01-01

    A method for the functional immobilization of Na,K-ATPase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent field of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-ATPase was used, which had been labelled covalently within the ATP-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation bi