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Sample records for antibody-based time-resolved fluorescence

  1. Time-resolved fluorescence decay measurements for flowing particles

    DOEpatents

    Deka, C.; Steinkamp, J.A.

    1999-06-01

    Time-resolved fluorescence decay measurements are disclosed for flowing particles. An apparatus and method for the measurement and analysis of fluorescence for individual cells and particles in flow are described, wherein the rapid measurement capabilities of flow cytometry and the robust measurement and analysis procedures of time-domain fluorescence lifetime spectroscopy are combined. A pulse-modulated CW laser is employed for excitation of the particles. The characteristics and the repetition rate of the excitation pulses can be readily adjusted to accommodate for fluorescence decays having a wide range of lifetimes. 12 figs.

  2. Time-resolved fluorescence decay measurements for flowing particles

    DOEpatents

    Deka, Chiranjit; Steinkamp, John A.

    1999-01-01

    Time-resolved fluorescence decay measurements for flowing particles. An apparatus and method for the measurement and analysis of fluorescence for individual cells and particles in flow are described, wherein the rapid measurement capabilities of flow cytometry and the robust measurement and analysis procedures of time-domain fluorescence lifetime spectroscopy are combined. A pulse-modulated cw laser is employed for excitation of the particles. The characteristics and the repetition rate of the excitation pulses can be readily adjusted to accommodate for fluorescence decays having a wide range of lifetimes.

  3. Time Resolved Raman and Fluorescence Spectrometer for Planetary Mineralogy

    NASA Astrophysics Data System (ADS)

    Blacksberg, Jordana; Rossman, George

    2010-05-01

    Raman spectroscopy is a prime candidate for the next generation of planetary instruments, as it addresses the primary goal of mineralogical analysis which is structure and composition. It does not require sample preparation and provides unique mineral fingerprints, even for mixed phase samples. However, large fluorescence return from many mineral samples under visible light excitation can seriously compromise the quality of the spectra or even render Raman spectra unattainable. Fluorescence interference is likely to be a problem on Mars and is evident in Raman spectra of Martian Meteorites[1]. Our approach uses time resolution for elimination of fluorescence from Raman spectra, allowing for traditional visible laser excitation (532 nm). Since Raman occurs instantaneously with the laser pulse and fluorescence lifetimes vary from nsec to msec depending on the mineral, it is possible to separate them out in time. Complementary information can also be obtained simultaneously using the time resolved fluorescence data. The Simultaneous Spectral Temporal Adaptive Raman Spectrometer (SSTARS) is a planetary instrument under development at the Jet Propulsion Laboratory, capable of time-resolved in situ Raman and fluorescence spectroscopy. A streak camera and pulsed miniature microchip laser provide psec scale time resolution. Our ability to observe the complete time evolution of Raman and fluorescence in minerals provides a foundation for design of pulsed Raman and fluorescence spectrometers in diverse planetary environments. We will discuss the SSTARS instrument design and performance capability. We will also present time-resolved pulsed Raman spectra collected from a relevant set of minerals selected using available data on Mars mineralogy[2]. Of particular interest are minerals resulting from aqueous alteration on Mars. For comparison, we will present Raman spectra obtained using a commercial continuous wave (CW) green (514 nm) Raman system. In many cases using a CW laser

  4. Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

    PubMed Central

    Marriott, G; Clegg, R M; Arndt-Jovin, D J; Jovin, T M

    1991-01-01

    An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:1723311

  5. 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).

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

    PubMed

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

    2000-06-01

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

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

    PubMed

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

    2005-01-01

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

  8. Protein chip analysis by probing time-resolved UV fluorescence

    NASA Astrophysics Data System (ADS)

    Grigaravicius, Paulius; Dietrich, Rüdiger; Fritzsche, Wolfgang; Greulich, Karl Otto; Horn, Uwe; Knoll, Dietmar; Peters, Sven; Striebel, Hans-Martin; Schellenberg, Peter

    2007-07-01

    We describe a novel label-free method to analyse protein interactions on microarrays as well as in solution. By this technique the time resolved native protein fluorescence in the UV is probed. The method is based on alterations of the protein upon ligand binding, and, as a consequence, of alterations of the environment of the proteins' aromatic amino acids. These amino acids act as internal probes, and as a result, the fluorescence lifetime of the proteins change due to binding to a ligand partner such as another protein. We were able to demonstrate the feasibility of the method with many compounds, including protein-protein, protein-antibody, protein-nucleic acid and protein-small ligand pairs. Unlike to many other label-free techniques, the sensitivity of the method does not depend on the size of the counterbinding ligand and therefore is particularly suitable for drug monitoring, when small molecules are involved.

  9. Time-Resolved Synchronous Fluorescence for Biomedical Diagnosis

    PubMed Central

    Zhang, Xiaofeng; Fales, Andrew; Vo-Dinh, Tuan

    2015-01-01

    This article presents our most recent advances in synchronous fluorescence (SF) methodology for biomedical diagnostics. The SF method is characterized by simultaneously scanning both the excitation and emission wavelengths while keeping a constant wavelength interval between them. Compared to conventional fluorescence spectroscopy, the SF method simplifies the emission spectrum while enabling greater selectivity, and has been successfully used to detect subtle differences in the fluorescence emission signatures of biochemical species in cells and tissues. The SF method can be used in imaging to analyze dysplastic cells in vitro and tissue in vivo. Based on the SF method, here we demonstrate the feasibility of a time-resolved synchronous fluorescence (TRSF) method, which incorporates the intrinsic fluorescent decay characteristics of the fluorophores. Our prototype TRSF system has clearly shown its advantage in spectro-temporal separation of the fluorophores that were otherwise difficult to spectrally separate in SF spectroscopy. We envision that our previously-tested SF imaging and the newly-developed TRSF methods will combine their proven diagnostic potentials in cancer diagnosis to further improve the efficacy of SF-based biomedical diagnostics. PMID:26404289

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

    PubMed

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

    2010-01-01

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

  11. Exploiting Molecular Biology by Time-Resolved Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    Müller, Francis; Fattinger, Christof

    Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal fluorophore. With fluorescence lifetimes in the microsecond range and fluorescence quantum yield of 0.4 some water soluble complexes of Ruthenium like modified Ru(sulfobathophenanthroline) complexes fulfill these properties. They are outstanding fluorescent labels for ultrasensitive assays as illustrated in two examples, in drug discovery and in point of care testing.We discuss the fundamentals and the state-of-the-art of the most sensitive time-gated fluorescence assays. We reflect on how the imaging devices currently employed for readout of these assays might evolve in the future. Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal

  12. Time-resolved fluorescence spectra of arterial fluorescent compounds: reconstruction with the Laguerre expansion technique.

    PubMed

    Maarek, J M; Marcu, L; Snyder, W J; Grundfest, W S

    2000-02-01

    The time-resolved fluorescence spectra of the main arterial fluorescent compounds were retrieved using a new algorithm based on the Laguerre expansion of kernels technique. Samples of elastin, collagen and cholesterol were excited with a pulsed nitrogen laser and the emission was measured at 29 discrete wavelengths between 370 and 510 nm. The expansion of the fluorescence impulse response function on the Laguerre basis of functions was optimized to reproduce the observed fluorescence emission. Collagen lifetime (5.3 ns at 390 nm) was substantially larger than that of elastin (2.3 ns) and cholesterol (1.3 ns). Two decay components were identified in the emission decay of the compounds. For collagen, the decay components were markedly wavelength dependent and hydration dependent such that the emission decay became shorter at higher emission wavelengths and with hydration. The decay characteristics of elastin and cholesterol were relatively unchanged with wavelength and with hydration. The observed variations in the time-resolved spectra of elastin, collagen and cholesterol were consistent with the existence of several fluorophores with different emission characteristics. Because the compounds are present in different proportions in healthy and atherosclerotic arterial walls, characteristic differences in their time-resolved emission spectra could be exploited to assess optically the severity of atherosclerotic lesions. PMID:10687392

  13. Monitoring tissue metabolism via time-resolved laser fluorescence

    NASA Astrophysics Data System (ADS)

    Maerz, Holger K.; Buchholz, Rainer; Emmrich, Frank; Fink, Frank; Geddes, Clive L.; Pfeifer, Lutz; Raabe, Ferdinand; Marx, Uwe

    1999-05-01

    Most assays for drug screening are monitoring the metabolism of cells by detecting the NADH content, which symbolize its metabolic activity, indirectly. Nowadays, the performance of a LASER enables us to monitor the metabolic state of mammalian cells directly and on-line by using time-resolved autofluorescence detection. Therefore, we developed in combination with tissue engineering, an assay for monitoring minor toxic effects of volatile organic compounds (VOC), which are accused of inducing Sick Building Syndrome (SBS). Furthermore, we used the Laserfluoroscope (LF) for pharmacological studies on human bone marrow in vitro with special interest in chemotherapy simulation. In cancer research and therapy, the effect of chemostatica in vitro in the so-called oncobiogram is being tested; up to now without great success. However, it showed among other things that tissue structure plays a vital role. Consequently, we succeeded in simulating a chemotherapy in vitro on human bone marrow. Furthermore, after tumor ektomy we were able to distinguish between tumoric and its surrounding healthy tissue by using the LF. With its sensitive detection of metabolic changes in tissues the LF enables a wide range of applications in biotechnology, e.g. for quality control in artificial organ engineering or biocompatability testing.

  14. Time-resolved fluorescence spectroscopy for chemical sensors

    NASA Astrophysics Data System (ADS)

    Draxler, Sonja; Lippitsch, Max E.

    1996-07-01

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

  15. Time-resolved fluorescence spectroscopic study of flavin fluorescence in purified enzymes of bioluminescent bacteria

    NASA Astrophysics Data System (ADS)

    Vetrova, Elena; Kudryasheva, N.; Cheng, K.

    2006-10-01

    Time-resolved fluorescence intensity and anisotropy decay measurements have been used to study the environment and rotational mobility of endogenous flavin in two purified enzymes of bioluminescent bacteria, Luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri. We compared the time-resolved fluorescence parameters, intensity decay lifetimes, rotational correlation times, and their fractional contribution, of the endogeneous flavin fluorescence in each of the two enzymes in the presence or absence of quinones of different structures and redox potentials. The endogeneous flavin exhibited multi-exponential decay characteristics as compared to a single decay lifetime of around 5 ns for free flavin, suggesting a complex and heterogeneous environment of flavin bound to the enzyme. In addition, a significant increase in the rotational correlation time and a certain degree of ordering of the molecule were observed for endogenous flavin when compared to a single and fast rotational correlation time of 150 ps of free flavin. Quinone significantly altered both the lifetime and rotational characteristics of endogenous flavin suggesting specific interactions of quinones to the endogeneous flavin in the bacterial enzyme.

  16. Time-resolved fluorescence and photon migration studies in biomedical and model random media

    NASA Astrophysics Data System (ADS)

    Das, B. B.; Liu, Feng; Alfano, R. R.

    1997-02-01

    This review highlights time-resolved fluorescence kinetics and photon transport in tissues and other biomedical media with a special emphasis on ultrafast measurements of key optical parameters. Measurements of fluorescence decay lifetimes from human breast and atherosclerotic artery tissues in the uv and visible region are described after a brief description of fundamentals of fluorescence kinetics. A time-dependent diffusion model for photon migration and various ultrafast methods for time-resolved light scattering measurements to obtain key optical parameters of tissues and other model turbid media are presented. The usefulness of optical parameters as markers in optical diagnostics and imaging is considered. Time-gated measurements of ballistic and snake photons to obtain shadowgrams and an inverse numerical reconstruction of the interior map of a turbid medium from time-resolved data in the context of optical tomography are presented.

  17. Time-resolved laser-induced fluorescence study on dyes used in DNA sequencing

    SciTech Connect

    Chang, Kaisyang; Force, R.K. )

    1993-01-01

    Research on the time-resolved fluorescence of fluorescein isothiocyanate, NBD, tetramethylrhodamine isothiocyanate, and Texas Red - the dyes used for fluorescence-based DNA sequencing - is described. Mean fluorescence lifetiems in both aqueous buffer solution and 5.3%T, 4.8%C polyacrylamide gel were determined as a function of excitation wave-lengths at 337, 470, and 550 nm and were found to be 3.5, 1.1, 2.5, and 4.3 ns; the detection limits are 10, 200, 200 and 200 amol for FITC, NBD, TEMR, and T. Red, respectively. Comparisons of fluorescence parameters between the conjugated dyes and the free dyes are also reported. Results on the optimization of the excitation source wavelengths to improve sensitivity and reduce background scattering in polyacrylamide gel are also reported. Time-resolved fluorescence was successfully applied to resolve spectral overlapping of emissions in both solution and in polyacrylamide gel. 12 refs., 6 figs., 1 tab.

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

    PubMed Central

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

    2012-01-01

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

  19. Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase.

    PubMed Central

    Kim, S J; Chowdhury, F N; Stryjewski, W; Younathan, E S; Russo, P S; Barkley, M D

    1993-01-01

    The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spectral data were fitted to discrete and distributed lifetime models. The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60%. The emission spectra of both components are identical with maxima at 327 nm. The quantum yield is 0.31 +/- 0.01. The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol. The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution. The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A. Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile. The allosteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure. PMID:8369432

  20. Use of Time-Resolved Fluorescence to Monitor Bioactive Compounds in Plant Based Foodstuffs

    PubMed Central

    Lemos, M. Adília; Sárniková, Katarína; Bot, Francesca; Anese, Monica; Hungerford, Graham

    2015-01-01

    The study of compounds that exhibit antioxidant activity has recently received much interest in the food industry because of their potential health benefits. Most of these compounds are plant based, such as polyphenolics and carotenoids, and there is a need to monitor them from the field through processing and into the body. Ideally, a monitoring technique should be non-invasive with the potential for remote capabilities. The application of the phenomenon of fluorescence has proved to be well suited, as many plant associated compounds exhibit fluorescence. The photophysical behaviour of fluorescent molecules is also highly dependent on their microenvironment, making them suitable probes to monitor changes in pH, viscosity and polarity, for example. Time-resolved fluorescence techniques have recently come to the fore, as they offer the ability to obtain more information, coupled with the fact that the fluorescence lifetime is an absolute measure, while steady state just provides relative and average information. In this work, we will present illustrative time-resolved measurements, rather than a comprehensive review, to show the potential of time-resolved fluorescence applied to the study of bioactive substances. The aim is to help assess if any changes occur in their form, going from extraction via storage and cooking to the interaction with serum albumin, a principal blood transport protein. PMID:26132136

  1. Use of Time-Resolved Fluorescence to Monitor Bioactive Compounds in Plant Based Foodstuffs.

    PubMed

    Lemos, M Adília; Sárniková, Katarína; Bot, Francesca; Anese, Monica; Hungerford, Graham

    2015-01-01

    The study of compounds that exhibit antioxidant activity has recently received much interest in the food industry because of their potential health benefits. Most of these compounds are plant based, such as polyphenolics and carotenoids, and there is a need to monitor them from the field through processing and into the body. Ideally, a monitoring technique should be non-invasive with the potential for remote capabilities. The application of the phenomenon of fluorescence has proved to be well suited, as many plant associated compounds exhibit fluorescence. The photophysical behaviour of fluorescent molecules is also highly dependent on their microenvironment, making them suitable probes to monitor changes in pH, viscosity and polarity, for example. Time-resolved fluorescence techniques have recently come to the fore, as they offer the ability to obtain more information, coupled with the fact that the fluorescence lifetime is an absolute measure, while steady state just provides relative and average information. In this work, we will present illustrative time-resolved measurements, rather than a comprehensive review, to show the potential of time-resolved fluorescence applied to the study of bioactive substances. The aim is to help assess if any changes occur in their form, going from extraction via storage and cooking to the interaction with serum albumin, a principal blood transport protein. PMID:26132136

  2. Advances in ultrafast time resolved fluorescence physics for cancer detection in optical biopsy

    NASA Astrophysics Data System (ADS)

    Alfano, R. R.

    2012-03-01

    We discuss the use of time resolved fluorescence spectroscopy to extract fundamental kinetic information on molecular species in tissues. The temporal profiles reveal the lifetime and amplitudes associated with key active molecules distinguishing the local spectral environment of tissues. The femtosecond laser pulses at 310 nm excite the tissue. The emission profile at 340 nm from tryptophan is non-exponential due to the micro-environment. The slow and fast amplitudes and lifetimes of emission profiles reveal that cancer and normal states can be distinguished. Time resolved optical methods offer a new cancer diagnostic modality for the medical community.

  3. Time-resolved spectroscopy of charge-transfer fluorescent molecules in polymer matrices

    NASA Astrophysics Data System (ADS)

    Hofstraat, Johannes W.; Verhey, H. J.; Verhoeven, Jan W.; Kuemke, M.; McGown, Linda B.; Novikov, Eugene G.; van Hoek, Arie; Visser, Antonie J. W. G.

    1996-03-01

    Time-resolved fluorescence measurements have been carried out on charge-transfer fluorescent molecules incorporated in polymeric lattices, consisting of polystyrene cores and polyglycidylmethacrylate shells, and in polymethylmethacrylate thin films. New approaches to the analysis of fluorescence lifetime data obtained for molecules in polymer matrices had to be applied, since conventional analysis methods appeared not suitable for such strongly heterogeneous systems. The polymer lattices could be characterized by application of phase- resolved fluorescence lifetime measurements followed by maximum-entropy methods for data analysis. The thin films were studied using time-correlated single photon counting fluorescence lifetime measurements and data analysis with a home-built program based on stretched exponential decays. Interactions of the fluorescent guest molecules could be established by combined fluorescence lifetime and depolarization measurements. Suggestions for further improvements in fluorescence lifetime methods for characterization of polymeric materials have been made.

  4. Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy.

    PubMed Central

    Barzda, V; de Grauw, C J; Vroom, J; Kleima, F J; van Grondelle, R; van Amerongen, H; Gerritsen, H C

    2001-01-01

    Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood. PMID:11423435

  5. A fluorescence LIDAR sensor for hyper-spectral time-resolved remote sensing and mapping.

    PubMed

    Palombi, Lorenzo; Alderighi, Daniele; Cecchi, Giovanna; Raimondi, Valentina; Toci, Guido; Lognoli, David

    2013-06-17

    In this work we present a LIDAR sensor devised for the acquisition of time resolved laser induced fluorescence spectra. The gating time for the acquisition of the fluorescence spectra can be sequentially delayed in order to achieve fluorescence data that are resolved both in the spectral and temporal domains. The sensor can provide sub-nanometric spectral resolution and nanosecond time resolution. The sensor has also imaging capabilities by means of a computer-controlled motorized steering mirror featuring a biaxial angular scanning with 200 μradiant angular resolution. The measurement can be repeated for each point of a geometric grid in order to collect a hyper-spectral time-resolved map of an extended target. PMID:23787661

  6. 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

  7. Halide (Cl(super -)) Quenching of Quinine Sulfate Fluorescence: A Time-Resolved Fluorescence Experiment for Physical Chemistry

    ERIC Educational Resources Information Center

    Gutow, Jonathan H.

    2005-01-01

    The time-resolved fluorescence experiment investigating the halide quenching of fluorescence from quinine sulfate in water is described. The objectives of the experiment include reinforcing student understanding of the kinetics of competing pathways, making connections with microscopic theories of kinetics through comparison of experimental and…

  8. Optical characterization of Pseudomonas fluorescens on meat surfaces using time-resolved fluorescence

    NASA Astrophysics Data System (ADS)

    Bouchard, Alain; Frechette, Julie; Vernon, Marcia L.; Cormier, Jean-François; Beaulieu, Rene M.; Vallée, Réal; Mafu, Akier A.

    2006-01-01

    A scanning optical system for the detection of bacteria on meat surfaces based on fluorescence lifetime and intensity measurements is described. The system detects autofluorescent light emitted by naturally occurring fluorophores in bacteria. The technique only requires minimal sample preparation and handling, thus the chemical properties of the specimen are preserved. This work presents the preliminary results obtained from a time-resolved fluorescence imaging system for the characterization of a nonpathogenic gram-negative bacteria, Pseudomonas fluorescens. Initial results indicate that the combination of fluorescence lifetime and intensity measurements provides a means for characterizing biological media and for detecting microorganisms on surfaces.

  9. A 0.18-µm CMOS Array Sensor for Integrated Time-Resolved Fluorescence Detection

    PubMed Central

    Huang, Ta-chien D.; Sorgenfrei, Sebastian; Gong, Ping; Levicky, Rastislav; Shepard, Kenneth L.

    2010-01-01

    This paper describes the design of an active, integrated CMOS sensor array for fluorescence applications which enables time-gated, time-resolved fluorescence spectroscopy. The 64-by-64 array is sensitive to photon densities as low as 8.8 × 106 photons/cm2 with 64-point averaging and, through a differential pixel design, has a measured impulse response of better than 800 ps. Applications include both active microarrays and high-frame-rate imagers for fluorescence lifetime imaging microscopy. PMID:20436922

  10. CMOS Time-Resolved, Contact, and Multispectral Fluorescence Imaging for DNA Molecular Diagnostics

    PubMed Central

    Guo, Nan; Cheung, Ka Wai; Wong, Hiu Tung; Ho, Derek

    2014-01-01

    Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA) detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS) technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art. PMID:25365460

  11. Feasibility analysis of an epidermal glucose sensor based on time-resolved fluorescence

    NASA Astrophysics Data System (ADS)

    Katika, Kamal M.; Pilon, Laurent

    2007-06-01

    The goal of this study is to test the feasibility of using an embedded time-resolved fluorescence sensor for monitoring glucose concentration. Skin is modeled as a multilayer medium with each layer having its own optical properties and fluorophore absorption coefficients, lifetimes, and quantum yields obtained from the literature. It is assumed that the two main fluorophores contributing to the fluorescence at these excitation and emission wavelengths are nicotinamide adenine dinucleotide (NAD)H and collagen. The intensity distributions of excitation and fluorescent light in skin are determined by solving the transient radiative transfer equation by using the modified method of characteristics. The fluorophore lifetimes are then recovered from the simulated fluorescence decays and compared with the actual lifetimes used in the simulations. Furthermore, the effect of adding Poissonian noise to the simulated decays on recovering the lifetimes was studied. For all cases, it was found that the fluorescence lifetime of NADH could not be recovered because of its negligible contribution to the overall fluorescence signal. The other lifetimes could be recovered to within 1.3% of input values. Finally, the glucose concentrations within the skin were recovered to within 13.5% of their actual values, indicating a possibility of measuring glucose concentrations by using a time-resolved fluorescence sensor.

  12. Time-Resolved Fluorescence in Lipid Bilayers: Selected Applications and Advantages over Steady State

    PubMed Central

    Amaro, Mariana; Šachl, Radek; Jurkiewicz, Piotr; Coutinho, Ana; Prieto, Manuel; Hof, Martin

    2014-01-01

    Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements. PMID:25517142

  13. Time-resolved fluorescence microscopy of gunshot residue: an application to forensic science.

    PubMed

    Bird, Damian K; Agg, Kent M; Barnett, Neil W; Smith, Trevor A

    2007-04-01

    Time-resolved fluorescence microscopy has rapidly emerged as the technique of choice for many researchers aiming to gain specific insights into the dynamics of intricate biological systems. Although the unique advantages the technique provides over other methods have proven to be particularly useful in the biosciences, to date they have been largely unexploited by other research disciplines. In this paper, we demonstrate the capacity of time-resolved fluorescence microscopy as a practical analytical tool in the forensic sciences via the imaging of gunshot residues that are expelled when a firearm is discharged. This information may prove to be useful for determination of the true sequence of events that took place in a firearm related crime. PMID:17381705

  14. BHHST: An improved lanthanide chelate for time-resolved fluorescence applications

    NASA Astrophysics Data System (ADS)

    Connally, Russell; Jin, Dayong; Piper, James

    2005-04-01

    The detection of the waterborne pathogens Giardia lamblia and Cryptosporidium parvum in environmental water bodies requires concentration of large volumes of water due to the low dose required for infection. The highly concentrated (10,000-fold) water sample is often rich in strongly autofluorescent algae, organic debris and mineral particles that can obscure immunofluorescently labeled (oo)cysts during analysis. Time-resolved fluorescence techniques exploit the long fluorescence lifetimes of lanthanide chelates (ms) to differentiate target fluorescence from background autofluorescence (ns). Relatively simple instrumentation can be used to enhance the signal-to-noise ratio (S/N) of labelled target. Time-resolved fluorescence techniques exploit the large difference in lifetime by briefly exciting fluorescence from the sample using a pulsed excitation source. Capture of the resulting fluorescence emission is delayed until the more rapidly decaying autofluorescence has faded beyond detection, whereon the much stronger and slower fading emission from labelled target is collected. BHHCT is a tetradentate beta-diketone chelate that is activated to bind with protein (antibody) as the chlorosulfonate. The high activity of this residue makes conjugations difficult to control and can lead to the formation of unstable immunoconjugates. To overcome these limitations a 5-atom hydrophylic molecular tether was attached to BHHCT via the chlorosulfonate and the BHHCT derivative was then activated to bind to proteins as the succinimide. The new compound (BHHST) could be prepared in high purity and was far more stable than the chlorosulfonate on storage. A high activity immunocojugate was prepared against Cryptosporidium that yielded an 8-fold increase in SNR using a lab-built time-resolved fluorescence microscope.

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

    NASA Astrophysics Data System (ADS)

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

    1994-08-01

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

  16. Time-resolvable fluorescent conjugates for the detection of pathogens in environmental samples containing autofluorescent material

    NASA Astrophysics Data System (ADS)

    Connally, Russell; Veal, Duncan; Piper, James A.

    2003-07-01

    Water is routinely monitored for environmental pathogens such a Cryptosporidium and Giardia using immunofluorescence microscopy (IFM). Autofluorescence can greatly diminish an operators capacity to resolve labeled pathogens from non-specific background. Naturally fluorescing components (autofluorophores) encountered in biological samples typically have fluorescent lifetimes (τ) of less than 100 nanoseconds and their emissions may be excluded through use of time-resolved fluorescence microscopy (TRFM). TRFM relies on the large differences in τ between autofluorescent molecules and long-lived lanthanide chelates. In TRFM, targets labeled with a time-resolvable fluorescent immunoconjugate are excited by an intense (UV) light pulse. A short delay is imposed to permit the decay of autofluorescence before capture of luminescence from the excited chelate using an image intensified CCD camera. In our experience, autofluorescence can be reduced to insignificant levels with a consequent 30-fold increase in target visibility using TRFM techniques. We report conjugation of a novel europium chelate to a monoclonal antibody specific for Giardia lamblia and use of the immunoconjugate for TRFM studies. Initial attempts to conjugate the same chelate to a monoclonal antibody directed against Cryptosporidium parvum led to poorly fluorescent constructs that were prone to denature and precipitate. We successfully conjugated BHHCT to anti-mouse polyvalent immunoglobulin and used this construct to overcome the difficulties in direct labeling of the anti-Cryptosporidium antibody. Both Giardia and Cryptosporidium were labeled using the anti-mouse protocol with a subsequent 20-fold and 6.6-fold suppression of autofluorescence respectively. A rapid protocol for conjugating and purifying the immunoconjugate was found and methods of quantifying the fluorescence to protein ratio determined. Performance of our TRFM was dependent on the quality and brightness of the immunoconjugate and

  17. Molecular diffusivity measurement through an alumina membrane using time-resolved fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Kennard, Raymond; DeSisto, William J.; Mason, Michael D.

    2010-11-01

    We present a simple fluorescence imaging method for measuring the time-resolved concentration of a fluorescent molecule diffusing through an anodic alumina membrane with a pore diameter of 20 nm. From the concentration breakthrough curve, the molecular diffusivity of the fluorophore was extracted. The experimentally determined diffusivity was three orders of magnitude lower than reported bulk values. Due to the relative simplicity and ease of use, this method can be applied to provide fundamental information for biomolecular separations applications. One feature of this method is the high sensitivity at intercellular volumes broadening its application to drug delivery and controlled cell growth.

  18. Time-resolved and steady-state fluorescence spectroscopy from bacteria subjected to bactericidal agents

    NASA Astrophysics Data System (ADS)

    Katz, Alvin; Alimova, Alexandra; Siddique, Masood; Savage, Howard E.; Shah, Mahendra; Rosen, Richard; Alfano, Robert

    2004-03-01

    The time-resolved and steady-state changes in fluorescence were investigated from one spore-forming (Bacillus subtilis) and four non-spore forming (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) bacteria subjected to different bactericidal agents. The bactericidal agents were sodium hypochlorite (bleach) hydrogen peroxide, formaldehyde, and UV light exposure. Application of sodium hypochlorite resulted in an almost total lose of fluorescence signal and large decrease in the optical density of the bacterial suspension. Addition of hydrogen peroxide resulted in a 35% decrease in emission intensity fom the Sa and an 85-95% decrease for the other bacteria. Ultraviolet light exposure resulted in a 5-35% decrease in the emission intensity of the tryptophan band. The addition of formaldehyde to the bacteria did not result in significant changes in the steady-state emission intensity, but did shift the tryptophan emission peak position to shorter wavelengths by 3 to 5 nm. Time-resolved fluorescence measurements showed that the fluorescence lifetime of tryptophan in the bacteria could not be described by a single exponential decay, and was similar to that of tryptophan in neutral aqueous solution. Upon addition of formaldehyde to the Gram positive bacteria (Bs and Sa) the strength of the short lifetime component increased dramatically, while for the Gram negative bacteria, a smaller increase was observed. These fluorescence changes reflect the different mechanisms of the bactericidal agents and may provide a useful tool to monitor the effectiveness of disinfectants.

  19. Time-resolved fluorescence monitoring of cholesterol in peripheral blood mononuclear cells

    NASA Astrophysics Data System (ADS)

    Martinakova, Z.; Horilova, J.; Lajdova, I.; Marcek Chorvatova, A.

    2014-12-01

    Precise evaluation of intracellular cholesterol distribution is crucial for improving diagnostics of diseased states associated with cholesterol alteration. Time-resolved fluorescence techniques are tested for non-invasive investigation of cholesterol in living cells. Fluorescent probe NBD attached to cholesterol was employed to evaluate cholesterol distribution in peripheral blood mononuclear cells (PBMC) isolated from the human blood. Fluorescence Lifetime Imaging Microscopy (FLIM) was successfully applied to simultaneously monitor the spatial distribution and the timeresolved characteristics of the NBD-cholesterol fluorescence in PBMC. Gathered data are the first step in the development of a new perspective non-invasive diagnostic method for evaluation of cholesterol modifications in diseases associated with disorders of lipid metabolism.

  20. Exciplex liquid-phase thermometer using time-resolved laser-induced fluorescence.

    PubMed

    Parigger, C; Plemmons, D H; Litchford, R J; Jeng, S M

    1998-01-01

    Pulsed photoexcitation of hydrocarbon fuels doped with organic molecules exhibits a temperature-dependent fluorescence spectrum that is used as the basis for a weakly intrusive optical thermometer. By use of pulsed excitation from a 308-nm 8-ns XeCl excimer laser with gated detection of the fluorescence emissions from doped n -heptane, we demonstrate that time-resolved measurement of the excited monomer and the redshifted excited-state complex (exciplex) fluorescence emissions can yield sub-1 degrees accuracy for temperatures ranging from 440 K to the vicinity of the critical temperature (540 K). The experiments also show that the exciplex fluorescence spectrum is pressure independent below and above supercritical pressure. PMID:18084417

  1. Fluorescence polarization spectroscopy and time-resolved fluorescence kinetics of native cancerous and normal rat kidney tissues.

    PubMed Central

    Tata, D B; Foresti, M; Cordero, J; Tomashefsky, P; Alfano, M A; Alfano, R R

    1986-01-01

    Steady state fluorescence polarization spectra and time-resolved emission decay kinetics have been measured in vitro from malignant and normal rat kidney tissue. The degrees of polarization and emission lifetimes from the cancerous and normal systems are different. The spectroscopic differences are attributed to environmental transformations local to the native flavin and porphyrin fluorophors' binding sites. PMID:3489490

  2. Simulation modelling of a micro-system for time-resolved fluorescence measurements

    NASA Astrophysics Data System (ADS)

    Repich, Marina; Stoppa, David; Rae, Bruce R.; Henderson, Robert K.; Dalla Betta, Gian-Franco

    2010-04-01

    This paper presents the simulation modelling of a typical experimental setup for time-resolved fluorescence measurement. The developed model takes into account the setup geometry, characteristics of light source, detector and fluorescent sample as well as the adopted measurement technique. A qualitative verification of the model has been reported before. In this paper, we present a quantitative analysis and verification of the system versatility. For this we conducted time-resolved fluorescence measurements using a two-chip based micro-system, including a blue micro-LED array as a light source and a CMOS SPAD array as a detector. The sample of interest (CdSe/ZnS quantum dots in toluene) in a micro-cavity slide and an excitation filter were placed in the gap between the excitation and detection planes. A time-correlated single photon counting module was used to build fluorescence decay curves. A range of experiments with different excitation light pulse widths and using several setups have been performed. The simulated data are in good agreement with measured results and the model proves to be flexible enough to simulate different light sources and detector quenching/recharging circuits. This model can be used to predict qualitative and quantitative results for specific experimental setups, supporting the explanations of observed effects and allowing the realisation of virtual experiments.

  3. Intracellular Monitoring of AS1411 Aptamer by Time-Resolved Microspectrofluorimetry and Fluorescence Imaging.

    PubMed

    Kočišová, Eva; Praus, Petr; Bok, Jiří; Bonneau, Stéphanie; Sureau, Franck

    2015-09-01

    Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one. PMID:26179074

  4. Drug/protein interactions studied by time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Gustavsson, Thomas; Markovitsi, Dimitra; Vayá, Ignacio; Bonancía, Paula; Jiménez, M. C.; Miranda, Miguel A.

    2014-09-01

    We report here on a recent time-resolved fluorescence study [1] of the interaction between flurbiprofen (FBP), a chiral non-steroidal anti-inflammatory drug, and human serum albumin (HSA), the main transport protein in the human body. We compare the results obtained for the drug-protein complex with those of various covalently linked flurbiprofentryptophan dyads having well-defined geometries. In all cases stereoselective dynamic fluorescence quenching is observed, varying greatly from one system to another. In addition, the fluorescence anisotropy decays also display a clear stereoselectivity. For the drug-protein complexes, this can be interpreted in terms of the protein microenvironment playing a significant role in the conformational relaxation of FBP, which is more restricted in the case of the (R)- enantiomer.

  5. Time-resolved tryptophan fluorescence in photosynthetic reaction centers from Rhodobacter sphaeroides

    NASA Technical Reports Server (NTRS)

    Godik, V. I.; Blankenship, R. E.; Causgrove, T. P.; Woodbury, N.

    1993-01-01

    Tryptophan fluorescence of reaction centers isolated from Rhodobacter sphaeroides, both stationary and time-resolved, was studied. Fluorescence kinetics were found to fit best a sum of four discrete exponential components. Half of the initial amplitude was due to a component with a lifetime of congruent to 60 ps, belonging to Trp residues, capable of efficient transfer of excitation energy to bacteriochlorophyll molecules of the reaction center. The three other components seem to be emitted by Trp ground-state conformers, unable to participate in such a transfer. Under the influence of intense actinic light, photooxidizing the reaction centers, the yield of stationary fluorescence diminished by congruent to 1.5 times, while the number of the kinetic components and their life times remained practically unchanged. Possible implications of the observed effects for the primary photosynthesis events are considered.

  6. Efficient signal processing for time-resolved fluorescence detection of nitrogen-vacancy spins in diamond

    NASA Astrophysics Data System (ADS)

    Gupta, A.; Hacquebard, L.; Childress, L.

    2016-03-01

    Room-temperature fluorescence detection of the nitrogen-vacancy center electronic spin typically has low signal to noise, requiring long experiments to reveal an averaged signal. Here, we present a simple approach to analysis of time-resolved fluorescence data that permits an improvement in measurement precision through signal processing alone. Applying our technique to experimental data reveals an improvement in signal to noise equivalent to a 14% increase in photon collection efficiency. We further explore the dependence of the signal to noise ratio on excitation power, and analyze our results using a rate equation model. Our results provide a rubric for optimizing fluorescence spin detection, which has direct implications for improving precision of nitrogen-vacancy-based sensors.

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

    PubMed

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

    2015-12-15

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

  8. A low cost time-resolved Raman spectroscopic sensing system enabling fluorescence rejection.

    PubMed

    Sinfield, Joseph V; Colic, Oliver; Fagerman, Daniel; Monwuba, Chike

    2010-02-01

    This paper describes a novel, compact, fiber-coupled, time-resolved Raman spectroscopy system that takes advantage of recent developments in diode laser and data acquisition technology to exploit the natural temporal separation between Raman and fluorescence phenomena and thereby limits the influence of fluorescence on Raman observations. The unit has been designed to be particularly low cost and is intended to provide the foundation for a wide range of in-line or fieldable sensing devices that can enhance the potential and affordability of in situ chemical analyses. The system operating principles, design, and performance are discussed along with its advantages and tradeoffs relative to traditional continuous wave (CW) Raman techniques. The system relies on a 6.4 kHz repetition rate 900 ps pulsed diode laser operating in the visible wavelength range (532 nm) to enhance the quality of Raman observations relative to CW and infrared systems, particularly for analytes examined in the presence of fluorophores. Time-resolved photon counting, achieved through a combination of off-the-shelf and custom hardware and software, limits the influence of fluorescence on Raman observations under pulsed excitation. The paper presents examples of the quality of Raman signatures that can be obtained with the system for a variety of compounds such as trichloroethylene, benzene, an aqueous nitrate solution, and olive oil. Further, the paper demonstrates an approximately 15-fold improvement in signal-to-noise ratio when comparing long- and short-gated time-resolved photon counting acquisition scenarios for a neat benzene sample doped with rhodamine 6G at a concentration of 1 x 10(-4) M. The system's versatility and effectiveness in the assessment of complex mixtures representative of industrial or field settings is demonstrated through analysis of a gasoline sample. Additional discussion outlines how efficient signal averaging over extended observation periods can enable low

  9. Advanced Time-Resolved Fluorescence Microscopy Techniques for the Investigation of Peptide Self-Assembly

    NASA Astrophysics Data System (ADS)

    Anthony, Neil R.

    The ubiquitous cross beta sheet peptide motif is implicated in numerous neurodegenerative diseases while at the same time offers remarkable potential for constructing isomorphic high-performance bionanomaterials. Despite an emerging understanding of the complex folding landscape of cross beta structures in determining disease etiology and final structure, we lack knowledge of the critical initial stages of nucleation and growth. In this dissertation, I advance our understanding of these key stages in the cross-beta nucleation and growth pathways using cutting-edge microscopy techniques. In addition, I present a new combined time-resolved fluorescence analysis technique with the potential to advance our current understanding of subtle molecular level interactions that play a pivotal role in peptide self-assembly. Using the central nucleating core of Alzheimer's Amyloid-beta protein, Abeta(16 22), as a model system, utilizing electron, time-resolved, and non-linear microscopy, I capture the initial and transient nucleation stages of peptide assembly into the cross beta motif. In addition, I have characterized the nucleation pathway, from monomer to paracrystalline nanotubes in terms of morphology and fluorescence lifetime, corroborating the predicted desolvation process that occurs prior to cross-beta nucleation. Concurrently, I have identified unique heterogeneous cross beta domains contained within individual nanotube structures, which have potential bionanomaterials applications. Finally, I describe a combined fluorescence theory and analysis technique that dramatically increases the sensitivity of current time-resolved techniques. Together these studies demonstrate the potential for advanced microscopy techniques in the identification and characterization of the cross-beta folding pathway, which will further our understanding of both amyloidogenesis and bionanomaterials.

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

    PubMed

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

    2015-05-01

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

  11. Noninvasive multimodal evaluation of bioengineered cartilage constructs combining time-resolved fluorescence and ultrasound imaging.

    PubMed

    Fite, Brett Z; Decaris, Martin; Sun, Yinghua; Sun, Yang; Lam, Adrian; Ho, Clark K L; Leach, J Kent; Marcu, Laura

    2011-04-01

    A multimodal diagnostic system that integrates time-resolved fluorescence spectroscopy, fluorescence lifetime imaging microscopy, and ultrasound backscatter microscopy is evaluated here as a potential tool for assessing changes in engineered tissue composition and microstructure nondestructively and noninvasively. The development of techniques capable of monitoring the quality of engineered tissue, determined by extracellular matrix (ECM) content, before implantation would alleviate the need for destructive assays over multiple time points and advance the widespread development and clinical application of engineered tissues. Using a prototype system combining time-resolved fluorescence spectroscopy, FLIM, and UBM, we measured changes in ECM content occurring during chondrogenic differentiation of equine adipose stem cells on 3D biodegradable matrices. The optical and ultrasound results were validated against those acquired via conventional techniques, including collagen II immunohistochemistry, picrosirius red staining, and measurement of construct stiffness. Current results confirm the ability of this multimodal approach to follow the progression of tissue maturation along the chondrogenic lineage by monitoring ECM production (namely, collagen type II) and by detecting resulting changes in mechanical properties of tissue constructs. Although this study was directed toward monitoring chondrogenic tissue maturation, these data demonstrate the feasibility of this approach for multiple applications toward engineering other tissues, including bone and vascular grafts. PMID:21303258

  12. Time-resolved imaging of fluorescent inclusions in optically turbid medium — phantom study

    NASA Astrophysics Data System (ADS)

    Kacprzak, M.; Liebert, A.; Sawosz, P.; Żołek, N.; Milej, D.; Maniewski, R.

    2010-03-01

    We present results of application of a time-resolved optical system for imaging of fluorescence excited in an inclusion containing indocyanine green (ICG), and located in optically turbid medium. The developed imaging system enabled simultaneous acquisition of fluorescence and diffusive reflectance. Eight independent time-resolved measurement channels based on time-correlated single photon counting technique were applied. In four of these channels, used for the fluorescence detection, sets of filters were applied in order to block the excitation light. Fast optomechanical switches allowed us to illuminate sequentially nine different spots on the surface of the studied object and finally 4×4 pixels maps at excitation and emission wavelengths were obtained. A liquid phantom used in this study consists of the fish tank filed with a solution ofmilk and water with black ink added to obtain optical properties in the range of the optical properties typical for the living tissue. A gel ball of a diameter of 5 mm with precisely controlled concentration of ICG was immersed in the liquid. The measurements were performed for inclusion located at different depths and for various ICG concentrations in the gel ball and in the surrounding liquid. The recorded distributions of times of arrival (DTA) of fluorescence photons and times of flight (DTOF) of diffusely reflected photons were analyzed by calculation of their statistical moments. We observed specific changes in moments of the measured DTAs as a function of depth of immersion of the fluorescent inclusion in the medium. We noted also that the changes of moments depend significantly on concentration of the dye in the fluorescence inclusion as well as in the surrounding liquid.

  13. High-performance time-resolved fluorescence by direct waveform recording

    NASA Astrophysics Data System (ADS)

    Muretta, Joseph M.; Kyrychenko, Alexander; Ladokhin, Alexey S.; Kast, David J.; Gillispie, Gregory D.; Thomas, David D.

    2010-10-01

    We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1 μJ/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 105 times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats.

  14. Analysis of hydrocarbon-bearing fluid inclusions (HCFI) using time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Przyjalgowski, Milosz A.; Ryder, Alan G.; Feely, Martin; Glynn, Thomas J.

    2005-06-01

    Hydrocarbon-bearing fluid inclusions (HCFI) are microscopic cavities within rocks that are filled with petroleum oil, the composition of which may not have changed since the trapping event. Thus, the composition of that entrapped oil can provide information about the formation and evolution of the oil reservoir. This type of information is important to the petroleum production and exploration industries. Crude oil fluorescence originates from the presence of cyclic aromatic compounds and the nature of the emission is governed by the chemical composition of the oil. Fluorescence based methods are widely used for analysis of crude oil because they offer robust, non-contact and non-destructive measurement options. The goal of our group is the development of a non-destructive analytical method for HCFI using time-resolved fluorescence methods. In broad terms, crude oil fluorescence behavior is governed by the concentration of quenching species and the distribution of fluorophores. For the intensity averaged fluorescence lifetime, the best correlations have been found between polar or alkane concentrations, but these are not suitable for robust, quantitative analysis. We have recently started to investigate another approach for characterizing oils by looking at Time-resolved Emission Spectra (TRES). TRES are constructed from intensities sampled at discrete times during the fluorescence decay of the sample. In this study, TRES, from a series of 10 crude oils from the Middle East, have been measured at discrete time gates (0.5 ns, 1 ns, 2 ns, 4 ns) over the 450-700 nm wavelength range. The spectral changes in TRES, such as time gate dependent Stokes' shift and spectral broadening, are analyzed in the context of energy transfer rates. In this work, the efficacy of using TRES for fingerprinting individual oils and HCFI is also demonstrated.

  15. [Characterization of Time-Resolved Laser-Induced Fluorescence from Crude Oil Samples].

    PubMed

    Liu, De-qing; Luan, Xiao-ning; Han, Xiao-shuang; Guo, Jin-jia; An, Ju-bai; Zheng, Rong-er

    2015-06-01

    To evaluate the feasibility of laser induced time-resolved fluorescence technique for in-situ detection of underwater suspended oil spill, extensive investigations have been carried out with different densities of crude oil samples from six different wells of Shengli Oilfield in this work. It was found that the fluorescence emission durations of these crude oil samples were almost the same, the Gate Pulse Delay of DDG (Digital Delay Generator) in the ICCD started at 52ns and ended at 82ns with a width (FWHM) of 10 ns. It appears that the peak location and lifetime of fluorescence for different crude oil samples varied with their densities, and those with similar densities shared a similar lifespan with the closer peak locations of fluorescence. It is also observed that the peak of fluorescence remained the same location before reaching the maximum intensity, subsequently shift to longer wavelength as fluorescence attenuated from maximum intensity with a red shift among 17-30 nm varied with samples. This demonstrated that the decay rate of fluorescent components in the crude oils was different, and energy transfer between these components might exist. It is hoped that those obtained results and characteristics could be the useful information for identification of suspended spilled-oil underwater. PMID:26601371

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

    PubMed Central

    2015-01-01

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

  17. 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

  18. The excited-state chemistry of protochlorophyllide a: a time-resolved fluorescence study.

    PubMed

    Dietzek, Benjamin; Kiefer, Wolfgang; Yartsev, Arkady; Sundström, Villy; Schellenberg, Peter; Grigaravicius, Paulius; Hermann, Gudrun; Popp, Jürgen; Schmitt, Michael

    2006-08-11

    The excited-state processes of protochlorophyllide a, the precursor of chlorophyll a in chlorophyll biosynthesis, are studied using picosecond time-resolved fluorescence spectroscopy. Following excitation into the Soret band, two distinct fluorescence components, with emission maxima at 640 and 647 nm, are observed. The 640 nm emitting component appears within the time resolution of the experiment and then decays with a time constant of 27 ps. In contrast, the 647 nm emitting component is built up with a 3.5 ps rise time and undergoes a subsequent decay with a time constant of 3.5 ns. The 3.5 ps rise kinetics are attributed to relaxations in the electronically excited state preceding the nanosecond fluorescence, which is ascribed to emission out of the thermally equilibrated S(1) state. The 27 ps fluorescence, which appears within the experimental response of the streak camera, is suggested to originate from a second minimum on the excited-state potential-energy surface. The population of the secondary excited state is suggested to reflect a very fast motion out of the Franck-Condon region along a reaction coordinate different from the one connecting the Franck-Condon region with the S(1) potential-energy minimum. The 27 ps-component is an emissive intermediate on the reactive excited-state pathway, as its decay yields the intermediate photoproduct, which has been identified previously (J. Phys. Chem. B 2006, 110, 4399-4406). No emission of the photoproduct is observed. The results of the time-resolved fluorescence study allow a detailed spectral characterization of the emission of the excited states in protochlorophyllide a, and the refinement of the kinetic model deduced from ultrafast absorption measurements. PMID:16841352

  19. Energy dissipation in matrix-isolated silver atoms: A time-resolved fluorescence study

    NASA Astrophysics Data System (ADS)

    Wiggenhauser, H.; Schroeder, W.; Kolb, D. M.

    1988-03-01

    The fluorescence from optically excited Ag atoms in Ar, Kr, and Xe matrices has been investigated in a time-resolved synchrotron-radiation study. A detailed energy dissipation model could be established from a systematic analysis of rise and decay times of all the observed fluorescence bands after pulsed excitation into the Ag (4d105p)2P1/2,3/2 levels, and by setting time windows between the excitation pulses in emission and emission-yield spectroscopy. Although the overall wavelength dependence of the decay time follows the λ3 law, the decay time is independent of λ within a given emission band. Finally, the role of energy transfer between Ag atoms and dimers for the evaluation of decay times is briefly addressed.

  20. Time-resolved fluorescence spectroscopic investigation of cationic polymer/DNA complex formation

    NASA Astrophysics Data System (ADS)

    D'Andrea, Cosimo; Bassi, Andrea; Taroni, Paola; Pezzoli, Daniele; Volonterio, Alessandro; Candiani, Gabriele

    2011-07-01

    Since DNA is not internalized efficiently by cells, the success of gene therapy depends on the availability of carriers to efficiently deliver genetic material into target cells. Gene delivery vectors can be broadly categorized into viral and non-viral ones. Non-viral gene delivery systems are represented by cationic lipids and polymers rely on the basics of supramolecular chemistry termed "self-assembling": at physiological pH, they are cations and spontaneously form lipoplexes (for lipids) and polyplexes (for polymers) complexing nucleic acids. In this scenario, cationic polymers are commonly used as non-viral vehicles. Their effectiveness is strongly related to key parameters including DNA binding ability and stability in different environments. Time-resolved fluorescence spectroscopy of SYBR Green I (DNA dye) was carried out to characterize cationic polymer/DNA complex (polyplex) formation dispersed in aqueous solution. Both fluorescence amplitude and lifetime proved to be very sensitive to the polymer/DNA ratio (N/P ratio, +/-).

  1. Glucose sensing by time-resolved fluorescence of sol-gel immobilized glucose oxidase.

    PubMed

    Esposito, Rosario; Della Ventura, Bartolomeo; De Nicola, Sergio; Altucci, Carlo; Velotta, Raffaele; Mita, Damiano Gustavo; Lepore, Maria

    2011-01-01

    A monolithic silica gel matrix with entrapped glucose oxidase (GOD) was constructed as a bioactive element in an optical biosensor for glucose determination. Intrinsic fluorescence of free and immobilised GOD was investigated in the visible range in presence of different glucose concentrations by time-resolved spectroscopy with time-correlated single-photon counting detector. A three-exponential model was used for analysing the fluorescence transients. Fractional intensities and mean lifetime were shown to be sensitive to the enzymatic reaction and were used for obtaining calibration curve for glucose concentration determination. The sensing system proposed achieved high resolution (up to 0.17 mM) glucose determination with a detection range from 0.4 mM to 5 mM. PMID:22163807

  2. Steady state and time-resolved fluorescence spectroscopic characterization of normal and cancerous urine

    NASA Astrophysics Data System (ADS)

    Rajasekaran, Ramu; Aruna, Prakasa Rao; Balu David, Munusamy; Koteeswaran, Dornadula; Muthuvelu, Kulandaivel; Rai, R.; Ganesan, Singaravelu

    2013-03-01

    Urine is one of the diagnostically important bio fluids, as it has many metabolites and some of them are native fluorophores. There may be a variation in the distribution and the physiochemical properties of the fluorophores during any metabolic change and pathologic conditions. Native fluorescence spectroscopy has been considered as a promising tool to characterize the fluorophores present in the urine. In this study, we aimed at characterizing the urine of both normal and patients with confirmed cancer using steady state and time-resolved fluorescence spectroscopy at 280 nm and 350 nm excitation. It is observed that the metabolites indoxyl sulphate and neopterin and its derivatives are responsible for altered spectral signatures at 280 nm, and 350 nm excitation. The overall spectral data were subjected to Principal Component Analysis and the resultant components were used as input in the linear discriminant analysis. As a total, 84% and 81.8% of samples were correctly classified at 280 nm and 350 nm respectively.

  3. Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity.

    PubMed

    Ferrer, Marc; Zuck, Paul; Kolodin, Garrett; Mao, Shi Shan; Peltier, Richard R; Bailey, Carolyn; Gardell, Stephen J; Strulovici, Berta; Inglese, James

    2003-06-01

    An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z(')>0.5) assay for high-throughput screening. PMID:12729605

  4. Multimodal imaging of vascular grafts using time-resolved fluorescence and ultrasound

    NASA Astrophysics Data System (ADS)

    Fatakdawala, Hussain; Griffiths, Leigh G.; Wong, Maelene L.; Humphrey, Sterling; Marcu, Laura

    2015-02-01

    The translation of engineered tissues into clinic requires robust monitoring of tissue development, both in vitro and in vivo. Traditional methods for the same are destructive, inefficient in time and cost and do not allow time-lapse measurements from the same sample or animal. This study reports on the ability of time-resolved fluorescence and ultrasound measurements for non-destructive characterization of explanted tissue engineered vascular grafts. Results show that TRFS and FLIm are able to assess alterations in luminal composition namely elastin, collagen and cellular (hyperplasia) content via changes in fluorescence lifetime values between normal and grafted tissue. These observations are complemented by structural changes observed in UBM pertaining to graft integration and intimal thickness over the grafted region. These results encourage the future application of a catheter-based technique that combines these imaging modalities for non-destructive characterization of vascular grafts in vivo.

  5. A Novel Europium Chelate Coated Nanosphere for Time-Resolved Fluorescence Immunoassay

    PubMed Central

    Shen, Yifeng; Xu, Shaohan; He, Donghua

    2015-01-01

    A novel europium ligand 2, 2’, 2’’, 2’’’-(4, 7-diphenyl-1, 10-phenanthroline-2, 9-diyl) bis (methylene) bis (azanetriyl) tetra acetic acid (BC-EDTA) was synthesized and characterized. It shows an emission spectrum peak at 610 nm when it is excited at 360 nm, with a large Stock shift (250 nm). It is covalently coated on the surface of a bare silica nanosphere containi free amino groups, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-Hydroxysuccinimide. We also observed an interesting phenomenon that when BC-EDTA is labeled with a silica nanosphere, the chelate shows different excitation spectrum peaks of about 295 nm. We speculate that the carboxyl has a significant influence on its excitation spectrum. The BC-EDTA/Eu3+coated nanosphere could be used as a fluorescent probe for time-resolved fluorescence immunoassay. We labeled the antibody with the fluorescent nanosphere to develop a nanosphere based hepatitis B surface antigen as a time-resolved fluorescence immunoassay reagent, which is very easy to operate and eliminates potential contamination of Eu3+ contained in the environment. The analytical and functional sensitivities are 0.0037 μg/L and 0.08 μg/L (S/N≥2.0) respectively. The detection range is 0.08-166.67 μg/L, which is much wider than that of ELISA (0.2-5μg/L). It is comparable to the commercial dissociation-enhanced lanthanide fluoro-immunoassay system (DELFIA) reagents (0.2-145μg/L). We propose that it can fulfill clinical applications. PMID:26056826

  6. [Discrimination of Crude Oil Samples Using Laser-Induced Time-Resolved Fluorescence Spectroscopy].

    PubMed

    Han, Xiao-shuang; Liu, De-qing; Luan, Xiao-ning; Guo, Jin-jia; Liu, Yong-xin; Zheng, Rong-er

    2016-02-01

    The Laser-induced fluorescence spectra combined with pattern recognition method has been widely applied in discrimination of different spilled oil, such as diesel, gasoline, and crude oil. However, traditional three-dimension fluorescence analysis method, which is not adapted to requirement of field detection, is limited to laboratory investigatio ns. The development of oil identification method for field detection is significant to quick response and operation of oil spill. In this paper, a new method based on laser-induced time-resolved fluorescence combined with support vector machine (SVM) model was introduced to discriminate crude oil samples. In this method, time-resolved spectra data was descended into two dimensions with selecting appropriate range in time and wavelength domains respectively to form a SVM data base. It is found that the classification accurate rate increased with an appropriate selection. With a selected range from 54 to 74 ns in time domain, the classification accurate rate has been increased from 83.3% (without selection) to 88.1%. With a selected wavelength range of 387.00~608.87 nm, the classification accurate rate of suspect oil was improved from 84% (without selection) to 100%. Since the detection delay of fluorescence lidar fluctuates due to wave and platform swing, the identification method with optimizing in both time and wavelength domains could offer a better flexibility for field applications. It is hoped that the developed method could provide some useful reference with data reduction for classification of suspect crude oil in the future development. PMID:27209747

  7. Time-resolved fluorescence spectroscopy for intraoperative assistance of thyroid surgery

    NASA Astrophysics Data System (ADS)

    Bachmann, L.; Brandao, M. P.; Iwakura, R.; Basilio, F. S.; Haleplian, K.; Ito, A. S.; Conti de Freitas, L. C.

    2016-03-01

    Searching for new methods to provide information of biochemical composition and structure is critical to improve the prognosis of thyroid diseases. The use of time-resolved fluorescence techniques to detect biochemical composition and tissue structure alterations could help develop a portable, minimally invasive, and non-destructive method to assist during surgical procedures. This research looks for employ a fluorescence technique based on lifetime measurements to differentiate healthy and benign lesions from malignant thyroid tissue. We employ a wide range of excitation and chose a more appropriate region for this work: 298-300 nm; and the fluorescence decay was measured at 340-450 nm. We observed fluorescence lifetimes at 340 nm emission of 0.80+/-0.26 and 3.94+/-0.47 ns for healthy tissue; 0.90+/-0.24 and 4.05+/-0.46 ns for benign lesions; and 1.21+/-0.14 and 4.63+/-0.25 ns for malignant lesions. For 450 nm emissions, we obtain lifetimes of 0.25+/-0.18 and 3.99+/-0.39 ns for healthy tissue, 0.24+/-0.17 and 4.20+/-0.48 ns for benign lesions, 0.33+/-0.32 and 4.55+/-0.55 ns for malignant lesions. We successfully demonstrated that fluorescence lifetimes at 340 nm emission can differentiate between thyroid malignant and healthy/benign tissues.

  8. 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.

  9. Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology

    PubMed Central

    Gullapalli, Ramachandra R.; Tabouillot, Tristan; Mathura, Rishi; Dangaria, Jhanvi H.; Butler, Peter J.

    2011-01-01

    Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction. PMID:17343487

  10. A Vertically Integrated CMOS Microsystem for Time-Resolved Fluorescence Analysis.

    PubMed

    Rae, Bruce R; Jingbin Yang; McKendry, Jonathan; Zheng Gong; Renshaw, David; Girkin, John M; Erdan Gu; Dawson, Martin D; Henderson, R K

    2010-12-01

    We describe a two-chip micro-scale time-resolved fluorescence analyzer integrating excitation, detection, and filtering. A new 8×8 array of drivers realized in standard low-voltage 0.35-μm complementary metal-oxide semiconductor is bump-bonded to AlInGaN blue micro-pixellated light-emitting diodes (micro-LEDs). The array is capable of producing sample excitation pulses with a width of 777 ps (FWHM), enabling short lifetime fluorophores to be investigated. The fluorescence emission is detected by a second, vertically-opposed 16 × 4 array of single-photon avalanche diodes (SPADs) fabricated in 0.35-μm high-voltage CMOS technology with in-pixel time-gated photon counting circuitry. Captured chip data are transferred to a PC for further processing, including histogramming, lifetime extraction, calibration and background/noise compensation. This constitutes the smallest reported solid-state microsystem for fluorescence decay analysis, replacing lasers, photomultiplier tubes, bulk optics, and discrete electronics. The system is demonstrated with measurements of fluorescent colloidal quantum dot and Rhodamine samples. PMID:23853381

  11. Energy transfer in Anabaena variabilis filaments under nitrogen depletion, studied by time-resolved fluorescence.

    PubMed

    Onishi, Aya; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

    2015-08-01

    Some filamentous cyanobacteria (including Anabaena) differentiate into heterocysts under nitrogen-depleted conditions. During differentiation, the phycobiliproteins and photosystem II in the heterocysts are gradually degraded. Nitrogen depletion induces changes in the pigment composition of both vegetative cells and heterocysts, which affect the excitation energy transfer processes. To investigate the changes in excitation energy transfer processes of Anabaena variabilis filaments grown in standard medium (BG11) and a nitrogen-free medium (BG110), we measured their steady-state absorption spectra, steady-state fluorescence spectra, and time-resolved fluorescence spectra (TRFS) at 77 K. TRFS were measured with a picosecond time-correlated single photon counting system. The pigment compositions of the filaments grown in BG110 changed throughout the growth period; the relative phycocyanin levels monotonically decreased, whereas the relative carotenoid (Car) levels decreased and then recovered to their initial value (at day 0), with formation of lower-energy Cars. Nitrogen starvation also altered the fluorescence kinetics of PSI; the fluorescence maximum of TRFS immediately after excitation occurred at 735, 740, and 730 nm after 4, 8, and 15 days growth in BG110, respectively. Based on these results, we discuss the excitation energy transfer dynamics of A. variabilis filaments under the nitrogen-depleted condition throughout the growth period. PMID:25596847

  12. Distinction of brain tissue, low grade and high grade glioma with time-resolved fluorescence spectroscopy.

    PubMed

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

    2006-01-01

    Neuropathology frozen section diagnoses are difficult in part because of the small tissue samples and the paucity of adjunctive rapid intraoperative stains. This study aims to explore the use of time-resolved laser-induced fluorescence spectroscopy as a rapid adjunctive tool for the diagnosis of glioma specimens and for distinction of glioma from normal tissues intraoperatively. Ten low grade gliomas, 15 high grade gliomas without necrosis, 6 high grade gliomas with necrosis and/or radiation effect, and 14 histologically uninvolved "normal" brain specimens are spectroscopicaly analyzed and contrasted. Tissue autofluorescence was induced with a pulsed Nitrogen laser (337 nm, 1.2 ns) and the transient intensity decay profiles were recorded in the 370-500 nm spectral range with a fast digitized (0.2 ns time resolution). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site were used for tissue characterization. A linear discriminant analysis diagnostic algorithm was used for tissue classification. Both low and high grade gliomas can be distinguished from histologically uninvolved cerebral cortex and white matter with high accuracy (above 90%). In addition, the presence or absence of treatment effect and/or necrosis can be identified in high grade gliomas. Taking advantage of tissue autofluorescence, this technique facilitates a direct and rapid investigation of surgically obtained tissue. PMID:16368511

  13. Time-resolved spectroscopy of the intrinsic fluorescence of nucleic acid species

    NASA Astrophysics Data System (ADS)

    Daniels, Malcolm; Hart, Lucas P.; Ho, Paul S.; Ballini, Jean-Pierre; Vigny, Paul

    1990-05-01

    Polarization and lifetime studies have shown that the fluorescence from nucleic acid species is complex, both at the individual chromophore level and because of the effect of stacking interactions on the electronic states. Recent work aimed at elucidating some aspects of this behavior by decay analysis and time-resolved spectroscopy is surveyed. Experimental work has been carried out using the ACO synchrotron at LURE, France) with time-correlated single photon counting, or a frequency-doubled N2-pumped dye laser, pulse width 700 ps, with fast-gated (100 ps width) analog detection and signal averaging. Decay curves are treated by global analysis using the Marquardt non-linear least-squares algorithm (synchrotron data) or the SPLMOD program (EMBO), which carries out a non-linear leastsquares minimization using cubic splines, for the laser data. Resolution of the decay data gives a model-based estimate of the number of components and their lifetimes. This information is then used to deconvolute timewindowed spectra (time-delayed spectra) into the time-resolved spectra. It is a particular feature of the combination of delayed photon counting with the continuous wavelength distribution of pulsed synchrotron radiation that excitation spectra correlating with emissions of different lifetimes can be obtained by uninterrupted repetitive scanning over a wide range of exciting wavelengths, in the present work from 230 nm to 354 urn. Such time-delayed excitation spectra can also be deconvoluted into components corresponding to the various time-resolved emission spectra. Examples of these three types of information viz resolved lifetimes, time-resolved emission spectra and their excitation spectra are presented and discussed for the following systems. I. adenosine; 6N, 6N-dimethyladenosine; protonated adenosine; this work shows the role of rotamers in the excited state behavior of this chromophore and demonstrates the forbidden nature of the lowest excited state. II. d(AT); d

  14. A CMOS Time-Resolved Fluorescence Lifetime Analysis Micro-System.

    PubMed

    Rae, Bruce R; Muir, Keith R; Gong, Zheng; McKendry, Jonathan; Girkin, John M; Gu, Erdan; Renshaw, David; Dawson, Martin D; Henderson, Robert K

    2009-01-01

    We describe a CMOS-based micro-system for time-resolved fluorescence lifetime analysis. It comprises a 16 × 4 array of single-photon avalanche diodes (SPADs) fabricated in 0.35 μm high-voltage CMOS technology with in-pixel time-gated photon counting circuitry and a second device incorporating an 8 × 8 AlInGaN blue micro-pixellated light-emitting diode (micro-LED) array bump-bonded to an equivalent array of LED drivers realized in a standard low-voltage 0.35 μm CMOS technology, capable of producing excitation pulses with a width of 777 ps (FWHM). This system replaces instrumentation based on lasers, photomultiplier tubes, bulk optics and discrete electronics with a PC-based micro-system. Demonstrator lifetime measurements of colloidal quantum dot and Rhodamine samples are presented. PMID:22291564

  15. Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence.

    PubMed

    Farino, Zachary J; Morgenstern, Travis J; Vallaghe, Julie; Gregor, Nathalie; Donthamsetti, Prashant; Harris, Paul E; Pierre, Nicolas; Freyberg, Robin; Charrier-Savournin, Fabienne; Javitch, Jonathan A; Freyberg, Zachary

    2016-01-01

    Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment. PMID:26849707

  16. A CMOS Time-Resolved Fluorescence Lifetime Analysis Micro-System

    PubMed Central

    Rae, Bruce R.; Muir, Keith R.; Gong, Zheng; McKendry, Jonathan; Girkin, John M.; Gu, Erdan; Renshaw, David; Dawson, Martin D.; Henderson, Robert K.

    2009-01-01

    We describe a CMOS-based micro-system for time-resolved fluorescence lifetime analysis. It comprises a 16 × 4 array of single-photon avalanche diodes (SPADs) fabricated in 0.35 μm high-voltage CMOS technology with in-pixel time-gated photon counting circuitry and a second device incorporating an 8 × 8 AlInGaN blue micro-pixellated light-emitting diode (micro-LED) array bump-bonded to an equivalent array of LED drivers realized in a standard low-voltage 0.35 μm CMOS technology, capable of producing excitation pulses with a width of 777 ps (FWHM). This system replaces instrumentation based on lasers, photomultiplier tubes, bulk optics and discrete electronics with a PC-based micro-system. Demonstrator lifetime measurements of colloidal quantum dot and Rhodamine samples are presented. PMID:22291564

  17. Estimation of crude oil grade using time-resolved fluorescence spectra.

    PubMed

    Hegazi, E; Hamdan, A

    2002-04-01

    Time-resolved fluorescence (TRF) spectra of six crude oils from the eastern province of Saudi Arabia were excited using a pulsed laser radiation at 250 nm and measured at specific time gates (TG) within the leading and trailing edges of the laser temporal pulse. The spectra showed the presence of a shoulder near 380 nm that systematically decreased in intensity from high-grade to low-grade crudes, and also from earlier to later TGs. The intensities of these shoulders are shown to be useful in estimating the grades of crude oils, particularly when the TRF spectra are measured at TGs within the leading edge of the laser temporal pulse. Contour diagrams depicting the shapes of the TRF spectra as function of TG (within the leading and trailing edges) are also presented to serve as true fingerprints of the crudes. PMID:18968578

  18. Time-Resolved Fluorescence Depolarization Study Of Lamellar To Inverted Cylindrical Micellar Phase

    NASA Astrophysics Data System (ADS)

    Cheng, Kwan H.

    1989-05-01

    The orientational order and rotational dynamics of 2-(3-(diphenyl-hexatrienyl) propanoy11-3-palmitoyl-L-a-phosphatidylcholine (DPH-PC) embedded in dioleoplphosphatidylethanolamine (DOPE) were studied by time-resolved fluorescence depolarization technique. Upon increasing the temperature, the wobbling diffusion constant D⊥ of DPH-PC was found to decrease at the lamellar (Lα) to inverted cylindrical (HII) phase transition (12°C). The calculated ratio of order parameter in the La phase to that in the HII phase was close to the theoretical value of 2.0 as predicted from the change in packing symmetry. The effects of butylated hydroxytoluene, cholesterol and phosphatidylchollne on this phase transition were also examined.

  19. Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

    PubMed Central

    Vallaghe, Julie; Gregor, Nathalie; Donthamsetti, Prashant; Harris, Paul E.; Pierre, Nicolas; Freyberg, Robin; Charrier-Savournin, Fabienne; Javitch, Jonathan A.; Freyberg, Zachary

    2016-01-01

    Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment. PMID:26849707

  20. Non-contact characterization of bacteria by time-resolved fluorescence

    NASA Astrophysics Data System (ADS)

    Bouchard, Alain; Frechette, Julie; Long, William F.; Vernon, Marcia; Cormier, Jean-Francois; Vallee, Real; Mafu, Akier A.; Lemay, Marie-Josee

    2004-07-01

    Accurate real-time methods for the detection of pathogenic microorganisms in the agri-food industry would represent an improvement over standard methods of analysis. We are currently developing a non-contact, scanning optical system for the detection of bacteria on meat surfaces based on fluorescence lifetime and intensity measurements. The system detects autofluorescent light emitted by the naturally occurring fluorophores in bacteria. Potential expected advantages of this system include accurate and efficient 2D real-time mapping of bacterial contamination of surfaces, and elimination of sample-to-sample cross-contamination. Furthermore, as the technique only requires minimal sample preparation and handling, the chemical properties of the specimen are preserved. This article presents the preliminary results obtained from a time-resolved fluorescence imaging system for the characterization of a non-pathogenic gram-negative bacteria, Pseudomonas fluorescens. Additionally we present a particular application of the system of interest to the agri-food industry, demonstrating its potential as a real-time macroscopic imaging system for mapping bacterial contamination on meat surfaces. Initial results indicate that the combination of fluorescence lifetime and intensity measurements provides a means for characterizing biological media and for detecting microorganisms on surfaces.

  1. Detection of cancer cells in prostate tissue with time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Gerich, C. E.; Opitz, J.; Toma, M.; Sergon, M.; Füssel, S.; Nanke, R.; Fehre, J.; Wirth, M.; Baretton, G.; Schreiber, J.

    2011-03-01

    Goals: Improving cancer diagnosis is one of the important challenges at this time. The precise differentiation between benign and malignant tissue is in the oncology and oncologic surgery of the utmost significance. A new diagnostic system, that facilitates the decision which tissue has to be removed, would be appreciated. In previous studies many attempts were made to use tissue fluorescence for cancer recognition. However, no clear correlation was found between tissue type and fluorescence parameters like time and wavelength dependent fluorescence intensity I(t, λ). The present study is focused on cooperative behaviour of cells in benign or malignant prostates tissue reflecting differences in their metabolism. Material and Methods: 50 prostate specimens were obtained directly after radical prostatectomy and from each specimen 6 punch biopsies were taken. Time-resolved fluorescence spectra were recorded for 4 different measurement points for each biopsy. The pathologist evaluated each measurement point separately. An algorithm was developed to determine a relevant parameter of the time dependent fluorescence data (fractal dimension DF ). The results of the finding and the DF -value were correlated for each point and then analysed with statistical methods. Results: A total of 1200 measurements points were analysed. The optimal algorithm and conditions for discrimination between malignant and non-malignant tissue areas were found. The correct classification could be stated in 93.4% of analysed points. The ROC-curve (AUC = 0.94) confirms the chosen statistical method as well as it informs about the specificity (0.94) and sensitivity (0.90). Conclusion: The new method seems to offer a very helpful diagnostic tool for pathologists as well as for surgery.

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

    PubMed

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

    2010-01-01

    The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8+/-0.3 ns) and 460 nm (lifetime=0.8+/-0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7+/-0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm; lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens. PMID:20459282

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  4. A homogeneous time-resolved fluorescence resonance energy transfer assay for phosphatidylserine exposure on apoptotic cells.

    PubMed

    Gasser, Jean-Philippe; Hehl, Michaela; Millward, Thomas A

    2009-01-01

    A simple, "mix-and-measure" microplate assay for phosphatidylserine (PtdSer) exposure on the surface of apoptotic cells is described. The assay exploits the fact that annexin V, a protein with high affinity and specificity for PtdSer, forms trimers and higher order oligomers on binding to membranes containing PtdSer. The transition from soluble monomer to cell-bound oligomer is detected using time-resolved fluorescence resonance energy transfer from europium chelate-labeled annexin V to Cy5-labeled annexin V. PtdSer detection is achieved by a single addition of a reagent mix containing labeled annexins and calcium ions directly to cell cultures in a 96-well plate, followed by a brief incubation before fluorescence measurement. The assay can be used to quantify PtdSer exposure on both suspension cells and adherent cells in situ. This method is simpler and faster than existing annexin V binding assays based on flow cytometry or microscopy, and it yields precise data with Z' values of 0.6-0.7. PMID:18835236

  5. The singlet-oxygen-sensitized delayed fluorescence in mammalian cells: a time-resolved microscopy approach.

    PubMed

    Scholz, Marek; Biehl, Anna-Louisa; Dědic, Roman; Hála, Jan

    2015-04-01

    The present work provides a proof-of-concept that the singlet oxygen-sensitized delayed fluorescence (SOSDF) can be detected from individual living mammalian cells in a time-resolved microscopy experiment. To this end, 3T3 mouse fibroblasts incubated with 100 μM TPPS4 or TMPyP were used and the microsecond kinetics of the delayed fluorescence (DF) were recorded. The analysis revealed that SOSDF is the major component of the overall DF signal. The microscopy approach enables precise control of experimental conditions - the DF kinetics are clearly influenced by the presence of the (1)O2 quencher (sodium azide), H2O/D2O exchange, and the oxygen concentration. Analysis of SOSDF kinetics, which was reconstructed as a difference DF kinetics between the unquenched and the NaN3-quenched samples, provides a cellular (1)O2 lifetime of τΔ = 1-2 μs and a TPPS4 triplet lifetime of τT = 22 ± 5 μs in agreement with previously published values. The short SOSDF acquisition times, typically in the range of tens of seconds, enable us to study the dynamic cellular processes. It is shown that SOSDF lifetimes increase during PDT-like treatment, which may provide valuable information about changes of the intracellular microenvironment. SOSDF is proposed and evaluated as an alternative tool for (1)O2 detection in biological systems. PMID:25591544

  6. The use of time-resolved fluorescence in gel-based proteomics for improved biomarker discovery

    NASA Astrophysics Data System (ADS)

    Sandberg, AnnSofi; Buschmann, Volker; Kapusta, Peter; Erdmann, Rainer; Wheelock, Åsa M.

    2010-02-01

    This paper describes a new platform for quantitative intact proteomics, entitled Cumulative Time-resolved Emission 2-Dimensional Gel Electrophoresis (CuTEDGE). The CuTEDGE technology utilizes differences in fluorescent lifetimes to subtract the confounding background fluorescence during in-gel detection and quantification of proteins, resulting in a drastic improvement in both sensitivity and dynamic range compared to existing technology. The platform is primarily designed for image acquisition in 2-dimensional gel electrophoresis (2-DE), but is also applicable to 1-dimensional gel electrophoresis (1-DE), and proteins electroblotted to membranes. In a set of proof-of-principle measurements, we have evaluated the performance of the novel technology using the MicroTime 100 instrument (PicoQuant GmbH) in conjunction with the CyDye minimal labeling fluorochromes (GE Healthcare, Uppsala, Sweden) to perform differential gel electrophoresis (DIGE) analyses. The results indicate that the CuTEDGE technology provides an improvement in the dynamic range and sensitivity of detection of 3 orders of magnitude as compared to current state-of-the-art image acquisition instrumentation available for 2-DE (Typhoon 9410, GE Healthcare). Given the potential dynamic range of 7-8 orders of magnitude and sensitivities in the attomol range, the described invention represents a technological leap in detection of low abundance cellular proteins, which is desperately needed in the field of biomarker discovery.

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

    PubMed

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

    2015-08-01

    Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed. PMID:25577254

  8. Photobleaching of arterial fluorescent compounds: characterization of elastin, collagen and cholesterol time-resolved spectra during prolonged ultraviolet irradiation.

    PubMed

    Marcu, L; Grundfest, W S; Maarek, J M

    1999-06-01

    To study the photobleaching of the main fluorescent compounds of the arterial wall, we repeatedly measured the time-resolved fluorescence of elastin, collagen and cholesterol during 560 s of excitation with nitrogen laser pulses. Three fluence rate levels were used: 0.72, 7.25 and 21.75 microW/mm2. The irradiation-related changes of the fluorescence intensity and of the time-resolved fluorescence decay constants were characterized for the emission at 390, 430 and 470 nm. The fluorescence intensity at 390 nm decreased by 25-35% when the fluence delivered was 4 mJ/mm2, a common value in fluorescence studies of the arterial wall. Cholesterol fluorescence photobleached the most, and elastin fluorescence photobleached the least. Photobleaching was most intense at 390 nm and least intense at 470 nm such that the emission spectra of the three compounds were markedly distorted by photobleaching. The time-resolved decay constants and the fluorescence lifetime were not altered by irradiation when the fluence was below 4 mJ/mm2. The spectral distortions associated with photobleaching complicate the interpretation of arterial wall fluorescence in terms of tissue content in elastin, collagen and cholesterol. Use of the time-dependent features of the emission that are not altered by photobleaching should increase the accuracy of arterial wall analysis by fluorescence spectroscopy. PMID:10378012

  9. Time-Resolved Light Scattering and Fluorescence Spectroscopy in Biomedical and Model Random Media

    NASA Astrophysics Data System (ADS)

    Das, Bidyut Baran

    Optical spectroscopy, light scattering and ultrafast time-gated imaging have been shown to offer novel approaches to study the optical characteristics of various biomedical and other random media. Fluorescence spectra from human malignant and nonmalignant breast tissues were measured at 300 nm excitation and a significant spectral difference was found between the two tissue types by using the ratio of fluorescence intensities at 340 and 440 nm. Optical density measurements on thin breast tissues show that the scattering cross-sections of breast tissues are relatively constant over the visible and the uv region. Transport mean free paths and the absorption lengths for various tissues and model random media were measured using time-resolved transmission. The scattering coefficients for human breast and chicken tissues were found to remain relatively constant in 570-630 nm wavelength region while they change significantly at 1064 nm. Chicken breast and fat tissues were found to be good models for human breast tissues as the values of the optical parameters of the two tissue types are about the same. The less scattering observed at 1064 nm makes tissues more transparent in the NIR region making it easier to image in thick tissues. Time-resolved backscattering measurements show that the scattering and the absorption parameters of a random medium can be obtained accurately in a two-fiber configuration as long as the radial distance is more than about seven times the transport mean free path of the sample. The single point source-detection configuration provides a tool to diagnose breast malignancy though it fails to give accurate values of the optical parameters of tissues. This failure is attributed to the invalidity of the diffusion approximation in this experimental configuration. A 2.5 mm thin chicken fat strip was imaged inside a 40 mm thick chicken breast tissue using snake photons at 625 nm with ultrafast time-gated detection. A simple model to describe the effect

  10. Uranium and nitrate remote sensing in the nuclear fuel cycle by time-resolved laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Moulin, Christophe; Couston, Laurent; Decambox, Pierre; Mauchien, Patrick; Pouyat, Dominique

    1994-12-01

    Time-Resolved Laser-Induced Fluorescence has been used for uranium and nitrate remote sensing in the nuclear fuel cycle. Advantages of this technique are aside sensitivity and selectivity, its ability to perform remote measurements via fiber optics and optode. Uranium is usually determined by the standard addition method but by applying a fluorescence model taking into account complexation and absorption phenomena, it is possible to directly determine uranium concentration. Nitrate concentration is determined after spectral deconvolution of the uranium fluorescence spectrum.

  11. 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).

  12. Full time-resolved diffuse fluorescence tomography accelerated with parallelized Fourier-series truncated diffusion approximation

    NASA Astrophysics Data System (ADS)

    Yi, Xi; Wang, Bingyuan; Wan, Wenbo; Wang, Yihan; Zhang, Yanqi; Zhao, Huijuan; Gao, Feng

    2015-05-01

    Of the three measurement schemes established for diffuse fluorescence tomography (DFT), the time-domain scheme is well known to provide the richest information about the distribution of the targeting fluorophore in living tissues. However, the explicit use of the full time-resolved data usually leads to a considerably lengthy time for image reconstruction, limiting its applications to three-dimensional or small-volume imaging. To cope with the adversity, we propose herein a computationally efficient scheme for DFT image reconstruction where the time-dependent photon density is expanded to a Fourier-series and calculated by solving the independent frequency-domain diffusion equations at multiple sampling frequencies with the support of a combined multicore CPU-based coarse-grain and multithread GPU-based fine-grain parallelization strategy. With such a parallelized Fourier-series truncated diffusion approximation, both the time- and frequency-domain inversion procedures are developed and validated for their effectiveness and accuracy using simulative and phantom experiments. The results show that the proposed method can generate reconstructions comparable to the explicit time-domain scheme, with significantly reduced computational time.

  13. Adsorption of Uranyl on Gibbsite: A Time-Resolved Laser-Induced Fluorescence Spectroscopy Study

    SciTech Connect

    Chang, Hyun-shik; Korshin, Gregory V.; Wang, Zheming; Zachara, John M.

    2006-02-15

    Uranyl adsorbed on gibbsite at pH 4.0-8.0 and ionic strengths (ISs) 0.001-0.4 M (NaClO4) in the absence of carbonate was studied using time-resolved laser-induced fluorescence spectroscopy (TRLIFS) under cryogenic conditions. TRLIFS data showed the presence of several distinct emission components. Their contributions were determined using the evolving factor analysis approach. Four components denoted as species A, B, C, and D were discerned. Each of them was characterized by a characteristic response to pH and IS changes and also by a unique combination of the values of the fundamental transition energy E0,0, vibronic spacing E, and half-width of the vibronic lines W. Species A and B were major contributors to the overall emission. They were mainly affected by the pH and predominated below and above pH 5.0, respectively. In contrast with that, the contribution of species C was noticeable only at IS = 0.001 M, while it was suppressed or absent at high IS values. It was concluded that species A and B are likely to correspond to inner-sphere surface aluminol complexes AlO-(UO2)+ and AlO-(UO2)OH, while species C was hypothesized to correspond to electrostatically bound uranyl complexes (predominantly [UO2(OH)3]-).

  14. Simultaneous detection of sulfamethazine and sulfaquinoxaline using a dual-label time-resolved fluorescence immunoassay.

    PubMed

    Le, Tao; Yan, Peifeng; Liu, Jin; Wei, Shu

    2013-01-01

    A dual-label time-resolved fluoroimmunoassay (TRFIA) was introduced for the simultaneous quantification of sulfamethazine (SM2) and sulfaquinoxaline (SQX). Lanthanide (Eu(3+) and Sm(3+))-labelled antibodies were used because lanthanides have higher stabilities and narrower emission spectra than most fluorescent dyes. The sensitivity of the TRFIA for SM2 was 0.02 ng ml(-1), and the average recoveries and the intra- and inter-assay CVs were 77.2-107.6%, 5.4-10.5%, and 6.0-11.2%, respectively. The sensitivity of the TRFIA for SQX was 0.04 ng ml(-1); and the average recoveries and the intra- and inter-assay CVs were 74.1-102.8%, 4.6-10.9%, and 8.7-11.2%, respectively. The method was used to analyse chicken tissue and egg samples, and the results agreed well with the results of HPLC and enzyme-linked immunosorbent assay (ELISA) analyses, with correlation coefficients (R(2)) of 0.9415-0.9724. The TRFIA developed is a simple, fast and sensitive method for the high-throughput simultaneous screening of SM2 and SQX in edible animal tissues. PMID:23782396

  15. A homogeneous time-resolved fluorescence assay to identify inhibitors of HIV-1 fusion.

    PubMed

    Smeulders, Liesbet; Bunkens, Lieve; Vereycken, Inge; Van Acker, Koen; Holemans, Pascale; Gustin, Emmanuel; Van Loock, Marnix; Dams, Géry

    2013-01-01

    The human immunodeficiency virus type 1 (HIV-1) initiates infection through sequential interactions with CD4 and chemokine coreceptors unmasking the gp41 subunit of the viral envelope protein. Consequently, the N-terminal heptad repeats of gp41 form a trimeric coiled-coil groove in which the C-terminal heptad repeats collapse, generating a stable six-helix bundle. This brings the viral and cell membrane in close proximity enabling fusion and the release of viral genome in the cytosol of the host cell. In this chapter, we describe a homogeneous time-resolved fluorescence assay to identify inhibitors of HIV-1 fusion, based on the ability of soluble peptides, derived from the N- and C-terminal domains of gp41, to form a stable six-helix bundle in vitro. Labeling of the peptides with allophycocyanin and the lanthanide europium results in a Föster resonance energy transfer (FRET) signal upon formation of the six-helix bundle. Compounds interfering with the six-helix bundle formation inhibit the HIV-1 fusion process and suppress the FRET signal. PMID:23821256

  16. Validation and evaluation of a novel time-resolved laser-induced fluorescence technique

    NASA Astrophysics Data System (ADS)

    Durot, C. J.; Gallimore, A. D.; Smith, T. B.

    2014-01-01

    We present a novel technique to measure time-resolved laser-induced fluorescence signals in plasma sources that have a relatively constant Fourier spectrum of oscillations in steady-state operation, but are not periodically pulsed, e.g., Hall thrusters. The technique uses laser modulation of the order of MHz and recovers signal via a combination of band-pass filtering, phase-sensitive detection, and averaging over estimated transfer functions calculated for many different cycles of the oscillation. Periodic discharge current oscillations were imposed on a hollow cathode. Measurements were validated by comparison with independent measurements from a lock-in amplifier and by comparing the results of the transfer function average to an independent analysis technique triggering averaging over many oscillation cycles in the time domain. The performance of the new technique is analyzed and compared to prior techniques, and it is shown that this new technique has a niche in measurements where the analog photomultiplier signal has a nonwhite noise spectral density and cycles of oscillation are not sufficiently repeatable to allow for reliable triggering or a meaningful average waveform in the time domain.

  17. Validation and evaluation of a novel time-resolved laser-induced fluorescence technique.

    PubMed

    Durot, C J; Gallimore, A D; Smith, T B

    2014-01-01

    We present a novel technique to measure time-resolved laser-induced fluorescence signals in plasma sources that have a relatively constant Fourier spectrum of oscillations in steady-state operation, but are not periodically pulsed, e.g., Hall thrusters. The technique uses laser modulation of the order of MHz and recovers signal via a combination of band-pass filtering, phase-sensitive detection, and averaging over estimated transfer functions calculated for many different cycles of the oscillation. Periodic discharge current oscillations were imposed on a hollow cathode. Measurements were validated by comparison with independent measurements from a lock-in amplifier and by comparing the results of the transfer function average to an independent analysis technique triggering averaging over many oscillation cycles in the time domain. The performance of the new technique is analyzed and compared to prior techniques, and it is shown that this new technique has a niche in measurements where the analog photomultiplier signal has a nonwhite noise spectral density and cycles of oscillation are not sufficiently repeatable to allow for reliable triggering or a meaningful average waveform in the time domain. PMID:24517766

  18. Steady-State and Time-Resolved Studies into the Origin of the Intrinsic Fluorescence of G-Quadruplexes.

    PubMed

    Sherlock, Madeline E; Rumble, Christopher A; Kwok, Chun Kit; Breffke, Jens; Maroncelli, Mark; Bevilacqua, Philip C

    2016-06-16

    Stretches of guanines in DNA and RNA can fold into guanine quadruplex structures (GQSs). These structures protect telomeres in DNA and regulate gene expression in RNA. GQSs have an intrinsic fluorescence that is sensitive to different parameters, including loop sequence and length. However, the dependence of GQS fluorescence on solution and sequence parameters and the origin of this fluorescence are poorly understood. Herein we examine effects of dangling nucleotides and cosolute conditions on GQS fluorescence using both steady-state and time-resolved fluorescence spectroscopy. The quantum yield of dGGGTGGGTGGGTGGG, termed "dG3T", is found to be modest at ∼2 × 10(-3). Nevertheless, dG3T and its variants are significantly brighter than the common nucleic acid fluorophore 2-aminopurine (2AP) largely due to their sizable extinction coefficients. Dangling 5'-end nucleotides generally reduce emission and blue-shift the resultant spectrum, whereas dangling 3'-end nucleotides slightly enhance fluorescence, particularly on the red side of the emission band. Time-resolved fluorescence decays are broadly distributed in time and require three exponential components for accurate fits. Time-resolved emission spectra suggest the presence of two emitting populations centered at ∼330 and ∼390 nm, with the redder component being a well-defined long-lived (∼1 ns) entity. Insights into GQS fluorescence obtained here should be useful in designing brighter intrinsic RNA and DNA quadruplexes for use in label-free biotechnological applications. PMID:27267433

  19. Time-Resolved Fluorescent Immunochromatography of Aflatoxin B1 in Soybean Sauce: A Rapid and Sensitive Quantitative Analysis

    PubMed Central

    Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Zhang, Wen

    2016-01-01

    Rapid and quantitative sensing of aflatoxin B1 with high sensitivity and specificity has drawn increased attention of studies investigating soybean sauce. A sensitive and rapid quantitative immunochromatographic sensing method was developed for the detection of aflatoxin B1 based on time-resolved fluorescence. It combines the advantages of time-resolved fluorescent sensing and immunochromatography. The dynamic range of a competitive and portable immunoassay was 0.3–10.0 µg·kg−1, with a limit of detection (LOD) of 0.1 µg·kg−1 and recoveries of 87.2%–114.3%, within 10 min. The results showed good correlation (R2 > 0.99) between time-resolved fluorescent immunochromatographic strip test and high performance liquid chromatography (HPLC). Soybean sauce samples analyzed using time-resolved fluorescent immunochromatographic strip test revealed that 64.2% of samples contained aflatoxin B1 at levels ranging from 0.31 to 12.5 µg·kg−1. The strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique in food safety analysis. PMID:27428975

  20. Time-Resolved Fluorescent Immunochromatography of Aflatoxin B1 in Soybean Sauce: A Rapid and Sensitive Quantitative Analysis.

    PubMed

    Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Zhang, Wen

    2016-01-01

    Rapid and quantitative sensing of aflatoxin B1 with high sensitivity and specificity has drawn increased attention of studies investigating soybean sauce. A sensitive and rapid quantitative immunochromatographic sensing method was developed for the detection of aflatoxin B1 based on time-resolved fluorescence. It combines the advantages of time-resolved fluorescent sensing and immunochromatography. The dynamic range of a competitive and portable immunoassay was 0.3-10.0 µg·kg(-1), with a limit of detection (LOD) of 0.1 µg·kg(-1) and recoveries of 87.2%-114.3%, within 10 min. The results showed good correlation (R² > 0.99) between time-resolved fluorescent immunochromatographic strip test and high performance liquid chromatography (HPLC). Soybean sauce samples analyzed using time-resolved fluorescent immunochromatographic strip test revealed that 64.2% of samples contained aflatoxin B1 at levels ranging from 0.31 to 12.5 µg·kg(-1). The strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique in food safety analysis. PMID:27428975

  1. Dynamics and Flexibility of Human Aromatase Probed by FTIR and Time Resolved Fluorescence Spectroscopy

    PubMed Central

    Sadeghi, Sheila J.; Castrignanò, Silvia; Mei, Giampiero; Di Venere, Almerinda; Nicolai, Eleonora; Allegra, Paola; Gilardi, Gianfranco

    2013-01-01

    Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min−1 for the inhibitor-bound enzyme to 0.12±0.02 min−1 for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor

  2. Highly sensitive detection of human papillomavirus type 16 DNA using time-resolved fluorescence microscopy and long lifetime probes

    NASA Astrophysics Data System (ADS)

    Wang, Xue F.; Periasamy, Ammasi; Wodnicki, Pawel; Siadat-Pajouh, M.; Herman, Brian

    1995-04-01

    We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using

  3. Comparison of beetroot extracts originating from several sites using time-resolved laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Rabasović, M. S.; Šević, D.; Terzić, M.; Marinković, B. P.

    2012-05-01

    Beetroot (Beta vulgaris) juice contains a large number of fluorophores which can fluoresce. There is a growing interest in beetroot extracts analysis. In contrast, there is only limited information about beetroot obtained without sample preparation and/or extraction of components from the sample. In this work, we continue our previous study (Rabasović et al 2009 Acta Phys. Pol. A 116 570-2), analyzing and comparing beetroot extracts from several sites, using the time-resolved laser-induced fluorescence technique to measure the fluorescence of samples at different excitation wavelengths (340-470 nm) and for different sample dilutions.

  4. A time-resolved fluorescence study of matrix-isolated Ag 2

    NASA Astrophysics Data System (ADS)

    Hebert, T.; Kolb, D. M.; Rotermund, H. H.; Schriever, U.; Wiggenhauser, H.

    1990-02-01

    The nanosecond lifetimes of the A, B and C states of Ag 2 in Ar, Kr and Xe matrices were determined by time-resolved emission spectroscopy. From an analysis of the rise and decay times after pulsed optical excitation, the non-radiative relaxation channel between the B and A states was quantitatively established.

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

    NASA Astrophysics Data System (ADS)

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

    2007-05-01

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

  6. Time-resolved fluorescence and fluorescence anisotropy of fluorescein-labeled poly(N-isopropylacrylamide) incorporated in polymersomes.

    PubMed

    Lee, Jung Seok; Koehorst, Rob B M; van Amerongen, Herbert; Feijen, Jan

    2011-11-17

    The phase behavior of fluorescein isothiocyanate (FITC) labeled poly(N-isopropylacrylamide) (PNIPAAm) incorporated in polymersomes (Ps) was studied by monitoring the fluorescence lifetime (FL) and the time-resolved fluorescence anisotropy (TRFA) as a function of temperature at pH 7.4. Ps containing FITC-labeled PNIPAAm with a diameter less than 200 nm were prepared by injecting a THF solution of poly(ethylene glycol)-b-poly(d,l-lactide) (mPEG-PDLLA) and FITC tagged PNIPAAm (FITC-N) into phosphate buffered saline (PBS, pH 7.4). Solutions of free FITC (2 μM) and FITC-N (2 μM) in PBS were used as controls. The polarized fluorescence decay curves of FITC were fitted with one rotational correlation time (θ(1)) and the corresponding amplitude (β(1)), while those for FITC-N were fitted with two rotational correlation times (θ(1,2)) and their corresponding amplitudes (β(1,2)). Short rotational correlation times, θ(1), correspond with the rotation of the FITC molecule itself, whereas θ(2) corresponds to FITC-segmental rotation. FITC-N encapsulated in Ps (FITC-N/Ps) showed a decrease of the rotational motion upon increasing the temperature. The long rotational correlation time (θ(2)) of FITC-N increased 3 fold, going from 15 to 40 °C, reflecting a reduced rotational mobility. The residual anisotropy (β(∞)) of FITC-N/Ps at pH 7.4 showed a gradual increase, going from 15 to 25 °C followed by a gradual decrease at higher temperatures. These results are explained by a transition from coil to globule, a gradual increase of intermolecular aggregation, and possibly phase separation and hydrogel formation. PMID:21995555

  7. A Vinblastine Fluorescent Probe for Pregnane X Receptor in a Time-Resolved Fluorescence Resonance Energy Transfer Assay

    PubMed Central

    Lin, Wenwei; Chen, Taosheng

    2013-01-01

    The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug-drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and non-radioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand-PXR interactions. Here we report the characterization of BODIPY FL Vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a Kd value of 673 nM. We provide evidence that BODIPY FL Vinblastine is a unique chemical entity different from either vinblastine or the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene in its function as a high-affinity human PXR ligand. We describe a BODIPY FL Vinblastine-based human PXR Time-Resolved Fluorescence Resonance Energy Transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL Vinblastine–based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR. PMID:24044991

  8. Manganese-doped ZnSe quantum dots as a probe for time-resolved fluorescence detection of 5-fluorouracil.

    PubMed

    Zhu, Dong; Chen, Yun; Jiang, Liping; Geng, Jun; Zhang, Jianrong; Zhu, Jun-Jie

    2011-12-01

    Quantum dots (QDs) are generally used for the conventional fluorescence detection. However, it is difficult for the QDs to be applied in time-resolved fluorometry due to their short-lived emission. In this paper, high-quality Mn-doped ZnSe QDs with long-lived emission were prepared using a green and rapid microwave-assisted synthetic approach in aqueous solution. Fluorescence lifetime of the Mn-doped ZnSe QDs was extended as long as 400 μs, which was 10,000 times higher than that of conventional QDs such as CdS, CdSe, and CdTe. The QDs exhibited an excellent photostability over 35 h under continuous irradiation at 260 nm. Capped with mercaptopropionic acid (MPA), the Mn-doped ZnSe QDs were used for the time-resolved fluorescence detection of 5-fluorouracil (5-FU) with the detection limit of 128 nM. The relative standard deviation for seven independent measurements of 1.5 μM 5-FU was 3.8%, and the recovery ranged from 93% to 106%. The results revealed that the Mn-doped ZnSe QDs could be a good candidate as a luminescence probe for highly sensitive time-resolved fluorometry. PMID:22026809

  9. Measurements of hydroxyl concentrations and lifetimes in laminar flames using picosecond time-resolved laser-induced fluorescence.

    PubMed

    Reichardt, T A; Klassen, M S; King, G B; Laurendeau, N M

    1996-04-20

    Picosecond time-resolved laser-induced fluorescence (PITLIF) can potentially be used to obtain measurements of minor species concentrations in rapidly fluctuating flames. Previous studies demonstrated this potential for atomic sodium by monitoring the temporal fluorescence signal with both an equivalent-time and a real-time sampling method. In this developmental study, PITLIF is used to determine hydroxyl concentrations in laminar CH(4)-O(2)-N(2) flames by the measurement of both the integrated fluorescence signal and the fluorescence lifetime. The quenching environment can be monitored with real-time sampling, and thus the necessary quenching rate coefficient is obtained in 348 us, which is fast enough for use in many turbulent flows. Fluorescence lifetimes of OH are also measured at different equivalence ratios in laminar flames by the use of the equivalent-time sampling technique. These results compare favorably with predicted lifetimes based on relevant quenching cross sections and calculated species concentrations. PMID:21085341

  10. Time-resolved fluorescence spectroscopy of matrix-isolated silver atoms after pulsed excitation of inner-shell transitions

    NASA Astrophysics Data System (ADS)

    Hebert, T.; Wiggenhauser, H.; Schriever, U.; Kolb, D. M.

    1990-02-01

    The energy dissipation in matrix-isolated silver atoms after pulsed vacuum ultraviolet (VUV) excitation of 4d-5p transitions has been studied by time-resolved fluorescence spectroscopy. The decay behavior of the various fluorescence bands has been analyzed and a model for the relaxation process proposed within the framework of a two-dimensional configuration-coordinate diagram. If minute quantities of Ag2 are present in the matrix, the analysis requires consideration of energy transfer between silver atoms and dimers.

  11. Computational modeling of time-resolved fluorescence transport in turbid media for non-invasive clinical diagnostics

    NASA Astrophysics Data System (ADS)

    Vishwanath, Karthik

    Fluorescence spectroscopy and imaging methods, including fluorescence lifetime sensing, are being developed for a variety of non-invasive clinical diagnostic procedures, including applications to early cancer diagnosis. Here, both the theoretical developments and experimental validations of a versatile, numerical Monte Carlo code that models photon migration in turbid media to include simulations of time-resolved fluorescence transport are presented. The developed numerical model was used to study, for the first time, the dependence of time-resolved fluorescence signals emanating from turbid media on the optical transport coefficients, fluorophore properties and source-detector configurations in single-layered turbid media as well as more complex multi-layered turbid media. The numerical codes presented here can be adapted to model a wide range of experimental techniques measuring the optical responses of biological tissues to laser irradiation and are demonstrated here for two specific applications (a) to model time-resolved fluorescence dynamics in human colon tissues and (b) to extract the frequency-dependent optical responses of a model adult human head to an incident laser-source whose intensity was harmonically modulated i.e. simulating frequency-domain measurements. Specifically, measurements of time-resolved fluorescence decays from a previous clinical study aimed toward detecting differences in tissue pathologies in patients undergoing gastro-intestinal endoscopy were simulated using the Monte Carlo model and results demonstrated that variations in tissue optical transport coefficients (absorption and scattering) alone could not account for the fluorescence decay differences detected between tissue pathologies in vivo. However, variations in fluorescence decay time as large as those detected clinically between normal and pre-malignant tissues (of 2 ns) could be accounted for by simulated variations in tissue morphology or biochemistry while intrinsic

  12. Time-resolved fluorescence of thioredoxin single-tryptophan mutants: modeling experimental results with minimum perturbation mapping

    NASA Astrophysics Data System (ADS)

    Silva, Norberto D., Jr.; Haydock, Christopher; Prendergast, Franklyn G.

    1994-08-01

    The time-resolved fluorescence decay of single tryptophan (Trp) proteins is typically described using either a distribution of lifetimes or a sum of two or more exponential terms. A possible interpretation for this fluorescence decay heterogeneity is the existence of different isomeric conformations of Trp about its (chi) +1) and (chi) +2) dihedral angles. Are multiple Trp conformations compatible with the remainder of the protein in its crystallographic configuration or do they require repacking of neighbor side chains? It is conceivable that isomers of the neighbor side chains interconvert slowly on the fluorescence timescale and contribute additional lifetime components to the fluorescence intensity. We have explored this possibility by performing minimum perturbation mapping simulations of Trp 28 and Trp 31 in thioredoxin (TRX) using CHARMm 22. Mappings of Trp 29 and Trp 31 give the TRX Trp residue energy landscape as a function of (chi) +1) and (chi) +2) dihedral angles. Time-resolved fluorescence intensity and anisotropy decay of mutant TRX (W28F and W31F) are measured and interpreted in light of the above simulations. Relevant observables, like order parameters and isomerization rates, can be derived from the minimum perturbation maps and compared with experiment.

  13. Persistent luminescence nanoprobe for biosensing and lifetime imaging of cell apoptosis via time-resolved fluorescence resonance energy transfer.

    PubMed

    Zhang, Lei; Lei, Jianping; Liu, Jintong; Ma, Fengjiao; Ju, Huangxian

    2015-10-01

    Time-resolved fluorescence technique can reduce the short-lived background luminescence and auto-fluorescence interference from cells and tissues by exerting the delay time between pulsed excitation light and signal acquisition. Here, we prepared persistent luminescence nanoparticles (PLNPs) to design a universal time-resolved fluorescence resonance energy transfer (TR-FRET) platform for biosensing, lifetime imaging of cell apoptosis and in situ lifetime quantification of intracellular caspase-3. Three kinds of PLNPs-based nanoprobes are assembled by covalently binding dye-labeled peptides or DNA to carboxyl-functionalized PLNPs for the efficient detection of caspase-3, microRNA and protein. The peptides-functionalized nanoprobe is also employed for fluorescence lifetime imaging to monitor cell apoptosis, which shows a dependence of cellular fluorescence lifetime on caspase-3 activity and thus leads to an in situ quantification method. This work provides a proof-of-concept for PLNPs-based TR-FRET analysis and demonstrates its potential in exploring dynamical information of life process. PMID:26232881

  14. Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

    NASA Astrophysics Data System (ADS)

    Kim, Moon S.; Cho, Byoung-Kwan; Lefcourt, Alan M.; Chen, Yud-Ren; Kang, Sukwon

    2008-04-01

    We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.

  15. Evaluating steady-state and time-resolved fluorescence as a tool to study the behavior of asphaltene in toluene.

    PubMed

    Zhang, Hui Ting; Li, Rui; Yang, Zixin; Yin, Cindy-Xing; Gray, Murray R; Bohne, Cornelia

    2014-06-01

    A combination of steady-state fluorescence, fluorescence lifetime measurements and the determination of time-resolved emission spectra were employed to characterize asphaltene toluene solutions. Lifetime measurements were shown to be insensitive to the source of asphaltene or the alkane solvent from which asphaltene was precipitated. This insensitivity suggests that either the composition of Athabasca and Cold Lake asphaltene is very similar or that the fluorescence behavior is dominated by the same sub-set of fluorophores for the different samples. These results highlight the limitations in using fluorescence to characterize asphaltene solutions. Different dependencies were observed for the average lifetimes with the asphaltene concentration when measured at two different emission wavelengths (420 nm and 520 nm). This result suggests that different fluorophores underwent diverse interactions with other asphaltene molecules as the asphaltene concentration was raised, suggesting that models for asphaltene aggregation need to include molecular diversity. PMID:24722727

  16. Time-resolved fluorescence for breast cancer detection using an octreotate-indocyanine green derivative dye conjugate

    NASA Astrophysics Data System (ADS)

    Sordillo, Laura A.; Das, B. B.; Pu, Yang; Liang, Kexian; Milione, Giovanni; Sordillo, Peter P.; Achilefu, Sam; Alfano, R. R.

    2013-03-01

    Time-resolved fluorescence was used to investigate malignant and normal adjacent breast tissues stained with a conjugate of indocyanine green and octreotate. A marked increase in fluorescence lifetime intensity was seen in the breast cancer sample compared to the normal sample. The fluorescent lifetimes were also investigated and showed similar fluorescence decay curves in stained malignant and normal breast tissue. These results confirm that somatostatin receptors occur on human breast carcinomas, suggest that the presence of somatostatin receptors should be investigated as a marker of breast cancer aggressiveness, and suggest that this conjugate might be used to detect the presence of residual breast cancer after surgery, allowing better assessment of tumor margins and reducing the need for second or repeat biopsies in selected patients. These results may also provide clues for designing future treatment options for breast cancer patients.

  17. Picosecond time-resolved fluorescence spectroscopy of K-590 in the bacteriorhodopsin photocycle.

    PubMed Central

    Atkinson, G H; Blanchard, D; Lemaire, H; Brack, T L; Hayashi, H

    1989-01-01

    The fluorescence spectrum of a distinct isometric and conformational intermediate formed on the 10(-11) s time scale during the bacteriorhodopsin (BR) photocycle is observed at room temperature using a two laser, pump-probe technique with picosecond time resolution. The BR photocycle is initiated by pulsed (8 ps) excitation at 565 nm, whereas the fluorescence is generated by 4-ps laser pulses at 590 nm. The unstructured fluorescence extends from 650 to 880 nm and appears in the same general spectral region as the fluorescence spectrum assigned to BR-570. The transient fluorescence spectrum can be distinguished from that assigned to BR-570 by a larger emission quantum yield (approximately twice that of BR-570) and by a maximum intensity near 731 nm (shifted 17 nm to higher energy from the maximum of the BR-570 fluorescence spectrum). The fluorescence spectrum of BR-570 only is measured with low energy, picosecond pulsed excitation at 590 nm and is in good agreement with recent data in the literature. The assignment of the transient fluorescence spectrum to the K-590 intermediate is based on its appearance at time delays longer than 40 ps. The K-590 fluorescence spectrum remains unchanged over the entire 40-100-ps interval. The relevance of these fluorescence data with respect to the molecular mechanism used to model the primary processes in the BR photocycle also is discussed. PMID:2713439

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  19. Time-resolved multicolor two-photon excitation fluorescence microscopy of cells and tissues

    NASA Astrophysics Data System (ADS)

    Zheng, Wei

    2014-11-01

    Multilabeling which maps the distribution of different targets is an indispensable technique in many biochemical and biophysical studies. Two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with conventional fluorescence labeling techniques such as genetically encoded fluorescent protein (FP) and fluorescent dyes staining could be a powerful tool for imaging living cells. However, the challenge is that the excitation and emission wavelength of these endogenous fluorophores and fluorescent labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores, fluorescent proteins and fluorescent dyes were excited in their optimal wavelengths simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and wavelength domains. Cellular organelles such as nucleus, mitochondria, microtubule and endoplasmic reticulum, were clearly revealed in the TPEF images. The simultaneous imaging of multiple fluorophores of cells will greatly aid the study of sub-cellular compartments and protein localization.

  20. Discrimination of human coronary artery atherosclerotic lipid-rich lesions by time-resolved laser-induced fluorescence spectroscopy.

    PubMed

    Marcu, L; Fishbein, M C; Maarek, J M; Grundfest, W S

    2001-07-01

    Lesion composition plays a significant role in atherosclerotic lesion instability and rupture. Current clinical techniques cannot fully characterize lesion composition or accurately identify unstable lesions. This study investigates the use of time-resolved fluorescence spectroscopy for unstable atherosclerotic lesion diagnosis. The fluorescence of human coronary artery samples was induced with nitrogen laser and detected in the 360- to 510-nm wavelength range. The samples were sorted into 7 groups according to the AHA classification: normal wall and types I, II(a) (fatty streaks), III (preatheroma), IV (atheroma), V(a) (fibrous), and V(b) (calcified) lesions. Spectral intensities and time-dependent parameters [average lifetime tau(f); decay constants: tau(1) (fast-term), tau(2) (slow-term), A(1) (fast-term amplitude contribution)] derived from the time-resolved spectra of coronary samples were used for tissue characterization. We determined that a few intensity values at longer wavelengths (>430 nm) and time-dependent parameters at peak emission region (390 nm) discriminate between all types of arterial samples except between normal wall and type I lesions. The lipid-rich lesions (more unstable) can be discriminated from fibrous lesions (more stable) on the basis of time-dependent parameters (lifetime and fast-term decay). We inferred that features of lipid fluorescence are reflected on lipid-rich lesion emission. Our results demonstrate that analysis of the time-resolved spectra may be used to enhance the discrimination between different grades of atherosclerotic lesions and provide a means of discrimination between lipid-rich and fibrous lesions. PMID:11451759

  1. Understanding THz and IR signals beneath time-resolved fluorescence from excited-state ab initio dynamics.

    PubMed

    Petrone, Alessio; Donati, Greta; Caruso, Pasquale; Rega, Nadia

    2014-10-22

    The detailed interpretation of time-resolved spectroscopic signals in terms of the molecular rearrangement during a photoreaction or a photophysical event is one of the most important challenges of both experimental and theoretical chemistry. Here we simulate a time-resolved fluorescence spectrum of a dye in aqueous solution, the N-methyl-6-oxyquinolinium betaine, and analyze it in terms of far IR and THz frequency contributions, providing a direct connection to specific molecular motions. To obtain this result, we build up an innovative and general approach based on excited state ab-initio molecular dynamics and a wavelet-based time-dependent frequency analysis of nonstationary signals. We obtain a nice agreement with key parameters of the solvent dynamics, such as the total Stokes shift and the Stokes shift relaxation times. As an important finding, we observe a strong change of specific solute-solvent interactions upon the electronic excitation, with the migration of about 1.5 water molecules from the first solvation shell toward the bulk. In spite of this event, the Stokes shift dynamics is ruled by collective solvent motions in the THz and far IR, which guide and modulate the strong rearrangement of the dye microsolvation. By the relaxation of THz and IR contributions to the emission signal, we can follow and understand in detail the molecularity of the process. The protocol presented here is, in principle, transferable to other time-resolved spectroscopic techniques. PMID:25243826

  2. Transient Absorption and Time-Resolved Fluorescence Studies of Solvated Ruthenium Di-Bipyridine Pseudo-Halide Complexes

    NASA Astrophysics Data System (ADS)

    Compton, R.; Weidinger, D.; Owrutsky, J. C.

    2012-06-01

    Time-resolved IR and fluorescence measurements were performed to probe the vibrational and electronic properties, respectively, of ruthenium di-bipyridine pseudo-halide (Ru(Bpy){_2}(X){_2} (where X = CN, N{_3} or NCS)) complexes. Vibrational energy relaxation (VER) times were determined for the complexes dissolved in dimethyl sulfoxide (DMSO) with a trend in VER time of NCS > CN > N{_3}. A similar trend and comparable absolute rates for NCS- and N3- were previously observed by our group and others for simple inorganic anions in solution, suggesting a minimal contribution due to complexation. Measurements of the VER time of the CN complex in various solvents provide VER times in ethanol (42.3 ps) and DMSO (53.3 ps), which shows that protic solvents promote the relaxation. Time-resolved fluorescence measurements indicate a strong ligand dependence, with a factor of five decrease in the excited electronic state decay time from the CN (215 ns) to the NCS (39 ns) complex. A solvent dependence of the CN complex reveals a nearly 3-fold increase in the fluorescence decay time from acetonitrile (70 ns) to DMSO (215 ns).

  3. Picosecond-nanosecond time-resolved fluorescence of weak EDA systems of TCNB-mesitylene (toluene) under restricted diffusion conditions

    NASA Astrophysics Data System (ADS)

    Kojer, R.; Dresner, J.; Prochorow, J.; Deperasińska, I.

    1996-04-01

    In this paper the results of studies of the time-resolved fluorescence spectra of a weak electron-donor-acceptor systems of tetracyanobenzene with mesitylene (and toluene) are presented. A strong dependence of observed spectra at room and low temperature (under restricted diffusion conditions) and the lack of interrelation between decay parameters determined for different spectral regions are discussed in terms of existence of this EDA systems in a number of different orientational isomers. The results of computations of potential energy surfaces of the ground and excited state for TCNB-mesitylene system are supporting this hypothesis.

  4. LDS-750 as a probe of solvation dynamics: a femtosecond time-resolved fluorescence study in liquid aniline

    NASA Astrophysics Data System (ADS)

    Smith, Neil A.; Meech, Stephen R.; Rubtsov, Igor V.; Yoshihara, Keitaro

    1999-04-01

    The dynamics of the fluorescence Stokes shift of the styryl dye LDS-750 have been measured in liquid aniline with sub-100 fs time resolution. The shape of the time-resolved spectra are time dependent, which is not consistent with the predictions of a solvation dynamics mechanism. However, the measured spectral shift correlation function is reasonably well described by the dynamical mean spherical approximation model of solvation dynamics. It is suggested that these observations are consistent if solvent dynamics is the rate controlling process in both solvation of the increased dipole moment of the excited state of LDS-750 and the stabilisation of a distribution of solute conformers in the excited state.

  5. Steady-state and time-resolved fluorescence studies of stripped Borage oil.

    PubMed

    Smyk, Bogdan; Amarowicz, Ryszard; Szabelski, Mariusz; Gryczynski, Ignacy; Gryczynski, Zygmunt

    2009-07-30

    In this study we explored the spectroscopic properties of Borage oil, particularly the use of fluorescence techniques to investigate the presence of conjugated fatty acids (CFAs). This research has important health and dietary applications. The absorption and fluorescence spectra of different CFAs and Borage oil in ethanol were measured. Time-domain fluorescence was employed to establish the life times of the samples. We found that Borage oil contains 1.2x10(-3) mol L(-1) of alpha-eleostearic acid or its isomer (i.e., a conjugated triene), 1.6x10(-4) mol L(-1) of cis-parinaric acid (i.e., a conjugated tetraene) and 1.1x10(-5) mol L(-1) of c-COPA (i.e., a conjugated pentaene). Because of the three-exponential fluorescence intensity decay for Borage oil, other fatty acids with a four conjugated double bond system could not be excluded. PMID:19523559

  6. Ultrafast solvent dynamics: Connection between time resolved fluorescence and optical Kerr measurements

    NASA Astrophysics Data System (ADS)

    Cho, Minhaeng; Rosenthal, Sandra J.; Scherer, Norbert F.; Ziegler, Lawrence D.; Fleming, Graham R.

    1992-04-01

    The vibrational characteristics of liquid dynamics are used to describe the ultrafast relaxations observed in time-dependent fluorescence Stokes shift [J. Chem. Phys. 95, 4715 (1991)] and heterodyne detected optical Kerr effect measurements on acetonitrile, via a Brownian oscillator model. Introducing a frequency distribution of vibrational modes makes it possible to compare the two experiments. The ultrafast decays observed in the fluorescence Stokes shift and optical Kerr signals are produced by destructive superposition of the high frequency, underdamped modes.

  7. ANG-2 for quantitative Na(+) determination in living cells by time-resolved fluorescence microscopy.

    PubMed

    Roder, Phillip; Hille, Carsten

    2014-12-01

    Sodium ions (Na(+)) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na(+) concentrations ([Na(+)]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na(+)-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na(+)-sensitivity appropriate for recordings in living cells. The Na(+)-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na(+)]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na(+)]i rise in cockroach salivary gland cells, which was dependent on a Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems. PMID:25311309

  8. Cyclodextrin supramolecular inclusion-enhanced pyrene excimer switching for time-resolved fluorescence detection of biothiols in serum.

    PubMed

    Zhang, Qier; Deng, Ting; Li, Jishan; Xu, Weijian; Shen, Guoli; Yu, Ruqin

    2015-06-15

    We report here an efficient pyrene excimer signaling-based time-resolved fluorescent sensor for the measurement of biothiols (cysteine (Cys), homocysteine (Hcy), glutathione (GSH)) in human serum based on thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and the inclusion interaction of cyclodextrin. The sensing mechanism of the approach is based on the competitive ligation of Hg(2+) ions by Hcy/Cys/GSH and T-T mismatches in a bis-pyrene-labeled DNA strand with the self-complementary 5' and 3' ends. The introduction of γ-cyclodextrin can provide cooperation for the molecular level space proximity of the two labeled pyrene molecules, moreover the hydrophobic cavity of γ-cyclodextrin can also offer protection for the pyrene dimer's emission from the quenching effect of environmental conditions and enhance the fluorescence intensity of the pyrene excimer. When the biothiols are not presented, the sensing ensemble is in the "off" state due to the long distance between the two labeled pyrene molecules resulted from the formation of a more stable T-Hg(2+)-T structure. While in the presence of biothiols, Hg(2+) interacts very strongly with thiol groups and the T-Hg(2+)-T structure is dehybridized, and then the pyrene excimer will be formed due to the self-complementary 5' and 3' ends of the DNA probe and the cooperation interaction of γ-cyclodextrin to pyrene dimer, thus resulting in switching the sensing ensemble to the "on" state. In the optimum conditions described, the linear concentration range of 1.0-100 μM with the limit of detection (LOD) of 0.36 μM for GSH was obtained. Moreover, due to the much longer lifetime of the pyrene excimer fluorescence than those of the ubiquitous endogenous fluorescent components, the time-resolved fluorescence technique has been successfully used for application in complicated biological samples. PMID:25590970

  9. Time-resolved fluorescence spectroscopic study of crude petroleum oils: influence of chemical composition.

    PubMed

    Ryder, Alan G

    2004-05-01

    The fluorescence of crude petroleum oils is sensitive to changes in chemical composition and many different fluorescence methods have been used to characterize crude oils. The use of fluorescence lifetimes to quantitatively characterize oil composition has practical advantages over steady-state measurements, but there have been comparatively few studies in which the lifetime behavior is correlated with gross chemical compositional data. In this study, the fluorescence lifetimes for a series of 23 crude petroleum oils with American Petroleum Institute (API) gravities of between 10 and 50 were measured at several emission wavelengths (450-785 nm) using a 380 nm light emitting diode (LED) excitation source. It was found that the intensity average fluorescence lifetime (tau) at any emission wave-length does not correlate well with either API gravity or aromatic concentration. However, it was found that tau is strongly negatively correlated with both the polar and sulfur concentrations and positively correlated with the corrected alkane concentration. This indicates that the fluorescence behavior of crude petroleum oils is governed primarily by the concentration of quenching species. All the strong lifetime-concentration correlations are nonlinear and show a high degree of scatter, especially for medium to light oils with API gravities of between 25 and 40. The degree of scatter is greatest for oils where the concentrations (wt %) of the polar fraction is approximately 10 +/- 4%, the asphaltene component is approximately 1 +/- 0.5%, and sulfur is 0.5 +/- 0.4%. This large degree of scatter precludes the use of average fluorescence lifetime data obtained with 380 nm excitation for the accurate prediction of the common chemical compositional parameters of crude petroleum oils. PMID:15165340

  10. Novel flashlamp-based time-resolved fluorescence microscope reduces autofluorescence for 30-fold contrast enhancement in environmental samples

    NASA Astrophysics Data System (ADS)

    Connally, Russell; Veal, Duncan; Piper, James A.

    2003-07-01

    The abundance of naturally fluorescing components (autofluorophors) encountered in environmentally sourced samples can greatly hinder the detection and identification of fluorescently labeled target using fluorescence microscopy. Time-resolved fluorescence microscopy (TRFM) is a technique that reduces the effects of autofluorescence through precisely controlled time delays. Lanthanide chelates have fluorescence lifetimes many orders of magnitude greater than typical autofluorophors, and persist in their luminescence long after autofluorescence has ceased. An intense short pulse of (UV) light is used to excite fluorescence in the sample and after a short delay period the longer persisting fluorescence from the chelate is captured with an image-intensified CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flashlamp with a short pulse duration was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, flash output decays with an approximate lifetime of 18μs and the TRFM requires a long-lived chelate to ensure probe fluorescence is still visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the water-borne parasite Giardia lamblia. Fluorescence lifetime of the construct was determined to be 339μs +/- 14μs and provided a 45-fold enhancement of labeled Giardia over background using a gate delay of 100μs. Despite the sub-optimal decay characteristics of the light pulse, flashlamps have many advantages compared to optical chopper wheels and modulated lasers. Their low cost, lack of vibration, ease of interface and small footprint are important factors to consider in TRFM design.

  11. Role of polyplex intermediate species on gene transfer efficiency: polyethylenimine-DNA complexes and time-resolved fluorescence spectroscopy.

    PubMed

    Ketola, Tiia-Maaria; Hanzlíková, Martina; Urtti, Arto; Lemmetyinen, Helge; Yliperttula, Marjo; Vuorimaa, Elina

    2011-03-01

    Polyethylenimine (PEI) is a cationic DNA condensing polymer that facilitates gene transfer into the mammalian cells. The highest gene transfer with branched PEI is obtained at high nitrogen/phosphate (N/P) ratios with free PEI present. The small molecular weight PEI alone is not able to mediate DNA transfection. Here, we used recently developed time-resolved fluorescence spectroscopic method to study the mechanism of PEI-DNA complex formation and to investigate how free PEI, mean molecular weight, and branching of PEI affect the complexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that for both linear and branched high-molecular-weight PEI the complexation takes place in two steps, but the small-molecular-weight branched PEI complexed DNA at a single step. According to the binding constants obtained from time-resolved spectroscopic measurements, the affinity of N/P complexation per nitrogen atom is highest for LPEI and weakest for BPEI, whereas SPEI-DNA complexation showed intermediate values. Thus, the binding constant alone does not give adequate measure for transfection efficiency. On the other hand, the presence of intermediate states during the polyplex formation seems to be favorable for the gene transfection. Free PEI had no impact on the physical state of PEI-DNA complexes, even though it was essential for gene transfection in the cell culture. In conclusion, the molecular size and topology of PEI have direct influence on the DNA complexation but the free PEI does not. Free PEI must facilitate transfection at the cellular level and not via indirect effects on the PEI-DNA complexes. PMID:21291220

  12. Time-resolved excitation density dependent fluorescence of R-phycoerythrin single crystal

    NASA Astrophysics Data System (ADS)

    Wang, H. Z.; Zheng, X. G.; Zhao, F. L.; Gao, Z. L.; Yu, Z. X.; Zhu, J. C.; Jiang, L. J.; Zhang, J. P.; Liang, D. C.

    1994-05-01

    The fluorescence kinetics of a new single crystal, R-phycoerythrin (R-PE), has been studied by picosecond laser spectroscopy. An excitonic band, which is much more narrow than that of the molecular fluorescnece, is observed. At high pump density, superradiance of excitons in the bulk pure single crystal is recorded. The experimental results of fluorescence kinetics and exciton superradiance of R-PE crystal demonstrate that exciton energy transfer is natural, effective and rapid. It is concluded that excitons play an important role in energy transfer in the antennae of photosynthetic systems.

  13. The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA.

    PubMed

    D'Andrea, Cosimo; Pezzoli, Daniele; Malloggi, Chiara; Candeo, Alessia; Capelli, Giulio; Bassi, Andrea; Volonterio, Alessandro; Taroni, Paola; Candiani, Gabriele

    2014-12-01

    Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency. PMID:25308511

  14. A time-resolved fluorescence immunoassay for insulin in rodent plasma.

    PubMed

    Daijo, J E; Sportsman, J R

    1999-03-01

    We describe a time-resolved fluoroimmunoassay (TR-FIA) for quantification of insulin in rodent serum and plasma in the picomolar levels typical of these samples. The method is a solid-phase, sequential saturation assay based on competition of unlabeled insulin and biotinamidocaproyl-labeled insulin for anti-insulin antibody. Europium-labeled streptavidin allows the DELFIA system (Wallac) to be used for detection. The assay is sensitive (0.1 fmol detection limit, EC50 = 58 +/- 3 pM), accurate ( > 95% recovery of 88-880 pM insulin added to the samples), and simple enough to be automated in a 96-well microtiter plate format. Blood samples of 5 microl can be quickly processed and analyzed within a working concentration range of 40-200 pM, allowing direct measurement of insulin levels in rodents from a tail bleed. We used the TR-FIA to assess insulin levels in mouse and rat samples. In studies of streptozotocin-induced diabetes, as well as glucose load experiments, the assay gave results consistent with known literature. The measured insulin levels correlated significantly with values obtained by radioimmunoassay (R2 = 0.996). The intra-assay and inter-assay coefficients of variation were 2.3% and 15%, respectively. We compared results of this assay with an enzyme-linked immunosorbent assay (ELISA) method. The TR-FIA method was comparable to the ELISA but had higher sensitivity and required only one-tenth as much sample. The assay can be performed using commercially available reagents that allow for high sensitivity and practicability. PMID:10704099

  15. Time-resolved fluorescence imaging (TRFI) for direct immunofluorescence of PSA and alpha-1-antichymotrypsin in prostatic tissue sections.

    PubMed

    Bjartell, A; Siivola, P; Hulkko, S; Pettersson, K; Rundt, K; Lilja, H; Lövgren, T

    1999-05-01

    We have developed a direct immunofluorescence technique utilising chelates of the lanthanide ions europium and terbium conjugated to monoclonal IgGs (Mabs) against prostate-specific antigen (PSA) and alpha-1-antichymotrypsin (ACT) for the detection and quantification on the same tissue section. Strong signals without disturbance from tissue autofluorescence were demonstrated in paraffin sections of ten benign and six malignant prostate tissue specimens. The signal intensity increased linearly with the amount of labelled Mab until epitope saturation began. The highest concentrations of bound IgG in tissue sections were 27.3 fmol/pixel for ACT and 7.2 for PSA. Time-resolved fluorescence imaging (TRFI) offers an attractive method for histochemical studies based on specific and quantitative detection of fluorescent lanthanide chelates. PMID:12496823

  16. Interaction between certain porphyrins and CdS colloids: A steady state and time resolved fluorescence quenching study

    NASA Astrophysics Data System (ADS)

    Jhonsi, M. Asha; Kathiravan, A.; Renganathan, R.

    2008-12-01

    The interaction between porphyrins namely, meso-tetrakis (4-methoxyphenyl)porphyrin (TMeOPP), protoporphyrin IX (PPIX) and Zinc(II) meso-tetraphenylporphyrin (ZnTPP) with colloidal CdS has been studied by using steady state and time resolved fluorescence quenching measurements. The porphyrins adsorbed on the surface of colloidal CdS due to electrostatic interaction. This adsorption leads to changes in the absorption spectra related to the complex formation. The apparent association constant ( Kapp) was in the order of 4.34-5.58 × 10 5 M -1 from the effect of colloidal CdS on the absorption spectra and 0.64-1.6 × 10 5 M -1 from fluorescence quenching data. Quenching is attributable mainly to static mechanism through ground state complex formation as confirmed by lifetime measurements.

  17. Global and time-resolved monitoring of crop photosynthesis with chlorophyll fluorescence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Global monitoring of agricultural productivity is critical in a world under a continuous increase of food demand. Here we have used new spaceborne retrievals of chlorophyll fluorescence, an emission quantity intrinsically linked to photosynthesis, to derive spatially explicit photosynthetic uptake r...

  18. Conformational heterogeneity of the copper binding site in azurin. A time-resolved fluorescence study.

    PubMed Central

    Szabo, A G; Stepanik, T M; Wayner, D M; Young, N M

    1983-01-01

    Comparison of the fluorescence spectra and the effect of temperature on the quantum yields of fluorescence of Azurin (from Pseudomonas fluorescens ATCC-13525-2) and 3-methylindole (in methylcyclohexane solution) provides substantive evidence that the tryptophan residue in azurin is completely inaccessible to solvent molecules. The quantum yields of azurin (CuII), azurin (CuI), and apoazurin (lambda ex = 291 nm) were 0.052, 0.054, and 0.31, respectively. Other evidence indicates that there is no energy transfer from tyrosine to tryptophan in any of these proteins. The fluorescence decay behavior of each of the azurin samples was found to be invariant with emission wavelength. The fluorescences of azurin (CuII) and azurin (CuI) decay with dual exponential kinetics (tau 1 = 4.80 ns, tau 2 = 0.18 ns) while that of apoazurin obeys single exponential decay kinetics (tau = 4.90). The ratio of pre-exponentials of azurin (CuII), alpha 1/alpha 2, is found to be 0.25, and this ratio increases to 0.36 on reduction to azurin (CuI). The results are interpreted as originating from different interactions of the tryptophan with two conformers of the copper-ligand complex in azurin. PMID:6404322

  19. A dissociative fluorescence enhancement technique for one-step time-resolved immunoassays

    PubMed Central

    Mukkala, Veli-Matti; Hakala, Harri H. O.; Mäkinen, Pauliina H.; Suonpää, Mikko U.; Hemmilä, Ilkka A.

    2010-01-01

    The limitation of current dissociative fluorescence enhancement techniques is that the lanthanide chelate structures used as molecular probes are not stable enough in one-step assays with high concentrations of complexones or metal ions in the reaction mixture since these substances interfere with lanthanide chelate conjugated to the detector molecule. Lanthanide chelates of diethylenetriaminepentaacetic acid (DTPA) are extremely stable, and we used EuDTPA derivatives conjugated to antibodies as tracers in one-step immunoassays containing high concentrations of complexones or metal ions. Enhancement solutions based on different β-diketones were developed and tested for their fluorescence-enhancing capability in immunoassays with EuDTPA-labelled antibodies. Characteristics tested were fluorescence intensity, analytical sensitivity, kinetics of complex formation and signal stability. Formation of fluorescent complexes is fast (5 min) in the presented enhancement solution with EuDTPA probes withstanding strong complexones (ethylenediaminetetra acetate (EDTA) up to 100 mM) or metal ions (up to 200 μM) in the reaction mixture, the signal is intensive, stable for 4 h and the analytical sensitivity with Eu is 40 fmol/L, Tb 130 fmol/L, Sm 2.1 pmol/L and Dy 8.5 pmol/L. With the improved fluorescence enhancement technique, EDTA and citrate plasma samples as well as samples containing relatively high concentrations of metal ions can be analysed using a one-step immunoassay format also at elevated temperatures. It facilitates four-plexing, is based on one chelate structure for detector molecule labelling and is suitable for immunoassays due to the wide dynamic range and the analytical sensitivity. Figure   PMID:21161513

  20. Photosystem II Does Not Possess a Simple Excitation Energy Funnel: Time-Resolved Fluorescence Spectroscopy Meets Theory

    PubMed Central

    2013-01-01

    The experimentally obtained time-resolved fluorescence spectra of photosystem II (PS II) core complexes, purified from a thermophilic cyanobacterium Thermosynechococcus vulcanus, at 5–180 K are compared with simulations. Dynamic localization effects of excitons are treated implicitly by introducing exciton domains of strongly coupled pigments. Exciton relaxations within a domain and exciton transfers between domains are treated on the basis of Redfield theory and generalized Förster theory, respectively. The excitonic couplings between the pigments are calculated by a quantum chemical/electrostatic method (Poisson-TrEsp). Starting with previously published values, a refined set of site energies of the pigments is obtained through optimization cycles of the fits of stationary optical spectra of PS II. Satisfactorily agreement between the experimental and simulated spectra is obtained for the absorption spectrum including its temperature dependence and the linear dichroism spectrum of PS II core complexes (PS II-CC). Furthermore, the refined site energies well reproduce the temperature dependence of the time-resolved fluorescence spectrum of PS II-CC, which is characterized by the emergence of a 695 nm fluorescence peak upon cooling down to 77 K and the decrease of its relative intensity upon further cooling below 77 K. The blue shift of the fluorescence band upon cooling below 77 K is explained by the existence of two red-shifted chlorophyll pools emitting at around 685 and 695 nm. The former pool is assigned to Chl45 or Chl43 in CP43 (Chl numbering according to the nomenclature of Loll et al. Nature2005, 438, 1040) while the latter is assigned to Chl29 in CP47. The 695 nm emitting chlorophyll is suggested to attract excitations from the peripheral light-harvesting complexes and might also be involved in photoprotection. PMID:23537277

  1. Characterization of the Dynamics of an Essential Helix in the U1A Protein by Time-Resolved Fluorescence Measurements†

    PubMed Central

    Anunciado, Divina; Agumeh, Michael; Kormos, Bethany L.; Beveridge, David L.; Knee, Joseph L.; Baranger, Anne M.

    2008-01-01

    The RNA recognition motif (RRM), one of the most common RNA-binding domains, recognizes single-stranded RNA. A C-terminal helix that undergoes conformational changes upon binding is often an important contributor to RNA recognition. The N-terminal RRM of the U1A protein contains a C-terminal helix (helix C) that interacts with the RNA-binding surface of a β-sheet in the free protein (closed conformation), but is directed away from this β-sheet in the complex with RNA (open conformation). The dynamics of helix C in the free protein have been proposed to contribute to binding affinity and specificity. We report here a direct investigation of the dynamics of helix C in the free U1A protein on the nanosecond time scale using time-resolved fluorescence anisotropy. The results indicate that helix C is dynamic on a 2–3 ns time scale within a 20° range of motion. Steady-state fluorescence experiments and molecular dynamics simulations suggest that the dynamical motion of helix C occurs within the closed conformation. Mutation of a residue on the β-sheet that contacts helix C in the closed conformation dramatically destabilizes the complex (Phe56Ala) and alters the steady-state fluorescence, but not the time-resolved fluorescence anisotropy, of a Trp in helix C. Mutation of Asp90 in the hinge region between helix C and the remainder of the protein to Ala or Gly subtly alters the dynamics of the U1A protein and destabilizes the complex. Together these results show that helix C maintains a dynamic closed conformation that is stable to these targeted protein modifications and does not equilibrate with the open conformation on the nanosecond time scale. PMID:18293956

  2. Time-resolved experimental and computational study of two-photon laser-induced fluorescence in a hydrogen plasma

    PubMed

    van Der Heijden HW; Boogaarts; Mazouffre; van Der Mullen JA; Schram

    2000-04-01

    The time profile of the fluorescence light emission of atomic hydrogen in an expanding plasma beam after pulsed excitation with a nanosecond laser is studied, both experimentally and computationally. Ground state H atoms in an expanding Ar-H cascaded arc plasma are excited to the p=3 level using two-photon laser excitation at 205 nm. The resulting fluorescence is resolved in time with a fast photomultiplier tube to investigate the occurrence of quenching. A fluorescence decay time of (10+/-0.5) ns is measured under all circumstances, indicating that there is a complete l mixing of the p=3 sublevels. A time-resolved collisional radiative model is developed to model pulsed laser induced fluorescence for a large range of plasma parameters. The model calculations agree well with the experimental results over the entire range of conditions and indicate that two-photon LIF can strongly influence the local electron and ion densities, resulting in a "self-quenching" of the laser-induced H fluorescence. PMID:11088238

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

    NASA Astrophysics Data System (ADS)

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

    2007-09-01

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

  4. Application of time-resolved fluorescence to the determination of metabolites.

    PubMed

    Murillo Pulgarín, J A; Alañón Molina, A; Martínez Ferreras, F

    2014-07-15

    A simple fluorescent methodology for the simultaneous determination of two major metabolites of acetylsalicylic acid--salicylic and gentisic acids--in pharmaceutical preparations and human urine is proposed. Due to the overlapping between the fluorescence spectra of both analytes, the use of the more selective fluorescence decay curves is proposed. Values of dependent instrumental variables affecting the signal-to-noise ratio were fixed with a simplex optimization procedure. A calibration matrix of thirteen standards plus two blank samples was processed using a partial least-squares (PLS) analysis. To assess the goodness of the proposed method, a prediction set of nine synthetic samples was analyzed, obtaining recovery percentages between 95% and 106%. Limits of detection, calculated by means of a new criterion, were 3.49 μg L(-1) and 1.66 μg L(-1) for salicylic and gentisic acids, respectively. The method was also tested in three pharmaceutical preparations containing salicylic acid, obtaining recovery percentages close to 100%. Finally, the simultaneous determination of both analytes in human urine samples was successfully carried out by the PLS-analysis of a matrix of thirteen standards plus five analyte blanks. Although spectra of analytes and urine overlap strongly, no extraction method neither prior separation of the analytes were needed. PMID:24662756

  5. Application of time-resolved fluorescence to the determination of metabolites

    NASA Astrophysics Data System (ADS)

    Murillo Pulgarín, J. A.; Alañón Molina, A.; Martínez Ferreras, F.

    2014-07-01

    A simple fluorescent methodology for the simultaneous determination of two major metabolites of acetylsalicylic acid - salicylic and gentisic acids - in pharmaceutical preparations and human urine is proposed. Due to the overlapping between the fluorescence spectra of both analytes, the use of the more selective fluorescence decay curves is proposed. Values of dependent instrumental variables affecting the signal-to-noise ratio were fixed with a simplex optimization procedure. A calibration matrix of thirteen standards plus two blank samples was processed using a partial least-squares (PLS) analysis. To assess the goodness of the proposed method, a prediction set of nine synthetic samples was analyzed, obtaining recovery percentages between 95% and 106%. Limits of detection, calculated by means of a new criterion, were 3.49 μg L-1 and 1.66 μg L-1 for salicylic and gentisic acids, respectively. The method was also tested in three pharmaceutical preparations containing salicylic acid, obtaining recovery percentages close to 100%. Finally, the simultaneous determination of both analytes in human urine samples was successfully carried out by the PLS-analysis of a matrix of thirteen standards plus five analyte blanks. Although spectra of analytes and urine overlap strongly, no extraction method neither prior separation of the analytes were needed.

  6. Time-resolved imaging system for fluorescence-guided surgery with lifetime imaging capability

    NASA Astrophysics Data System (ADS)

    Powolny, F.; Homicsko, K.; Sinisi, R.; Bruschini, Claudio E.; Grigoriev, E.; Homulle, H.; Prior, John O.; Hanahan, D.; Dubikovskaya, E.; Charbon, E.

    2014-05-01

    We present a single-photon camera for fluorescence imaging, with a time resolution better than 100ps, capable of providing both intensity and lifetime images. the camera was fabricated in standard CMOS technology. With this FluoCam we show the possibility to study sub-nanosecond fluorescence mechanisms. The FluoCam was used to characterize a near-infrared probe, indocyanine green, conjugated with multimeric cyclic pentapeptide (cRGD). The fluorescent probe-conjugated was used to target and mark tumors with better specificity, in particular aiming at targeting the integrins αvβ3 and αvβ5. As a first step towards clinical studies, preliminary results obtained in-vivo are presented. The first envisioned clinical application would be image-guided surgical oncology to help the surgeon to remove tumor tissue by a better discrimination from normal tissues and also to improve the detection of metastatic lymph nodes. A further application could be the in-vivo determination of the αvβ3 and αvβ5 targets to select patients for therapy with RGD chemotherapy conjugates.

  7. Time-resolved fluorescence studies of a transmembrane peptide sequence of the dopamine D2 receptor

    NASA Astrophysics Data System (ADS)

    Williams, Valerie L.; Courtney, Scott H.; Schuster, David I.; Murphy, Randall B.

    1994-08-01

    Highly hydrophobic peptides in small unilamellar vesicles can be used to model membrane-embedded proteins such as the dopamine D2 receptor. The transmembrane domains of the dopamine D2 receptor are known to contain residues corresponding to the binding sites for natural receptor ligands. We have developed a model system consisting of a peptide whose sequence was taken from the transmembrane region of the dopamine D2 receptor and incorporated it into phospholipid bilayers. This polypeptide sequence, NH2-D-V-L-Y-S-A-F-T-W-L-G-Y-V-N-S-A-V-N-P-I-I-Y-T- T-F-N-V-CO2H, contains a single tryptophan residue, whose fluorescence properties provides an intrinsic probe of the microenvironment of the peptide within the bilayer. Purification of this highly hydrophobic peptide required the development of a novel alcohol-based reversed-phase HPLC solvent system. The vesicles were produces by cosonication of the peptide with dimyristoylphosphatidylcholine lipid and were characterized by electron microscopy and fluorescence spectroscopy. Time- correlated single photon counting was sued to measure the fluorescence anisotropy of the system as a function of temperature across the lipid phase transition range and as a function of the peptide/lipid ratio.

  8. Coherent photon interference elimination and spectral correction in femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy

    NASA Astrophysics Data System (ADS)

    Dang, Wei; Mao, Pengcheng; Weng, Yuxiang

    2013-07-01

    We report an improved setup of femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy (FNOPAS) with a 210 fs temporal response. The system employs a Cassegrain objective to collect and focus fluorescence photons, which eliminates the interference from the coherent photons in the fluorescence amplification by temporal separation of the coherent photons and the fluorescence photons. The gain factor of the Cassegrain objective-assisted FNOPAS is characterized as 1.24 × 105 for Rhodamine 6G. Spectral corrections have been performed on the transient fluorescence spectra of Rhodamine 6G and Rhodamine 640 in ethanol by using an intrinsic calibration curve derived from the spectrum of superfluorescence, which is generated from the amplification of the vacuum quantum noise. The validity of spectral correction is illustrated by comparisons of spectral shape and peak wavelength between the corrected transient fluorescence spectra of these two dyes acquired by FNOPAS and their corresponding standard reference spectra collected by the commercial streak camera. The transient fluorescence spectra of the Rhodamine 6G were acquired in an optimized phase match condition, which gives a deviation in the peak wavelength between the retrieved spectrum and the reference spectrum of 1.0 nm, while those of Rhodamine 640 were collected in a non-optimized phase match condition, leading to a deviation in a range of 1.0-3.0 nm. Our results indicate that the improved FNOPAS can be a reliable tool in the measurement of transient fluorescence spectrum for its high temporal resolution and faithfully corrected spectrum.

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

    NASA Astrophysics Data System (ADS)

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

    1992-04-01

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

  10. Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes

    PubMed Central

    Neely, Robert K.; Daujotyte, Dalia; Grazulis, Saulius; Magennis, Steven W.; Dryden, David T. F.; Klimašauskas, Saulius; Jones, Anita C.

    2005-01-01

    DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures. PMID:16340006

  11. A novel luminescent terbium-3-carboxycoumarin probe for time-resolved fluorescence sensing of pesticides methomyl, aldicarb and prometryne

    NASA Astrophysics Data System (ADS)

    Azab, Hassan A.; Duerkop, Axel; Saad, E. M.; Awad, F. K.; Abd El Aal, R. M.; Kamel, Rasha M.

    2012-11-01

    The luminescence arising from lanthanide cations offers several advantages over organic fluorescent molecules: sharp, distinctive emission bands allow for easy resolution between multiple lanthanide signals; long emission lifetimes (μs-ms) make them excellent candidates for time-resolved measurements; and high resistance to photo bleaching allow for long or repeated experiments. A time-resolved (gated) luminescence-based method for determination of pesticides methomyl, aldicarb and prometryne in microtiterplate format using the long-lived terbium-3-carboxycoumarin in 1:3 metal:ligand ratio has been developed. The limit of detection is 1.20 × 106, 5.19 × 105 and 2.74 × 106 ng L-1 for methomyl, prometryne and aldicarb, respectively. The quantum yield (QY = 0.08) of Tb(III)-3-carboxycoumarin was determined using 3-(2-benzothiazolyl)-7-diethylamino-coumarin (coumarin 6). Stern-volmer studies at different temperatures indicate that collisional quenching dominates for methomyl, aldicarb and prometryne. Binding constants were determined at 303, 308 and 313 K by using Lineweaver-Burk equation. A thermodynamic analysis showed that the reaction is spontaneous with negative ΔG. Effect of some relevant interferents on the detection of pesticides has been investigated.

  12. Cellular Oxygen and Nutrient Sensing in Microgravity Using Time-Resolved Fluorescence Microscopy

    NASA Technical Reports Server (NTRS)

    Szmacinski, Henryk

    2003-01-01

    Oxygen and nutrient sensing is fundamental to the understanding of cell growth and metabolism. This requires identification of optical probes and suitable detection technology without complex calibration procedures. Under this project Microcosm developed an experimental technique that allows for simultaneous imaging of intra- and inter-cellular events. The technique consists of frequency-domain Fluorescence Lifetime Imaging Microscopy (FLIM), a set of identified oxygen and pH probes, and methods for fabrication of microsensors. Specifications for electronic and optical components of FLIM instrumentation are provided. Hardware and software were developed for data acquisition and analysis. Principles, procedures, and representative images are demonstrated. Suitable lifetime sensitive oxygen, pH, and glucose probes for intra- and extra-cellular measurements of analyte concentrations have been identified and tested. Lifetime sensing and imaging have been performed using PBS buffer, culture media, and yeast cells as a model systems. Spectral specifications, calibration curves, and probes availability are also provided in the report.

  13. Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay.

    PubMed

    Röthlein, Christoph; Miettinen, Markus S; Borwankar, Tejas; Bürger, Jörg; Mielke, Thorsten; Kumke, Michael U; Ignatova, Zoya

    2014-09-26

    The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered β-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure. PMID:25092288

  14. Architecture of Polyglutamine-containing Fibrils from Time-resolved Fluorescence Decay

    PubMed Central

    Röthlein, Christoph; Miettinen, Markus S.; Borwankar, Tejas; Bürger, Jörg; Mielke, Thorsten; Kumke, Michael U.; Ignatova, Zoya

    2014-01-01

    The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered β-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure. PMID:25092288

  15. Time-resolved spectroscopy and fluorescence resonance energy transfer in the study of excimer laser damage of chromatin

    NASA Astrophysics Data System (ADS)

    Radu, L.; Mihailescu, I.; Radu, S.; Gazdaru, D.

    2007-09-01

    The analysis of chromatin damage produced by a 248 nm excimer laser radiation, for doses of 0.3-3 MJ/m 2 was carried out by time-resolved spectroscopy and fluorescence resonance energy transfer (FRET). The chromatin was extracted from a normal and a tumoral tissue of Wistar rats. The decrease with laser dose of the relative contribution of the excited state lifetimes of ethidium bromide (EtBr) bounded to chromatin constitutes an evidence of the reduction of chromatin deoxyribonucleic acid (DNA) double-strand structure. FRET was performed from dansyl chloride to acridine orange, both coupled to chromatin. The increase of the average distance between these ligands, under the action of laser radiation, reflects a loosening of the chromatin structure. The radiosensitivity of tumor tissue chromatin is higher than that of a normal tissue. The determination of the chromatin structure modification in an excimer laser field can be of interest in laser therapy.

  16. Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

    PubMed

    Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

    2016-06-01

    G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. PMID:26954998

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

    SciTech Connect

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

    2013-07-01

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

  18. Global and time-resolved monitoring of crop photosynthesis with chlorophyll fluorescence

    PubMed Central

    Guanter, Luis; Zhang, Yongguang; Jung, Martin; Joiner, Joanna; Voigt, Maximilian; Berry, Joseph A.; Frankenberg, Christian; Huete, Alfredo R.; Zarco-Tejada, Pablo; Lee, Jung-Eun; Moran, M. Susan; Ponce-Campos, Guillermo; Beer, Christian; Camps-Valls, Gustavo; Buchmann, Nina; Gianelle, Damiano; Klumpp, Katja; Cescatti, Alessandro; Baker, John M.; Griffis, Timothy J.

    2014-01-01

    Photosynthesis is the process by which plants harvest sunlight to produce sugars from carbon dioxide and water. It is the primary source of energy for all life on Earth; hence it is important to understand how this process responds to climate change and human impact. However, model-based estimates of gross primary production (GPP, output from photosynthesis) are highly uncertain, in particular over heavily managed agricultural areas. Recent advances in spectroscopy enable the space-based monitoring of sun-induced chlorophyll fluorescence (SIF) from terrestrial plants. Here we demonstrate that spaceborne SIF retrievals provide a direct measure of the GPP of cropland and grassland ecosystems. Such a strong link with crop photosynthesis is not evident for traditional remotely sensed vegetation indices, nor for more complex carbon cycle models. We use SIF observations to provide a global perspective on agricultural productivity. Our SIF-based crop GPP estimates are 50–75% higher than results from state-of-the-art carbon cycle models over, for example, the US Corn Belt and the Indo-Gangetic Plain, implying that current models severely underestimate the role of management. Our results indicate that SIF data can help us improve our global models for more accurate projections of agricultural productivity and climate impact on crop yields. Extension of our approach to other ecosystems, along with increased observational capabilities for SIF in the near future, holds the prospect of reducing uncertainties in the modeling of the current and future carbon cycle. PMID:24706867

  19. Global and Time-Resolved Monitoring of Crop Photosynthesis with Chlorophyll Fluorescence

    NASA Technical Reports Server (NTRS)

    Guanter, Luis; Zhang, Yongguang; Jung, Martin; Joiner, Joanna; Voigt, Maximilian; Berry, Joseph A.; Frankenberg, Christian; Huete, Alfredo R.; Zarco-Tejada, Pablo; Lee, Jung-Eun; Moran, M. Susan; Ponce-Campos, Guillermo; Beer, Christian; Camps-Valls, Gustavo; Buchmann, Nina; Gianelle, Damiano; Klumpp, Katja; Cescatti, Alessandro; Baker, John M.; Griffis, Timothy J.

    2014-01-01

    Photosynthesis is the process by which plants harvest sunlight to produce sugars from carbon dioxide and water. It is the primary source of energy for all life on Earth; hence it is important to understand how this process responds to climate change and human impact. However, model-based estimates of gross primary production (GPP, output from photosynthesis) are highly uncertain, in particular over heavily managed agricultural areas. Recent advances in spectroscopy enable the space-based monitoring of sun-induced chlorophyll fluorescence (SIF) from terrestrial plants. Here we demonstrate that spaceborne SIF retrievals provide a direct measure of the GPP of cropland and grassland ecosystems. Such a strong link with crop photosynthesis is not evident for traditional remotely sensed vegetation indices, nor for more complex carbon cycle models. We use SIF observations to provide a global perspective on agricultural productivity. Our SIF-based crop GPP estimates are 50-75% higher than results from state-of-the-art carbon cycle models over, for example, the US Corn Belt and the Indo-Gangetic Plain, implying that current models severely underestimate the role of management. Our results indicate that SIF data can help us improve our global models for more accurate projections of agricultural productivity and climate impact on crop yields. Extension of our approach to other ecosystems, along with increased observational capabilities for SIF in the near future, holds the prospect of reducing uncertainties in the modeling of the current and future carbon cycle.

  20. Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics.

    PubMed

    Sandberg, AnnSofi; Buschmann, Volker; Kapusta, Peter; Erdmann, Rainer; Wheelock, Åsa M

    2016-03-15

    Limitations in the sensitivity and dynamic range of two-dimensional gel electrophoresis (2-DE) are currently hampering its utility in global proteomics and biomarker discovery applications. In the current study, we present proof-of-concept analyses showing that introducing time-resolved fluorescence in the image acquisition step of in-gel protein quantification provides a sensitive and accurate method for subtracting confounding background fluorescence at the photon level. In-gel protein detection using the minimal difference gel electrophoresis workflow showed improvements in lowest limit of quantification in terms of CyDye molecules per pixel of 330-fold in the blue-green region (Cy2) and 8000-fold in the red region (Cy5) over conventional state-of-the-art image acquisition instrumentation, here represented by the Typhoon 9400 instrument. These improvements make possible the detection of low-abundance proteins present at sub-attomolar levels, thereby representing a quantum leap for the use of gel-based proteomics in biomarker discovery. These improvements were achieved using significantly lower laser powers and overall excitation times, thereby drastically decreasing photobleaching during repeated scanning. The single-fluorochrome detection limits achieved by the cumulative time-resolved emission two-dimensional electrophoresis (CuTEDGE) technology facilitates in-depth proteomics characterization of very scarce samples, for example, primary human tissue materials collected in clinical studies. The unique information provided by high-sensitivity 2-DE, including positional shifts due to post-translational modifications, may increase the chance to detect biomarker signatures of relevance for identification of disease subphenotypes. PMID:26854653

  1. On the possibility of ephedrine detection: time-resolved fluorescence resonance energy transfer (FRET)-based approach.

    PubMed

    Varriale, Antonio; Marzullo, Vincenzo Manuel; Di Giovanni, Stefano; Scala, Andrea; Capo, Alessandro; Majoli, Adelia; Pennacchio, Angela; Staiano, Maria; D'Auria, Sabato

    2016-09-01

    Ephedrine is one of the main precursor compounds used in the illegal production of amphetamines and related drugs. Actually, conventional analytical methods such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and gas chromatography-mass spectrometry (GC-MS) are used for the detection of ephedrine; sadly, these methods require qualified personnel and are time-consuming and expensive. In order to overcome these problems, in recent years, different methods have been developed based on the surface plasmon resonance (SPR) and electrochemical method. In this work, we present a simple, rapid, and effective method to detect the presence of ephedrine in solution, based on competitive fluorescence resonance energy transfer (FRET) assay. The antibody anti-ephedrine and ephedrine derivative were produced and labeled respectively, with two different fluorescent probes (donor and acceptor). The change in FRET signal intensity between donor and acceptor ephedrine compounds gives the possibility of detecting ephedrine traces of at least 0.81 ± 0.04 ppm (LOD). Graphical abstract A new Time-resolved Fluorescence Resonance Energy Transfer (FRET) assay for ephedrine detection. PMID:27395357

  2. Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy.

    PubMed

    Sun, Yang; Park, Jesung; Stephens, Douglas N; Jo, Javier A; Sun, Lei; Cannata, Jonathan M; Saroufeem, Ramez M G; Shung, K Kirk; Marcu, Laura

    2009-06-01

    We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 microm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque. PMID:19566223

  3. Full Genotyping of a Highly Polymorphic Human Gene Trait by Time-Resolved Fluorescence Resonance Energy Transfer

    PubMed Central

    Totè, Edoardo; Lamperti, Marco; Bondani, Maria; Salerno, Domenico; Cassina, Valeria; Nardo, Luca

    2014-01-01

    The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA) system. The polymorphisms of the trait of the DQB1 gene including codons 52–57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52–57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52–57 trait of DQB1 (8 homozygous and 28 heterozygous). We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes. PMID:25215592

  4. Picosecond time-resolved absorption and fluorescence dynamics in the artificial bacteriorhodopsin pigment BR6.11.

    PubMed Central

    Brack, T. L.; Delaney, J. K.; Atkinson, G. H.; Albeck, A.; Sheves, M.; Ottolenghi, M.

    1993-01-01

    The picosecond molecular dynamics in an artificial bacteriorhodopsin (BR) pigment containing a structurally modified all-trans retinal chromphore with a six-membered ring bridging the C11=C12-C13 positions (BR6.11) are measured by picosecond transient absorption and picosecond time-resolved fluorescence spectroscopy. Time-dependent intensity and spectral changes in absorption in the 570-650-nm region are monitored for delays as long as 5 ns after the 7-ps, 573-nm excitation of BR6.11. Two intermediates, J6.11 and K6.11/1, both with enhanced absorption to the red (> 600 nm) of the BR6.11 spectrum are observed within approximately 50 ps. The J6.11 intermediate decays with a time constant of 12 +/- 3 ps to form K6.11/1. The K6.11/1 intermediate decays with an approximately 100-ps time constant to form a third intermediate, K6.11/2, which is observed through diminished 650-nm absorption (relative to that of K6.11/1). No other transient absorption changes are found during the remainder of the initial 5-ns period of the BR6.11 photoreaction. Fluorescence in the 650-900-nm region is observed from BR6.11, K6.11/1, and K6.11/2, but no emission assignable to J6.11 is found. The BR6.11 fluroescence spectrum has a approximately 725-nm maximum which is blue-shifted by approximately 15 nm relative to that of native BR-570 and is 4.2 +/- 1.5 times larger in intensity (same sample optical density). No differences in the profile of the fluorescence spectra of BR6.11 and the intermediates K6.11/1 and K6.11/2 are observed. Following ground-state depletion of the BR6.11 population, the time-resolved fluroescence intensity monitored at 725 nm increases with two time constants, 12 +/- 3 and approximately 100 ps, both of which correlate well with changes in the picosecond transient absorption data. The resonance Raman spectrum of ground-state BR6.11, measured with low-energy, 560-nm excitation, is significantly different from the spectrum of native BR-570, thus confirming that the

  5. Identifiability analysis of rotational diffusion tensor and electronic transition moments measured in time-resolved fluorescence depolarization experiment

    SciTech Connect

    Szubiakowski, Jacek P.

    2014-06-14

    The subject of this paper is studies of the deterministic identifiability of molecular parameters, such as rotational diffusion tensor components and orientation of electronic transition moments, resulting from the time-resolved fluorescence anisotropy experiment. In the most general case considered, a pair of perpendicularly polarized emissions enables the unique determination of all the rotational diffusion tensor's principal components. The influence of the tensor's symmetry and the associated degeneration of its eigenvalues on the identifiability of the electronic transitions moments is systematically investigated. The analysis reveals that independently of the rotational diffusion tensor's symmetry, the transition moments involved in photoselection and emission processes cannot be uniquely identified without a priori information about their mutual orientation or their orientation with respect to the principal axes of the tensor. Moreover, it is shown that increasing the symmetry of the rotational diffusion tensor deteriorates the degree of the transition moments identifiability. To obtain these results analytically, a novel approach to solve bilinear system of equations for Markov parameters is applied. The effect of the additional information, obtained from fluorescence measurements for different molecular mobilities, to improve the identifiability at various levels of analysis is shown. The effectiveness and reliability of the target analysis method for experimental determination of the molecular parameters is also discussed.

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

  7. A time-resolved fluorescence immunoassay for the ultrasensitive determination of diethylstilbestrol based on the double-codified gold nanoparticles.

    PubMed

    Wang, Longjun; Zhang, Yuanfu; Liu, Guofu; Zhang, Chunyan; Wang, Shuhao

    2014-11-01

    An ultrasensitive and selective method is presented for the determination of diethylstilbestrol (DES) using time-resolved fluorescence immunoassay (TRFIA) based on double-codified gold nanoparticles (DC-AuNPs). In this system, the DC-AuNPs, that are gold nanoparticles (AuNPs) modified with anti-DES antibody and SH-dsDNA-biotin, was regarded as signal amplifier. A competitive immunoreaction was performed on polystyrene microtitration plates, where the DES compete with the immobilized DES-ovalbumin on polystyrene microtitration plates to bind to anti-DES antibodies on DC-AuNPs, and the europium(III)-labeled streptavidin was added to link to the SH-dsDNA-biotin as a tracer. Fluorescence signal was amplified via the AuNPs and the biotin-streptavidin double amplification systems. Under the optimized condition, DES can be quantified by TRFIA. The linear range and the limit of detection of DES were 1.0×10(-6)-10ngmL(-1) and 0.4fgmL(-1), respectively. This method was applied to determine DES in beef sample, with the recoveries ranging from 88% to 105%. PMID:25091151

  8. Limitations of Time-Resolved Fluorescence Suggested by Molecular Simulations: Assessing the Dynamics of T cell Receptor Binding Loops

    PubMed Central

    Scott, Daniel R.; Vardeman, Charles F.; Corcelli, Steven A.; Baker, Brian M.

    2012-01-01

    Time-resolved fluorescence anisotropy (TRFA) has a rich history in evaluating protein dynamics. Yet as often employed, TRFA assumes that the motional properties of a covalently tethered fluorescent probe accurately portray the motional properties of the protein backbone at the probe attachment site. In an extensive survey using TRFA to study the dynamics of the binding loops of a αβ T cell receptor, we observed multiple discrepancies between the TRFA data and previously published results that led us to question this assumption. We thus simulated several of the experimentally probed systems using a protocol that permitted accurate determination of probe and protein time correlation functions. We found excellent agreement in the decays of the experimental and simulated correlation functions. However, the motional properties of the probe were poorly correlated with those of the backbone of both the labeled and unlabeled protein. Our results warrant caution in the interpretation of TRFA data and suggest further studies to ascertain the extent to which probe dynamics reflect those of the protein backbone. Meanwhile, the agreement between experiment and computation validates the use of molecular dynamics simulations as an accurate tool for exploring the molecular motion of T cell receptors and their binding loops. PMID:23260055

  9. A multicolor time-resolved fluorescence aptasensor for the simultaneous detection of multiplex Staphylococcus aureus enterotoxins in the milk.

    PubMed

    Huang, Yukun; Zhang, Hui; Chen, Xiujuan; Wang, Xiaole; Duan, Nuo; Wu, Shijia; Xu, Baocai; Wang, Zhouping

    2015-12-15

    Food safety is one of the most important public health issues worldwide. Foodborne illnesses caused by Staphylococcus aureus enterotoxins (SEs) commonly occur, affecting both developing and developed countries. In this study, multicolor lanthanide-doped time-resolved fluorescence nanoparticles labeled with aptamers were used as bioprobes, and graphene oxide (GO) was employed as a resonance energy acceptor. Based on the "turn down" strategy, the simultaneous detection of multiplex SEs was realized in a homogeneous solution. Under the optimal conditions, the developed method exhibited high sensitivity and selectivity to three serological types of enterotoxins, including type A, B, C1, with limits of detection below 1 ng mL(-1). The application of this bioassay in milk analysis with no sample dilution was also investigated, and the results of recovery rates covered from 92.76% to 114.58%, revealing that the developed method was accurate. Therefore, this detection aptasnesor can be a good candidate for multiplex analysis and screening with simple and effective operations. PMID:26141103

  10. Characterization of energetic and thermalized sputtered atoms in pulsed plasma using time-resolved tunable diode-laser induced fluorescence

    SciTech Connect

    Desecures, M.; Poucques, L. de; Easwarakhanthan, T.; Bougdira, J.

    2014-11-03

    In this work, a time-resolved tunable diode-laser (DL) induced fluorescence (TR-TDLIF) method calibrated by absorption spectroscopy has been developed in order to determine atom and flux velocity distribution functions (AVDF and FVDF) of the energetic and the thermalized atoms in pulsed plasmas. The experimental set-up includes a low-frequency (∼3 Hz) and high spectral-resolution DL (∼0.005 pm), a fast rise-time pulse generator, and a high power impulse magnetron sputtering (HiPIMS) system. The induced TR-TDLIF signal is recorded every 0.5 μs with a digital oscilloscope of a second-long trace. The technique is illustrated with determining the AVDF and the FVDF of a metastable state of the sputtered neutral tungsten atoms in the HiPIMS post-discharge. Gaussian functions describing the population of the four W isotopes were used to fit the measured TR-TDLIF signal. These distribution functions provide insight into transition from the energetic to thermalized regimes from the discharge onset. This technique may be extended with appropriate DLs to probe any species with rapidly changing AVDF and FVDF in pulsed and strongly oscillating plasmas.

  11. Quantitative Time-Resolved Fluorescence Imaging of Androgen Receptor and Prostate-Specific Antigen in Prostate Tissue Sections.

    PubMed

    Krzyzanowska, Agnieszka; Lippolis, Giuseppe; Helczynski, Leszek; Anand, Aseem; Peltola, Mari; Pettersson, Kim; Lilja, Hans; Bjartell, Anders

    2016-05-01

    Androgen receptor (AR) and prostate-specific antigen (PSA) are expressed in the prostate and are involved in prostate cancer (PCa). The aim of this study was to develop reliable protocols for reproducible quantification of AR and PSA in benign and malignant prostate tissue using time-resolved fluorescence (TRF) imaging techniques. AR and PSA were detected with TRF in tissue microarrays from 91 PCa patients. p63/ alpha-methylacyl-CoA racemase (AMACR) staining on consecutive sections was used to categorize tissue areas as benign or cancerous. Automated image analysis was used to quantify staining intensity. AR intensity was significantly higher in AMACR+ and lower in AMACR- cancer areas as compared with benign epithelium. The PSA intensity was significantly lower in cancer areas, particularly in AMACR- glands. The AR/PSA ratio varied significantly in the AMACR+ tumor cells as compared with benign glands. There was a trend of more rapid disease progression in patients with higher AR/PSA ratios in the AMACR- areas. This study demonstrates the feasibility of developing reproducible protocols for TRF imaging and automated image analysis to study the expression of AR and PSA in benign and malignant prostate. It also highlighted the differences in AR and PSA protein expression within AMACR- and AMACR+ cancer regions. PMID:27026295

  12. Time-Resolved Laser-Induced Fluorescence Measurements of the Ion Velocity Distribution in the H6 Hall Thruster Plume

    NASA Astrophysics Data System (ADS)

    Durot, Christopher; Gallimore, Alec

    2013-10-01

    We developed a technique to recover time-resolved laser-induced fluorescence signals from strong background emission in plasma sources that have a relatively constant spectrum of oscillations in steady-state operation but are not periodically pulsed, such as Hall thrusters. The system was previously validated using a hollow cathode plasma source with forced discharge current oscillations. We present the first results using the new technique to capture oscillations in a Hall thruster. The ion velocity distribution function in the plume of the H6 Hall thruster is interrogated during breathing mode oscillations, which are characterized by an oscillating depletion and replenishment of neutrals at a frequency of 10-25 kHz. We use laser modulation on the order of megahertz, well above the time scale of interest (about 0.1 ms). A combination of band-pass filtering, phase-sensitive detection (with a time constant on the order of microseconds), and averaging over transfer functions is used to recover the signal. This technique has advantages such as a shorter dwell time than other techniques and the lack of a need for triggering averaging in the time domain. The ultimate bandwidth of the system that we implemented is approximately 1 MHz, limited by the speed of the AOM and signal photon rate collected. This work was supported by AFOSR and AFRL through the MACEEP center of excellence grant number FA9550-09-1-0695.

  13. Europium Nanospheres-Based Time-Resolved Fluorescence for Rapid and Ultrasensitive Determination of Total Aflatoxin in Feed.

    PubMed

    Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Ding, Xiaoxia; Zhang, Wen

    2015-12-01

    Immunochromatographic (IC) assays are considered suitable diagnostic tools for the determination of mycotoxins. A europium nanospheres-based time-resolved fluorescence immunoassay (Eu-Nano-TRFIA), based on a monoclonal antibody and a portable TRFIA reader, was developed to determine total aflatoxin (including aflatoxins B1, B2, G1, and G2) levels in feed samples. Under optimized conditions, the Eu-Nano-TRFIA method detected total aflatoxin within 12 min. It showed good linearity (R(2) > 0.985), LOD of 0.16 μg/kg, a wide dynamic range of 0.48-30.0 μg/kg, recovery rates of 83.9-113.9%, and coefficients of variation (CVs) of 3.5-8.8%. In the 397 samples from company and livestock farms throughout China, the detection rate was 78.3%, concentrations were 0.50-145.30 μg/kg, the highest total aflatoxin content was found in cottonseed meal, and corn was found to be the most commonly contaminated feed. This method could be a powerful alternative for the rapid and ultrasensitive determination of total aflatoxin in quality control and meet the required Chinese maximum residue limits. PMID:26565941

  14. Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening

    PubMed Central

    Shukla, Sunita J.; Nguyen, Dac-Trung; MacArthur, Ryan; Simeonov, Anton; Frazee, William J.; Hallis, Tina M.; Marks, Bryan D.; Singh, Upinder; Eliason, Hildegard C.; Printen, John; Austin, Christopher P.; Inglese, James

    2009-01-01

    Abstract The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug–drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z′-factors of ≥0.5. Seven- to 15-point concentration–response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format. PMID:19505231

  15. Time-resolved fluorescence anisotropy and fluctuation correlation analysis of major histocompatibility complex class I proteins in fibroblast cells.

    PubMed

    Heikal, Ahmed A

    2014-03-15

    Major histocompatibility complex class I proteins, MHC(I), are expressed in almost all nucleated cells and synthesized in the endoplasmic reticulum (ER). The orientation and mobility of these complexes are crucial in their biological function in the immune system, i.e., the cytosolic pathogen peptides loading and their presentation to T-cell receptors at the plasma membrane, where cell destruction is triggered. Here, we investigate the structural flexibility and associations of GFP-encoded MHC(I) alleles (H2L(d)), namely H2L(d)GFPin and H2L(d)GFPout, in cultured mouse fibroblast cells. Time-resolved fluorescence anisotropy of H2L(d)GFPin in the ER indicates a dominant overall tumbling motion of 56±7 ns (ER), with a fast conformational flexibility, as compared with a restricted rotation of H2L(d)GFPout. At the single-molecule level, the diffusion coefficient of H2L(d)GFPin and H2L(d)GFPout in the ER is (1.8±0.5)×10(-9) and (2.1±0.6)×10(-9) cm(2)/s, respectively, as revealed by fluorescence correlation spectroscopy. A complementary immunoblotting of H2L(d)GFP constructs, isolated from mouse fibroblast cells, reveals band at 75 kDa as compared with 29 kDa of the free EGFP. These real-time dynamics provide new insights into the structural flexibility and intracellular associations of GFP-labeled MHC(I) alleles (H2L(d)) in living cells. PMID:23811298

  16. Multi-channel lock-in amplifier assisted femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy with efficient rejection of superfluorescence background

    NASA Astrophysics Data System (ADS)

    Mao, Pengcheng; Wang, Zhuan; Dang, Wei; Weng, Yuxiang

    2015-12-01

    Superfluorescence appears as an intense background in femtosecond time-resolved fluorescence noncollinear optical parametric amplification spectroscopy, which severely interferes the reliable acquisition of the time-resolved fluorescence spectra especially for an optically dilute sample. Superfluorescence originates from the optical amplification of the vacuum quantum noise, which would be inevitably concomitant with the amplified fluorescence photons during the optical parametric amplification process. Here, we report the development of a femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectrometer assisted with a 32-channel lock-in amplifier for efficient rejection of the superfluorescence background. With this spectrometer, the superfluorescence background signal can be significantly reduced to 1/300-1/100 when the seeding fluorescence is modulated. An integrated 32-bundle optical fiber is used as a linear array light receiver connected to 32 photodiodes in one-to-one mode, and the photodiodes are further coupled to a home-built 32-channel synchronous digital lock-in amplifier. As an implementation, time-resolved fluorescence spectra for rhodamine 6G dye in ethanol solution at an optically dilute concentration of 10-5M excited at 510 nm with an excitation intensity of 70 nJ/pulse have been successfully recorded, and the detection limit at a pump intensity of 60 μJ/pulse was determined as about 13 photons/pulse. Concentration dependent redshift starting at 30 ps after the excitation in time-resolved fluorescence spectra of this dye has also been observed, which can be attributed to the formation of the excimer at a higher concentration, while the blueshift in the earlier time within 10 ps is attributed to the solvation process.

  17. Multi-channel lock-in amplifier assisted femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy with efficient rejection of superfluorescence background

    SciTech Connect

    Mao, Pengcheng; Wang, Zhuan; Dang, Wei; Weng, Yuxiang

    2015-12-15

    Superfluorescence appears as an intense background in femtosecond time-resolved fluorescence noncollinear optical parametric amplification spectroscopy, which severely interferes the reliable acquisition of the time-resolved fluorescence spectra especially for an optically dilute sample. Superfluorescence originates from the optical amplification of the vacuum quantum noise, which would be inevitably concomitant with the amplified fluorescence photons during the optical parametric amplification process. Here, we report the development of a femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectrometer assisted with a 32-channel lock-in amplifier for efficient rejection of the superfluorescence background. With this spectrometer, the superfluorescence background signal can be significantly reduced to 1/300–1/100 when the seeding fluorescence is modulated. An integrated 32-bundle optical fiber is used as a linear array light receiver connected to 32 photodiodes in one-to-one mode, and the photodiodes are further coupled to a home-built 32-channel synchronous digital lock-in amplifier. As an implementation, time-resolved fluorescence spectra for rhodamine 6G dye in ethanol solution at an optically dilute concentration of 10{sup −5}M excited at 510 nm with an excitation intensity of 70 nJ/pulse have been successfully recorded, and the detection limit at a pump intensity of 60 μJ/pulse was determined as about 13 photons/pulse. Concentration dependent redshift starting at 30 ps after the excitation in time-resolved fluorescence spectra of this dye has also been observed, which can be attributed to the formation of the excimer at a higher concentration, while the blueshift in the earlier time within 10 ps is attributed to the solvation process.

  18. Multi-channel lock-in amplifier assisted femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy with efficient rejection of superfluorescence background.

    PubMed

    Mao, Pengcheng; Wang, Zhuan; Dang, Wei; Weng, Yuxiang

    2015-12-01

    Superfluorescence appears as an intense background in femtosecond time-resolved fluorescence noncollinear optical parametric amplification spectroscopy, which severely interferes the reliable acquisition of the time-resolved fluorescence spectra especially for an optically dilute sample. Superfluorescence originates from the optical amplification of the vacuum quantum noise, which would be inevitably concomitant with the amplified fluorescence photons during the optical parametric amplification process. Here, we report the development of a femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectrometer assisted with a 32-channel lock-in amplifier for efficient rejection of the superfluorescence background. With this spectrometer, the superfluorescence background signal can be significantly reduced to 1/300-1/100 when the seeding fluorescence is modulated. An integrated 32-bundle optical fiber is used as a linear array light receiver connected to 32 photodiodes in one-to-one mode, and the photodiodes are further coupled to a home-built 32-channel synchronous digital lock-in amplifier. As an implementation, time-resolved fluorescence spectra for rhodamine 6G dye in ethanol solution at an optically dilute concentration of 10(-5)M excited at 510 nm with an excitation intensity of 70 nJ/pulse have been successfully recorded, and the detection limit at a pump intensity of 60 μJ/pulse was determined as about 13 photons/pulse. Concentration dependent redshift starting at 30 ps after the excitation in time-resolved fluorescence spectra of this dye has also been observed, which can be attributed to the formation of the excimer at a higher concentration, while the blueshift in the earlier time within 10 ps is attributed to the solvation process. PMID:26724012

  19. Isolation of Mammalian Oogonial Stem Cells by Antibody-Based Fluorescence-Activated Cell Sorting.

    PubMed

    Navaroli, Deanna M; Tilly, Jonathan L; Woods, Dori C

    2016-01-01

    The ability to isolate and subsequently culture mitotically active female germ cells from adult ovaries, referred to as either oogonial stem cells (OSCs) or adult female germline stem cells (aFGSCs), has provided a robust system to study female germ cell development under multiple experimental conditions, and in many species. Flow cytometry or fluorescence-activated cell sorting (FACS) is an integral part of many isolation and characterization protocols. Here, we provide methodological details for antibody-based flow cytometric isolation of OSCs using antibodies specific for external epitopes of the proteins Ddx4 or Ifitm3, alone or in combination with the use of fluorescent reporter mice. Beginning with sample preparation, we provide point-by-point instructions to guide researchers on how to isolate OSCs using flow cytometry. PMID:27557587

  20. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence.

    PubMed

    Stoddard, Robyn A; Quinn, Conrad P; Schiffer, Jarad M; Boyer, Anne E; Goldstein, Jason; Bagarozzi, Dennis A; Soroka, Stephen D; Dauphin, Leslie A; Hoffmaster, Alex R

    2014-06-01

    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)μM (0.551ng/ml) for PA83 and 2.51×10(-5)μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

  1. Quantitative determination of major capsaicinoids in serum by ELISA and time-resolved fluorescent immunoassay based on monoclonal antibodies.

    PubMed

    Yang, Qingqing; Zhu, Jianguo; Ma, Fei; Li, Peiwu; Zhang, Liangxiao; Zhang, Wen; Ding, Xiaoxia; Zhang, Qi

    2016-07-15

    To monitor capsaicinoids in serum on-site, three new monoclonal antibodies (mAbs) were firstly proposed using a conjugate of 4-[(4-hydroxy-3-methoxybenzyl) amino]-4-oxobutanoic acid as the immunogen. Among them, the YQQD8 mAb showed the highest sensitivity and cross-reactivity to major capsaicinoids, such as capsaicin, dihydrocapsaicin and N-vanillylnonanamide. A competitive indirect enzyme-linked immunosorbent assay (icELISA) and a time-resolved fluorescent immunochromatographic assay (TRFICA) were established based on this mAb. The linear range was 1.1-27.0ngmL(-1) for icELISA and 1.9-62.5ngmL(-1) for TRFICA and the limit of detection (LOD) of TRFICA was 1.5ngmL(-1). To decrease the interference of sample components and increase accuracy, serum samples were diluted four times before assays. As a result, the linear range of serum samples was 4.6-107.9ngmL(-1) for icELISA and 7.6-250.0ngmL(-1) for TRFICA. Both icELISA and TRFICA showed good recoveries (91.0-112.8% for icELISA and 87.6-111.5% for TRFICA) and concordant results in spiked experiments. Overall, this is the first report of immunoassay based on the mAbs for quantitative determination of major capsaicinoids, and the results demonstrate that both methods can meet the demands of rapid on-site assay for capsaicinoids in serum samples. PMID:26954788

  2. Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine.

    PubMed

    Huynh Nhat, Kim Phuong; Watanabe, Takayoshi; Yoshikoshi, Kensuke; Hohsaka, Takahiro

    2016-08-01

    Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules. PMID:26896314

  3. Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector.

    PubMed

    Rojalin, Tatu; Kurki, Lauri; Laaksonen, Timo; Viitala, Tapani; Kostamovaara, Juha; Gordon, Keith C; Galvis, Leonardo; Wachsmann-Hogiu, Sebastian; Strachan, Clare J; Yliperttula, Marjo

    2016-01-01

    In this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the

  4. Effect of ouabain on metabolic oxidative state in living cardiomyocytes evaluated by time-resolved spectroscopy of endogenous NAD(P)H fluorescence

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Elzwiei, Fathia; Mateasik, Anton; Chorvat, Dusan

    2012-10-01

    Time-resolved spectrometry of endogenous nicotinamide dinucleotide phosphate [NAD(P)H] fluorescence is a useful method to evaluate metabolic oxidative state in living cells. Ouabain is a well-known pharmaceutical drug used in the treatment of cardiovascular disease, the effects of which on myocardial metabolism were recently demonstrated. Mechanisms implicated in these actions are still poorly understood. We investigate the effect of ouabain on the metabolic oxidative state of living cardiac cells identified by time-resolved fluorescence spectroscopy of mitochondrial NAD(P)H. Spectral unmixing is used to resolve individual NAD(P)H fluorescence components. Ouabain decreased the integral intensity of NAD(P)H fluorescence, leading to a reduced component amplitudes ratio corresponding to a change in metabolic state. We also noted that lactate/pyruvate, affecting the cytosolic NADH gradient, increased the effect of ouabain on the component amplitudes ratio. Cell oxidation levels, evaluated as the percentage of oxidized NAD(P)H, decreased exponentially with rising concentrations of the cardiac glycoside. Ouabain also stimulated the mitochondrial NADH production. Our study sheds a new light on the role that ouabain plays in the regulation of metabolic state, and presents perspective on a noninvasive, pharmaceutical approach for testing the effect of drugs on the mitochondrial metabolism by means of time-resolved fluorescence spectroscopy in living cells.

  5. Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

    PubMed Central

    Alleti, Ramesh; Vagner, Josef; Dehigaspitiya, Dilani Chathurika; Moberg, Valerie E.; Elshan, N. G. R. D.; Tafreshi, Narges K.; Brabez, Nabila; Weber, Craig S.; Lynch, Ronald M.; Hruby, Victor J.; Gillies, Robert J.; Morse, David L.; Mash, Eugene A.

    2013-01-01

    Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9 nM and 4.2±0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for

  6. Monitoring changes of cellular metabolism and microviscosity in vitro based on time-resolved endogenous fluorescence and its anisotropy decay dynamics

    NASA Astrophysics Data System (ADS)

    Zheng, Wei; Li, Dong; Qu, Jianan Y.

    2010-05-01

    Reduced nicotinamide adenine dinucleotide (NADH) is a well-known metabolic coenzyme and endogenous fluorophore. In this study, we develop a system that simultaneously measures time- and wavelength-resolved fluorescence to extract free and protein-bound NADH signals from total cellular fluorescence. We analyze temporal characteristics of NADH fluorescence in a mixture of NADH and lactate dehydrogenase (LDH) as well as in living cell samples. The results show that in both the NADH/LDH mixture and cell samples, a fraction of free NADH and protein-bound components can be identified. The extracted free and bound NADH signals are confirmed by time-resolved measurement of anisotropy decay of NADH fluorescence, based on the fact that free NADH is a small fluorescent molecule with much shorter rotational diffusion time than bound NADH. The ratio of free NADH signal to bound NADH signal is very different between normal and cancer cervical epithelial cells. In addition, the ratio changes significantly when the cell samples are treated with a mitochondrial inhibitor or uncoupler, demonstrating that the method is sensitive to monitor cellular metabolic activity. Finally, we demonstrate that the microviscosity for relatively small molecules such as NADH in cells could be extracted from wavelength- and time-resolved NADH fluorescence of living cell samples.

  7. Time-Resolved Laser-Induced Fluorescence Measurements of Ion Velocity Distribution in the Plume of a 6 kW Hall Thruster with Unperturbed Discharge Oscillations

    NASA Astrophysics Data System (ADS)

    Durot, Christopher; Gallimore, Alec

    2014-10-01

    We present laser-induced fluorescence (LIF) measurements of the time-resolved ion velocity distribution in the plume of a 6 kW laboratory Hall thruster. To our knowledge, these are the first measurements of time-resolved ion velocity distribution on completely unperturbed Hall thruster operating conditions. To date, time-resolved LIF measurements have been made on Hall thrusters with oscillations driven or perturbed to be amenable to averaging techniques that assume a periodic oscillation. Natural Hall thruster breathing and spoke oscillations, however, are not periodic due to chaotic variations in amplitude and frequency. Although the system averages over many periods of nonperiodic oscillation, it recovers the time-resolved signal in part by assuming that a constant transfer function exists relating discharge current and LIF signal and averaging over the transfer function itself (http://dx.doi.org/10.1063/1.4856635). The assumption of a constant transfer function has been validated for a Hall thruster and the technique is now applied to a Hall thruster for the first time.

  8. A novel cell-based duplex high-throughput screening assay combining fluorescent Ca(2+) measurement with homogeneous time-resolved fluorescence technology.

    PubMed

    Kiss, László; Cselenyák, Attila; Varga, Ágnes; Visegrády, András

    2016-08-15

    Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm. PMID:27235172

  9. Picosecond time-resolved fluorescence study on solute-solvent interaction of 2-aminoquinoline in room-temperature ionic liquids: aromaticity of imidazolium-based ionic liquids.

    PubMed

    Iwata, Koichi; Kakita, Minoru; Hamaguchi, Hiro-o

    2007-05-10

    Time-resolved fluorescence spectra and fluorescence anisotropy decay of 2-aminoquinoline (2AQ) have been measured in eight room-temperature ionic liquids, including five imidazolium-based aromatic ionic liquids and three nonaromatic ionic liquids. The same experiments have also been carried out in several ordinary molecular liquids for comparison. The observed time-resolved fluorescence spectra indicate the formation of pi-pi aromatic complexes of 2AQ in some of the aromatic ionic liquids but not in the nonaromatic ionic liquids. The fluorescence anisotropy decay data show unusually slow rotational diffusion of 2AQ in the aromatic ionic liquids, suggesting the formation of solute-solvent complexes. The probe 2AQ molecule is likely to be incorporated in the possible local structure of ionic liquids, and hence the anisotropy decays only through the rotation of the whole local structure, making the apparent rotational diffusion of 2AQ slow. The rotational diffusion time decreases rapidly by adding a small amount of acetonitrile to the solution. This observation is interpreted in terms of the local structure formation in the aromatic ionic liquids and its destruction by acetonitrile. No unusual behavior upon addition of acetonitrile has been found for the nonaromatic ionic liquids. It is argued that the aromaticity of the imidazolium cation plays a key role in the local structure formation in imidazolium-based ionic liquids. PMID:17428083

  10. An integrated logic system for time-resolved fluorescent "turn-on" detection of cysteine and histidine base on terbium (III) coordination polymer-copper (II) ensemble.

    PubMed

    Xue, Shi-Fan; Lu, Ling-Fei; Wang, Qi-Xian; Zhang, Shengqiang; Zhang, Min; Shi, Guoyue

    2016-09-01

    Cysteine (Cys) and histidine (His) both play indispensable roles in many important biological activities. An enhanced Cys level can result in Alzheimer's and cardiovascular diseases. Likewise, His plays a significant role in the growth and repair of tissues as well as in controlling the transmission of metal elements in biological bases. Therefore, it is meaningful to detect Cys and His simultaneously. In this work, a novel terbium (III) coordination polymer-Cu (II) ensemble (Tb(3+)/GMP-Cu(2+)) was proposed. Guanosine monophosphate (GMP) can self-assemble with Tb(3+) to form a supramolecular Tb(3+) coordination polymer (Tb(3+)/GMP), which can be suited as a time-resolved probe. The fluorescence of Tb(3+)/GMP would be quenched upon the addition of Cu(2+), and then the fluorescence of the as-prepared Tb(3+)/GMP-Cu(2+) ensemble would be restored again in the presence of Cys or His. By incorporating N-Ethylmaleimide and Ni(2+) as masking agents, Tb(3+)/GMP-Cu(2+) was further exploited as an integrated logic system and a specific time-resolved fluorescent "turn-on" assay for simultaneously sensing His and Cys was designed. Meanwhile it can also be used in plasma samples, showing great potential to meet the need of practical application. PMID:27343597

  11. Time-Resolved Down-Conversion of 2-Aminopurine in a DNA Hairpin: Fluorescence Anisotropy and Solvent Effects

    NASA Astrophysics Data System (ADS)

    Tourón Touceda, Patricia; Gelot, Thomas; Crégut, Olivier; Léonard, Jérémie; Haacke, Stefan

    2013-03-01

    Femtosecond fluorescence anisotropy decay measured by type II difference frequency generation provides new insight into the local structural dynamics of ΔP(-)PBS fragments of the HIV- 1 DNA primary binding sequence, labeled with 2-aminopurine.

  12. Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study.

    PubMed Central

    Chen, L X; Longworth, J W; Fleming, G R

    1987-01-01

    The tryptophyl fluorescence of ribonuclease T1 decays monoexponentially at pH 5.5, tau = 4.04 ns but on increasing pH, a second short-lived component of 1.5 ns appears with a midpoint between pH 6.5 and 7.0. Both components have the same fluorescence spectrum. Acrylamide quenches both fluorescence components, and the short-lived component is quenched fivefold faster than the predominant long component. Binding of the substrate analogue 2'-guanylic acid at pH 5.5 quenches the fluorescence by 20% and introduces a second decay component, tau = 1.16 ns. Acrylamide quenches both tryptophyl decay components, with similar quenching rates. The fluorescence anisotropy decay of ribonuclease T1 was consistent with a molecule the size of ribonuclease T1 surrounded by a single layer of water at pH 7.4, even though the anisotropy decay at pH 5.5 deviated from Stokes-Einstein behavior. The fluorescence data were interpreted with a model where the tryptophyl residue exists in two conformations, remaining in a hydrophobic pocket. The acrylamide quenching is interpreted with electron transfer theory and suggests that one conformer has the nearest atom approximately 3 A from the protein surface, and the other, approximately 2 A. PMID:3038204

  13. Time-resolved detection of aromatic compounds on planetary surfaces by ultraviolet laser induced fluorescence and Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Eshelman, E.; Daly, M. G.; Slater, G.; Cloutis, E.

    2015-12-01

    Raman spectroscopic instruments are highly capable in the search for organics on Mars due to the potential to perform rapid and nondestructive measurements on unprepared samples. Upcoming and future Raman instruments are likely to also incorporate laser-induced fluorescence (LIF) capabilities, which can be added for modest cost and complexity. We demonstrate that it is possible to obtain sub-ns fluorescence lifetime measurements of Mars-relevant organics and minerals if a fast time-gating capability is used with an intensified detector and a short ultraviolet laser pulse. This serves a primary purpose of discriminating mineral from short-lived (less than 10 ns) organic fluorescence, considered a potential biosignature. Additionally, lifetime measurements may assist in determining if more than one fluorescing species is present and provide information concerning the molecular structure as well as the local environment. Fast time-gating is also useful at longer visible or near-IR wavelengths, as this approach increases the sensitivity of the instrument to organic material by removing the majority of the fluorescence background from the Raman signal and reducing the effect of ambient light.

  14. Time-resolved multi-channel optical system for assessment of brain oxygenation and perfusion by monitoring of diffuse reflectance and fluorescence

    NASA Astrophysics Data System (ADS)

    Milej, D.; Gerega, A.; Kacprzak, M.; Sawosz, P.; Weigl, W.; Maniewski, R.; Liebert, A.

    2014-03-01

    Time-resolved near-infrared spectroscopy is an optical technique which can be applied in tissue oxygenation assessment. In the last decade this method is extensively tested as a potential clinical tool for noninvasive human brain function monitoring and imaging. In the present paper we show construction of an instrument which allows for: (i) estimation of changes in brain tissue oxygenation using two-wavelength spectroscopy approach and (ii) brain perfusion assessment with the use of single-wavelength reflectometry or fluorescence measurements combined with ICG-bolus tracking. A signal processing algorithm based on statistical moments of measured distributions of times of flight of photons is implemented. This data analysis method allows for separation of signals originating from extra- and intracerebral tissue compartments. In this paper we present compact and easily reconfigurable system which can be applied in different types of time-resolved experiments: two-wavelength measurements at 687 and 832 nm, single wavelength reflectance measurements at 760 nm (which is at maximum of ICG absorption spectrum) or fluorescence measurements with excitation at 760 nm. Details of the instrument construction and results of its technical tests are shown. Furthermore, results of in-vivo measurements obtained for various modes of operation of the system are presented.

  15. Luminescence of the Cr3+ ion in sodium β- and β''-alumina: Site selection by time-resolved fluorescence line narrowing

    NASA Astrophysics Data System (ADS)

    Rossi, F.; Mariotto, G.; Montagna, M.; Ferrari, M.

    1994-03-01

    Fluorescence of Cr3+ ions in the two different octahedral sites of the spinel block of β- and β''-alumina have been measured by time-resolved fluorescence line-narrowing spectroscopy. For the excitation either a pulsed or a chopped cw-dye laser was used. Lifetimes and splittings of the ground state (4A2) and of the 2E state have been measured by tuning the excitation energy within the inhomogeneous profile of the R1 and R2 lines of the split 2E-4A2 transition. In sodium β''-alumina, we have isolated several inequivalent emitting centers, which are related to different configurations of the stabilizing Mg2+ ions.

  16. Simultaneous determination of nabumetone and its principal metabolite in medicines and human urine by time-resolved fluorescence.

    PubMed

    Murillo Pulgarín, José Antonio; Alañón Molina, Aurelia; Martínez Ferreras, Fernando

    2012-11-01

    A simple fluorescent methodology for the simultaneous determination of nabumetone and its main metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA), in pharmaceutical preparations and human urine is proposed. Due to the strong overlapping between the fluorescence spectra of both analytes, the use of fluorescence decay curves to resolve their mixture is proposed, since these curves are more selective. Values of dependent instrumental variables affecting the signal-to-noise ratio were fixed using a simplex optimization procedure. A factorial design with three levels per factor coupled to a central composite design was selected to obtain a calibration matrix of thirteen standards plus one blank sample that was processed using a partial least-squares (PLS) analysis. In order to assess the goodness of the proposed method, a prediction set of ten synthetic samples was analyzed, obtaining recovery percentages between 97 and 105%. Limits of detection, calculated by means of a new criterion, were 0.96 μg L(-1) and 0.88 μg L(-1) for nabumetone and 6-MNA, respectively. The method was also tested in the pharmaceutical preparation Relif, which contains nabumetone, obtaining recovery percentages close to 100%. Finally, the simultaneous determination of both analytes in human urine samples was successfully carried out by the PLS-analysis of a matrix of fifteen standards plus four analyte blanks and the use of the standard addition technique. Although urine shows native fluorescence, no extraction method or prior separation of the analytes was needed. PMID:22977877

  17. A high-throughput time-resolved mini-silicon photomultiplier with embedded fluorescence lifetime estimation in 0.13 μm CMOS.

    PubMed

    Tyndall, David; Rae, Bruce R; Li, David Day-Uei; Arlt, Jochen; Johnston, Abigail; Richardson, Justin A; Henderson, Robert K

    2012-12-01

    We describe a miniaturized, high-throughput, time-resolved fluorescence lifetime sensor implemented in a 0.13 m CMOS process, combining single photon detection, multiple channel timing and embedded pre-processing of fluorescence lifetime estimations on a single device. Detection is achieved using an array of single photon avalanche diodes (SPADs) arranged in a digital silicon photomultiplier (SiPM) architecture with 400 ps output pulses and a 10% fill-factor. An array of time-to-digital converters (TDCs) with ≈50 ps resolution records up to 8 photon events during each excitation period. Data from the TDC array is then processed using a centre-of-mass method (CMM) pre-calculation to produce fluorescence lifetime estimations in real-time. The sensor is believed to be the first reported implementation of embedded fluorescence lifetime estimation. The system is demonstrated in a practical laboratory environment with measurements of a variety of fluorescent dyes with different single exponential lifetimes, successfully showing the sensor's ability to overcome the classic pile-up limitation of time-correlated single photon counting (TCSPC) by over an order of magnitude. PMID:23853257

  18. Time-resolved laser-induced fluorescence measurement of ion and neutral dynamics in a Hall thruster during ionization oscillations

    NASA Astrophysics Data System (ADS)

    Lucca Fabris, Andrea; Young, Christopher V.; Cappelli, Mark A.

    2015-12-01

    The paper presents spatially and temporally resolved laser-induced fluorescence (LIF) measurements of the xenon ion and neutral velocity distribution functions in a 400 W Hall thruster during natural ionization oscillations at 23 kHz, the so-called "breathing mode." Strong fluctuations in measured axial ion velocity throughout the discharge current cycle are observed at five spatial locations and the velocity maxima appear in the low current interval. The spatio-temporal evolution of the ion velocity distribution function suggests a propagating acceleration front undergoing periodic motion between the thruster exit plane and ˜1 cm downstream into the plume. The ion LIF signal intensity oscillates almost in phase with the discharge current, while the neutral fluorescence signal appears out of phase, indicating alternating intervals of strong and weak ionization.

  19. Time-resolved spectroscopic study of photofragment fluorescence in methane/air mixtures and its diagnostic implications

    NASA Astrophysics Data System (ADS)

    Jonsson, Malin; Borggren, Jesper; Aldén, Marcus; Bood, Joakim

    2015-09-01

    In this work 80-picosecond laser pulses of 266-nm wavelength with intensities up to (2.0 ± 0.5) × 1011 W/cm2 were used for fragmentation of methane/air gas mixtures at ambient pressure and temperature. Emission spectra are, for the first time, studied with ultrahigh temporal resolution using a streak camera. Fluorescence spectra from CH(A2Δ-X2Π, B2Σ--X2Π, C2Σ+-X2Π), CN(B2Σ+-X2Σ+, Δ v = 0 and Δ v = ±1), NH(A3Π--X3Σ-), OH(A2Σ+-X2Π) and N2 +(B2Σu + X2Σg + were recorded and analyzed. By fitting simulated spectra to high-resolution experimental spectra, rotational and vibrational temperatures are estimated, showing that CH(C), CN(B), NH(A), and OH(A) are formed in highly excited vibrational and rotational states. The fluorescence signal dependencies on laser intensity and CH4/air equivalence ratio were investigated as well as the fluorescence lifetimes. All fragments observed are formed within 200 ps after the arrival of the laser pulse and their fluorescence lifetimes are shorter than 1 ns, except for CN(B-X) Δ v = 0 whose lifetime is 2.0 ns. The CN(B-X) Δ v = 0 fluorescence was studied temporally under high spectral resolution, and it was found that the vibrational levels are not populated simultaneously, but with a rate that decreases with increasing vibrational quantum number. This observation indicates that the rate of the chemical reaction that forms the CN(B) fragments is decreasing with increasing vibrational state of the product. The results provide vital information for the application of laser diagnostic techniques based on strong UV excitation, as they show that such methods might not be entirely non-intrusive and suffering from spectral interferences, unless the laser intensity is kept sufficiently low. Finally, equivalence ratios were determined from "unknown" spectra using multivariate analysis, showing a good agreement with theoretical compositions with an error of 4 %. The method is expected to be a useful diagnostic tool for

  20. Density relaxation and particle motion characteristics in a non-ionic deep eutectic solvent (acetamide + urea): Time-resolved fluorescence measurements and all-atom molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Das, Anuradha; Das, Suman; Biswas, Ranjit

    2015-01-01

    Temperature dependent relaxation dynamics, particle motion characteristics, and heterogeneity aspects of deep eutectic solvents (DESs) made of acetamide (CH3CONH2) and urea (NH2CONH2) have been investigated by employing time-resolved fluorescence measurements and all-atom molecular dynamics simulations. Three different compositions (f) for the mixture [fCH3CONH2 + (1 - f)NH2CONH2] have been studied in a temperature range of 328-353 K which is ˜120-145 K above the measured glass transition temperatures (˜207 K) of these DESs but much lower than the individual melting temperature of either of the constituents. Steady state fluorescence emission measurements using probe solutes with sharply different lifetimes do not indicate any dependence on excitation wavelength in these metastable molten systems. Time-resolved fluorescence anisotropy measurements reveal near-hydrodynamic coupling between medium viscosity and rotation of a dissolved dipolar solute. Stokes shift dynamics have been found to be too fast to be detected by the time-resolution (˜70 ps) employed, suggesting extremely rapid medium polarization relaxation. All-atom simulations reveal Gaussian distribution for particle displacements and van Hove correlations, and significant overlap between non-Gaussian (α2) and new non-Gaussian (γ) heterogeneity parameters. In addition, no stretched exponential relaxations have been detected in the simulated wavenumber dependent acetamide dynamic structure factors. All these results are in sharp contrast to earlier observations for ionic deep eutectics with acetamide [Guchhait et al., J. Chem. Phys. 140, 104514 (2014)] and suggest a fundamental difference in interaction and dynamics between ionic and non-ionic deep eutectic solvent systems.

  1. Density relaxation and particle motion characteristics in a non-ionic deep eutectic solvent (acetamide + urea): time-resolved fluorescence measurements and all-atom molecular dynamics simulations.

    PubMed

    Das, Anuradha; Das, Suman; Biswas, Ranjit

    2015-01-21

    Temperature dependent relaxation dynamics, particle motion characteristics, and heterogeneity aspects of deep eutectic solvents (DESs) made of acetamide (CH3CONH2) and urea (NH2CONH2) have been investigated by employing time-resolved fluorescence measurements and all-atom molecular dynamics simulations. Three different compositions (f) for the mixture [fCH3CONH2 + (1 - f)NH2CONH2] have been studied in a temperature range of 328-353 K which is ∼120-145 K above the measured glass transition temperatures (∼207 K) of these DESs but much lower than the individual melting temperature of either of the constituents. Steady state fluorescence emission measurements using probe solutes with sharply different lifetimes do not indicate any dependence on excitation wavelength in these metastable molten systems. Time-resolved fluorescence anisotropy measurements reveal near-hydrodynamic coupling between medium viscosity and rotation of a dissolved dipolar solute. Stokes shift dynamics have been found to be too fast to be detected by the time-resolution (∼70 ps) employed, suggesting extremely rapid medium polarization relaxation. All-atom simulations reveal Gaussian distribution for particle displacements and van Hove correlations, and significant overlap between non-Gaussian (α2) and new non-Gaussian (γ) heterogeneity parameters. In addition, no stretched exponential relaxations have been detected in the simulated wavenumber dependent acetamide dynamic structure factors. All these results are in sharp contrast to earlier observations for ionic deep eutectics with acetamide [Guchhait et al., J. Chem. Phys. 140, 104514 (2014)] and suggest a fundamental difference in interaction and dynamics between ionic and non-ionic deep eutectic solvent systems. PMID:25612718

  2. Density relaxation and particle motion characteristics in a non-ionic deep eutectic solvent (acetamide + urea): Time-resolved fluorescence measurements and all-atom molecular dynamics simulations

    SciTech Connect

    Das, Anuradha; Das, Suman; Biswas, Ranjit

    2015-01-21

    Temperature dependent relaxation dynamics, particle motion characteristics, and heterogeneity aspects of deep eutectic solvents (DESs) made of acetamide (CH{sub 3}CONH{sub 2}) and urea (NH{sub 2}CONH{sub 2}) have been investigated by employing time-resolved fluorescence measurements and all-atom molecular dynamics simulations. Three different compositions (f) for the mixture [fCH{sub 3}CONH{sub 2} + (1 − f)NH{sub 2}CONH{sub 2}] have been studied in a temperature range of 328-353 K which is ∼120-145 K above the measured glass transition temperatures (∼207 K) of these DESs but much lower than the individual melting temperature of either of the constituents. Steady state fluorescence emission measurements using probe solutes with sharply different lifetimes do not indicate any dependence on excitation wavelength in these metastable molten systems. Time-resolved fluorescence anisotropy measurements reveal near-hydrodynamic coupling between medium viscosity and rotation of a dissolved dipolar solute. Stokes shift dynamics have been found to be too fast to be detected by the time-resolution (∼70 ps) employed, suggesting extremely rapid medium polarization relaxation. All-atom simulations reveal Gaussian distribution for particle displacements and van Hove correlations, and significant overlap between non-Gaussian (α{sub 2}) and new non-Gaussian (γ) heterogeneity parameters. In addition, no stretched exponential relaxations have been detected in the simulated wavenumber dependent acetamide dynamic structure factors. All these results are in sharp contrast to earlier observations for ionic deep eutectics with acetamide [Guchhait et al., J. Chem. Phys. 140, 104514 (2014)] and suggest a fundamental difference in interaction and dynamics between ionic and non-ionic deep eutectic solvent systems.

  3. Silver nanoparticles-enhanced time-resolved fluorescence sensor for VEGF(165) based on Mn-doped ZnS quantum dots.

    PubMed

    Zhu, Dong; Li, Wei; Wen, Hong-Mei; Yu, Sheng; Miao, Zhao-Yi; Kang, An; Zhang, Aihua

    2015-12-15

    A silver nanoparticles (AgNPs)-enhanced time-resolved fluorescence (TR-FL) sensor based on long-lived fluorescent Mn-doped ZnS quantum dots (QDs) is developed for the sensitive detection of vascular endothelial growth factor-165 (VEGF165), a predominant cancer biomarker in cancer angiogenesis. The aptamers bond with the Mn-doped ZnS QDs and the BHQ-2 quencher-labelling strands hybridized in duplex are coupled with streptavidin (SA)-functionalized AgNPs to form the AgNPs-enhanced TR-FL sensor, showing lower fluorescence intensity in the duplex state due to the fluorescence resonance energy transfer (FRET) between the Mn-doped ZnS QDs and quenchers. Upon the addition of VEGF165, the BHQ-2 quencher-labelling strands of the duplex are displaced, leading to the disruption of the FRET. As a result, the fluorescence of the Mn-doped QDs within the proximity of the AgNPs is recovered. The FL signal can be measured free of the interference of short-lived background by setting appropriate delay time and gate time, which offers a signal with high signal-to-noise ratio in photoluminescent biodetection. Compared with the bare TR-FL sensor, the AgNPs-based TR-FL sensor showed a huge improvement in fluorescence based on metal-enhanced fluorescence (MEF) effect, and the sensitivity increased 11-fold with the detection limit of 0.08 nM. In addition, the sensor provided a wide range of linear detection from 0.1 nM to 16 nM. PMID:26276542

  4. Interaction of quinine sulfate with anionic micelles of sodium dodecylsulfate: A time-resolved fluorescence spectroscopy at different pH

    NASA Astrophysics Data System (ADS)

    Joshi, Sunita; Pant, Debi D.

    2015-09-01

    Photophysical behavior and rotational relaxation dynamics of quinine sulfate (QS) in anionic surfactant, sodium dodecylsulfate (SDS) at different pH have been studied using steady state and time resolved fluorescence spectroscopy. It has been observed that the cationic form of quinine sulfate (at pH 2) forms a fluorescent ion pair complex with the surfactant molecules at lower concentrations of surfactant. However, for higher concentrations of SDS, the probe molecules bind strongly with the micelles and reside at the water-micelle interface. At pH 7, QS is singly protonated in bulk aqueous solution. At lower concentrations of SDS aggregation between probe and surfactant molecules has been observed. However, for higher concentrations of SDS, an additional fluorescence peak corresponding to dicationic form of QS appears and this has been attributed to double protonation of the QS molecule in micellar solution. At pH 7, in the presence of SDS micelles, the photophysical properties of QS showed substantial changes compared to that in the bulk water solution. At pH 12, an increase in fluorescence intensity and lifetime has been observed and this has been attributed to the increase in radiative rate due to the incorporation of QS at the micelle-water interface. The local pH at micellar surface has been found different from the pH of bulk solution.

  5. Interaction of quinine sulfate with anionic micelles of sodium dodecylsulfate: A time-resolved fluorescence spectroscopy at different pH.

    PubMed

    Joshi, Sunita; Pant, Debi D

    2015-09-01

    Photophysical behavior and rotational relaxation dynamics of quinine sulfate (QS) in anionic surfactant, sodium dodecylsulfate (SDS) at different pH have been studied using steady state and time resolved fluorescence spectroscopy. It has been observed that the cationic form of quinine sulfate (at pH 2) forms a fluorescent ion pair complex with the surfactant molecules at lower concentrations of surfactant. However, for higher concentrations of SDS, the probe molecules bind strongly with the micelles and reside at the water-micelle interface. At pH 7, QS is singly protonated in bulk aqueous solution. At lower concentrations of SDS aggregation between probe and surfactant molecules has been observed. However, for higher concentrations of SDS, an additional fluorescence peak corresponding to dicationic form of QS appears and this has been attributed to double protonation of the QS molecule in micellar solution. At pH 7, in the presence of SDS micelles, the photophysical properties of QS showed substantial changes compared to that in the bulk water solution. At pH 12, an increase in fluorescence intensity and lifetime has been observed and this has been attributed to the increase in radiative rate due to the incorporation of QS at the micelle-water interface. The local pH at micellar surface has been found different from the pH of bulk solution. PMID:25863459

  6. Modification of energy-transfer processes in the cyanobacterium, Arthrospira platensis, to adapt to light conditions, probed by time-resolved fluorescence spectroscopy.

    PubMed

    Akimoto, Seiji; Yokono, Makio; Aikawa, Shimpei; Kondo, Akihiko

    2013-11-01

    In cyanobacteria, the interactions among pigment-protein complexes are modified in response to changes in light conditions. In the present study, we analyzed excitation energy transfer from the phycobilisome and photosystem II to photosystem I in the cyanobacterium Arthrospira (Spirulina) platensis. The cells were grown under lights with different spectral profiles and under different light intensities, and the energy-transfer characteristics were evaluated using steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopy techniques. The fluorescence rise and decay curves were analyzed by global analysis to obtain fluorescence decay-associated spectra. The direct energy transfer from the phycobilisome to photosystem I and energy transfer from photosystem II to photosystem I were modified depending on the light quality, light quantity, and cultivation period. However, the total amount of energy transferred to photosystem I remained constant under the different growth conditions. We discuss the differences in energy-transfer processes under different cultivation and light conditions. PMID:23605291

  7. Identification of two quenching sites active in the regulation of photosynthetic light-harvesting studied by time-resolved fluorescence

    NASA Astrophysics Data System (ADS)

    Holzwarth, Alfred R.; Miloslavina, Yuliya; Nilkens, Manuela; Jahns, Peter

    2009-12-01

    The regulation of light-harvesting (called non-photochemical quenching, NPQ) is an essential photoprotective mechanism active in plants. Total NPQ is dependent on PsbS, a pH-sensing protein, and on the action of the xanthophyll carotenoid zeaxanthin (Zx). Using ultrafast fluorescence on intact leaves we demonstrate two independent NPQ quenching sites in vivo which depend differently on the actions of PsbS and Zx. The first site is formed in the functionally detached major light-harvesting complex of PS II and depends strictly on PsbS. The second site is in the minor antennae of photosystem (PS) II and quenching depends on the presence of Zx.

  8. Energy transfer in the primary stages of the photosynthetic process investigated by picosecond time resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Pellegrino, F.

    The fate of the absorbed light energy in the primary stages of the photosynthetic process was studied. In particular, the energy transfer in the accessory pigment complex consisting of carotenoids, Chl. a and Chl. b in higher green plants and phycobiliproteins in blue-green algae were investigated. These accessory pigments are responsible for the highly efficient transfer of the excitation energy to the photochemically active reaction center traps. The risetime, decay time, fluorescence depolarization, temperature and intensity dependence of the fluoresence emission from higher green plant and algal photosystems were directly measured. Excitation was provided by single picosecond laser pulses, as well as a train of pulses at 530 nm, within an intensity range of 10 to the 12th power to 10 to the 16th power photons/sq cm per pulse.

  9. Steady-state and time-resolved fluorescence studies of reverse micelles in liquids and supercritical solvents

    SciTech Connect

    Zhang, Jing.

    1992-01-01

    The authors investigate the effects of temperature, salt concentration, and water loading on the internal dynamics of AOT Aerosol-OT, sodium bis (2-ethylhexyl sulfosuccinate) micelles in liquid heptane, using ANS-like (anilino-naphthalene sulfate) fluorescent probes. The important results from these experiments are that: (1) the molecular geometry of the probe is the predominant factor controlling partitioning even at high water loadings; (2) the photophysics of ANS is strongly dependent on the water content and temperature and corresponds to changes in local polarity and viscosity; (3) addition of electrolytes changes the dynamic fluorescence which is in turn related to the changes in internal microenvironments; and (4) a nanosecond solvent relaxation process occurs within reverse micelles. It was wondered if the continuous phase (alkane) density could be used to control the internal dynamics within a reverse micelle. To answer this question, research focused on: (1) the effects of water loading, temperature, and fluid density on solute partitioning and determination of the density effects on micellar aggregates; (2) the effects of solute structure on the distribution of probe molecules within reverse micelles; and (3) the effects of fluid density, water concentration, and temperature on the reorganizational dynamics within AOT reverse micelles. Simple thermodynamic measurements and nanosecond solvent relaxation experiments are used to account for this partitioning and water reorganization in AOT reverse micelles, respectively. Results on excited-state deprotonation reactions in AOT reverse micelles maintained in sub-critical propane, provides a useful model for density-controlled deprotonation reactions within reverse micelles. Preliminary work shows that the continuous phase density can be used to control reactions within reverse micelles formed in near- and supercritical alkanes.

  10. Role of Coherent Low-Frequency Motion in Excited-State Proton Transfer of Green Fluorescent Protein Studied by Time-Resolved Impulsive Stimulated Raman Spectroscopy.

    PubMed

    Fujisawa, Tomotsumi; Kuramochi, Hikaru; Hosoi, Haruko; Takeuchi, Satoshi; Tahara, Tahei

    2016-03-30

    Green fluorescent protein (GFP) from jellyfish Aequorea victoria, an essential bioimaging tool, luminesces via excited-state proton transfer (ESPT) in which the phenolic proton of the p-hydroxybenzylideneimidazolinone chromophore is transferred to Glu222 through a hydrogen-bond network. In this process, the ESPT mediated by the low-frequency motion of the chromophore has been proposed. We address this issue using femtosecond time-resolved impulsive stimulated Raman spectroscopy. After coherently exciting low-frequency modes (<300 cm(-1)) in the excited state of GFP, we examined the excited-state structural evolution and the ESPT dynamics within the dephasing time of the low-frequency vibration. A clear anharmonic vibrational coupling is found between one high-frequency mode of the chromophore (phenolic CH bend) and a low-frequency mode at ∼104 cm(-1). However, the data show that this low-frequency motion does not substantially affect the ESPT dynamics. PMID:26943852

  11. Time-resolved fluorescence sensing of pesticides chlorpyrifos, crotoxyphos and endosulfan by the luminescent Eu(III)-8-allyl-3-carboxycoumarin probe

    NASA Astrophysics Data System (ADS)

    Azab, Hassan A.; Khairy, Gasser M.; Kamel, Rasha M.

    2015-09-01

    This work describes the application of time resolved fluorescence in microtiter plates for investigating the interactions of europium-allyl-3-carboxycoumarin with pesticides chlorpyrifos, endosulfan and crotoxyphos. Stern-Volmer studies at different temperatures for chlorpyrifos and crotoxyphos shows dynamic and static quenching mechanisms respectively. Direct methods for the determination of the pesticides under investigation have been developed using the luminescence variations of the probe in solution. The detection limits are 6.53, 0.004, 3.72 μmol/L for chlorpyrifos, endosulfan, and crotoxyphos, respectively. The binding constants and thermodynamic parameters of the pesticides with probe were evaluated. A thermodynamic analysis showed that the reaction is spontaneous with negative ΔG. Effect of some relevant interferents on the detection of pesticides has been investigated. The new method was applied to the determination of the pesticides in different types of water samples (tap, mineral, and waste water).

  12. Differences in excitation energy transfer of Arthrospira platensis cells grown in seawater medium and freshwater medium, probed by time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Arba, Muhammad; Aikawa, Shimpei; Niki, Kenta; Yokono, Makio; Kondo, Akihiko; Akimoto, Seiji

    2013-11-01

    Excitation energy transfer of Arthrospira platensis cells grown in f/2 medium (a high salinity medium) and SOT medium (a control) was investigated by steady-state and time-resolved spectroscopies. Growth in f/2 medium induced changes in absorption and fluorescence spectra as well as in the energy transfer pathways. Excitation energy captured by phycobilisome (PBS) was transferred directly to photosystem (PS) I, instead of being first transferred to an intermediate (PBS → PSII → PSI), as observed in SOT medium. The respiration rate increased while photosynthetic rate reduced in f/2 medium. Possible causes of the differences in light-harvesting and energy-transfer processes between the two media are discussed.

  13. Conformational States of the Rapana thomasiana Hemocyanin and Its Substructures Studied by Dynamic Light Scattering and Time-Resolved Fluorescence Spectroscopy

    PubMed Central

    Georgieva, Dessislava; Schwark, Daniel; Nikolov, Peter; Idakieva, Krassimira; Parvanova, Katja; Dierks, Karsten; Genov, Nicolay; Betzel, Christian

    2005-01-01

    Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another. PMID:15533921

  14. Tracking Local Conformational Changes of Ribonuclease A Using Picosecond Time-Resolved Fluorescence of the Six Tyrosine Residues

    PubMed Central

    Noronha, Melinda; Lima, João C.; Paci, Emanuele; Santos, Helena; Maçanita, António L.

    2007-01-01

    The six tyrosine residues of ribonuclease A (RNase A) are used as individual intrinsic probes for tracking local conformational changes during unfolding. The fluorescence decays of RNase A are well described by sums of three exponentials with decay times (τ1 = 1.7 ns, τ2 = 180 ps, and τ3 = 30 ps) and preexponential coefficients (A1 = 1, A2 = 1, and A3 = 4) at pH 7, 25°C. The decay times are controlled by photo-induced electron transfer from individual tyrosine residues to the nearest disulphide (–SS–), bridge, which is distance (R) dependent. We assign τ1 to Tyr-76 (R = 12.8 Å), τ2 to Tyr-115 (R = 6.9 Å), and τ3 to Tyr-25, Tyr-73, Tyr-92, and Tyr-97 (all four at R = 5.5 ± 0.3 Å) at 23°C. On the basis of this assignment, the results show that, upon thermal or chemical unfolding only Tyr-25, Tyr-92, and Tyr-76 undergo significant displacement from their nearest –SS– bridge. Despite reporting on different regions of the protein, the concordance between the transition temperatures, Tm, obtained from Tyr-76 (Tm = 59.2°C) and Tyr-25 and Tyr-92 (Tm = 58.2°C) suggests a single unfolding event in this temperature range that affects all these regions similarly. PMID:17384067

  15. Segmental dynamics of the cytoplasmic domain of erythrocyte band 3 determined by time-resolved fluorescence anisotropy: sensitivity to pH and ligand binding.

    PubMed

    Thevenin, B J; Periasamy, N; Shohet, S B; Verkman, A S

    1994-03-01

    Interactions between the erythrocyte membrane and its skeleton are mediated primarily by binding of cytoskeletal components to a conformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3). To examine the nanosecond segmental motions of cdb3, band 3 was labeled selectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3 about which pH-dependent conformational changes occur. Time-resolved anisotropy of labeled cdb3 in isolated form and in stripped erythrocyte membranes was measured by parallel-acquisition frequency-domain microfluorimetry. Samples had a single-component fluorescein lifetime of approximately 4 ns. Multifrequency phase and modulation data (5-200 MHz) fitted well to a segmental motion model containing two correlation times (tau 1c and tau 2c) and two limiting anisotropies (r1infinity and r2infinity). Measurements in protease-cleaved and denatured samples indicated that tau 1c (100-150 ps) corresponded to rapid rotation of bound fluorescein and tau 2c (2-5 ns) corresponded to segmental motion of cdb3. Both motions were hindered as quantified by nonzero r1infinity and r2infinity. The strong pH dependence of segmental motion correlated with that of cdb3 conformation measured by intrinsic tryptophan fluorescence. Significant changes in cdb3 segmental motion occurred upon interactions with the small ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymes known to bind to the N terminus of band 3. These time-resolved fluorescence measurements of the nanosecond segmental dynamics of a labeled membrane protein provide evidence for the sensitivity of cdb3 conformation to ligand binding and suggest long-range structural communication through cdb3. PMID:8127875

  16. Time-resolved optical fluorescence spectroscopy of heterogeneous turbid media with special emphasis on brain tissue structures including diseased regions: A sensitivity analysis

    NASA Astrophysics Data System (ADS)

    Vaudelle, Fabrice; L'huillier, Jean-Pierre

    2013-09-01

    Fluorescence-enhanced optical imaging based on near-infrared light provides a promising tool to differentiate diseased lesions from normal tissue. However, the measurement sensitivity of the fluorescence signals acquired at the output surface of the tissue is greatly influenced by the tissue structure, the optical properties, the location and the size of the target. In this paper, we present a numerical model based on the Monte Carlo method that allows to simulate time-resolved reflectance signals acquired on the surface of the scalp of a human head model bearing a fluorescent diseased region (tumor, glioma). The influence of tumor depth, tumor size and tumor shape evolution on the computed signals are analyzed by taking into account the multi-layered tissue structure. The simulations show that the mean-time-of-flight and the difference between two mean-times acquired at two source-detector distances are both relevant to this problem type. Furthermore, the simulations suggest that the use of the difference between mean-flight-times may be interesting to probe scattering changes that occur in the cerebrospinal fluid (CSF).

  17. Steady-state and time-resolved two-photon fluorescence microscopy: a versatile tool for probing cellular environment and function

    NASA Astrophysics Data System (ADS)

    Denicke, Stefan; Ehlers, Jan-Eric; Niesner, Raluca; Quentmeier, Stefan; Gericke, Karl-Heinz

    2007-09-01

    In the last decade, the two-photon fluorescence laser-scanning microscopy (TPLSM) has become an indispensable tool for the bioscientific and biomedical research. TPLSM techniques as well as their applications are currently experiencing a dramatic evolution and represent the focus of many biophysical research projects. In this work, we compare in detail two steady-state TPLSM techniques, i.e. single-beam scanning microscopy combined with point-detection (SB-PMT) and multi-beam scanning microscopy combined with synchronous detection (MB-CCD), as far as their technical characteristics relevant for the bioscientific research are concerned, i.e. optical performance and imaging speed. We demonstrate that the SB-PMT technique is more adequate for deep-tissue imaging (few 100 μm depth) than the MB-CCD technique, whereas only the MB-CCD technique enables high-speed imaging for characterizing the dynamics of fast biological phenomena. Novel applications of these techniques are additionally discussed. Moreover, we employ a time-resolved TPLSM technique, i.e. biexponential fluorescence lifetime imaging based on the cellular fluorescence of the nicotinamide pyridine dinucleotides NADH and NADPH, which allows us to probe for the first time the redox cellular metabolism of MIN6 cells (mutated insulin producing pancreatic β-cells) as well as to show the potential of this method for the specific and dynamic investigation of NADH- and NADPH-dependent cellular processes.

  18. ESIPT and photodissociation of 3-hydroxychromone in solution: photoinduced processes studied by static and time-resolved UV/Vis, fluorescence, and IR spectroscopy.

    PubMed

    Chevalier, Katharina; Grün, Anneken; Stamm, Anke; Schmitt, Yvonne; Gerhards, Markus; Diller, Rolf

    2013-11-01

    The spectral properties of fluorescence sensors such as 3-hydroxychromone (3-HC) and its derivatives are sensitive to interaction with the surrounding medium as well as to substitution. 3-HC is a prototype system for other derivatives because it is the basic unit of all flavonoides undergoing ESIPT and is not perturbed by a substituent. In this study, the elementary processes and intermediate states in the photocycle of 3-HC as well as its anion were identified and characterized by the use of static and femtosecond time-resolved spectroscopy in different solvents (methylcyclohexane, acetonitrile, ethanol, and water at different pH). Electronic absorption and fluorescence spectra and lifetimes of the intermediate states were obtained for the normal, tautomer and anionic excited state, while mid-IR vibrational spectra yielded structural information on ground and excited states of 3-HC. A high sensitivity on hydrogen-bonding perturbations was observed, leading to photoinduced anion formation in water, while in organic solvents, different processes are suggested, including slow picosecond ESIPT and contribution of the trans-structure excited state or a different stable solvation state with different direction of OH. The formation of the latter could be favored by the lack of a substituent increasing contact points for specific solute-solvent interactions at the hydroxyl group compared to substituted derivatives. The effect of substituents has to be considered for the design of future fluorescence sensors based on 3-HC. PMID:24083478

  19. In vivo validation of a bimodal technique combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy for diagnosis of oral carcinoma

    NASA Astrophysics Data System (ADS)

    Sun, Yang; Xie, Hongtao; Liu, Jing; Lam, Matthew; Chaudhari, Abhijit J.; Zhou, Feifei; Bec, Julien; Yankelevich, Diego R.; Dobbie, Allison; Tinling, Steven L.; Gandour-Edwards, Regina F.; Monsky, Wayne L.; Gregory Farwell, D.; Marcu, Laura

    2012-11-01

    Tissue diagnostic features generated by a bimodal technique integrating scanning time-resolved fluorescence spectroscopy (TRFS) and ultrasonic backscatter microscopy (UBM) are investigated in an in vivo hamster oral carcinoma model. Tissue fluorescence is excited by a pulsed nitrogen laser and spectrally and temporally resolved using a set of filters/dichroic mirrors and a fast digitizer, respectively. A 41-MHz focused transducer (37-μm axial, 65-μm lateral resolution) is used for UBM scanning. Representative lesions of the different stages of carcinogenesis show that fluorescence characteristics complement ultrasonic features, and both correlate with histological findings. These results demonstrate that TRFS-UBM provide a wealth of co-registered, complementary data concerning tissue composition and structure as it relates to disease status. The direct co-registration of the TRFS data (sensitive to surface molecular changes) with the UBM data (sensitive to cross-sectional structural changes and depth of tumor invasion) is expected to play an important role in pre-operative diagnosis and intra-operative determination of tumor margins.

  20. In vivo validation of a bimodal technique combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy for diagnosis of oral carcinoma.

    PubMed

    Sun, Yang; Xie, Hongtao; Liu, Jing; Lam, Matthew; Chaudhari, Abhijit J; Zhou, Feifei; Bec, Julien; Yankelevich, Diego R; Dobbie, Allison; Tinling, Steven L; Gandour-Edwards, Regina F; Monsky, Wayne L; Farwell, D Gregory; Marcu, Laura

    2012-11-01

    Tissue diagnostic features generated by a bimodal technique integrating scanning time-resolved fluorescence spectroscopy (TRFS) and ultrasonic backscatter microscopy (UBM) are investigated in an in vivo hamster oral carcinoma model. Tissue fluorescence is excited by a pulsed nitrogen laser and spectrally and temporally resolved using a set of filters/dichroic mirrors and a fast digitizer, respectively. A 41-MHz focused transducer (37-μm axial, 65-μm lateral resolution) is used for UBM scanning. Representative lesions of the different stages of carcinogenesis show that fluorescence characteristics complement ultrasonic features, and both correlate with histological findings. These results demonstrate that TRFS-UBM provide a wealth of co-registered, complementary data concerning tissue composition and structure as it relates to disease status. The direct co-registration of the TRFS data (sensitive to surface molecular changes) with the UBM data (sensitive to cross-sectional structural changes and depth of tumor invasion) is expected to play an important role in pre-operative diagnosis and intra-operative determination of tumor margins. PMID:23117798

  1. Effect of Ca2+ on the Steady-State and Time-Resolved Emission Properties of the Genetically Encoded Fluorescent Sensor CatchER

    PubMed Central

    2015-01-01

    We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ. PMID:24836743

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

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

  3. Fulvic acid complexation of Eu(III) and Cm(III) at elevated temperatures studied by time-resolved laser fluorescence spectroscopy.

    PubMed

    Fröhlich, Daniel R; Skerencak-Frech, Andrej; Gast, Michael; Panak, Petra J

    2014-11-01

    The interaction of Eu(III) and Cm(III) with three different aquatic fulvic acids (FA) was studied as a function of the temperature (T = 20-80 °C) in 0.1 M NaCl solution by time-resolved laser fluorescence spectroscopy. The speciation of both trivalent metal ions was determined by peak deconvolution of the recorded fluorescence spectra. For each studied metal ion-FA system only one complexed species is formed under the given experimental conditions. The stability constants at 20, 40, 60 and 80 °C (log β'(T)) were determined according to the charge neutralization model. The log β' (20 °C) for the different FAs show similar values (log β(20 °C) = 5.60-6.29). The stability constants increase continuously with increasing temperature by approximately 0.3-1.0 orders of magnitude. The reaction enthalpies and entropies are derived from the integrated Van't Hoff equation. The results show that all investigated complexation reactions are endothermic and entropy-driven. PMID:25207846

  4. Rotational and translational dynamics of rhodamine 6G in a pyrrolidinium ionic liquid: a combined time-resolved fluorescence anisotropy decay and NMR study.

    PubMed

    Guo, Jianchang; Han, Kee Sung; Mahurin, Shannon M; Baker, Gary A; Hillesheim, Patrick C; Dai, Sheng; Hagaman, Edward W; Shaw, Robert W

    2012-07-12

    NMR spectroscopy and time-resolved fluorescence anisotropy decay (TRFAD) are two of the most commonly used methods to study solute-solvent interactions. However, only a few studies have been reported to date using a combined NMR and TRFAD approach to systematically investigate the overall picture of diffusional and rotational dynamics of both the solute and solvent. In this paper, we combined NMR and TRFAD to probe fluorescent rhodamine dye in a pyrrolidinium-based room temperature ionic liquid (RTIL), an emergent environmentally friendly solvent type used in several energy-related applications. A specific interaction of the R6G cation and [Tf2N] anion was identified, resulting in near-stick boundary condition rotation of R6G in this RTIL. The diffusional rates of the R6G solute and [C4mpyr][Tf2N] solvent derived from (1)H NMR suggest the rates are proportional to their corresponding hydrodynamic radii. The (1)H and (13)C NMR studies of self-rotational dynamics of [C4mpyr][Tf2N] showed that the self-rotational correlation time of [C4mpyr](+) is 47 ± 2 ps at 300 K. At the same temperature, we find that the correlation time for N-CH3 rotation in [C4mpyr](+) is 77 ± 2 ps, comparable to overall molecular reorientation. This slow motion is attributed to properties of the cation structure. PMID:22690897

  5. Rotational and Translational Dynamics of Rhodamine 6G in a Pyrrolidinium Ionic Liquid: A Combined Time-Resolved Fluorescence Anisotropy Decay and NMR Study

    SciTech Connect

    Guo, Jianchang; Han, Kee Sung; Mahurin, Shannon Mark; Baker, Gary A; Hillesheim, Patrick C; Dai, Sheng; Hagaman, Edward {Ed} W; Shaw, Robert W

    2012-01-01

    NMR spectroscopy and time-resolved fluorescence anisotropy decay (TRFAD) are two of the most commonly used methods to study solute-solvent interactions. However, only a few studies have been reported to date using a combined NMR and TRFAD approach to systematically investigate the overall picture of diffusional and rotational dynamics of both the solute and solvent. In this paper, we combined NMR and TRFAD to probe fluorescent rhodamine dyes in a pyrrolidinium-based room temperature ionic liquid (RTIL), an emergent environmentally-friendly solvent type used in several energy-related applications. A specific interaction of the R6G cation and [Tf2N]- anion was identified, resulting in near-stick boundary condition rotation of R6G in this RTIL. The diffusional rates of the R6G solute and [C4mpyr][Tf2N] solvent derived from 1H NMR suggest the rates are proportional to their corresponding hydrodynamic radii. The 1H and 13C NMR studies of self-rotational dynamics of [C4mpyr][Tf2N] showed that the self-rotational correlation time of [C4mpyr]+ is 47 2 ps at 300 K. At the same temperature, we find that the correlation time for N-CH3 rotation in [C4mpyr]+ is 77 2 ps, comparable to overall molecular reorientation. This slow motion is attributed to properties of the cation structure.

  6. Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study.

    PubMed Central

    Bastiaens, P I; van Hoek, A; Benen, J A; Brochon, J C; Visser, A J

    1992-01-01

    Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar

  7. NI-49SMART SUCKER: NEXT GENERATION SMART SURGICAL TOOL FOR INTRAOPERATIVE BRAIN TUMOR RESECTION USING TIME RESOLVED LASER INDUCED FLUORESCENCE SPECTROSCOPY

    PubMed Central

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

    2014-01-01

    Primary brain tumors are highly lethal tumors where surgical resection is the primary treatment of choice. It has been shown that survival rate is directly related to the extent of tumor resection. In order to aid the surgeon in achieving near-complete resection, novel technologies are required. Time-resolved laser induced fluorescence spectroscopy (TRLIFS) promises to be one such technology, where the tissue is excited using an ultra-short laser and the corresponding fluorescence intensity decay is captured. Based on the fluorescence spectrum and the decay characteristics at various color bands from TRLIFS, differentiation of tumor from the normal brain tissue is possible in real-time. We built a portable TRLIFS system using custom optics and hardware (laser excitation: 355nm, 400ps pulse width, 5 uJ/pulse; PMT detector: Photek, rise time 80 picoseconds; digitizer: 7 Giga-samples per second) which is capable of providing the results in real time (every 50 milliseconds). We have designed a custom probe which is attached to a Roton sucker "Smart sucker" to collect the data during surgical resection from patients at Cedars-Sinai Medical Center. The histopathological diagnosis of the site under study with TRLIFS is confirmed with a biopsy and H-E staining. We will present our preliminary data from human brain tumor samples collected in-vivo. Our preliminary study shows that TRLIFS is capable of classifying low grade tumors with high sensitivity and specificity. This study will also demonstrate the potential of using the TRLIFS system to enhance the surgical instrumentation, aiding surgeons in near-complete excision of tumors and bringing these instruments into the next generation of smart tools.

  8. Time-resolved, 3D, laser-induced fluorescence measurements of fine-structure passive scalar mixing in a tubular reactor

    NASA Astrophysics Data System (ADS)

    Van Vliet, E.; Van Bergen, S. M.; Derksen, J. J.; Portela, L. M.; Van den Akker, H. E. A.

    A three-dimensional, time-resolved, laser-induced fluorescence (3D-LIF) technique was developed to measure the turbulent (liquid-liquid) mixing of a conserved passive scalar in the wake of an injector inserted perpendicularly into a tubular reactor with Re=4,000. In this technique, a horizontal laser sheet was traversed in its normal direction through the measurement section. Three-dimensional scalar fields were reconstructed from the 2D images captured at consecutive, closely spaced levels by means of a high-speed CCD camera. The ultimate goal of the measurements was to assess the downstream development of the 3D scalar fields (in terms of the full scalar gradient vector field and its associated scalar energy dissipation rate) in an industrial flow with significant advection velocity. As a result of this advection velocity, the measured 3D scalar field is artificially ``skewed'' during a scan period. A method to correct for this skewing was developed, tested and applied. Analysis of the results show consistent physical behaviour.

  9. Homogeneous time-resolved fluorescence assays for the detection of activity and inhibition of phosphatase enzymes employing phosphorescently labeled peptide substrates.

    PubMed

    O'Shea, Desmond J; O'Riordan, Tomás C; O'Sullivan, Paul J; Papkovsky, Dmitri B

    2007-02-01

    A rapid, homogenous, antibody-free assay for phosphatase enzymes was developed using the phosphorescent platinum (II)-coproporphyrin label (PtCP) and time-resolved fluorescent detection. An internally quenched decameric peptide substrate containing a phospho-tyrosine residue, labeled with PtCP-maleimide and dabcyl-NHS at its termini was designed. Phosphatase catalysed dephosphorylation of the substrate resulted in a minor increase in PtCP signal, while subsequent cleavage by chymotrypsin at the dephosphorylated Tyr-Leu site provided a 3.5 fold enhancement of PtCP phosphorescence. This phosphorescence phosphatase enhancement assay was optimized to a 96 well plate format with detection on a commercial TR-F plate reader, and applied to measure the activity and inhibition of alkaline phosphatase, recombinant human CD45, and tyrosine phosphatases in Jurkat cell lysates within 40 min. Parameters of these enzymatic reactions such as Km's, limits of detection (L.O.D's) and IC50 values for the non-specific inhibitor sodium orthovanadate were also determined. PMID:17386566

  10. Antenna structure and excitation dynamics in photosystem I. I. Studies of detergent-isolated photosystem I preparations using time-resolved fluorescence analysis.

    PubMed Central

    Owens, T G; Webb, S P; Alberte, R S; Mets, L; Fleming, G R

    1988-01-01

    The temporal and spectral properties of fluorescence decay in isolated photosystem I (PS I) preparations from algae and higher plants were measured using time-correlated single photon counting. Excitations in the PS I core antenna decay with lifetimes of 15-40 ps and 5-6 ns. The fast decay results from efficient photochemical quenching by P700, whereas the slow decay is attributed to core antenna complexes lacking a trap. Samples containing core and peripheral antenna complexes exhibited an additional intermediate lifetime (150-350 ps) decay. The PS I core antenna is composed of several spectral forms of chlorophyll a that are not temporally resolved in the decays. Analysis of the temporal and spectral properties of the decays provides a description of the composition, structure, and dynamics of energy transfer and trapping reactions in PS I. The core antenna size dependence of the spectral properties and the contributions of the spectral forms to the time-resolved decays show that energy is not concentrated in the longest wavelength absorbing pigments but is nearly homogenized among the spectral forms. These data suggest that the "funnel" description of antenna structure and energy transfer (Seely, G. R. 1973. J. Theor. Biol. 40:189-199) may not be applicable to the PS I core antenna. PMID:3134059

  11. A high-throughput screening-compatible homogeneous time-resolved fluorescence assay measuring the glycohydrolase activity of human poly(ADP-ribose) glycohydrolase.

    PubMed

    Stowell, Alexandra I J; James, Dominic I; Waddell, Ian D; Bennett, Neil; Truman, Caroline; Hardern, Ian M; Ogilvie, Donald J

    2016-06-15

    Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition. PMID:27036617

  12. Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms.

    PubMed

    Haleyur Giri Setty, Mohan Kumar; Liu, Jikun; Mahtani, Prerna; Zhang, Panhe; Du, Bingchen; Ragupathy, Viswanath; Devadas, Krishnakumar; Hewlett, Indira K

    2016-06-01

    Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody. PMID:26978478

  13. Antenna structure and excitation dynamics in photosystem I. I. Studies of detergent-isolated photosystem I preparations using time-resolved fluorescence analysis.

    PubMed

    Owens, T G; Webb, S P; Alberte, R S; Mets, L; Fleming, G R

    1988-05-01

    The temporal and spectral properties of fluorescence decay in isolated photosystem I (PS I) preparations from algae and higher plants were measured using time-correlated single photon counting. Excitations in the PS I core antenna decay with lifetimes of 15-40 ps and 5-6 ns. The fast decay results from efficient photochemical quenching by P700, whereas the slow decay is attributed to core antenna complexes lacking a trap. Samples containing core and peripheral antenna complexes exhibited an additional intermediate lifetime (150-350 ps) decay. The PS I core antenna is composed of several spectral forms of chlorophyll a that are not temporally resolved in the decays. Analysis of the temporal and spectral properties of the decays provides a description of the composition, structure, and dynamics of energy transfer and trapping reactions in PS I. The core antenna size dependence of the spectral properties and the contributions of the spectral forms to the time-resolved decays show that energy is not concentrated in the longest wavelength absorbing pigments but is nearly homogenized among the spectral forms. These data suggest that the "funnel" description of antenna structure and energy transfer (Seely, G. R. 1973. J. Theor. Biol. 40:189-199) may not be applicable to the PS I core antenna. PMID:3134059

  14. Applications of immunomagnetic capture and time-resolved fluorescence to detect outbreak Escherichia coli O157 and Salmonella in alfalfa sprouts

    NASA Astrophysics Data System (ADS)

    Tu, Shu-I.; Gordon, Marsha; Fett, William F.; Gehring, Andrew G.; Irwin, Peter L.

    2004-03-01

    Commercially available alfalfa seeds were inoculated with low levels (~ 4 CFU/g) of pathogenic bacteria. The inoculated seeds were then allowed to sprout in sterile tap water at 22°C. After 48 hours, the irrigation water and sprouts were separately transferred to bovine heart infusion (BHI) media. The microbes in the BHI samples were allowed to grow for 4 hours at 37°C and 160 rpm. Specific immunomagnetic beads (IMB) were then applied to capture the E.coli O157 and/or Salmonella in the growth media. Separation and concentration of IMB-captured pathogens were achieved using magnetic separators. The captured E. coli O157:H7 and Salmonella spp were further tagged with europium (Eu) labeled anti-E. coli O157 antibodies and samarium (Sm) labeled anti-Salmonella antibodies, respectively. After washing, the lanthanide labels were extracted out from the complexes by specific chelators to form strongly fluorescent chelates. The specific time-resolved fluorescence (TRF) associated with Eu or Sm was measured to estimate the extent of capture of the E. coli O157 and Salmonella, respectively. The results indicated that the approach could detect E. coli O157 and Salmonella enterica from the seeds inoculated with ~ 4 CFU/g of the pathogens. Non-targeted bacteria, e.g., Aeromonas and Citrobacter exhibited essentially no cross reactivity. Since the pathogen detection from the sprouts was achieved within 6 hours, the developed methodology could be use as a rapid, sensitive and specific screening process for E. coli O157 and Salmonella enterica in this popular salad food.

  15. Picosecond time-resolved fluorescence studies on excitation energy transfer in a histidine 117 mutant of the D2 protein of photosystem II in Synechocystis 6803.

    PubMed

    Vasil'ev, S; Bruce, D

    2000-11-21

    The role of the peripheral reaction center chlorophyll a molecule associated with His117 of the D2 polypeptide in photosystem II was investigated in Synechocystis sp. PCC 6803 using a combination of steady state, pump-probe, and picosecond time-resolved fluorescence spectroscopy. Data were obtained from intact cells and isolated thylakoid membranes of a control mutant and a D2-H117T mutant, both of which lacked photosystem I. Excitation energy transfer and trapping were investigated by analyzing the data with a kinetic model that used an exact numerical solution of the Pauli master equation, taking into account available photosystem II spectral and structural information. The results of our kinetic analysis revealed the observed difference in excited-state dynamics between the H117T mutant and the control to be consistent with a retardation of the rate of excitation energy transfer from the peripheral chlorophyll of D2 (Chl at His117) to the electron-transfer pigments and an increase of the rate constant for charge recombination in the H117T mutant. The kinetic model was able to account for the experimentally observed changes in absorption cross section and fluorescence decay kinetics between the control and mutant by invoking changes in only these two rate constants. The results rule out quenching of excitation by a chlorophyll cation radical as a mechanism responsible for the lower efficiency of excitation energy utilization in the H117T mutant. Our work also demonstrates the importance of the chlorophyll associated with His117 of the D2 protein for excitation energy transfer to the PSII electron-transfer pigments and for the effective stabilization of the primary radical pair. PMID:11087370

  16. Ultrafast Time-Resolved Emission and Absorption Spectra of meso-Pyridyl Porphyrins upon Soret Band Excitation Studied by Fluorescence Up-Conversion and Transient Absorption Spectroscopy.

    PubMed

    Venkatesh, Yeduru; Venkatesan, M; Ramakrishna, B; Bangal, Prakriti Ranjan

    2016-09-01

    A comprehensive study of ultrafast molecular relaxation processes of isomeric meso-(pyridyl) porphyrins (TpyPs) has been carried out by using femtosecond time-resolved emission and absorption spectroscopic techniques upon pumping at 400 nm, Soret band (B band or S2), in 4:1 dichloromethane (DCM) and tetrahydrofuran (THF) solvent mixture. By combined studies of fluorescence up-conversion, time-correlated single photon counting, and transient absorption spectroscopic techniques, a complete model with different microscopic rate constants associated with elementary processes involved in electronic manifolds has been reported. Besides, a distinct coherent nuclear wave packet motion in Qy state is observed at low-frequency mode, ca. 26 cm(-1) region. Fluorescence up-conversion studies constitute ultrafast time-resolved emission spectra (TRES) over the whole emission range (430-710 nm) starting from S2 state to Qx state via Qy state. Careful analysis of time profiles of up-converted signals at different emission wavelengths helps to reveal detail molecular dynamics. The observed lifetimes are as indicated: A very fast decay component with 80 ± 20 fs observed at ∼435 nm is assigned to the lifetime of S2 (B) state, whereas being a rise component in the region of between 550 and 710 nm emission wavelength pertaining to Qy and Qx states, it is attributed to very fast internal conversion (IC) occurring from B → Qy and B → Qx as well. Two distinct components of Qy emission decay with ∼200-300 fs and ∼1-1.5 ps time constants are due to intramolecular vibrational redistribution (IVR) induced by solute-solvent inelastic collisions and vibrational redistribution induced by solute-solvent elastic collision, respectively. The weighted average of these two decay components is assigned as the characteristic lifetime of Qy, and it ranges between 0.3 and 0.5 ps. An additional ∼20 ± 2 ps rise component is observed in Qx emission, and it is assigned to the formation time of

  17. FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye

    PubMed Central

    Klemm, Matthias; Schweitzer, Dietrich; Peters, Sven; Sauer, Lydia; Hammer, Martin; Haueisen, Jens

    2015-01-01

    Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX’s applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation. PMID:26192624

  18. Development and implementation of a miniaturized high-throughput time-resolved fluorescence energy transfer assay to identify small molecule inhibitors of polo-like kinase 1.

    PubMed

    Sharlow, Elizabeth R; Leimgruber, Stephanie; Shun, Tong Ying; Lazo, John S

    2007-12-01

    Polo-like kinase (Plk) 1 is a key enzyme involved in regulating the mammalian cell cycle that is also a validated anticancer drug target. Nonetheless, there are relatively few readily available potent and selective small molecule inhibitors of Plk1. To increase the availability of pharmacologically valuable Plk1 inhibitors, we describe herein the development, variability assessment, validation, and implementation of a 384-well automated, miniaturized high-throughput time-resolved fluorescence energy transfer screening assay designed to identify Plk1 kinase inhibitors. Using a small molecule library of pharmaceutically active compounds to gauge high-throughput assay robustness and reproducibility, we found nine general kinase inhibitors, including H-89, which was selected as the minimum control. We then interrogated a 97,101 compound library from the National Institutes of Health repository for small molecule inhibitors of Plk1 kinase activity. The initial primary hit rate in a single 10 microM concentration format was 0.21%. Hit compounds were subjected to concentration-response confirmation and interference assays. Identified in the screen were seven compounds with 50% inhibitory concentration (IC50) values below 1 microM, 20 compounds with IC50 values between 1 microM and 5 microM, and eight compounds with IC50 values between 5 and 10 microM, which could be assigned to seven distinct chemotype classes. Hit compounds were also examined for their ability to inhibit other kinases such as protein kinase D, focal adhesion kinase, rho-associated coiled coil protein kinase 2, c-jun NH2-terminal kinase 3, and protein kinase A via experimentation or data-mining. These compounds should be useful as probes for the biological activity of Plk1 and as leads for the development of new selective inhibitors of Plk1. PMID:18181689

  19. Homogeneous Time-Resolved Fluorescence-Based Assay to Monitor Extracellular Signal-Regulated Kinase Signaling in a High-Throughput Format

    PubMed Central

    Ayoub, Mohammed Akli; Trebaux, Julien; Vallaghe, Julie; Charrier-Savournin, Fabienne; Al-Hosaini, Khaled; Gonzalez Moya, Arturo; Pin, Jean-Philippe; Pfleger, Kevin D. G.; Trinquet, Eric

    2014-01-01

    The extracellular signal-regulated kinases (ERKs) are key components of multiple important cell signaling pathways regulating diverse biological responses. This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We report the development of a new cell-based Phospho-ERK1/2 assay (designated Phospho-ERK), which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently, or stably expressing the different receptors. The validation was performed for agonists, antagonists, and inhibitors in dose–response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z′-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signaling. Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling PMID:25002860

  20. Insight into the factors influencing the backbone dynamics of three homologous proteins, dendrotoxins I and K, and BPTI: FTIR and time-resolved fluorescence investigations.

    PubMed

    Hollecker, Michelle; Vincent, Michel; Gallay, Jacques; Ruysschaert, Jean-Marie; Goormaghtigh, Erik

    2002-12-24

    Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, combined with hydrogen/deuterium exchange technique and time-resolved fluorescence spectroscopy, has been used to investigate the changes in structure and dynamics that underlie the thermodynamic stability differences observed for three closely homologous proteins: dendrotoxins I and K, and bovine pancreatic trypsin inhibitor (BPTI). The experiments were performed on proteins under their native state and a modified form, obtained by selective reduction of a disulfide bond at the surface of the molecule, increasing slightly the backbone flexibility without changing the average structure. The data confirmed the high local as well as global rigidity of BPTI. In protein K, the exchange process was slow during the first 2 h of exchange, presumably reflecting a compact three-dimensional conformation, and then increased rapidly, the internal amide protons of the beta-strands exchanging 10-fold faster than in BPTI or protein I. The most probable destabilizing element was identified as Pro32, in the core of the beta-sheet. Protein I was found to present a 10% more expanded volume than protein K or BPTI, and there is a possible correlation between the resulting increased flexibility of the molecule and the lower thermodynamic stability observed for this protein. Interestingly, the interior amide protons of the beta-sheet structure were found to be as protected against exchange in protein I as in BPTI, suggesting that, although globally more flexible than that of Toxin K or BPTI, the structure of Toxin I could be locally quite rigid. The structural factors suspected to be responsible for the differences in internal flexibility of the two toxins could play a significant role in determining their functional properties. PMID:12484765

  1. Photosynthetic dioxygen formation studied by time-resolved delayed fluorescence measurements--method, rationale, and results on the activation energy of dioxygen formation.

    PubMed

    Buchta, Joachim; Grabolle, Markus; Dau, Holger

    2007-06-01

    The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 mus to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O-O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019-1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 mus (at 20 degrees C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 degrees C, pH 6.4). In the second part of the study, the temperature dependence (-2.7 to 27.5 degrees C) of the rate constant of dioxygen formation (600/s at 20 degrees C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained. PMID:17543884

  2. Spatial proximity of the HIV-1 nucleocapsid protein zinc fingers investigated by time-resolved fluorescence and fluorescence resonance energy transfer.

    PubMed

    Mély, Y; Jullian, N; Morellet, N; De Rocquigny, H; Dong, C Z; Piémont, E; Roques, B P; Gérard, D

    1994-10-11

    The three-dimensional structure of peptides encompassing the two zinc-saturated finger motifs of the nucleocapsid protein NCp7 of HIV-1 has been reported by several groups. Whereas the folded structures of the finger motifs were in good agreement, discrepancies existed concerning their spatial relationship since the fingers were found either close to each other [Morellet, N., Jullian, N., De Rocquigny, H., Maigret, B., Darlix, J. L., & Roques, B. P. (1992) Embo J. 11, 3059-3065] or independently folded [Omichinski, J. G., Clore, G. M., Sakaguchi, K., Appella, E., & Gronenborn, A. M. (1991) FEBS Lett. 292, 25-30, Summers, M. F., Henderson, L. E., Chance, M. R., Bess, J. W., Jr., South, T. L., Blake, P. R., Sagi, I., Perez-Alvarado, G., Sowder, R.C., III, Hare, D.R., & Arthur, L. O. (1992) Protein Sci. 1, 563-574]. As in the interacting finger model, Phe16 in the NH2-terminal finger and Trp37 in the COOH-terminal finger were found to be spatially close, the fluorescence properties of the aromatic residues at positions 16 and 37 in the wild-type and two conservatively substituted (12-53) NCp7 peptides were investigated and compared with those of three negative control derivatives where the finger motifs were not in close contact. Direct distance measurements by Tyr-Trp fluorescence resonance energy transfer of the former derivatives yielded a 7-12 A interchromophore distance range which is clearly inconsistent with the 12.5-18 A range measured for the negative controls and thus a random orientation of the zinc finger motifs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7918429

  3. The dependence of the ultrafast relaxation kinetics of the S2 and S1 states in β-carotene homologs and lycopene on conjugation length studied by femtosecond time-resolved absorption and Kerr-gate fluorescence spectroscopies

    NASA Astrophysics Data System (ADS)

    Kosumi, Daisuke; Fujiwara, Masazumi; Fujii, Ritsuko; Cogdell, Richard J.; Hashimoto, Hideki; Yoshizawa, Masayuki

    2009-06-01

    The ultrafast relaxation kinetics of all-trans-β-carotene homologs with varying numbers of conjugated double bonds n(n =7-15) and lycopene (n =11) has been investigated using femtosecond time-resolved absorption and Kerr-gate fluorescence spectroscopies, both carried out under identical excitation conditions. The nonradiative relaxation rates of the optically allowed S2(1Bu+1) state were precisely determined by the time-resolved fluorescence. The kinetics of the optically forbidden S1(2Ag-1) state were observed by the time-resolved absorption measurements. The dependence of the S1 relaxation rates upon the conjugation length is adequately described by application of the energy gap law. In contrast to this, the nonradiative relaxation rates of S2 have a minimum at n =9 and show a reverse energy gap law dependence for values of n above 11. This anomalous behavior of the S2 relaxation rates can be explained by the presence of an intermediate state (here called the Sx state) located between the S2 and S1 states at large values of n (such as n =11). The presence of such an intermediate state would then result in the following sequential relaxation pathway S2→Sx→S1→S0. A model based on conical intersections between the potential energy curves of these excited singlet states can readily explain the measured relationships between the decay rates and the energy gaps.

  4. Development of a Time-Resolved Fluorescence Immunoassay for Epstein–Barr Virus Zta IgA Antibodies in Human Serum

    PubMed Central

    Chen, Juanjuan; Liu, Tiancai; Chen, Zhenhua; Hou, Jingyuan

    2015-01-01

    Abstract The Epstein–Barr virus (EBV) transactivator protein (ZEBRA) is an immediate–early protein that plays an important role in the switch from latency to productive cycle in EBV virus. ZEBRA is an important marker of EBV reactivation. In order to diagnose EBV infection status correctly and timely, a novel immunoassay was developed based on an indirect time-resolved fluoroimmunoassay (TRFIA) for Zta IgA, which used recombinant Zta antigen as solid-phase antigen and Eu3+-labeled mouse antihuman IgA as corresponding probe. The precision, sensitivity, specificity test, and stability of the TRFIA kit were evaluated, and comparison with the traditional enzyme-linked immunosorbent assay (ELISA) was also investigated. The cutoff value for the TRFIA was 2.5. Intra- and interassay coefficients of variation for the TRFIA were 2.45–3.30% and 3.38–4.61% respectively. There was no cross-reactivity with the antibodies of cytomegalovirus (CMV) or herpes simplex virus (HSV) types 1 and 2, or other potential interferences. The established assay kit also behaved better in sensitivity and stability than the ELISA one. Additionally, the results in 382 serum samples using two analytical methods showed there was good agreement between the TRFIA and commercial ELISA kit. In the current study, the results demonstrated that the TRFIA that was developed for Zta IgA detection was more sensitive and reliable for the diagnosis of EBV infection and had potential value in automation and high-throughput screening. PMID:25651045

  5. Multiemission wavelength picosecond time-resolved fluorescence decay data obtained on the millisecond time scale: application to protein:DNA interactions and protein-folding reactions

    NASA Astrophysics Data System (ADS)

    Beechem, Joseph M.

    1992-04-01

    One of the major aspects of fluorescence spectroscopy which differentiates this technique from many other spectroscopic approaches is the inherent multidimensional nature of the data. For instance, the basic pulsed-laser fluorescence data set is characterized by fluorescence versus: emission wavelength, polarization state (parallel and perpendicular intensities), time of emission (picoseconds to nanoseconds), and time of biological reaction (milliseconds to minutes). Usually, this six-dimensional data set is obtained piecemeal, single dimension at a time; often complete data sets are not even collected. This is especially true of the biological time scale axis. Data acquisition times for picosecond decay data are typically seconds to minutes, and, therefore, it has not been generally possible to perform this experiment in a kinetic mode. What is described in this report is the construction of a parallel multichannel time-correlated single-photon counting (TCSPC) fluorometer which is capable of simultaneous collection of: fluorescence vs. picosecond to nanosecond time vs. emission wavelength vs. polarization state vs. millisecond to second time. Use is made of two multi-anode microchannel plate detectors, each obtaining data at two different polarization states, six different emission wavelengths, along 12 independent TCSPC channels. This instrument is interfaced to a three-syringe stepper motor controlled stop-flow apparatus, and picosecond decay data along all of these channels is stored and collected by two 33 MHz 80486 computers at rates approaching 1200 - 12000 data sets per second.

  6. Effect of nanosize micelles of ionic and neutral surfactants on the photophysics of protonated 6-methoxyquinoline: Time-resolved fluorescence study

    NASA Astrophysics Data System (ADS)

    Tej Varma, Y.; Joshi, Sunita; Pant, Debi D.

    2015-03-01

    The excited state dynamic studies have been carried out to investigate the effects of micellar surface charge on the photophysics of protonated 6-methoxyquinoline (6MQ+) in anionic, sodium dodecylsulphate (SDS), cationic, cetyltrimethylammonium bromide (CTAB) and neutral, triton X-100 (TX100) surfactant at premicellar, micellar and postmicellar concentrations in aqueous phase at room temperature. At premicellar concentrations of SDS, there is a slight decrease in emission intensity and at micellar and postmicellar concentrations, increase in emission intensity and blue shift of spectrum has been observed. The blue shift in fluorescence spectrum and slight increase in quantum yield are attributed to incorporation of solute molecule to the micelles. Edge excitation red shift (EERS) in fluorescence maximum of 6MQ+ has been observed in all the surfactant solutions studied. The EERS has been ascribed in terms of solvent relaxation process. In SDS surfactant system, due to heterogeneous restricted motion of solvent molecules, the solvent viscosity increases which results in an increase in net magnitude of EERS. The fluorescence decay components of 6MQ+ fit with multi exponential functions in all the micellar systems studied. The location of the probe molecule in micellar systems is justified by a variety of spectral parameters such as refractive index, dielectric constant, ET (30), EERS, average fluorescence decay time, radiative and non radiative rate constants, and rotational relaxation time.

  7. Revealing the radiative and non-radiative relaxation rates of the fluorescent dye Atto488 in a λ/2 Fabry-Pérot-resonator by spectral and time resolved measurements

    NASA Astrophysics Data System (ADS)

    Konrad, Alexander; Metzger, Michael; Kern, Andreas M.; Brecht, Marc; Meixner, Alfred J.

    2016-07-01

    Using a Fabry-Pérot-microresonator with controllable cavity lengths in the λ/2-regime, we show the controlled modification of the vibronic relaxation dynamics of a fluorescent dye molecule in the spectral and time domain. By altering the photonic mode density around the fluorophores we are able to shape the fluorescence spectrum and enhance specifically the probability of the radiative transitions from the electronic excited state to distinct vibronic excited states of the electronic ground state. Analysis and correlation of the spectral and time resolved measurements by a theoretical model and a global fitting procedure allows us to reveal quantitatively the spectrally distributed radiative and non-radiative relaxation dynamics of the respective dye molecule under ambient conditions at the ensemble level.Using a Fabry-Pérot-microresonator with controllable cavity lengths in the λ/2-regime, we show the controlled modification of the vibronic relaxation dynamics of a fluorescent dye molecule in the spectral and time domain. By altering the photonic mode density around the fluorophores we are able to shape the fluorescence spectrum and enhance specifically the probability of the radiative transitions from the electronic excited state to distinct vibronic excited states of the electronic ground state. Analysis and correlation of the spectral and time resolved measurements by a theoretical model and a global fitting procedure allows us to reveal quantitatively the spectrally distributed radiative and non-radiative relaxation dynamics of the respective dye molecule under ambient conditions at the ensemble level. Electronic supplementary information (ESI) available. See DOI: 10.1039/C6NR02380K

  8. Effect of electric fields on the decay branching ratio of {sup 1}P{sup e} doubly excited states in helium measured by time-resolved fluorescence

    SciTech Connect

    Zitnik, Matjaz; Mihelic, A.; Bucar, K.; Penent, F.; Lablanquie, P.; Richter, R.; Alagia, M.; Stranges, S.

    2006-11-15

    We have measured the lifetimes of {sup 1}P{sup e} (n=9-12) doubly excited states in static electric fields (1-6 kV/cm) by observing the decay of the fluorescence signal as a function of time. The effects of the field on these helium states below the second ionization threshold are twofold: their excitation becomes possible due to the Stark mixing with the optically allowed {sup 1}P{sup o} series, and their lifetime is strongly modified by the opening of the autoionization channel, not accessible in zero field. Although the electric field represents only a tiny perturbation of the atomic potential, a substantial shortening of the lifetimes below 100 ps is observed. This is the simplest quantum system where the ratio of autoionization to fluorescence decay probability can be effectively controlled by an electric field of moderate strength.

  9. Time-resolved fluorescence spectroscopy investigation of the effect of 4-hydroxynonenal on endogenous NAD(P)H in living cardiac myocytes

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Aneba, Swida; Mateasik, Anton; Chorvat, Dusan; Comte, Blandine

    2013-06-01

    Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.

  10. A time-resolved laser induced fluorescence study on the ion velocity distribution function in a Hall thruster after a fast current disruption

    SciTech Connect

    Mazouffre, S.; Gawron, D.; Sadeghi, N.

    2009-04-15

    The temporal characteristics of the Xe{sup +} ion axial velocity distribution function (VDF) were recorded in the course of low-frequency discharge current oscillations ({approx}14 kHz) of the 5 kW class PPS X000 Hall thruster. The evolution in time of the ion axial velocity component is monitored by means of a laser induced fluorescence diagnostic tool with a time resolution of 100 ns. As the number of fluorescence photons is very low during such a short time period, a homemade pulse-counting lock-in system was used to perform real-time discrimination between background photons and fluorescence photons. The evolution in time of the ion VDF was observed at three locations along the thruster channel axis after a fast shutdown of the thruster power. The anode discharge current is switched off at 2 kHz during 5 {mu}s without any synchronization with the current oscillation cycle. This approach allows to examine the temporal behavior of the ion VDF during decay and ignition of the discharge as well as during forced and natural plasma oscillations. Measurements show that the distribution function of the axial component of the Xe{sup +} ion does change periodically in time with a frequency close to the current oscillation frequency in both forced and natural cases. The ion density and the mean velocity are found to oscillate, whereas the velocity dispersion stays constant, which indicates that ionization and acceleration layers have identical dynamics. Finally, variations over time in the electric field are for the first time experimentally evidenced in a crossed-field discharge.