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Sample records for antibody-based time-resolved fluorescence

  1. Time resolved fluorescence of naproxen in organogel medium

    NASA Astrophysics Data System (ADS)

    Burguete, M. Isabel; Izquierdo, M. Angeles; Galindo, Francisco; Luis, Santiago V.

    2008-07-01

    The interaction between non-steroidal anti-inflammatory drug naproxen and the self assembled fibrillar network created by a low molecular weight organogelator has been probed by means of time resolved fluorescence spectroscopy.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. Motor Oil Classification Based on Time-Resolved Fluorescence

    PubMed Central

    Mu, Taotao; Chen, Siying; Zhang, Yinchao; Guo, Pan; Chen, He; Meng, Fandong

    2014-01-01

    A time-resolved fluorescence (TRF) technique is presented for classifying motor oils. The system is constructed with a third harmonic Nd:YAG laser, a spectrometer, and an intensified charge coupled device (ICCD) camera. Steady-state and time-resolved fluorescence (TRF) measurements are reported for several motor oils. It is found that steady-state fluorescence is insufficient to distinguish the motor oil samples. Then contour diagrams of TRF intensities (CDTRFIs) are acquired to serve as unique fingerprints to identify motor oils by using the distinct TRF of motor oils. CDTRFIs are preferable to steady-state fluorescence spectra for classifying different motor oils, making CDTRFIs a particularly choice for the development of fluorescence-based methods for the discrimination and characterization of motor oils. The two-dimensional fluorescence contour diagrams contain more information, not only the changing shapes of the LIF spectra but also the relative intensity. The results indicate that motor oils can be differentiated based on the new proposed method, which provides reliable methods for analyzing and classifying motor oils. PMID:24988439

  7. Time resolved fluorescence of cow and goat milk powder

    NASA Astrophysics Data System (ADS)

    Brandao, Mariana P.; de Carvalho dos Anjos, Virgílio; Bell., Maria José V.

    2017-01-01

    Milk powder is an international dairy commodity. Goat and cow milk powders are significant sources of nutrients and the investigation of the authenticity and classification of milk powder is particularly important. The use of time-resolved fluorescence techniques to distinguish chemical composition and structure modifications could assist develop a portable and non-destructive methodology to perform milk powder classification and determine composition. This study goal is to differentiate milk powder samples from cows and goats using fluorescence lifetimes. The samples were excited at 315 nm and the fluorescence intensity decay registered at 468 nm. We observed fluorescence lifetimes of 1.5 ± 0.3, 6.4 ± 0.4 and 18.7 ± 2.5 ns for goat milk powder; and 1.7 ± 0.3, 6.9 ± 0.2 and 29.9 ± 1.6 ns for cow's milk powder. We discriminate goat and cow powder milk by analysis of variance using Fisher's method. In addition, we employed quadratic discriminant analysis to differentiate the milk samples with accuracy of 100%. Our results suggest that time-resolved fluorescence can provide a new method to the analysis of powder milk and its composition.

  8. Portable optical oxygen sensor based on time-resolved fluorescence.

    PubMed

    Chu, Cheng-Shane; Chu, Ssu-Wei

    2014-11-10

    A new, simple signal processing, low-cost technique for the fabrication of a portable oxygen sensor based on time-resolved fluorescence is described. The sensing film uses the oxygen sensing dye platinum meso-tetra (pentfluorophenyl) porphyrin (PtTFPP) embedded in a polymer matrix. The ratio τ0100 measures sensitivity of the sensing film, where τ0 and τ100 represent the detected fluorescence lifetimes from the sensing film exposed to 100% nitrogen and 100% oxygen, respectively. The experimental results reveal that the PtTFPP-doped oxygen sensor has a sensitivity of 2.2 in the 0%-100% range. A preparation procedure for coating the photodiodes with the oxygen sensor film that produces repetitive and reliable sensing devices is proposed. The developed time-resolved optical oxygen sensor is portable, low-cost, has simple signal processing, and lacks optical filter elements. It is a cost-effective alternative to traditional electrochemical-based oxygen sensors and provides a platform for other optical based sensors.

  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 spectroscopy of the fluorescence quenching of a donor — acceptor pair by halothane

    NASA Astrophysics Data System (ADS)

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

    1992-04-01

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

  11. 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.

  12. Time-resolved fluorescence study of all-trans-retinal

    NASA Astrophysics Data System (ADS)

    Erez, Yuval; Presiado, Itay; Gepshtein, Rinat; Simkovitch, Ron; Huppert, Dan

    2014-11-01

    UV-vis steady-state and time-resolved emission techniques were employed to study the ultrafast relaxation path of all-trans-retinal. We found that the steady-state emission spectrum consists mainly of two bands that we assign to the allowed transition from the ? state and the forbidden transition from the ?(ππ*) state. The time-resolved emission signal is dependent on the excitation wavelength, and is composed of three decay components. The short-time component of less than 80 fs, irrespective of the solvent, is assigned to the transition from the ? state. The intermediate-time decay component is assigned to the transition from the ?(ππ*) state, depends on the solvent's polarity and not on the existence of hydrogen bonds between the solute and the solvent or the viscosity of the latter. It has a lifetime of ~1 ps in polar solvents, and of 0.6 and 0.4 ps in the non-polar solvents n-octane and cyclohexane, respectively.

  13. Polar plot representation of time-resolved fluorescence.

    PubMed

    Eichorst, John Paul; Wen Teng, Kai; Clegg, Robert M

    2014-01-01

    Measuring changes in a molecule's fluorescence emission is a common technique to study complex biological systems such as cells and tissues. Although the steady-state fluorescence intensity is frequently used, measuring the average amount of time that a molecule spends in the excited state (the fluorescence lifetime) reveals more detailed information about its local environment. The lifetime is measured in the time domain by detecting directly the decay of fluorescence following excitation by short pulse of light. The lifetime can also be measured in the frequency domain by recording the phase and amplitude of oscillation in the emitted fluorescence of the sample in response to repetitively modulated excitation light. In either the time or frequency domain, the analysis of data to extract lifetimes can be computationally intensive. For example, a variety of iterative fitting algorithms already exist to determine lifetimes from samples that contain multiple fluorescing species. However, recently a method of analysis referred to as the polar plot (or phasor plot) is a graphical tool that projects the time-dependent features of the sample's fluorescence in either the time or frequency domain into the Cartesian plane to characterize the sample's lifetime. The coordinate transformations of the polar plot require only the raw data, and hence, there are no uncertainties from extensive corrections or time-consuming fitting in this analysis. In this chapter, the history and mathematical background of the polar plot will be presented along with examples that highlight how it can be used in both cuvette-based and imaging applications.

  14. 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.

  15. Instrument response standard in time-resolved fluorescence.

    PubMed

    Luchowski, Rafal; Gryczynski, Zygmunt; Sarkar, Pabak; Borejdo, Julian; Szabelski, Mariusz; Kapusta, Peter; Gryczynski, Ignacy

    2009-03-01

    The fluorescence of LDS 798 dye in aqueous solution has a very short lifetime of 24 ps, independent of excitation wavelength. The time response of common photon counting detectors depends on the wavelength of the registered photon. In lifetime measurements, the instrument response function (IRF) is usually approximated by the temporal profile of the scattered excitation light. Because lambda(Exc) is typically much shorter than lambda(Em), a systematic error may be present in these measurements. We demonstrate that the fluorescence decay of LDS 798 is a better approximation of IRF, in particular, for avalanche photodiodes used in the near infrared spectral region.

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

    NASA Astrophysics Data System (ADS)

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

    2001-05-01

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

  17. Time-resolved fluorescence spectroscopy for chemical sensors.

    PubMed

    Draxler, S; Lippitsch, M E

    1996-07-20

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

  18. 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.

  19. Phase-locked 10 MHz reference signal for frequency domain time-resolved fluorescence measurements

    NASA Astrophysics Data System (ADS)

    Smith, Trevor A.; Bird, Damian K.; Nuske, John W.

    2007-05-01

    A complete electronic system that is suitable for use in megahertz frequency domain time-resolved fluorescence instruments based on mode-locked lasers is described. The circuit produces a 10MHz signal, phase locked to the mode-locked laser pulse frequency, which is required by many commercial frequency synthesizers as the external reference signal. This device is particularly useful in conjunction with ultrafast gated intensified charge coupled device cameras capable of being frequency modulated for time-resolved fluorescence imaging.

  20. Time-resolved fluorescence measurements of actin-phalloidin interactions

    NASA Astrophysics Data System (ADS)

    Helms, Michael K.; French, Todd E.

    2000-03-01

    Compounds that interact with the cytoskeleton affect mobility and division, making them useful for treatment of certain types of cancer. Actin binding drugs such as the phallotoxins (small, bicyclic peptides) bind to and stabilize actin polymers (F-actin) without binding to actin monomers (G-actin). It has been shown that the intensity of fluorescently labeled phallotoxins such as fluorescein- phalloidin and rhodamine-phalloidin increases upon bind F- actin. We used LJL BioSystems' new FLAReTM technology to measure excited state lifetime changes of fluorescein- phalloidin and rhodamine-phalloidin upon binding to F- actin.

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

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

  2. Monte Carlo algorithm for efficient simulation of time-resolved fluorescence in layered turbid media.

    PubMed

    Liebert, A; Wabnitz, H; Zołek, N; Macdonald, R

    2008-08-18

    We present an efficient Monte Carlo algorithm for simulation of time-resolved fluorescence in a layered turbid medium. It is based on the propagation of excitation and fluorescence photon bundles and the assumption of equal reduced scattering coefficients at the excitation and emission wavelengths. In addition to distributions of times of arrival of fluorescence photons at the detector, 3-D spatial generation probabilities were calculated. The algorithm was validated by comparison with the analytical solution of the diffusion equation for time-resolved fluorescence from a homogeneous semi-infinite turbid medium. It was applied to a two-layered model mimicking intra- and extracerebral compartments of the adult human head.

  3. 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.

  4. Time-resolved principal component imaging analysis of chlorophyll fluorescence induction for monitoring leaf water stress.

    PubMed

    Kobori, Hikaru; Tsuchikawa, Satoru

    2013-06-01

    Chlorophyll fluorescence induction is widely applied to investigate plant growth conditions by calculating the ratio of its intensity at oxidized and reduced states. We examined the applicability of a time-resolved profile of chlorophyll fluorescence induction with the aid of multivariate analysis to monitor the leaf water stress. Principal component (PC) analysis of time-resolved images of chlorophyll fluorescence induction and their score images were reconstructed. Control leaves (non-stressed leaves) and water-stressed leaves could be classified by normalized PC3 score images. This technique has the potential to monitor the water stress condition of plants by using a simple device.

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

    PubMed

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

    2009-11-15

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

  6. X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals.

    PubMed

    Perozzo, M A; Ward, K B; Thompson, R B; Ward, W W

    1988-06-05

    The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P6122 or P6522 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallel-epipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, beta = 108 degrees, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Time-resolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein's structure-function relationship.

  7. Plastique: A synchrotron radiation beamline for time resolved fluorescence in the frequency domain

    NASA Astrophysics Data System (ADS)

    De Stasio, Gelsomina; Zema, N.; Antonangeli, F.; Savoia, A.; Parasassi, T.; Rosato, N.

    1991-06-01

    PLASTIQUE is the only synchrotron radiation beamline in the world that performs time resolved fluorescence experiments in frequency domain. These experiments are extremely valuable sources of information on the structure and dynamics of molecules. We describe the beamline and some initial data.

  8. 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

  9. 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

    2015-06-26

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2004-07-01

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

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

    PubMed

    Otosu, Takuhiro; Nishimoto, Etsuko; Yamashita, Shoji

    2009-03-26

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

  12. 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.

  13. 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

  14. 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…

  15. 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…

  16. 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

  17. 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

  18. Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

    PubMed Central

    Gohar, Ali Vaziri; Cao, Ruofan; Jenkins, Patrick; Li, Wenyan; Houston, Jessica P.; Houston, Kevin D.

    2013-01-01

    Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime. PMID:24010001

  19. 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.

  20. 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

  1. 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.

  2. Spectra-resolved technique of a sensitive time-resolved fluorescence immunoassay instrument

    NASA Astrophysics Data System (ADS)

    Guo, Zhouyi; Tian, Zhen; Jia, Yali

    2004-07-01

    The lanthanide trivalence ion and its chelates are used for marking substance in time-resolved fluorescence immunoassay (TRFIA), marking the protein, hormone, antibody, nucleic acid probe or biologica alive cell, to measure the concentration of the analysis substance inside the reaction system with time-resolved fluorometry after the reaction system occurred, and attain the quantitative analysis's purpose. TRFIA has been become a kind of new and more sensitive measure method after radioisotope marking, enzymatic marking, chemiluminescence, electrochemiluminescence, it primarily is decided by the special physics and chemistry characteristic of lanthanide trivalence ion and its chelates. In this paper, the result of spectroscopic evaluation of europium trivalence ion and its chelate, and the principle of spectra-resolved technology and a sensitive time-resolved fluorescence immunoassay instrument made by ourselves are reported. In the set, a high frequency Xenon pulsed-light was adopted as exciting light, and two special filters was utilized according to spectra-resolved technique. Thus the influence of scattering light and short-lifetime fluorescence was removed. And the sensitivity is 10-12mol/L (when Eu3+ was used for marking substance), examination repeat is CV <= 5%, examination linearity is from 10-8mol/L to 10-12mol/L, correlation coefficient r >= 95% (p < 0.01).

  3. 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.

  4. Time-resolved fluorescence immunoassay for C-reactive protein using colloidal semiconducting nanoparticles.

    PubMed

    Härmä, Harri; Toivonen, Juha; Soini, Juhani T; Hänninen, Pekka; Parak, Wolfgang J

    2011-01-01

    Besides the typical short-lived fluorescence with decay times in the nanosecond range, colloidal II/VI semiconductor nanoparticles dispersed in buffer also possess a long-lived fluorescence component with decay times in the microsecond range. Here, the signal intensity of the long-lived luminescence at microsecond range is shown to increase 1,000-fold for CdTe nanoparticles in PBS buffer. This long-lived fluorescence can be conveniently employed for time-gated fluorescence detection, which allows for improved signal-to-noise ratio and thus the use of low concentrations of nanoparticles. The detection principle is demonstrated with a time-resolved fluorescence immunoassay for the detection of C-reactive protein (CRP) using CdSe-ZnS nanoparticles and green light excitation.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. Mechanism of response of potential-sensitive dyes studied by time-resolved fluorescence

    PubMed Central

    Das, Tapan K.; Periasamy, N.; Krishnamoorthy, G.

    1993-01-01

    The mechanism of response of two potential-sensitive dyes, diOC2(5) (3,3′-diethyloxadicarbocyanine iodide) and oxonol V (bis-[3-phenyl-5-oxoisoxazol-4-yl]pentamethine oxonol), were studied by using steady-state and time-resolved fluorescence techniques. The lipid concentration dependence of the Δψ (membrane potential)-induced change in total fluorescence intensity was quite different for these two dyes. Time-resolved fluorescence measurements showed that the fluorescence decay of these dyes in membranes could be resolved into at least three exponentials. Δψ-induced changes in the levels of these three populations were also measured under a variety of conditions. In the case of diOC2(5) an inside negative Δψ increased the levels of the bound forms. This shows that diOC2(5) responds to Δψ mainly by an “on-off” mechanism whereby Δψ perturbs the membrane-water partition coefficient of the dye. The Δψ-induced changes approached zero when the dye was totally membrane bound. In contrast, the Δψ-induced response of oxonol V increased with increased membrane binding. An inside negative Δψ decreased the level of the bound form with a longer lifetime. This shows that the mechanism of response of oxonol V is a Δψ-induced shift in the equilibrium between bound forms of the dye. PMID:19431883

  10. 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.

  11. Revisiting the photophysics of 9-fluorenone: Ultrafast time-resolved fluorescence and theoretical studies

    NASA Astrophysics Data System (ADS)

    Chang, Chih-Wei; Sølling, Theis I.; Diau, Eric Wei-Guang

    2017-10-01

    Ultrafast intersystem crossing dynamics of fluorenone in gas and condensed phases were investigated by time-resolved mass spectrometry and fluorescence up-conversion spectroscopy. The former shows the ultrafast Franck-Condon relaxation and the internal conversion dynamics of isolated fluorenone in the gas phase. The latter reveals that the vibrational relaxation time is 2.2 ps and a 110 ps fluorescence lifetime of fluorenone in hexane. The fluorescence lifetime in acetonitrile and dimethylsulfoxide is 16 ns and 15.1 ns, respectively. The potential energy surface along the Cdbnd O out of plane bending motion shows that this coordinate is important for ISC in both polar and non-polar solvents.

  12. 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.

  13. 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.

  14. 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.

  15. Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes.

    PubMed

    Neely, Robert K; Tamulaitis, Gintautas; Chen, Kai; Kubala, Marta; Siksnys, Virginijus; Jones, Anita C

    2009-11-01

    Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

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

    PubMed

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

    2015-05-01

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

  17. 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.

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

    PubMed

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

    2016-10-01

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

  19. [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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-08

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

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

    2014-01-01

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

  5. 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

  6. Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Pian, Qi; Yao, Ruoyang; Sinsuebphon, Nattawut; Intes, Xavier

    2017-07-01

    Spectrally resolved fluorescence lifetime imaging and spatial multiplexing have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (∼40 ps temporal resolution) using fewer than N2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Förster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.

  7. 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.

  8. Development of time resolved fluorescence resonance energy transfer-based assay for FXR antagonist discovery.

    PubMed

    Yu, Donna D; Lin, Wenwei; Chen, Taosheng; Forman, Barry M

    2013-07-15

    FXR (farnesoid X receptor, NRIH4), a nuclear receptor, plays a major role in the control of cholesterol metabolism. FXR ligands have been investigated in preclinical studies for targeted therapy against metabolic diseases, but have shown limitations. Therefore, there is a need for new agonist or antagonist ligands of FXR, both for potential clinical applications, as well as to further elucidate its biological functions. Here we describe the use of the X-ray crystal structure of FXR complexed with the potent small molecule agonist GW4064 to design and synthesize a novel fluorescent, high-affinity probe (DY246) for time resolved fluorescence resonance energy transfer (TR-FRET) assays. We then used the TR-FRET assay for high throughput screening of a library of over 5000 bioactive compounds. From this library, we identified 13 compounds that act as putative FXR transcriptional antagonists.

  9. Development of Time Resolved Fluorescence Resonance Energy Transfer-based Assay for FXR Antagonist Discovery

    PubMed Central

    Yu, Donna D.; Lin, Wenwei; Chen, Taosheng; Forman, Barry M.

    2013-01-01

    FXR (farnesoid X receptor, NRIH4), a nuclear receptor, plays a major role in the control of cholesterol metabolism. FXR ligands have been investigated in preclinical studies for targeted therapy against metabolic diseases, but have shown limitations. Therefore, there is a need for new agonist or antagonist ligands of FXR, both for potential clinical applications, as well as to further elucidate its biological functions. Here we describe the use of the X-ray crystal structure of FXR complexed with the potent small molecule agonist GW4064 to design and synthesize a novel fluorescent, high-affinity probe (DY246) for time resolved fluorescence resonance energy transfer (TR-FRET) assays. We then used the TR-FRET assay for high throughput screening of a library of over 5,000 bioactive compounds. From this library, we identified 13 compounds that act as putative FXR transcriptional antagonists. PMID:23688559

  10. 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, +/-).

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

    NASA Astrophysics Data System (ADS)

    Millar, David P.; Benkovic, Stephen J.

    1990-05-01

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

  12. 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.

  13. Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay.

    PubMed

    Aggerbeck, H; Nørgaard-Pedersen, B; Heron, I

    1996-04-19

    A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu(3+)-labelled toxoids and to 0.0035 IU/ml using Sm(3+)-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies.

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

    PubMed

    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 (0.2-5 μg/L). It is comparable to the commercial dissociation-enhanced lanthanide fluoro-immunoassay system (DELFIA) reagents (0.2-145 μg/L). We propose that it can fulfill clinical applications.

  15. Time-resolved spectroscopy of the probe fluorescence in the study of human blood protein dynamic structure on SR beam

    NASA Astrophysics Data System (ADS)

    Dobretsov, G. E.; Kurek, N. K.; Syrejshchikova, T. I.; Yakimenko, M. N.; Clarke, D. T.; Jones, G. R.; Munro, I. H.

    2000-06-01

    Time-resolved spectroscopy on the SRS of the Daresbury Laboratory was used for the study of the human serum lipoproteins and human blood albumins with fluorescent probes K-37 and K-35, developed in Russia. The probe K-37 was found sensitive to the difference in dynamic properties of the lipid objects. Two sets of the parameters were used for the description of lipid dynamic structure: (1) time-resolved fluorescence spectra and (2) time-resolved fluorescence depolarization as a function of rotational mobility of lipid molecules. Each measured dynamic parameter reflected the monotonous changes of dynamic properties in the range: lipid spheres-very low density lipoproteins-low density lipoproteins-high density lipoproteins-phospholipid liposomes. The range is characterized by the increase of the ratio polar/ nonpolar lipids. Thus, time-resolved fluorescence could be used to detect some structural modifications in lipoproteins related to atherosclerosis and subsequent cardiovascular diseases development.

  16. Time resolved laser induced fluorescence measurements: Considerations when using Nd:YAG based system

    NASA Astrophysics Data System (ADS)

    Rabasovic, Maja S.; Sevic, Dragutin; Terzic, Mira; Marinkovic, Bratislav P.

    2012-05-01

    Time-resolved laser-induced fluorescence (TR-LIF) and the laser induced breakdown spectroscopy (LIBS) have been shown to be methods which are fast and sensitive to provide information about the constituents in analyzed samples. TR-LIF and LIBS have similar hardware requirements. In this paper, we analyze some characteristics of TR-LIF/LIBS system implemented in our laboratory, considering the fact that the excitation part of the system is based on Nd:YAG laser and Optical Parametric Oscillator (OPO). The laser is more than powerful enough (365 mJ at 1064 nm, variable OPO output >5 mJ) for LIBS, but somehow slow (the length of fundamental laser harmonic output pulse is about 5 ns) for fluorescence measurements in our present area of interest, namely plants and food products. Fortunately, the pulse length of tunable OPO output (320-475 nm) is less then 1 ns, so by means of a correct deconvolution procedure it is possible to measure the fluorescence lifetimes in the range as small as a few nanoseconds. The fluorescence detection part of our system is based on picosecond streak camera. Using the fluorescent dyes (Rhodamine B and Fluorescein) ethanol solutions we verified the analyzing capabilities of our TR-LIF system.

  17. 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.

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2017-05-01

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

  20. Time-resolved remote Raman and fluorescence spectrometers for planetary exploration

    NASA Astrophysics Data System (ADS)

    Sharma, Shiv K.; Misra, Anupam K.; Acosta, Tayro E.; Lucey, Paul G.

    2012-06-01

    At the University of Hawaii, we have developed compact time-resolved (TR) Raman, and fluorescence spectrometers suitable for planetary exploration under NASA's Mars Instrument Development Program. The compact Raman and fluorescence spectrometers consist of custom miniature spectrographs based on volume holographic gratings, and custom miniature intensified CCD cameras. These spectrographs have been interfaced with a regular 50 mm camera lens as well as with a three and a half inch diameter telescope for remotely interrogating minerals, water, water-ice and dry ice. Using a small frequency-doubled Nd:YAG pulsed laser (35 mJ/pulse, 20 Hz) and 50 mm camera lens, TRRaman and LINF spectra of minerals, and bio-minerals can be measured within 30 s under super-critical CO2, and with 3.5-inch telescope these samples can be interrogated to 50 m radial distance during day time and nighttime. The fluorescence spectrograph is capable of measuring TR- laser-induced fluorescence excited with 355 nm laser in the spectral range 400-800 nm spectral range. The TR-fluorescence spectra allow measurement of LINF from rare-earths and transition-metal ions in time domain, and also assist in differentiating between abiogenic minerals from organic and biogenic materials based on the fluorescence lifetime. Biological materials are also identified from their characteristic short-lived (<10 ns) laser-induced fluorescence lifetime. These instruments will play important role in planetary exploration especially in NASA's future Mars Sample Return Mission, and lander and rover missions.

  1. 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.

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

    PubMed

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

    2011-01-01

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

  3. Versatility of homogeneous time-resolved fluorescence resonance energy transfer assays for biologics drug discovery.

    PubMed

    Rossant, Christine J; Matthews, Carl; Neal, Frances; Colley, Caroline; Gardener, Matthew J; Vaughan, Tristan

    2015-04-01

    Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability of donor and acceptor fluorophore-conjugated reagents for detection of multiple tags or fusion proteins. The time-resolved component of the technology reduces potential assay interference, allowing screening of a range of different crude sample types derived from the bacterial or mammalian cell expression systems often used for antibody discovery projects. Here we describe the successful application of this technology across multiple projects targeting soluble proteins and demonstrate how it has provided key information for the isolation of potential therapeutic antibodies with the desired activity profile.

  4. 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

  5. Nanoscale diffusion in the synaptic cleft and beyond measured with time-resolved fluorescence anisotropy imaging

    PubMed Central

    Zheng, Kaiyu; Jensen, Thomas P.; Savtchenko, Leonid P.; Levitt, James A.; Suhling, Klaus; Rusakov, Dmitri A.

    2017-01-01

    Neural activity relies on molecular diffusion within nanoscopic spaces outside and inside nerve cells, such as synaptic clefts or dendritic spines. Measuring diffusion on this small scale in situ has not hitherto been possible, yet this knowledge is critical for understanding the dynamics of molecular events and electric currents that shape physiological signals throughout the brain. Here we advance time-resolved fluorescence anisotropy imaging combined with two-photon excitation microscopy to map nanoscale diffusivity in ex vivo brain slices. We find that in the brain interstitial gaps small molecules move on average ~30% slower than in a free medium whereas inside neuronal dendrites this retardation is ~70%. In the synaptic cleft free nanodiffusion is decelerated by ~46%. These quantities provide previously unattainable basic constrains for the receptor actions of released neurotransmitters, the electrical conductance of the brain interstitial space and the limiting rate of molecular interactions or conformational changes in the synaptic microenvironment. PMID:28181535

  6. 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

  7. 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).

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

    PubMed Central

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

    2016-01-01

    Actinic cheilitis is a potentially malignant disorder of the lips. Its first cause is believed to be UV sun radiation. The lesion is highly heterogeneous, making the choice of area to be biopsied difficult. This study exploits the capabilities of time-resolved fluorescence spectroscopy for the identification of the most representative area to be biopsied. A preliminary study was performed on fourteen patients. A classification algorithm was used on data acquired on nine different biopsies. The algorithm discriminated between absent, mild, and moderate dysplasia with a sensitivity of 92.9%, 90.0%, and 80.0%, respectively. The false positive rate for healthy tissue (specificity) was 88.8%. PMID:27867726

  9. Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3.

    PubMed

    Karvinen, Jarkko; Hurskainen, Pertti; Gopalakrishnan, Sujatha; Burns, David; Warrior, Usha; Hemmilä, Ilkka

    2002-06-01

    In addition to kinases and G protein-coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/microl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.

  10. Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy

    PubMed Central

    2016-01-01

    Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ2 orientation factor arising from differences in donor–acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor–acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET. PMID:28217242

  11. Fluorophore conjugated silver nanoparticles: a time-resolved fluorescence correlation spectroscopic study

    NASA Astrophysics Data System (ADS)

    Ray, Krishanu; Zhang, Jian; Lakowicz, Joseph R.

    2009-02-01

    Fluorescence detection is a central component in biological research. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. A single-stranded oligonucleotide was chemically bound to a single 50 nm diameter silver particle and a Cy5-labeled complementary single-stranded oligonucleotide was hybridized with the particle-bound oligonucleotide. The bound Cy5 molecules on the silver particles were spatially separated from the silver surface by the hybridized DNA duplex chains, which were about 8 nm in length, to reduce the competitive quenching. We use fluorescence lifetime correlation spectroscopy (FLCS) with picosecond time-resolved detection to separate the fluorescence correlation spectroscopy (FCS) contributions from fluorophores and metal-conjugated fluorophores. The single Cy5-labeled 50 nm silver particles displayed a factor of 15-fold increase in emission signal and 5-fold decrease in emission lifetimes in solution relative to the Cy5-DNA in the absence of metal. Lifetime measurements support the near-field interaction mechanism between the fluorophore and silver nanoparticle. In this study, FLCS is being applied to a system where the brightness and the fluorescent lifetime of the emitting species are significantly different. Our measurements suggest that FLCS is a powerful method for investigating the metal-fluorophore interaction at the single molecule level and to separate two different species from a mixture solution emitting at the same wavelength. Additionally, the highly bright Cy5-DNA-Ag molecules offer to be excellent probes in high background biological samples.

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

    PubMed

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

    2015-08-01

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

  13. Tryptophan dynamics of the FK506 binding protein: time-resolved fluorescence and simulations.

    PubMed Central

    Silva, N D; Prendergast, F G

    1996-01-01

    The FK506-binding protein (FKBP12) is important in the immunosuppressant action of FK506 and rapamycin. We have investigated Trp side chain dynamics in FKBP12, with and without a bound immunosuppressant, by measuring the Trp time-resolved fluorescence anisotropy decay r(t). The r(t) for W59 in aqueous uncomplexed FKBP12 at 20 degrees C is well described by a single exponential with a recovered initial anisotropy, r(eff)o, of 0.192 and an overall rotational correlation time for the protein, phi p, of 4.7 ns; r(eff)o = 0.214 and phi p = 4.2 ns for the FKBP12/FK506 complex. Using an expression for the order parameter squared, namely S2 = r(eff)o/rTo, where rTo is the vitrified steady-state excitation anisotropy, we recovered an S2 of 0.75 for W59 fluorescence in uncomplexed FKBP12 and S2 approximately equal to 1 in the FKBP12/FK506 complex. Results obtained for the FKBP12/rapamycin complex are similar to those found for the FKBP12/FK506 complex. Minimum perturbation mapping simulations were performed on the free and complexed forms of FKBP12 and the results were generally in agreement with the experimental data. Images FIGURE 5 FIGURE 6 PMID:8785272

  14. Time-resolved measurements of short-wavelength fluorescence from x-ray-excited ions.

    PubMed

    Kapteyn, H C; Murnane, M M; Falcone, R W

    1987-09-01

    We demonstrate a novel technique for time-resolved spectroscopic studies of highly excited ions. The technique uses a laser-produced plasma as a short-pulse, soft-x-ray light source with a high repetition rate. A Nd:YAG laser with a pulse duration of 90 psec, a pulse energy of 70 microJ, and repetition rate of 10(4) pulses per second is focused onto a rotating metal target. Soft x rays from the resulting plasma photoionize a gas surrounding the target, and fluorescence from the gas is detected by using a spectrometer and a high-speed photodetector. Using the technique of time-correlated photon counting, we determined the radiative lifetime and collisional quenching rate of the Xe III 5s(0)5p(6)(1)S(0) state by observing its fluorescence at 108.9 nm. A time resolution of better than 400 psec was obtained. We also measured relative Auger decay yields of a core hole state in xenon using a higher-energy laser-produced plasma light source at a lower repetition rate.

  15. 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.

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

    PubMed

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

    2016-12-24

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-01-01

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

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

    PubMed

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

    2017-05-05

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

  19. Applications of immunomagnetic capture and time-resolved fluorescence detection for Salmonella enteriditis in liquid eggs

    NASA Astrophysics Data System (ADS)

    Tu, Shu-I.; Gehring, Andrew; Paoli, George

    2008-04-01

    An immuno sandwich method was evaluated for the detection of Salmonella in liquid eggs. Liquid eggs spiked with different out-break strains of Salmonella were mixed with proper enrichment media and incubated at 37 C for 4 to 20 h. After enrichment, immunomagnetic beads (IMB) coated with anti Salmonella antibodies were used to capture the bacteria. Samarium (Sm) labeled anti Salmonella antibodies were then used to form sandwiched complexes with IMB captured bacteria. Sandwiched Salmonella were then treated with Sm-chelator to allow the measurement of the released Sm by time-resolved fluorescence (TRF). The processes ranging from IMB capture to Sm chelation were performed using an automated KingFisher apparatus. With this approach, the presence of ~ 1 CFU of outbreak strains of Salmonella Enteritidis per egg (~50 g of liquid eggs) could be detected after enrichment for 20 h at 37 C. For higher levels of Salmonella Enteritidis contamination, e.g., 10 CFU per 50 g of liquid eggs, the enrichment time could be reduced to 5 h at 37 C. The results demonstrated that a combination of IMB capture and TRF measurement could be a rapid and sensitive method for Salmonella Enteritidis detection in liquid eggs.

  20. Microscopic dynamics of the glass transition investigated by time-resolved fluorescence measurements of doped chromophores

    NASA Astrophysics Data System (ADS)

    Ye, Jing Yong; Hattori, Toshiaki; Nakatsuka, Hiroki; Maruyama, Yoshihiro; Ishikawa, Mitsuru

    1997-09-01

    The microscopic dynamics of several monomeric and polymeric glass-forming materials has been investigated by time-resolved fluorescence measurements of doped malachite green molecules in a wide temperature region. For monomers, 1-propanol, propylene glycol, and glycerol, and a polymer without side chains, poly- butadiene, the temperature dependence of nonradiative decay time of doped malachite green molecules behaves in a similar way through the glass-transition region. Besides a kink around the calorimetric glass-transition temperature Tg, another crossover at a critical temperature Tc about 30-50 K above Tg has been clearly observed. This experimental finding is in agreement with the prediction of the mode-coupling theory that a dynamical transition exists well above Tg. On the other hand, for the complex polymers with side chains, poly(vinyl acetate), poly(methyl acrylate), and poly(ethyl methacrylate), the crossover at Tg is less pronounced than those for the monomers and the polymer without side chains. Moreover, although we could not distinguish any singularities above Tg for these complex polymers, we observed another kink below Tg, which may be attributed to the side-chain motions.

  1. Steady-state and time resolved fluorescence analysis on tyrosine-histidine model compounds.

    PubMed

    Voicescu, Mariana; Heinrich, Martine; Hellwig, Petra

    2009-03-01

    Four model compounds, for a tyrosine-histidine covalent bonding, 2-(5-imidazolyl)-4-methylphenol (C-C bonding in ortho-position at the phenyl group); 2'-(1-imidazolyl)-4-methylphenol (C-N bonding in ortho'-position at the phenyl group); 2-(5-imidazolyl)-4-H-phenol and 2-(5-imidazolyl)-4-H-phenol, at physiological pH have been studied by UV-Vis absorption, steady-state and time resolved fluorescence spectroscopy. Their absorption and emission properties are presented and discussed. The photophysical properties depend on the para-substituted phenyl group as well as on C-C/C-N bonding in the Phenol-Imidazole linkage. The N position, N1-N3/N1-N4, in the imidazole group was found to be relevant. The results are discussed with relevance to the redox processes of tyrosine and to better understand the role of a tyrosine-histidine covalent linkage as found in cytochrome c oxidase.

  2. Investigation of Prolactin Receptor Activation and Blockade Using Time-Resolved Fluorescence Resonance Energy Transfer

    PubMed Central

    Tallet, Estelle; Fernandez, Isabelle; Zhang, Chi; Salsac, Marion; Gregor, Nathalie; Ayoub, Mohammed Akli; Pin, Jean Philippe; Trinquet, Eric; Goffin, Vincent

    2011-01-01

    The prolactin receptor (PRLR) is emerging as a therapeutic target in oncology. Knowledge-based drug design led to the development of a pure PRLR antagonist (Del1-9-G129R-hPRL) that was recently shown to prevent PRL-induced mouse prostate tumorogenesis. In humans, the first gain-of-function mutation of the PRLR (PRLRI146L) was recently identified in breast tumor patients. At the molecular level, the actual mechanism of action of these two novel players in the PRL system remains elusive. In this study, we addressed whether constitutive PRLR activation (PRLRI146L) or PRLR blockade (antagonist) involved alteration of receptor oligomerization and/or of inter-chain distances compared to unstimulated and PRL-stimulated PRLR. Using a combination of various biochemical and spectroscopic approaches (co-IP, blue native electrophoresis, BRET1), we demonstrated that preformed PRLR homodimers are altered neither by PRL- or I146L-induced receptor triggering, nor by antagonist-mediated blockade. These findings were confirmed using a novel time-resolved fluorescence resonance energy transfer (TR-FRET) technology that allows monitoring distance changes between cell surface tagged receptors. This technology revealed that PRLR blockade or activation did not involve detectable distance changes between extracellular domains of receptor chains within the dimer. This study merges with our previous structural investigations suggesting that the mechanism of PRLR activation solely involves intermolecular contact adaptations leading to subtle intramolecular rearrangements. PMID:22649370

  3. Microscopic time-resolved imaging of singlet oxygen by delayed fluorescence in living cells.

    PubMed

    Scholz, Marek; Dědic, Roman; Hála, Jan

    2017-09-22

    Singlet oxygen is a highly reactive species which is involved in a number of processes, including photodynamic therapy of cancer. Its very weak near-infrared emission makes imaging of singlet oxygen in biological systems a long-term challenge. We address this challenge by introducing Singlet Oxygen Feedback Delayed Fluorescence (SOFDF) as a novel modality for semi-direct microscopic time-resolved wide-field imaging of singlet oxygen in biological systems. SOFDF has been investigated in individual fibroblast cells incubated with a well-known photosensitizer aluminium phthalocyanine tetrasulfonate. The SOFDF emission from the cells is several orders of magnitude stronger and much more readily detectable than the very weak near-infrared phosphorescence of singlet oxygen. Moreover, the analysis of SOFDF kinetics enables us to estimate the lifetimes of the involved excited states. Real-time SOFDF images with micrometer spatial resolution and submicrosecond temporal-resolution have been recorded. Interestingly, a steep decrease in the SOFDF intensity after the photodynamically induced release of a photosensitizer from lysosomes has been demonstrated. This effect could be potentially employed as a valuable diagnostic tool for monitoring and dosimetry in photodynamic therapy.

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

    PubMed

    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.

  5. A sensitive solid-phase time-resolved fluorescence immunoassay apparatus

    NASA Astrophysics Data System (ADS)

    Wang, Yun-lei; Song, Ke-fei; Zhang, Wei-lai; Li, Jun-ling

    2009-07-01

    In the device, a He-Ne laser of flash frequency 1-20 Hz was adopted as exciting light source, and three key technical problems have been solved successfully in order to enhance the detecting sensitivity and measuring stability of the device for time-resolved fluorimmunoassays(TRFIA)[1]. The first one is to design optimum exciting optical system, so that the exciting light beam excite the sample most effectively. The second one is to have a project spectrum filter which can reduce the affection of the background light to the photomultiplier tube and also ensure influence of the stray light and mixed diffusion light to the sample fluorescence to the least, the sample fluorescence through the integrating sphere and come to the grating monochromator, The right wavelength will be chosed through changing the angle of incidence of the grating monochromator. The third one is to simulate the principle of sample averaging of BOXCAR averager. In the device, SCM was used as primary controller and CPLD was used as timing controller. Through the preparation process, signal-to-noise ratio(SNR) will be improved, also adjust delay time, ampling frequency and sampling number arbitrarily. By testing, the sensitivity is 10-12mol/L(substance marked by Eu3+), examination repeat is <=2.5%, examination linearity is from 10-8mol/L to 10-12mol/L, correlation coefficient is 99.98%(p<=0.01). The instrument is advanced for ultrasensitive detection of antigen and antibody , and solve the tumor, genetic variation, the virus protein detection.

  6. Unfolding of Ubiquitin Studied by Picosecond Time-Resolved Fluorescence of the Tyrosine Residue

    PubMed Central

    Noronha, Melinda; Lima, João C.; Bastos, Margarida; Santos, Helena; Maçanita, António L.

    2004-01-01

    The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25°C) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and excited-state tyrosinate. Second, at pH 1.5, the water-exposed tyrosine of either thermally or chemically unfolded UBQ decays as a sum of two exponentials. The double-exponential decays were interpreted and analyzed in terms of excited-state intramolecular electron transfer from the phenol to the amide moiety, occurring in one of the three rotamers of tyrosine in UBQ. The values of the rate constants indicate the presence of different unfolded states and an increase in the mobility of the tyrosine residue during unfolding. Finally, from the pre-exponential coefficients of the fluorescence decays, the unfolding equilibrium constants (KU) were calculated, as a function of temperature or denaturant concentration. Despite the presence of different unfolded states, both thermal and chemical unfolding data of UBQ could be fitted to a two-state model. The thermodynamic parameters Tm = 54.6°C, ΔHTm = 56.5 kcal/mol, and ΔCp = 890 cal/mol//K, were determined from the unfolding equilibrium constants calculated accordingly, and compared to values obtained by differential scanning calorimetry also under the assumption of a two-state transition, Tm = 57.0°C, ΔHm= 51.4 kcal/mol, and ΔCp = 730 cal/mol//K. PMID:15454455

  7. A chloride ion nanosensor for time-resolved fluorimetry and fluorescence lifetime imaging.

    PubMed

    Ruedas-Rama, Maria J; Orte, Angel; Hall, Elizabeth A H; Alvarez-Pez, Jose M; Talavera, Eva M

    2012-03-21

    In this work, the first CdSe/ZnS quantum dot (QD) photoluminescence lifetime based chloride ion nanosensor is reported. The acridinium dication lucigenin was self-assembled on the surface of negatively charged mercaptopropionic acid capped QDs to achieve QD-lucigenin conjugates. Upon attachment, a drastic decrease of the photoluminescence lifetime of both QD nanoparticles and lucigenin is observed by virtue of a charge transfer mechanism. Since lucigenin is a chloride-sensitive indicator dye, the photoluminescence decay of QD-lucigenin conjugates changes by adding chloride ion. The photoluminescence lifetime of the QDs in the conjugate increases after reacting with Cl(-), but also shows a concomitant decrease in the lucigenin lifetime immobilized on the surface. The photoluminescence lifetime of QD-lucigenin nanosensors shows a linear response in the Cl(-) concentration range between 0.5 and 50 mM. Moreover, the ratio τ(ave)(QD)/τ(ave)(luc) can be used as an analytical signal since the lifetime ratio presents a linear response in the same Cl(-) concentration range. The system also shows good selectivity towards most of the main anions and molecules that can be found in biological fluids. These nanosensors have been satisfactorily applied for Cl(-) determination in simulated intracellular media with high sensitivity and high selectivity. Finally, we demonstrate the potential application of the proposed nanosensor in confocal fluorescence lifetime imaging (FLIM). These results show the promising application of the QD-lucigenin nanosensors in FLIM, particularly for intracellular sensing, with the invaluable advantages of the time-resolved fluorescence techniques.

  8. Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase.

    PubMed

    Ross, J B; Schmidt, C J; Brand, L

    1981-07-21

    The tryptophan fluorescence decay of horse liver alcohol dehydrogenase, at 10 degrees C in 0.1 M pH 7.4 sodium phosphate buffer, with excitation at 295 nm, is a double exponential with time constants of 3.8 and 7.2 ns. Within experimental error, the two lifetimes remain constant across the emission spectrum. Only the 3.8-ns lifetime is quenched in the NAD+-pyrazole ternary complex, and only the 7.2-ns lifetime is quenched by 0-0.05 M KI. On the basis of these results, we assign the 3.8-ns lifetime to the buried tryptophan, Trp-314, and the 7.2-ns lifetime to the exposed tryptophan, Trp-15. The steady-state lifetime-resolved emission spectrum of Trp-15 has a maximum at approximately 340 nm and that of Trp-15 is at approximately 325 nm. The total time-resolved emission, after 40 ns of decay, has a maximum between 338 and 340 nm and is primarily due to the Trp-15 emission. As a consequence of the wavelength dependence of the preexponential weighting factors, there is an increase in the average lifetime from the blue to the red edge of the emission. This increase reflects the change in the spectral contributions of Trp-15 and Trp-314. Consideration of the spectral overlap between the emission spectra of the two tryptophans and the absorption due to formation of the ternary complex, as well as the distances between the two residues and the bound NAD+, shows that the selective fluorescence quenching in the ternary complex can be accounted for entirely by singlet-single energy transfer. The decay of the fluorescence anisotropy was measured as a function of temperature from 10 to 40 degrees C and is well described by a monoexponential decay law. Over this temperature range the calculated hydrodynamic radius increases from 33.5 to 35.1 A. Evidently, the indole groups of Trp-15 and Trp-314 rotate with the protein as a whole, and there is some expansion of the protein matrix as the ambient temperature is increased.

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

    PubMed

    Smith, Adam W

    2015-01-01

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

  10. 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

  11. Rapid homogenous time-resolved fluorescence (HTRF) immunoassay for anthrax detection.

    PubMed

    Cohen, Noam; Mechaly, Adva; Mazor, Ohad; Fisher, Morly; Zahavy, Eran

    2014-05-01

    Infection with Bacillus anthracsis spores induces an acute anthrax disease that can cause casualties and death in untreated cases. Thus rapid diagnosis of anthrax at early stage of the disease is essential to allow an effective treatment. Here we present the development of rapid and sensitive homogenous time-resolved fluorescence (HTRF) immunoassays based on the energy transfer process of europium cryptate (EuK) donor to AlexaFluor647 acceptor. The energy transfer process is limited to d < 10 nm, making the HTRF an ideal assay for examination of homogenous and complex samples, since only mutual binding of the donor and acceptor antibodies to the analyte would result in positive signal. HTRF assay was developed for the detection of the bacterial Protective Antigen (PA) toxin, a serological marker that correlates with bacteremia in infected hosts, using two monoclonal anti-PA antibodies that specifically recognize two different epitopes on the PA molecule. The assay was sensitive enabling detection of 2 ng/ml PA in the serum of B. anthracsis-infected rabbits in only 15 min assay. Additionally, HTRF assay was developed for the detection of bacterial spores using polyclonal anti-spore antibodies that recognize many epitopes on the bacterial surface. The assay enabled the detection of 2 × 10(6) spores/ml in 30 min assay and was specific, showing no cross reactivity with closely related non-virulent bacillus cereus strain. This study describes the use of the HTRF assay for the detection of both singled-epitope (proteins) and multi-epitope (particles) as rapid, simple and sensitive method that can be used at the time that fast results are needed to allow an effective medical care.

  12. Detection of Rupture-Prone Atherosclerotic Plaques by Time-Resolved Laser Induced Fluorescence Spectroscopy

    PubMed Central

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

    2009-01-01

    Objective Plaque with dense inflammatory cells, including macrophages, thin fibrous cap and superficial necrotic/lipid core is thought to be prone-to-rupture. We report a time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) technique for detection of such markers of plaque vulnerability in human plaques. Methods The autofluorescence of carotid plaques (65 endarterectomy patients) induced by a pulsed laser (337 nm, 0.7 ns) was measured from 831 distinct areas. The emission was resolved spectrally- (360–550 nm range) and temporally- (0.3 ns resolution) using a prototype fiber-optic TR-LIFS apparatus. Lesions were evaluated microscopically and quantified as to the % of different components (fibrous cap, necrotic core, inflammatory cells, foam cells, mature and degraded collagen, elastic fibers, calcification, and smooth muscle cell of the vessel wall). Results We determined that the spectral intensities and time-dependent parameters at discrete emission wavelengths 1) allow for discrimination (sensitivity >81%, specificity >94%) of various compositional and pathological features associated with plaque vulnerability including infiltration of macrophages into intima and necrotic/lipid core under a thin fibrous cap, and 2) show a linear correlation with plaque biochemical content: elastin (P<0.008), collagen (P<0.02), inflammatory cells (P<0.003), necrosis (P<0.004). Conclusion Our results demonstrate the feasibility of TR-LIFS as a method for the identification of markers of plaque vulnerability. Current findings enable future development of TR-LIFS based clinical devices for rapid investigation of atherosclerotic plaques and detection of those at high-risk. PMID:18926540

  13. Discovery of novel aromatase inhibitors using a homogeneous time-resolved fluorescence assay

    PubMed Central

    Ji, Jin-zi; Lao, Ke-jing; Hu, Jie; Pang, Tao; Jiang, Zhen-zhou; Yuan, Hao-liang; Miao, Jing-shan; Chen, Xin; Ning, Shan-shan; Xiang, Hua; Guo, Yu-meng; Yan, Ming; Zhang, Lu-yong

    2014-01-01

    Aim: Aromatase is an important target for drugs to treat hormone-dependent diseases, including breast cancer. The aim of this study was to develop a homogeneous time-resolved fluorescence (HTRF) aromatase assay suitable for high-throughput screening (HTS). Methods: A 384-well aromatase HTRF assay was established, and used to screen about 7000 compounds from a compound library. Anti-proliferation activity of the hit was evaluated using alamarBlue(R) assay in a hormone-dependent breast cancer cell line T47D. Molecular docking was conducted to elucidate the binding mode of the hit using the Discovery Studio program. Results: The Z′ value and signal to background (S/B) ratio were 0.74 and 5.4, respectively. Among the 7000 compounds, 4 hits (XHN22, XHN26, XHN27 and triptoquinone A) were found to inhibit aromatase with IC50 values of 1.60±0.07, 2.76±0.24, 0.81±0.08 and 45.8±11.3 μmol /L, respectively. The hits XHN22, XHN26 and XHN27 shared the same chemical scaffold of 4-imidazolyl quinoline. Moreover, the most potent hit XHN27 at 10 and 50 μmol/L inhibited the proliferation of T47D cells by 45.3% and 35.2%, respectively. The docking study revealed that XHN27 docked within the active site of aromatase and might form a hydrogen bond and had a π-cation interaction with amino acid residues of the protein. Conclusion: XHN27, an imidazolyl quinoline derivative of flavonoid, is a potent aromatase inhibitor with anti-proliferation activity against breast cancer in vitro. The established assay can be used in HTS for discovering novel aromatase inhibitor. PMID:25047514

  14. Time-resolved microspectrofluorometry and fluorescence lifetime imaging using ps-pulsed diode lasers in laser scanning microscopes

    NASA Astrophysics Data System (ADS)

    Rueck, Angelika; Dolp, Frank; Scalfi-Happ, Claudia; Steiner, Rudolf W.; Beil, Michael

    2003-10-01

    A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for online detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting on a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module (TCSPC) was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). To prove and calibrate the system, the time-resolved fluorescence characteristics of the mitochondrial marker Rhodamine 123 and 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acidhexylester)- induced protoporphyrine IX (PPIX) were investigated in solution and in cell culture. Different lifetimes could be found in different cell compartiments. During illumination, the lifetimes decreased significantly. From photobleaching experiments different metabolites of 5-ALA could be correlated with different fluorescence lifetimes. In conclusion FLIM, using ps diode lasers and TCSPC techniques is a valuable method to selectively identify and localize various metabolites of fluorescent probes during laser scanning microscopy.

  15. 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

  16. Fluorescence quenching and time-resolved fluorescence studies of alpha-mannosidase from Aspergillus fischeri (NCIM 508).

    PubMed

    Shashidhara, K S; Gaikwad, Sushama M

    2007-11-01

    Apart from the vital role in glycoprotein biosynthesis and degradation, alpha-mannosidase is currently an important therapeutic target for the development of anticancer agents. Fluorescence quenching and time-resolved fluorescence of alpha-mannosidase, a multitryptophan protein from Aspergillus fischeri were carried out to investigate the tryptophan environment. The tryptophans were found to be differentially exposed to the solvent and were not fully accessible to the neutral quencher indicating heterogeneity in the environment. Quenching of the fluorescence by acrylamide was collisional. Surface tryptophans were found to have predominantly positively charged amino acids around them and differentially accessible to the ionic quenchers. Denaturation led to more exposure of tryptophans to the solvent and consequently in the significant increase in quenching with all the quenchers. The native enzyme showed two different lifetimes, tau (1) (1.51 ns) and tau (2) (5.99 ns). The average lifetime of the native protein (tau) (3.187 ns) was not affected much after denaturation (tau) (3.219 ns), while average lifetime of the quenched protein samples was drastically reduced (1.995 ns for acrylamide and 1.537 ns for iodide). This is an attempt towards the conformational studies of alpha-mannosidase.

  17. Characterization of type I, II, III, IV, and V collagens by time-resolved laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2000-04-01

    The relative proportions of genetically distinct collagen types in connective tissues vary with tissue type and change during disease progression, development, wound healing, aging. This study aims to 1) characterize the spectro- temporal fluorescence emission of fiber different types of collagen and 2) assess the ability of time-resolved laser- induced fluorescence spectroscopy to distinguish between collagen types. Fluorescence emission of commercially available purified samples was induced with nitrogen laser excitation pulses and detected with a MCP-PMT connected to a digital storage oscilloscope. The recorded time-resolved emission spectra displayed distinct fluorescence emission characteristics for each collagen type. The time domain information complemented the spectral domain intensity data for improved discrimination between different collagen types. Our results reveal that analysis of the fluorescence emission can be used to characterize different species of collagen. Also, the results suggest that time-resolved spectroscopy can be used for monitoring of connective tissue matrix composition changes due to various pathological and non-pathological conditions.

  18. Lanthanide labeling of a potent protease activated receptor-2 agonist for time-resolved fluorescence analysis

    PubMed Central

    Hoffman, Justin; Flynn, Andrea N.; Tillu, Dipti V.; Zhang, Zhenyu; Patek, Renata; Price, Theodore J.; Vagner, Josef; Boitano, Scott

    2012-01-01

    Protease activated receptor-2 (PAR2) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR2 can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR2 to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report we describe the synthesis and use of a modified PAR2 peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH2 (2-f-LIGRLO-dtpa), designed for lanthanide-based time resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR2 agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH2, 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH2, 2-at-LIGRL and; 6-aminonicotinyl-LIGRL-NH2, 6-an-LIGRL) and PAR2. A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH2, 3-ia-LIGRL) that does not activate PAR2 signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR2 binding. In summary, we have developed a Europium-containing PAR2 agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR2 ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR2 ligands. PMID:22994402

  19. Collisional deactivation of N2O(0001) studied by time-resolved infrared fluorescence

    NASA Astrophysics Data System (ADS)

    Poel, Kathleen L.; Alwahabi, Zeyad T.; King, Keith D.

    1996-07-01

    The time-resolved infrared fluorescence (IRF) technique has been used to study the vibrational deactivation of excited N2O by large polyatomic colliders at ambient temperature (295±2 K). N2O(0001) molecules were prepared by direct pumping with the P(18) line of a pulsed CO2 laser at 9.536 μm. The bimolecular rate constant for self-deactivation was determined to be (0.763±0.006)×103 Torr-1 s-1, in very good agreement with previous work. The rate constants for deactivation by Ar and H2 were found to be (0.103±0.003) and (4.89±0.52)×103 Torr-1 s-1, respectively. The deactivation rate constants for the large polyatomic molecules, c-C6H10, c-C6H12, C6H6, C6D6, C7H8, C7D8, C6H5F, p-C6H4F2, C6HF5 and C6F6, were found to be (176±10), (153±22), (115±4), (201±2), (127±11), (407±52), (144±14), (173±13), (129±8), and (48±9)×103 Torr-1 s-1, respectively. Experimental deactivation probabilities and average energies removed per collision are calculated and compared. There is little difference in deactivation probabilities between the acyclic ring compounds and their aromatic analogues and the partially-fluorinated benzenes but the perfluorinated compound, C6F6 is much less efficient than the other species. The perdeuterated species, C6D6 and C7D8, especially the latter, show enhanced deactivation relative to the other species, probably as a result of near-resonant intermolecular V-V energy transfer. The results are compared with our recent work on the deactivation of CO2(0001) by the same group of large polyatomic colliders [K. L. Poel, Z. T. Alwahabi, and K. D. King, Chem. Phys. 201, 263 (1995)].

  20. Time-resolved and steady-state fluorescence studies of excited-state proton-transfer reactions of proflavine

    NASA Astrophysics Data System (ADS)

    De Silvestri, S.; Laporta, P.

    1984-01-01

    Time-resolved and steady-state fluorescence studies of proflavine in aqueous solution are presented. The observation of a monoexponential fluorescence decay with a time constant decreasing with increasing pH and the presence of an anomalous red-shift in the fluorescence spectrum as a function of pH indicate the existence of a complex proton-transfer mechanism in the excited state. A reaction scheme is proposed and the corresponding proton-transfer rates are evaluated. An excited-state pK value of 12.85 is obtained for the equilibrium between the cationic form of proflavine and the same form dissociated at an amino group.

  1. 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.

  2. Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult's brain

    NASA Astrophysics Data System (ADS)

    Gerega, Anna; Milej, Daniel; Weigl, Wojciech; Botwicz, Marcin; Zolek, Norbert; Kacprzak, Michal; Wierzejski, Wojciech; Toczylowska, Beata; Mayzner-Zawadzka, Ewa; Maniewski, Roman; Liebert, Adam

    2012-08-01

    Optical technique based on diffuse reflectance measurement combined with indocyanine green (ICG) bolus tracking is extensively tested as a method for clinical assessment of brain perfusion in adults at the bedside. Methodology of multiwavelength and time-resolved detection of fluorescence light excited in the ICG is presented and advantages of measurements at multiple wavelengths are discussed. Measurements were carried out: 1. on a physical homogeneous phantom to study the concentration dependence of the fluorescence signal, 2. on the phantom to simulate the dynamic inflow of ICG at different depths, and 3. in vivo on surface of the human head. Pattern of inflow and washout of ICG in the head of healthy volunteers after intravenous injection of the dye was observed for the first time with time-resolved instrumentation at multiple emission wavelengths. The multiwavelength detection of fluorescence signal confirms that at longer emission wavelengths, probability of reabsorption of the fluorescence light by the dye itself is reduced. Considering different light penetration depths at different wavelengths, and the pronounced reabsorption at longer wavelengths, the time-resolved multiwavelength technique may be useful in signal decomposition, leading to evaluation of extra- and intracerebral components of the measured signals.

  3. Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult's brain.

    PubMed

    Gerega, Anna; Milej, Daniel; Weigl, Wojciech; Botwicz, Marcin; Zolek, Norbert; Kacprzak, Michal; Wierzejski, Wojciech; Toczylowska, Beata; Mayzner-Zawadzka, Ewa; Maniewski, Roman; Liebert, Adam

    2012-08-01

    Optical technique based on diffuse reflectance measurement combined with indocyanine green (ICG) bolus tracking is extensively tested as a method for clinical assessment of brain perfusion in adults at the bedside. Methodology of multiwavelength and time-resolved detection of fluorescence light excited in the ICG is presented and advantages of measurements at multiple wavelengths are discussed. Measurements were carried out: 1. on a physical homogeneous phantom to study the concentration dependence of the fluorescence signal, 2. on the phantom to simulate the dynamic inflow of ICG at different depths, and 3. in vivo on surface of the human head. Pattern of inflow and washout of ICG in the head of healthy volunteers after intravenous injection of the dye was observed for the first time with time-resolved instrumentation at multiple emission wavelengths. The multiwavelength detection of fluorescence signal confirms that at longer emission wavelengths, probability of reabsorption of the fluorescence light by the dye itself is reduced. Considering different light penetration depths at different wavelengths, and the pronounced reabsorption at longer wavelengths, the time-resolved multiwavelength technique may be useful in signal decomposition, leading to evaluation of extra- and intracerebral components of the measured signals.

  4. Time-Resolved Emission Spectra Of Tryptophan And Proteins From Frequency-Domain Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Szmacineki, Henryk; Lakowicz, Joseph R.; Johnson, Michael L.

    1988-06-01

    We report measurements of time-resolved emission spectra of N-acetyl-L-tryptophanamide (NATA), adrenocorticotropic hormone (ACTH, residues 1-24), and of S. Nuclease. These spectra were calculated from the frequency-response of the emission, measured at several wavelengths across the emission spectra. Measurements were performed on samples not quenched and quenched by acrylamide, the latter providing additional information on the short time events. The time-resolved center-of-gravity does not decay as a single exponential. At least two spectral relaxation times are needed to account for the present data. NATA and ACTH each display relaxation times near 50 and 800 ps, which may be characteristic of exposed tryptophan residues. S. nuclease displayed slower relaxation times near 0.5 and 10 ns, which probably reflect the dynamic protein matrix which surrounds the residue.

  5. In vivo detection of macrophages in a rabbit atherosclerotic model by time-resolved laser-induced fluorescence spectroscopy

    PubMed Central

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

    2007-01-01

    Accumulation of numerous macrophages in the fibrous cap is a key identifying feature of plaque inflammation and vulnerability. This study investigates the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a potential tool for detection of macrophage foam cells in the intima of atherosclerotic plaques. Experiments were conducted in vivo on 14 New Zealand rabbits (6 control, 8 hypercholesterolemic) following aortotomy to expose the intimal luminal surface of the aorta. Tissue autofluorescence was induced with a nitrogen pulse laser (337 nm, 1 ns). Lesions were histologically classified by the percent of collagen or macrophage foam cells as well as thickness of the intima. Using parameters derived from the time-resolved fluorescence emission of plaques, we determined that intima rich in macrophage foam cells can be distinguished from intima rich in collagen with high sensitivity (>85%) and specificity (>95%). This study demonstrates, for the first time, that a time-resolved fluorescence-based technique can differentiate and demark macrophage content versus collagen content in vivo. Our results suggest that TR-LIFS technique can be used in clinical applications for identification of inflammatory cells important in plaque formation and rupture. PMID:16039283

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

  7. 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.

  8. 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.

  9. Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes.

    PubMed

    Kress, Matthias; Meier, Thomas; Steiner, Rudolf; Dolp, Frank; Erdmann, Rainer; Ortmann, Uwe; Rück, Angelika

    2003-01-01

    This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging

  10. Steady-state and time-resolved fluorescence studies on the conjugation of Rose Bengal to gold nanorods

    NASA Astrophysics Data System (ADS)

    Gabudean, Ana-Maria; Groza, Raluca; Maniu, Dana; Astilean, Simion

    2014-09-01

    This work examines the fluorescence properties of Rose Bengal (RB) molecules conjugated to cetyltrimethylammonium bromide (CTAB) - coated gold nanorods (GNRs) by performing steady-state and time-resolved fluorescence measurements. We show that the quantum yield and fluorescence lifetime can be significantly modified by the electromagnetic coupling of RB to GNRs but the overall fluorescence signal depends also on the environmental conditions in which RB molecules reside - in solution or on solid substrate. For example, we have observed the increase of fluorescence intensity after binding RB to GNRs (RB@GNR) as result of both non-radiative rate decrease and contribution from the electromagnetic coupling of RB to GNRs. For RB@GNRs conjugates deposited on solid substrate we can provide evidence for a clear metal-enhanced fluorescence (MEF) mechanism by observing the decrease of fluorescence lifetime of RB from an average of 2.1 ± 0.36 ns, when complexed to CTAB solely, to 1.6 ± 0.26 ns, when conjugated to GNRs, together with the increase of fluorescence intensity. Our findings contribute to the development and evaluation of novel fluorescent tags based on plasmonic nanoparticles for biomedical applications.

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  12. 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.

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

    PubMed Central

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

    2011-01-01

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

  14. Time-resolved fluorescence spectra of cis-stilbene in hexane and acetonitrile

    NASA Astrophysics Data System (ADS)

    Sajadi, M.; Dobryakov, A. L.; Garbin, E.; Ernsting, N. P.; Kovalenko, S. A.

    2010-04-01

    Transient fluorescence spectra from cis-stilbene in solution are recorded with 0.24 ps instrument response by a Kerr-Shutter upon excitation at 283 and 267 nm. The fluorescence decay shows no dependence on the excitation wavelength and proceeds monoexponentially with 0.21 ps in acetonitrile and 0.75 ps in hexane. No spectral shift or distortion of the fluorescence band is observed during the decay. Fluorescence contribution from 4a,4b-dihydrophenanthrene (DHP), produced in a competing reaction channel, was not detectable. From comparison with trans-fluorescence, the emission oscillator strength of cis-stilbene is determined to be 0.19 in hexane and 0.21 in acetonitrile.

  15. Two-Dimensional Subpicosecond Time-Resolved Fluorescence Anisotropy: Optical Kerr-Gating with a Dynamic Polarization Excitation.

    NASA Astrophysics Data System (ADS)

    Fujiwara, Takashige; Romano, Natalie C.; Modarelli, David A.; Lim, Edward C.

    2013-06-01

    With an advent of ultrafast lasers, a number of applications are widely adopted to probe photophysical and photochemical properties of a molecule that occurs in an ultrafast (femtosecond to picosecond) time scale. Intramolecular charge transfer (ICT) or proton transfer in photoexcited electron donor-acceptor (EDA) molecules, for instance, has been a topic of very extensive time-resolved studies for several decades. Time-evolution of an anisotropic property can track dipole orientations or conformational changes in their photoexcited molecular systems, which is of extreme importance to examine its structure and excited-state dynamics rather than probing an isotropic "population change".With this respect, we recently developed a subpicosecond time-resolved 2-D fluorescence anisotropy (TRFA) in which method implements a dynamic alternation of laser polarizations to excite a sample using a photoelastic modulator (PEM). In the combination of an ultrafast optical shutter (Kerr-gating) and a spectrograph that is coupled with a CCD, two signal phases so-obtained dynamically, I_{∥}( t, λ) and I_{⊥}( t, λ), provide a 2-D mapped information on both a wide range for spectra and time-resolved kinetics of photoexcited molecules of interest. From the definition of an anisotropy 2-D TRFA, r (t, λ), is given instantly and even more reliably at a single measurement. In this paper we will present benchmark tests of some target samples to establish performance of TRFA.

  16. Time-resolved hyperspectral single-pixel camera implementation for compressive wide-field fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Pian, Qi; Yao, Ruoyang; Intes, Xavier

    2016-03-01

    Single-pixel imaging based on compressive sensing theory has been a highlighted technique in the biomedical imaging field for many years. This interest has been driven by the possibility of performing microscopic or macroscopic imaging based on low-cost detector arrays, increased SNR (signal-to-noise ratio) in the acquired data sets and the ability to perform high quality image reconstruction with compressed data sets by exploiting signal sparsity. In this work, we present our recent work in implementing this technique to perform time domain fluorescence-labeled investigations in preclinical settings. More precisely, we report on our time-resolved hyperspectral single-pixel camera for fast, wide-field mapping of molecular labels and lifetime-based quantification. The hyperspectral single-pixel camera implements a DMD (Digital micro-mirror device) to generate optical masks for modulating the illumination field before it is delivered onto the sample and focuses the emission light signals into a multi-anode hyperspectral time-resolved PMT (Photomultiplier tube) to acquire spatial, temporal and spectral information enriched 4-D data sets. Fluorescence dyes with lifetime and spectral contrast are embedded in well plates and thin tissues. L-1 norm based regularization or the least square method, is applied to solve the underdetermined inverse problem during image reconstruction. These experimental results prove the possibility of fast, wide-field mapping of fluorescent labels with lifetime and spectral contrast in thin media.

  17. A homogeneous europium cryptate-based assay for the diagnosis of mutations by time-resolved fluorescence resonance energy transfer

    PubMed Central

    Lopez-Crapez, E.; Bazin, H.; Andre, E.; Noletti, J.; Grenier, J.; Mathis, G.

    2001-01-01

    Oligonucleotide ligation assay (OLA) is considered to be a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied in the past to several scientific fields. This technique is well adapted to nucleic acid analysis such as DNA sequencing, DNA hybridization and polymerase chain reaction. We describe here a homogeneous format based on the use of a rare earth cryptate label as donor: tris-bipyridine-Eu3+. The long-lived fluorescence of this label makes it possible to reach a high sensitivity by using a time-resolved detection mode. A non-radiative energy transfer technology, known as time-resolved amplification of cryptate emission (TRACE®) characterized by a temporal and spectral selectivity has been developed. The TRACE® detection of characterized single nucleotide polymorphism using the OLA for allelic discrimination is proposed. We demonstrate the potentialities of this OLA–TRACE® methodology through the analysis of K-ras oncogene point mutations. PMID:11452039

  18. Self-association of the polyene antibiotic nystatin in dipalmitoylphosphatidylcholine vesicles: a time-resolved fluorescence study.

    PubMed Central

    Coutinho, A; Prieto, M

    1995-01-01

    The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, both in gel (T = 21 degrees C) and in liquid-crystalline (T = 45 degrees C) phases, was studied by steady-state and time-resolved fluorescence measurements by taking advantage of the intrinsic tetraene fluorophore present in this antibiotic. It was shown that Nystatin aggregates in aqueous solution with a critical concentration of 3 microM. The enhancement in the fluorescence intensity of the antibiotic was applied to study the membrane binding of Nystatin, and it was shown that the antibiotic had an almost fivefold higher partition coefficient for the vesicles in a gel (P = (1.4 +/- 0.1) x 10(3)) than in a liquid-crystalline phase (P = (2.9 +/- 0.1) x 10(2)). Moreover, a time-resolved fluorescence study was used to examine Nystatin aggregation in the membrane. The emission decay kinetics of Nystatin was described by three and two exponentials in the lipid membrane at 21 degrees C and 45 degrees C, respectively. Nystatin mean fluorescence lifetime is concentration-dependent in gel phase lipids, increasing steeply from 11 to 33 ns at an antibiotic concentration of 5-6 microM, but the fluorescence decay parameters of Nystatin were unvarying with the antibiotic concentration in fluid lipids. These results provide evidence for the formation of strongly fluorescent antibiotic aggregates in gel-phase membrane, an interpretation that is at variance with a previous study. However, no antibiotic self-association was detected in a liquid-crystalline lipid bilayer within the antibiotic concentration range studied (0-14 microM). PMID:8599661

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

    SciTech Connect

    Kojer, R.; Dresner, J.; Prochorow, J.; Deperasinska, 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.

  20. Characterization of time-resolved fluorescence response measurements for distributed optical-fiber sensing.

    PubMed

    Sinchenko, Elena; Gibbs, W E Keith; Davis, Claire E; Stoddart, Paul R

    2010-11-20

    A distributed optical-fiber sensing system based on pulsed excitation and time-gated photon counting has been used to locate a fluorescent region along the fiber. The complex Alq3 and the infrared dye IR-125 were examined with 405 and 780 nm excitation, respectively. A model to characterize the response of the distributed fluorescence sensor to a Gaussian input pulse was developed and tested. Analysis of the Alq3 fluorescent response confirmed the validity of the model and enabled the fluorescence lifetime to be determined. The intrinsic lifetime obtained (18.2±0.9 ns) is in good agreement with published data. The decay rate was found to be proportional to concentration, which is indicative of collisional deactivation. The model allows the spatial resolution of a distributed sensing system to be improved for fluorophores with lifetimes that are longer than the resolution of the sensing system.

  1. 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.

  2. Time-resolved fluorescence spectroscopy of high-lying electronic states of Zn-tetraphenylporphyrin

    NASA Astrophysics Data System (ADS)

    Gurzadyan, G. G.; Tran-Thi, T.-H.; Gustavsson, T.

    1998-01-01

    Depopulation of the S2 excited electronic state of the Zn-tetraphenylporphyrin (ZnTPP) was monitored by measuring the decay of S2→S0 and the rise of S1→S0 fluorescence using the up-conversion fluorescence technique with a time-resolution of 120 fs. The lifetime of the S2 electronic state, measured for ZnTPP in ethanol (τS2=2.35 ps) correlates with the risetime of S1→S0 fluorescence. This result demonstrates the depopulation of S2 to S1 via (vibrational) states with lifetimes much shorter than that of S2. The rise time of S2 fluorescence (τv=60-90 fs) was attributed to vibrational relaxation (in S2). Fluorescence anisotropy decay of the S2 state was also studied by measuring the parallel and perpendicular fluorescence components. The high initial anisotropy of r⩾0.7 is interpreted as due to the existence of a degenerate excited electronic state S2 and the corresponding fast decay time τ1=0.2 ps to the electronic dephasing of the degenerate level pair. The long component of the anisotropy decay (τ2≫10 ps) is due to rotational diffusion.

  3. 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.

  4. Time-resolved fluorescence and FCS studies of dye-doped DNA

    NASA Astrophysics Data System (ADS)

    Nicolaou, N.; Marsh, R. J.; Blacker, T.; Armoogum, D. A.; Bain, A. J.

    2009-08-01

    Fluorescence lifetime, anisotropy and intensity dependent single molecule fluorescence correlation spectroscopy (I-FCS) are used to investigate the mechanism of fluorescence saturation in a free and nucleotide bound fluorophore (NR6104) in an antioxidising ascorbate buffer. Nucleotide attachment does not appreciably affect the fluorescence lifetime of the probe and there is a decrease in the rate of intersystem crossing relative to that of triplet state deactivation. The triplet state fraction is seen to plateau at 72% (G-attached) and 80% (free fluorophore) in agreement with these observations. Measurements of translational diffusion times show no intensity dependence for excitation intensities between 1 and 105kW cm-2 and photobleaching is therefore negligible. The dominant mechanism of fluorescence saturation is thus triplet state formation. I-FCS measurements for Rhodamine 6G in water were compared with those in the ascorbate buffer. In water the triplet fraction was saturated at considerably higher powers (45% at ca. 1.5 × 103kW cm-2) than in the ascorbate buffer (55%ca. 1 1kW cm-2)

  5. The primary event in vision investigated by time-resolved fluorescence spectroscopy.

    PubMed Central

    Doukas, A G; Junnarkar, M R; Alfano, R R; Callender, R H; Balogh-Nair, V

    1985-01-01

    The picosecond fluorescence kinetics and quantum yield from bovine rhodopsin were measured in the 5-40 degrees K range. The fluorescence rise and decay times are faster than our resolution of 15 ps (full width at half maximum) over this entire temperature range. The size of the observed emission was also temperature independent, and we find that the upper limit of rhodopsin's fluorescence quantum yield to be phi f approximately equal to 10(-5). Replacing all of rhodopsin's exchangeable protons with deuterons by suspending rhodopsin in D2O had no effect on either the kinetics of the emission or the value of the quantum yield. Our data provide strong confirmation of the idea that the first step in the visual process is an excited-state cis-to-trans isomerization about the C11-C12 double bond of retinal. PMID:4016199

  6. 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.

  7. Time-autocorrelated two-photon counting technique for time-resolved fluorescence measurements

    NASA Astrophysics Data System (ADS)

    Borst, Walter L.; Liu, Lin-I.

    1999-01-01

    We describe a new instrumental technique for the excitation, acquisition, and analysis of fluorescence decays from a variety of substances, in the present case plastic scintillators. The fluorescence is excited by β particles from a radioactive source (100 μCi Sr-90). A random photon from the resulting fluorescence decay provides a trigger pulse to start a time-to-amplitude converter (TAC), while another random photon from the same β-excitation event provides the stop pulse. The optical components and geometry for detecting these two photons, i.e., the two photomultipliers (PMT), the filters, and the pulse counting system, are identical. As a consequence, the measured fluorescence signal is the autocorrelation function of the fluorescence decay from the sample. A delay line of 50 ns is inserted between the "stop" signal PMT and the TAC so that those "stop" pulses which arrive before "start pulses" also are recorded. Thus the acquired fluorescence signal versus time is symmetric about the delay time and contains twice as many counts as without delay. We call the new technique the "time-autocorrelated two-photon counting technique" (TATPC) in distinction to the conventional "time-correlated single-photon counting technique" (TCSPC). We compared both techniques with the same equipment and scintillators, where in the TCSPC case, a β particle is used for the start of the TAC instead of a random photon in the TATPC technique. We find that under similar experimental circumstances, the signal count rate with TATPC is about 50 times larger than with TCSPC. The new method is well suited for obtaining fluorescence decay times from plastic scintillators, which we use in this article to exemplify the technique. More generally, β-particle excitation in combination with TATPC should prove useful for materials with high energy levels or band gaps, which cannot be excited with pulsed lasers in the visible region. The length of our excitation pulse is less than 20 ps and is

  8. Site-specific structural dynamics of α-Synuclein revealed by time-resolved fluorescence spectroscopy: a review

    NASA Astrophysics Data System (ADS)

    Sahay, Shruti; Krishnamoorthy, G.; Maji, Samir K.

    2016-12-01

    Aggregation of α-Synuclein (α-Syn) into amyloid fibrils is known to be associated with the pathogenesis of Parkinson’s disease (PD). Several missense mutations of the α-Syn gene have been associated with rare, early onset familial forms of PD. Despite several studies done so far, the local/residue-level structure and dynamics of α-Syn in its soluble and aggregated fibril form and how these are affected by the familial PD associated mutations are still not clearly understood. Here, we review studies performed by our group as well as other research groups, where time-resolved fluorescence spectroscopy has been used to understand the site-specific structure and dynamics of α-Syn under physiological conditions as well as under conditions that alter the aggregation properties of the protein such as low pH, high temperature, presence of membrane mimics and familial PD associated mutations. These studies have provided important insights into the critical structural properties of α-Syn that may govern its aggregation. The review also highlights time-resolved fluorescence as a promising tool to study the critical conformational transitions associated with early oligomerization of α-Syn, which are otherwise not accessible using other commonly used techniques such as thioflavin T (ThT) binding assay.

  9. 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.

  10. Site-specific structural dynamics of α-Synuclein revealed by time-resolved fluorescence spectroscopy: a review.

    PubMed

    Sahay, Shruti; Krishnamoorthy, G; Maji, Samir K

    2016-10-11

    Aggregation of α-Synuclein (α-Syn) into amyloid fibrils is known to be associated with the pathogenesis of Parkinson's disease (PD). Several missense mutations of the α-Syn gene have been associated with rare, early onset familial forms of PD. Despite several studies done so far, the local/residue-level structure and dynamics of α-Syn in its soluble and aggregated fibril form and how these are affected by the familial PD associated mutations are still not clearly understood. Here, we review studies performed by our group as well as other research groups, where time-resolved fluorescence spectroscopy has been used to understand the site-specific structure and dynamics of α-Syn under physiological conditions as well as under conditions that alter the aggregation properties of the protein such as low pH, high temperature, presence of membrane mimics and familial PD associated mutations. These studies have provided important insights into the critical structural properties of α-Syn that may govern its aggregation. The review also highlights time-resolved fluorescence as a promising tool to study the critical conformational transitions associated with early oligomerization of α-Syn, which are otherwise not accessible using other commonly used techniques such as thioflavin T (ThT) binding assay.

  11. Time-resolved and temperature tuneable measurements of fluorescent intensity using a smartphone fluorimeter.

    PubMed

    Hossain, Md Arafat; Canning, John; Yu, Zhikang; Ast, Sandra; Rutledge, Peter J; Wong, Joseph K-H; Jamalipour, Abbas; Crossley, Maxwell J

    2017-05-30

    A smartphone fluorimeter capable of time-based fluorescence intensity measurements at various temperatures is reported. Excitation is provided by an integrated UV LED (λex = 370 nm) and detection obtained using the in-built CMOS camera. A Peltier is integrated to allow measurements of the intensity over T = 10 to 40 °C. All components are controlled using a smartphone battery powered Arduino microcontroller and a customised Android application that allows sequential fluorescence imaging and quantification every δt = 4 seconds. The temperature dependence of fluorescence intensity for four emitters (rhodamine B, rhodamine 6G, 5,10,15,20-tetraphenylporphyrin and 6-(1,4,8,11-tetraazacyclotetradecane)2-ethyl-naphthalimide) are characterised. The normalised fluorescence intensity over time of the latter chemosensor dye complex in the presence of Zn(2+) is observed to accelerate with an increasing rate constant, k = 1.94 min(-1) at T = 15 °C and k = 3.64 min(-1) at T = 30 °C, approaching a factor of ∼2 with only a change in temperature of ΔT = 15 °C. Thermally tuning these twist and bend associated rates to optimise sensor approaches and device applications is proposed.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Time-resolved fluorescence observation of di-tyrosine formation in horseradish peroxidase upon ultrasound treatment leading to enzyme inactivation

    NASA Astrophysics Data System (ADS)

    Tsikrika, Konstantina; Lemos, M. Adília; Chu, Boon-Seang; Bremner, David H.; Hungerford, Graham

    2017-02-01

    The application of ultrasound to a solution can induce cavitional phenomena and generate high localised temperatures and pressures. These are dependent of the frequency used and have enabled ultrasound application in areas such as synthetic, green and food chemistry. High frequency (100 kHz to 1 MHz) in particular is promising in food chemistry as a means to inactivate enzymes, replacing the need to use periods of high temperature. A plant enzyme, horseradish peroxidase, was studied using time-resolved fluorescence techniques as a means to assess the effect of high frequency (378 kHz and 583 kHz) ultrasound treatment at equivalent acoustic powers. This uncovered the fluorescence emission from a newly formed species, attributed to the formation of di-tyrosine within the horseradish peroxidase structure caused by auto-oxidation, and linked to enzyme inactivation.

  14. Solvation dynamics of coumarin 153 embedded in AOT + phenol organogels studied by time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Nishiyama, Katsura; Takata, Kei; Watanabe, Keiichi; Shigematsu, Hirotake

    2012-03-01

    We investigate solvation dynamics of organogel utilizing ps-ns fluorescence spectroscopy. The organogel studied in this Letter comprises bis(2-ethylhexyl) sulfosuccinate (AOT) and p-chlorophenol in the m-xylene solvent, that produce an organogel architecture with self-assembly. Within the organogel, an emitting probe, coumarin 153 (C153), is embedded. We then obtain dynamic response functions of solvation derived from the time-resolved fluorescence spectra of C153. We propose that total energy of the C153-organogel system relaxes with a relaxation time of 3.9 ns, whereas the entire rearrangement of the organogel structure around C153 is achieved with that of 6.1 ns, respectively.

  15. Time-resolved fluorescence resonance energy transfer as a versatile tool in the development of homogeneous cellular kinase assays.

    PubMed

    Saville, Lisa; Spais, Chrysanthe; Mason, Jennifer L; Albom, Mark S; Murthy, Seetha; Meyer, Sheryl L; Ator, Mark A; Angeles, Thelma S; Husten, Jean

    2012-12-01

    Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.

  16. 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

  17. Time-resolved fluorescence microspectroscopy for characterizing crude oils in bulk and hydrocarbon-bearing fluid inclusions.

    PubMed

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

    2004-09-01

    Time-resolved fluorescence data was collected from a series of 23 bulk crude petroleum oils and six microscopic hydrocarbon-bearing fluid inclusions (HCFI). The data was collected using a diode laser fluorescence lifetime microscope (DLFLM) over the 460-700 nm spectral range using a 405 nm excitation source. The correlation between intensity averaged lifetimes (tau) and chemical and physical parameters was examined with a view to developing a quantitative model for predicting the gross chemical composition of hydrocarbon liquids trapped in HCFI. It was found that tau is nonlinearly correlated with the measured polar and corrected alkane concentrations and that oils can be classified on this basis. However, these correlations all show a large degree of scatter, preventing accurate quantitative prediction of gross chemical composition of the oils. Other parameters such as API gravity and asphaltene, aromatic, and sulfur concentrations do not correlate well with tau measurements. Individual HCFI were analyzed using the DLFLM, and time-resolved fluorescence measurements were compared with tau data from the bulk oils. This enabled the fluid within the inclusions to be classified as either low alkane/high polar or high alkane/low polar. Within the high alkane/low polar group, it was possible to clearly discriminate HCFI from different locales and to see differences in the trapped hydrocarbon fluids from a single geological source. This methodology offers an alternative method for classifying the hydrocarbon content of HCFI and observing small variations in the trapped fluid composition that is less sensitive to fluctuations in the measurement method than fluorescence intensity based methods.

  18. Short-pulsed diode lasers as an excitation source for time-resolved fluorescence applications and confocal laser scanning microscopy in PDT

    NASA Astrophysics Data System (ADS)

    Kress, Matthias; Meier, Thomas H.; El-Tayeb, Tarek A. A.; Kemkemer, Ralf; Steiner, Rudolf W.; Rueck, Angelika C.

    2001-11-01

    This article describes a setup for subcellular time-resolved fluorescence spectroscopy and fluorescence lifetime measurements using a confocal laser scanning microscope in combination with a short pulsed diode laser for fluorescence excitation and specimen illumination. The diode laser emits pulses at 398 nm wavelength with 70 ps full width at half maximum (FWHM) duration. The diode laser can be run at a pulse repetition rate of 40 MHz down to single shot mode. For time resolved spectroscopy a spectrometer setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Subcellular fluorescence lifetime measurements were achieved using a time-correlated single photon counting (TCSPC) module instead of the spectrometer setup. The capability of the short pulsed diode laser for fluorescence imaging, fluorescence lifetime measurements and time-resolved spectroscopy in combination with laser scanning microscopy is demonstrated by fluorescence analysis of several photosensitizers on a single cell level.

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

    NASA Astrophysics Data System (ADS)

    Freiberg, Arvi; Pullerits, Tonu; Timpmann, Kou

    1990-05-01

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

  20. A time-resolved multi-wavelength fluorescence diffuse optical tomography system for small animal imaging

    NASA Astrophysics Data System (ADS)

    Montcel, Bruno; Chabrier, Renee; Poulet, Patrick

    2005-08-01

    A tomographic approach, relying on diffuse near infrared photons to image the optical properties of tissues and the inner distribution of fluorescent probes is described. The method should improve the spatial resolution and quantification of fluorescence signals, thanks to multiple-projection acquisitions and to a reconstruction procedure using the principles of diffuse optical tomography. The scanner assembled uses picosecond laser diodes, an eight-anode photo-multiplier tube (PMT) and time-correlated single photon counting. Two sets of laser heads, each operating at four wavelengths, are fitted with furcated optical fibers, providing two sequential sources of light positioned on the animal or object studied. Multimode optical fibers are used to detect light at eight output points on the animal or object. These fibers are connected to the PMT, with an air-gap allowing the insertion of an optical filter to reject the excitation wavelength. The light sources and detectors can be rotated to increase the number of projections recorded. For the reconstruction process, the coordinates of the body surface of the animal to be imaged are necessary. These are acquired by interferometry, using a conoscope and an XY scanning system, before the animal is entered in the scanner. The profiles measured at the excitation wavelengths are used to compute absorption and reduced scattering images and perfusion/oxygenation images of the animal. Fluorescence images, free from diffusion and absorption artefacts, can then be computed with a-priori knowledge of the optical images of the animal. The scanner, its performances and images of light-scattering and fluorescent phantoms are presented.

  1. 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.

  2. 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.

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

    PubMed

    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 × 10(5) 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.

  4. (Time-resolved fluorescence studies of surface recombination in CdSe electrodes)

    SciTech Connect

    Not Available

    1991-01-01

    The long range goal of our investigations is to understand the dynamics of heterogeneous electron transfer reactions. The primary method we use to monitor the carrier dynamics is the fluorescence of the bandgap emission. This all optical approach circumvents the limitations of photopotential and photocurrent methods. Before such studies on a reactive system can be informative, it is necessary to understand the dynamics of the photogenerated carriers under nonreactive conditions. Presently we are concentrating on carrier dynamics in the materials, cadmium selenide (CdSe) and cadmium sulfide (CdS). Under these conditions the carriers recombine either directly or through intragap electronic states. 2 refs., 1 fig.

  5. Photodynamics and time-resolved fluorescence of azobenzene in solution: a mixed quantum-classical simulation.

    PubMed

    Cusati, Teresa; Granucci, Giovanni; Persico, Maurizio

    2011-04-06

    We have simulated the photodynamics of azobenzene by means of the Surface Hopping method. We have considered both the trans → cis and the cis → trans processes, caused by excitation in the n → π* band (S(1) state). To bring out the solvent effects on the excited state dynamics, we have run simulations in four different environments: in vacuo, in n-hexane, in methanol, and in ethylene glycol. Our simulations reproduce very well the measured quantum yields and the time dependence of the intensity and anisotropy of the transient fluorescence. Both the photoisomerization and the S(1) → S(0) internal conversion require the torsion of the N═N double bond, but the N-C bond rotations and the NNC bending vibrations also play a role. In the trans → cis photoconversion the N═N torsional motion and the excited state decay are delayed by increasing the solvent viscosity, while the cis → trans processes are less affected. The analysis of the simulation results allows the experimental observations to be explained in detail, and in particular the counterintuitive increase of the trans → cis quantum yield with viscosity, as well as the relationship between the excited state dynamics and the solvent effects on the fluorescence lifetimes and depolarization.

  6. The fluorescence resonance energy transfer (FRET) gate: A time-resolved study

    PubMed Central

    Xu, Qing-Hua; Wang, Shu; Korystov, Dmitry; Mikhailovsky, Alexander; Bazan, Guillermo C.; Moses, Daniel; Heeger, Alan J.

    2005-01-01

    The two-step energy-transfer process in a self-assembled complex comprising a cationic conjugated polymer (CCP) and a dsDNA is investigated by using pump-dump-emission spectroscopy and time-correlated single-photon counting; energy is transferred from the CCP to an ethidium bromide (EB) molecule intercalated into the dsDNA through a fluorescein molecule linked to one terminus of the DNA. Time-dependent anisotropy measurements indicate that the inefficient direct energy transfer from the CCP to the intercalated EB results from the near orthogonality of their transition moments. These measurements also show that the transition moment of the fluorescein spans a range of angular distributions and lies between that of the CCP and EB. Consequently, the fluorescein acts as a fluorescence resonance energy-transfer gate to relay the excitation energy from the CCP to the EB. PMID:15642946

  7. 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.

  8. The fluorescence resonance energy transfer (FRET) gate: a time-resolved study.

    PubMed

    Xu, Qing-Hua; Wang, Shu; Korystov, Dmitry; Mikhailovsky, Alexander; Bazan, Guillermo C; Moses, Daniel; Heeger, Alan J

    2005-01-18

    The two-step energy-transfer process in a self-assembled complex comprising a cationic conjugated polymer (CCP) and a dsDNA is investigated by using pump-dump-emission spectroscopy and time-correlated single-photon counting; energy is transferred from the CCP to an ethidium bromide (EB) molecule intercalated into the dsDNA through a fluorescein molecule linked to one terminus of the DNA. Time-dependent anisotropy measurements indicate that the inefficient direct energy transfer from the CCP to the intercalated EB results from the near orthogonality of their transition moments. These measurements also show that the transition moment of the fluorescein spans a range of angular distributions and lies between that of the CCP and EB. Consequently, the fluorescein acts as a fluorescence resonance energy-transfer gate to relay the excitation energy from the CCP to the EB.

  9. 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

  10. 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.

  11. 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.

  12. Short-term light adaptation of a cyanobacterium, Synechocystis sp. PCC 6803, probed by time-resolved fluorescence spectroscopy.

    PubMed

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

    2014-08-01

    In photosynthetic organisms, the interactions among pigment-protein complexes change in response to light conditions. In the present study, we analyzed the transfer of excitation energy from the phycobilisome (PBS) and photosystem (PS) II to PSI in the cyanobacterium Synechocystis sp. PCC 6803. After 20 min of dark adaptation, Synechocystis cells were illuminated for 5 min with strong light with different spectral profiles, blue, green, two kinds of red, and white light. After illumination, the energy-transfer characteristics were evaluated using 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, followed by spectral component analysis. Under illumination with strong light, the contribution of the energy transfer from the PSII to PSI (spillover) became greater, and that of the energy transfer from the PBS to PSI decreased; the former change was larger than the latter. The energy transfer pathway to PSI was sensitive to red light. We discuss the short-term adaptation of energy-transfer processes in Synechocystis under strong-light conditions. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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

    PubMed

    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 1.20×10(6), 5.19×10(5) and 2.74×10(6)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. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. 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.

  15. 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; hide

    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.

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

    PubMed

    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

    2014-04-08

    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.

  17. 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

  18. Glossoscolex paulistus hemoglobin with fluorescein isothiocyanate: Steady-state and time-resolved fluorescence.

    PubMed

    Barros, Ana E B; Barioni, Marina B; Carvalho, Francisco A O; Ito, Amando Siuiti; Tabak, Marcel

    2017-05-01

    Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been followed, in the presence of urea, using fluorescein isothiocyanate (FITC). Binding of FITC to HbGp results in a significant quenching of probe fluorescence. Tryptophan emission decays present four characteristic lifetimes: two in the sub-nanosecond/picosecond, and two in the nanosecond time ranges. Tryptophan decays for pure HbGp and HbGp-FITC systems are similar. In the absence of denaturant, and up to 2.5mol/L of urea, the shorter lifetimes predominate. At 3.5 and 6.0mol/L of urea, the longer lifetimes increase significantly their contribution. Urea-induced unfolding process is characterized by protein oligomeric dissociation and denaturation of dissociated subunits. FITC emission decays for FITC-HbGp system are also multi-exponential with three lifetimes: two in the sub-nanosecond and one in the nanosecond range with a value similar to free probe in buffer. Increase of urea concentration leads to increase of the longer lifetime contribution, implying the removal of the quenching observed for the native HbGp-FITC system. Anisotropy decays are characterized by two rotational correlation times associated to re-orientational motions of the probe relative to protein. Our results suggest that FITC bound to HbGp is useful to monitor denaturant effects on the protein.

  19. 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.

  20. 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.

  1. Interactions of a lytic peptide with supported lipid bilayers investigated by time-resolved evanescent wave-induced fluorescence spectroscopy.

    PubMed

    Rapson, Andrew C; Gee, Michelle L; Clayton, Andrew H A; Smith, Trevor A

    2016-09-28

    We report investigations, using time-resolved and polarised evanescent wave-induced fluorescence methods, into the location, orientation and mobility of a fluorescently labelled form of the antimicrobial peptide, melittin, when it interacts with vesicles and supported lipid bilayers (SLBs). This melittin analogue, termed MK14-A430, was found to penetrate the lipid headgroup structure in pure, ordered-phase DPPC membranes but was located near the headgroup-water region when cholesterol was included. MK14-A430 formed lytic pores in SLBs, and an increase in pore formation with incubation time was observed through an increase in polarity and mobility of the probe. When associated with the Cholesterol-containing SLB, the probe displayed polarity and mobility that indicated a population distributed near the lipid headgroup-water interface with MK14-A430 arranged predominantly in a surface-aligned state. This study indicates that the lytic activity of MK14-A430 occurred through a pore-forming mechanism. The lipid headgroup environment experienced by the fluorescent label, where MK14-A430 displayed pore information, indicated that pore formation was best described by the toroidal pore model.

  2. Interactions of a lytic peptide with supported lipid bilayers investigated by time-resolved evanescent wave-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Rapson, Andrew C.; Gee, Michelle L.; Clayton, Andrew H. A.; Smith, Trevor A.

    2016-12-01

    We report investigations, using time-resolved and polarised evanescent wave-induced fluorescence methods, into the location, orientation and mobility of a fluorescently labelled form of the antimicrobial peptide, melittin, when it interacts with vesicles and supported lipid bilayers (SLBs). This melittin analogue, termed MK14-A430, was found to penetrate the lipid headgroup structure in pure, ordered-phase DPPC membranes but was located near the headgroup-water region when cholesterol was included. MK14-A430 formed lytic pores in SLBs, and an increase in pore formation with incubation time was observed through an increase in polarity and mobility of the probe. When associated with the Cholesterol-containing SLB, the probe displayed polarity and mobility that indicated a population distributed near the lipid headgroup-water interface with MK14-A430 arranged predominantly in a surface-aligned state. This study indicates that the lytic activity of MK14-A430 occurred through a pore-forming mechanism. The lipid headgroup environment experienced by the fluorescent label, where MK14-A430 displayed pore information, indicated that pore formation was best described by the toroidal pore model.

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

    NASA Astrophysics Data System (ADS)

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

    2009-06-01

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

  4. Phosphorylation of nucleoside diphosphate kinase at the active site studied by steady-state and time-resolved fluorescence.

    PubMed

    Deville-Bonne, D; Sellam, O; Merola, F; Lascu, I; Desmadril, M; Véron, M

    1996-11-19

    Nucleoside diphosphate (NDP) kinase is the enzyme responsible in the cell for the phosphorylation of nucleoside or deoxynucleoside diphosphates into the corresponding triphosphates at the expense of ATP. Transfer of the gamma-phosphate is very fast (turnover number above 1000 s-1) and involves the phosphorylation of a histidine residue at the active site of the enzyme. We have used intrinsic protein fluorescence of the single tryptophan of Dictyostelium discoideum NDP kinase as a sensitive probe for monitoring the interaction of the enzyme with its substrates. We demonstrate that the 20% quenching of steady-state fluorescence observed upon addition of ATP is due to formation of the phosphorylated intermediate. Time-resolved fluorescence indicates that the Trp-137 side chain is rigidly bound to the protein core with a unique lifetime of 4.5 ns for the free enzyme at 20 degrees C and that it remains tightly immobilized during the time course of the reaction. Phosphorylation of this catalytic residue (His-122) in the presence of ATP induces a similar decrease in mean lifetime, due to the splitting of the signal and the appearance of a shorter decay. This splitting is discussed in terms of a slow conformational equilibrium. We demonstrate that, in the wild-type enzyme, the conserved His-55 quenches the fluorescence of Trp-137 as the H55A mutant protein fluorescence displays an increase in quantum yield. Even though H55A mutant enzyme is active, the absence of the imidazole ring prevents the detection of the phosphorylated state of His-122 by Trp-137. We conclude that His-55 serves as a relay between His-122 and Trp-137.

  5. Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

    PubMed

    Yu, L S L; Reed, S A; Golden, M H

    2002-03-01

    An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.

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

    PubMed

    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.

  7. 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.

  8. Imaging time-resolved electrothermal atomization laser-excited atomic fluorescence spectrometry for determination of mercury in seawater.

    PubMed

    Le Bihan, Alain; Cabon, Jean-Yves; Deschamps, Laure; Giamarchi, Philippe

    2011-06-15

    In this study, direct determination of mercury at the nanogram per liter level in the complex seawater matrix by imaging time-resolved electrothermal atomization laser-excited atomic fluorescence spectrometry (ITR-ETA-LEAFS) is described. In the case of mercury, the use of a nonresonant line for fluorescence detection with only one laser excitation is not possible. For measurements at the 253.652 nm resonant line, scattering phenomena have been minimized by eliminating the simultaneous vaporization of salts and by using temporal resolution and the imaging mode of the camera. Electrothermal conditions (0.1 M oxalic acid as matrix modifier, low atomization temperature) have been optimized in order to suppress chemical interferences and to obtain a good separation of specific signal and seawater background signal. For ETA-LEAFS, a specific response has been obtained for Hg with the use of time resolution. Moreover, an important improvement of the detection limit has been obtained by selecting, from the furnace image, pixels collecting the lowest number of scattered photons. Using optimal experimental conditions, a detection limit of 10 ng L(-1) for 10 μL of sample, close to the lowest concentration level of total Hg in the open ocean, has been obtained.

  9. Time-resolved detection of the one- and two-photon excited fluorescence of single molecules of a folding enhanced green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Cotlet, Mircea; Goodwin, Peter M.; Waldo, Geoffrey S.; Werner, James H.

    2006-02-01

    We use time-resolved single molecule fluorescence detection (MSMD) to investigate the fluorescence dynamics of a mutant of the wild-type Green Fluorescent Protein (GFP) from Aequorea victoria, the folding enhanced GFP (FEGFP). The folding enhanced GFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. This variant shows increased thermal stability and the ability to retain its fluorescence when fused to poorly folding proteins. Here we apply one- (OPE) and two- (TPE) photon excitation on freely diffusing FEGFP molecules. Under OPE, single FEGFP molecules undergo fluorescence flickering in the time scale of μs and tens of μs due to triplet formation and ground-state protonation-deprotonation, respectively. OPE fluorescence lifetimes of single FEGFP molecules show evidence for the presence of different emitting species, the I and B forms of FEGFP chromophore. TPE single FEGFP molecules flicker in fluorescence in the time scale of μs due to singlet-triplet transitions of the chromophore. Two-photon excitation of single FEGFP molecules results in the creation of a photoconverted species with a fluorescence lifetime of 2.5 ns, a species which is bright enough to be detected at the single molecule level. Our results indicate FEGFP is a promising fusion reporter for intracellular applications when using OPE and TPE microscopy with single molecule sensitivity.

  10. Interaction of meso-tetrakis ( p-sulfonatophenyl) porphyrin (TSPP) with pyrimidines: A steady state and time-resolved fluorescence quenching study

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

    Fluorescence quenching of meso-tetrakis ( p-sulfonatophenyl) porphyrin (TSPP) by certain pyrimidines has been investigated by using steady state and time-resolved fluorescence techniques. The pyrimidines used are uracil, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 5-iodouracil and 5-aminouracil. The quenching was found to obey the Stern-Volmer equation and the corresponding Stern-Volmer plots were linear from both steady state and time-resolved measurements, indicating dynamic nature of quenching and the bimolecular quenching rate constants ( kq) agreed well. Electron transfer from TSPP to pyrimidines were confirmed by the calculation of free energy change ( ΔGet) using Rehm-Weller equation.

  11. Adaptation of light-harvesting systems of Arthrospira platensis to light conditions, probed by time-resolved fluorescence spectroscopy.

    PubMed

    Akimoto, Seiji; Yokono, Makio; Hamada, Fumiya; Teshigahara, Ayaka; Aikawa, Shimpei; Kondo, Akihiko

    2012-08-01

    Cyanobacteria change the quantity and/or quality of their pigment-protein complexes in response to light conditions. In the present study, we analyzed excitation relaxation dynamics in the cyanobacterium, Arthrospira (Spirulina) platensis, grown under lights exhibiting different spectral profiles, by means of steady-state absorption and picosecond time-resolved fluorescence spectroscopies. It was found that F760, which is the PSI red-chlorophyll characteristic of A. platensis, contributes to slower energy-transfer phase in the PSI of A. platensis. Excitation energy transfers in phycobilisome and those from PSII to PSI were modified depending on the light quality. Existence of quencher was suggested in PSI of the blue-light grown cells. Phycobilisomes in the green-light grown cells and the far-red-light grown cells transferred excitation energy from phycobilisome to chlorophyll without loss of energy. In these cells, excitation energy was shared between two photosystems. Fast energy transfer was established in phycobilisome under the yellow-light condition where only the phycobilisome can absorb the cultivation light. Differences in light-harvesting and energy-transfer processes under different cultivation-light conditions are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. 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.

  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

    2015-12-02

    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.

  14. 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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Identification of pregnane X receptor ligands using time-resolved fluorescence resonance energy transfer and quantitative high-throughput screening.

    PubMed

    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; Auld, Douglas S

    2009-04-01

    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 >or=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.

  16. 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.

  17. 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.

  18. Theoretical and experimental study on the difference of the time-resolved fluorescence spectrum between the tumor and the adjacent normal tissue

    NASA Astrophysics Data System (ADS)

    Li, Mingzhao; Xu, Tao

    2006-09-01

    The objective of this paper is to study the difference of the time-resolved fluorescence spectrum between the tumor and the adjacent normal tissue. The time-resolved spectrum of the s-180 sarcoma tumor and adjacent normal tissue mediated by the Palladium-porphyrin photosensilizer (Pd-TCPP) is detected by a homemade instrumentation. An improved Monte Carlo algorithm, taking into account the depth of the upper layer tissue (z I) and the delay of the fluorescence photon emission for the time (τ) Pd-TCPP electrons spend in the excited state, is developed to study the reason of the spectrum difference formation. It is found from the experiment that the decay constant T decay of the time-resolved fluorescence spectrum of the tumor is obviously larger than that of the adjacent normal tissue. And T decay increased with the tumor growth, from 554μs in the first day to 634μs in the eighth day while it keeps steady for normal tissue. The simulated spectrum at different z I and different τ reveal that both the increase of z I and τ can delay the spectrum decay. It is believed that T decay of the tumor tissue should be larger than that of adjacent normal tissue because of the hypoxia. The changes of tumor size and oxygen status cause the difference of the time-resolved fluorescence spectrum between the tumor and the adjacent normal tissue and this fact can be made use of in the tumor diagnoses.

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

    PubMed Central

    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.

    2015-01-01

    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 (0.551 ng/ml) for PA83 and 2.51 × 10−5 μM (1.58 ng/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 use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

  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. Published by Elsevier B.V.

  1. Time-resolved infrared fluorescence studies of the collisional deactivation of CO 2(00 01) by large polyatomic molecules

    NASA Astrophysics Data System (ADS)

    Poel, Kathleen L.; Alwahabi, Zeyad T.; King, Keith D.

    1995-12-01

    The time-resolved infrared fluorescence (IRF) technique has been used to study the vibrational deactivation of CO 2(00 01) by large polyatomic molecules at ambient temperature (295 ± 2 K).The excited CO 2 molecules were prepared by direct pumping with the P(21) line of a pulsed CO 2 laser at 10.6 μm. The bimolecular rate constant for deactivation by CO 2 was determined to be (0.353 ± 0.026) × 10 3 Torr -1 s -1, in excellent agreement with previous work. The rate constants for deactivation by the large polyatomic molecules, c-C 6H 10, c-C 6H 12, C 6H 6, C 6D 6, C 7H 8, C 7D 8, C 6H 5 F, p-C 6H 4F 2, C 6HF 5 and C 6F 6, were found to be (143 ± 18), (150 ± 12), (120 ± 4), (238 ± 9), (140 ± 5), (234 ± 15), (121 ± 7), (132 ± 23), (132 ± 12), and (94 ± 5) × 10 3 Torr -1 s -1, respectively. Experimental deactivation probabilities and average energies removed per collision are calculated and compared. There is little difference in deactivation probabilities between the acyclic ring compounds and their aromatic analogues but the perfluorinated compound, C 6F 6 is clearly less efficient than its hydrocarbon analogue, C 6H 6. The perdeuterated species, C 6D 6 and C 7D 8 show considerably enhanced deactivation relative to the other species, probably as a result of near-resonant intermolecular V-V energy transfer.

  2. Energy transfer in photosystem I. Time resolved fluorescence of the native photosystem I complex and its core complex

    NASA Astrophysics Data System (ADS)

    Pålsson, Lars-Olof; Tjus, Staffan E.; Andersson, Bertil; Gillbro, Tomas

    1995-05-01

    Energy transfer within isolated spinach photosystem I (PS I) complexes with different antenna size were studied using time-resolved picosecond and steady-state fluorescence spectroscopy. In both the native PS I complexes and the PS I core complexes lacking the outer chlorophyll a/ b antenna we observed a fast dominating emission component ≈ 35 ps at room temperature which is associated with the trapping process by the reaction centre. In the native PS I complex there also appears a 120 ps component which was not observed in the PS I core complex. This component most likely represents an energy transfer from low energy pigments in the light-harvesting complex I antenna and into the core. Due to a very fast energy equilibration (< 10 ps) it was not possible to resolve the energy transfer at room temperature. At 77 K, however, it was possible to follow the energy transfer from F690 to F720 with a transfer time of ≈ 35 ps within the native PS I complex and slightly longer, 78 ps, in the PS I core complex. The native PS I complex also exhibited in the region 700-740 nm a 102 ps component which originates from F720 and represents energy transfer from F720 to P700 at 77 K. At low temperatures the PS I core complex exhibited a component of 161 ps which is associated with F720 and has the same function as the 102 ps component of the native PS I complex. We conclude that the F720 emission originates from pigments in the core antenna system. This emission also increases at low temperature. In the native PS I complex there is an initial increase in the F720 emission as the temperature is lowered but at 77 K the F735 emission originating from LHC I dominates.

  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. Steady-state and time-resolved fluorescence spectroscopic studies on the interaction between bovine serum albumin and Ag-nanoparticles

    NASA Astrophysics Data System (ADS)

    Ye, Manping; Shi, Yarong; Chen, Huacai

    2016-10-01

    The interaction between bovine serum albumin(BSA) and Ag-nanoparticles was studied under a pH 7.4 buffer system by time-resolved fluorescence technique combined with the steady-state absorption and fluorescence spectrum. With Ag-nanoparticles, the BSA showed blue shift of fluorescence from 335nm to 332.5nm, accompanied by the fluorescence intensity decreasing. When adding the Ag-nanoparticles to the three fluorescent amino acids tryptophan(Trp), tyrosine(Tyr)and phenylalanine(Phe), only Trp displayed peak shift which from 346.5nm to 341nm. Strong interaction between BSA and the Ag-nanoparticles may come from Trp residue. Time-resolved fluorescence gave that BSA had only one fluorescence lifetime around 6ns from 308 to 313K. When adding Ag-nanoparticles, two fluorescence lifetimes appeared. One is a little above than 6ns and the other is around 3ns. The two Trp residues in 134th and 212th position may give contribution to the changes of the fluorescence lifetime. The 134th Trp residue is probably protected by BSA molecule structure and basically don't contact with Ag-nanoparticles, which shows little change of fluorescence lifetime. The 212th Trp residue is likely the target of the Ag-nanoparticles. The Ag-nanoparticles changed the microenvironment of BSA around the 212th Trp residue and therefore increases the exposure of the 212th Trp and the 134th Trp .

  5. Steady-state and time-resolved fluorescence studies on wild type and mutant chromatium vinosum high potential iron proteins: holo- and apo-forms.

    PubMed Central

    Sau, A K; Chen, C A; Cowan, J A; Mazumdar, S; Mitra, S

    2001-01-01

    Detailed circular dichroism (CD), steady-state and time-resolved tryptophan fluorescence studies on the holo- and apo- forms of high potential iron protein (HiPIP) from Chromatium vinosum and its mutant protein have been carried out to investigate conformational properties of the protein. CD studies showed that the protein does not have any significant secondary structure elements in the holo- or apo- HiPIP, indicating that the metal cluster does not have any effect on formation of secondary structure in the protein. Steady-state fluorescence quenching studies however, suggested that removal of the iron-sulfur ([Fe(4)S(4)](3+)) cluster from the protein leads to an increase in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. CD studies on the holo- and apo- HiPIP also showed that removal of the metal prosthetic group drastically affects the tertiary structure of the protein. Time-resolved fluorescence decay of the wild type protein was fitted to a four-exponentials model and that of the W80N mutant was fitted to a three-exponentials model. The time-resolved fluorescence decay was also analyzed by maximum entropy method (MEM). The results of the MEM analysis agreed with those obtained from discrete exponentials model analysis. Studies on the wild type and mutants helped to assign the fast picosecond lifetime component to the W80 residue, which exhibits fast fluorescence energy transfer to the [Fe(4)S(4)](3+) cluster of the protein. Decay-associated fluorescence spectra of each tryptophan residues were calculated from the time-resolved fluorescence results at different emission wavelengths. The results suggested that W80 is in the hydrophobic core of the protein, but W60 and W76 are partially or completely exposed to the solvent. PMID:11566801

  6. 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.

  7. Study of fluorescence interaction and conformational changes of bovine serum albumin with histamine H₁ -receptor--drug epinastine hydrochloride by spectroscopic and time-resolved fluorescence methods.

    PubMed

    Ariga, Girish G; Naik, Praveen N; Nandibewoor, Sharanappa T; Chimatadar, Shivamurti A

    2015-11-01

    The fluorescence, ultraviolet (UV) absorption, time resolved techniques, circular dichroism (CD), and infrared spectral methods were explored as tools to investigate the interaction between histamine H1 drug, epinastine hydrochloride (EPN), and bovine serum albumin (BSA) under simulated physiological conditions. The experimental results showed that the quenching of the BSA by EPN was static quenching mechanism and also confirmed by lifetime measurements. The value of n close to unity indicated that one molecule of EPN was bound to protein molecule. The binding constants (K) at three different temperatures were calculated (7.1 × 10(4), 5.5 × 10(4), and 3.9 × 10(4) M(-1)). Based on the thermodynamic parameters (ΔH(0), ΔG(0), and ΔS(0)), the nature of binding forces operating between drug and protein was proposed. The site of binding of EPN in the protein was proposed to be Sudlow's site I based on displacement experiments using site markers viz, warfarin, ibuprofen, and digitoxin. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (EPN) was evaluated and found to be 4.48 nm. The UV-visible, synchronous fluorescence, CD, and three-dimensional fluorescence spectral results revealed the changes in secondary structure of the protein upon its interaction with EPN.

  8. 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.

  9. Multiplexed fluorescence detection in microfabricated devices with both time-resolved and spectral-discrimination capabilities using near-infrared fluorescence.

    PubMed

    Zhu, Li; Stryjewski, Wieslaw J; Soper, Steven A

    2004-07-15

    We examined the feasibility of using a two-color time-resolved detection scheme with microdevices for DNA sequencing applications. A home-built dual-color optical-fiber-based time-resolved near-infrared (IR) fluorescence microscope successfully coupled lifetime discrimination with color discrimination, increasing fluorescence multiplexing capabilities. The instrument was constructed by using two pulsed-diode lasers (680/780-nm excitation) and two avalanche photodiodes as the basic building blocks. The data were processed using electronics configured in a time-correlated single-photon counting format. The use of near-IR fluorescence detection greatly simplified the hardware and allowed low detection limits (< 0.1nM). We examined the separation of a single-base tract on a microchip and compared the performance with that of conventional capillary gel electrophoresis. The microchip was fabricated in glass and contained an effective separation length of 7.0 cm. It was found that, without incorporating a solid-phase reversible immobilization cleanup procedure, the calculated lifetime of the dye label on the microchip was longer and the standard deviation was larger than those of the same sample analyzed using capillary electrophoresis. Using cleanup steps, the accuracy and precision of the measurements improved. Lifetimes of four near-IR dyes (AlexaFluor680, IRD700, IRD800, and IRD40) used in this study were determined to be 986 ps (RSD=2.1%), 1551 ps (RSD=1.8%), 520 ps (RSD=3.3%), and 788 ps (RSD=4.9%), respectively, in a microchannel filled with poly(dimethylacrylamide) (POP-6) gel. The lifetimes calculated using maximum likelihood estimators provided favorable precision on the microchip, where small numbers of photocounts were collected. An M13mp18 template was sequenced on the microchip using a two-color two-lifetime format with POP-6 as the sieving polymer. Read lengths of 294 bp with calling accuracies of 90.8 and 83.7% were achieved in each color channel. The

  10. 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.

  11. 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.

  12. Dual lanthanide-doped complexes: the development of a time-resolved ratiometric fluorescent probe for anthrax biomarker and a paper-based visual sensor.

    PubMed

    Wang, Qi-Xian; Xue, Shi-Fan; Chen, Zi-Han; Ma, Shi-Hui; Zhang, Shengqiang; Shi, Guoyue; Zhang, Min

    2017-03-16

    In this work, a novel time-resolved ratiometric fluorescent probe based on dual lanthanide (Tb: terbium, and Eu: europium)-doped complexes (Tb/DPA@SiO2-Eu/GMP) has been designed for detecting anthrax biomarker (dipicolinic acid, DPA), a unique and major component of anthrax spores. In such complexes-based probe, Tb/DPA@SiO2 can serve as a stable reference signal with green fluorescence and Eu/GMP act as a sensitive response signal with red fluorescence for ratiometric fluorescent sensing DPA. Additionally, the probe exhibits long fluorescence lifetime, which can significantly reduce the autofluorescence interferences from biological samples by using time-resolved fluorescence measurement. More significantly, a paper-based visual sensor for DPA has been devised by using filter paper embedded with Tb/DPA@SiO2-Eu/GMP, and we have proved its utility for fluorescent detection of DPA, in which only a handheld UV lamp is used. In the presence of DPA, the paper-based visual sensor, illuminated by a handheld UV lamp, would result in an obvious fluorescence color change from green to red, which can be easily observed with naked eyes. The paper-based visual sensor is stable, portable, disposable, cost-effective and easy-to-use. The feasibility of using a smartphone with easy-to-access color-scanning APP as the detection platform for quantitative scanometric assays has been also demonstrated by coupled with our proposed paper-based visual sensor. This work unveils an effective method for accurate, sensitive and selective monitoring anthrax biomarker with backgroud-free and self-calibrating properties.

  13. Advantages of time-resolved fluorescent nanobeads compared with fluorescent submicrospheres, quantum dots, and colloidal gold as label in lateral flow assays for detection of ractopamine.

    PubMed

    Hu, Li-Ming; Luo, Kai; Xia, Jun; Xu, Guo-Mao; Wu, Cheng-Hui; Han, Jiao-Jiao; Zhang, Gang-Gang; Liu, Miao; Lai, Wei-Hua

    2017-05-15

    Label selection is a critical factor for improving the sensitivity of lateral flow assay. Time-resolved fluorescent nanobeads, fluorescent submicrospheres, quantum dots, and colloidal gold-based lateral flow assay (TRFN-LFA, FM-LFA, QD-LFA, and CG-LFA) were first systematically compared for the quantitative detection of ractopamine in swine urine based on competitive format. The limits of detection (LOD) of TRFN-LFA, FM-LFA, QD-LFA, and CG-LFA were 7.2, 14.7, 23.6, and 40.1pg/mL in swine urine samples, respectively. The sensitivity of TRFN-LFA was highest. In the quantitative determination of ractopamine (RAC) in swine urine samples, TRFN-LFA exhibited a wide linear range of 5pg/mL to 2500pg/mL with a reliable coefficient of correlation (R(2)=0.9803). Relatively narrow linear ranges of 10-500pg/mL (FM-LFA) and 25-2500pg/mL (QD-LFA and CG-LFA) were acquired. Approximately 0.005µg of anti-RAC poly antibody (pAb) was used in each TRFN-LFA test strip, whereas 0.02, 0.054, and 0.15µg of pAb were used in each of the FM-LFA, QD-LFA, and CG-LFA test strips, respectively. In addition, TRFN-LFA required the least RAC-BSA antigens and exhibited the shortest detection time compared with the other lateral flow assays. Analysis of the RAC in swine urine samples showed that the result of TRFN-LFA was consistent with that of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a commercial enzyme-linked immunosorbent assay (ELISA) kit.

  14. A Dual Readout Assay Based on Fluorescence Polarization and Time-Resolved Fluorescence Resonance Energy Transfer to Screen for RSK1 Inhibitors.

    PubMed

    Jeong, Eun-mi; Lee, Mi Young; Lee, Jeong Hyun; Lee, Byung Ho; Oh, Kwang-Seok

    2016-01-01

    A dual readout assay based on fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) exhibits many advantages over single assay technology in terms of screening quality and efficiency. In this study, we developed a dual readout assay combining FP and TR-FRET to identify ribosomal S6 kinase 1 (RSK1) inhibitors. This dual readout assay can monitor both FP and TR-FRET signals from a single RSK1 kinase reaction by using the immobilized metal affinity for phosphochemical (IMAP)-based assay. The Z' value and signal to background (S/B) ratio were 0.85 and 4.0 using FP, and 0.79 and 10.6 using TR-FRET, which led to performance of a pilot library screening against the drug repositioning set consisting of 2320 compounds with a reasonable reproducibility. From this screening, we identified 16 compounds showing greater than 50% inhibition against RSK1 for both FP and TR-FRET; 6 compounds with greater than 50% inhibition only for FP; and 4 compounds with greater than 50% inhibition only for TR-FRET. In a cell-based functional assay to validate the hit compounds, 10 compounds identified only in a single assay had little effect on the RSK-mediated phosphorylation of liver kinase B1, whereas 5 compounds showing greater than 80% inhibition for both FP and TR-FRET reduced the phosphorylation of liver kinase B1. These results demonstrate that the dual readout assay can be used to identify hit compounds by subsequently monitoring both FP and TR-FRET signals from one RSK1 reaction.

  15. Imaging time-resolved fluorescence characteristics of organelle specific fluorophores and photosensitizers using ps pulsed diode lasers and TCSPC techniques

    NASA Astrophysics Data System (ADS)

    Scalfi-Happ, Claudia; Dolp, Frank; Forster, Florian; Rueck, Angelika

    2004-09-01

    A time-correlated single photon counting (TCSPC) module (SPC-730, Becker & Hickl, Germany) was connected to a laser scanning microscope (Zeiss, Germany) equipped with an ultrafast photomultiplier. Short pulse excitation was achieved with two laser diodes emitting at 398nm and 434nm with a pulse duration of 70ps and 60 ps (PicoQuant, Germany) to allow intracellular fluorescence lifetime imaging (FLIM). With this setup, fluorescence lifetime of the mitochondrial marker Rhodamine 123 could be studied in solution under the same instrumental conditions as used for fluorescence lifetime imaging of cell monolayers. With the same set of parameters, fluorescence lifetime of Rhodamine 123 was calculated with good reproducibility in mitochondria of living cells. We present here a comparison of different fitting routines, including a multiexponential fitting based on the method of Laplace transformation. Fluorescence lifetimes calculated with the multiexponential fitting routine proved to be particularly useful to study the distribution of 5-ALA metabolites in cell monolayers.

  16. Time-resolved studies of fluorescence and band-edge charge carrier dynamics in In1-xGaxP colloidal quantum dots

    NASA Astrophysics Data System (ADS)

    Mikhailovsky, Alexander A.; Diana, Frederic S.; Kim, Sangcheol; Kramer, Edward J.; Petroff, Pierre M.; Gerbec, Jeffrey; Strouse, Geoffrey

    2005-08-01

    In this submission, we report on the results of spectroscopic studies of charge carrier dynamics in colloidal In1-xGaxP quantum dots (QDs) with low levels of Ga doping (x~1%). These QDs exhibit large global Stokes shift of fluorescence (up to 300 meV) along with high emission yield (up to 30% in solution and 25% in films under blue excitation at 300 K) after post-synthesis photo-chemical treatment. In order to reveal the nature of large fluorescence Stokes shift and study the band-edge carriers dynamics, we performed time-resolved measurements of emission and photo-induced absorption changes in QDs with different sizes and surface passivation. The work was focused on the studies of differences between QDs subjected to photochemical surface passivation and bare nanoparticles. Time-resolved absorption spectroscopy indicates that holes' trapping strongly depends on passivation of surface trap states and can even suppress Auger multiparticle recombination in poorly passivated nanoparticles. Transient fluorescence measurements in well-passivated nanoparticles demonstrate that at short delays (<2 ns), emission Stokes shift is almost twice smaller than in steady-state measurements and matches the emission band in unpassivated QDs. At longer delays, time-resolved emission matches the spectra obtained with continuous wave (CW) excitation. We propose that initially photoluminescence occurs from quantum-confined state and subsequent hole relaxation onto surface/interface sites gives rise to emission with large global Stokes shift. In poorly passivated QDs, holes escape quickly to deep-trap states that leads to formation of low-efficiency broad emission band red-shifted with respect to the excitonic PL band.

  17. Development of a broadband picosecond infrared spectrometer and its incorporation into an existing ultrafast time-resolved resonance Raman, UV/visible, and fluorescence spectroscopic apparatus.

    PubMed

    Towrie, Michael; Grills, David C; Dyer, Joanne; Weinstein, Julia A; Matousek, Pavel; Barton, Robin; Bailey, Philip D; Subramaniam, Naresh; Kwok, Wai M; Ma, Chensheng; Phillips, David; Parker, Anthony W; George, Michael W

    2003-04-01

    We have constructed a broadband ultrafast time-resolved infrared (TRIR) spectrometer and incorporated it into our existing time-resolved spectroscopy apparatus, thus creating a single instrument capable of performing the complementary techniques of femto-/picosecond time-resolved resonance Raman (TR3), fluorescence, and UV/visible/infrared transient absorption spectroscopy. The TRIR spectrometer employs broadband (150 fs, approximately 150 cm(-1) FWHM) mid-infrared probe and reference pulses (generated by difference frequency mixing of near-infrared pulses in type I AgGaS2), which are dispersed over two 64-element linear infrared array detectors (HgCdTe). These are coupled via custom-built data acquisition electronics to a personal computer for data processing. This data acquisition system performs signal handling on a shot-by-shot basis at the 1 kHz repetition rate of the pulsed laser system. The combination of real-time signal processing and the ability to normalize each probe and reference pulse has enabled us to achieve a high sensitivity on the order of deltaOD approximately 10(-4) - 10(-5) with 1 min of acquisition time. We present preliminary picosecond TRIR studies using this spectrometer and also demonstrate how a combination of TRIR and TR3 spectroscopy can provide key information for the full elucidation of a photochemical process.

  18. 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.

  19. Selective nonpeptidic fluorescent ligands for oxytocin receptor: design, synthesis, and application to time-resolved FRET binding assay.

    PubMed

    Karpenko, Iuliia A; Margathe, Jean-François; Rodriguez, Thiéric; Pflimlin, Elsa; Dupuis, Elodie; Hibert, Marcel; Durroux, Thierry; Bonnet, Dominique

    2015-03-12

    The design and the synthesis of the first high-affinity fluorescent ligands for oxytocin receptor (OTR) are described. These compounds enabled the development of a TR-FRET based assay for OTR, readily amenable to high throughput screening. The validation of the assay was achieved by competition experiments with both peptide and nonpeptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor.

  20. Microviscosity in polyacrylamide gels with pendant triphenyl-methane leuco derivatives: picosecond time-resolved fluorescence study

    NASA Astrophysics Data System (ADS)

    Tamai, Naoto; Ishikawa, Masazumi; Kitamura, Noboru; Masuhara, Hiroshi

    1991-10-01

    Picosecond fluorescence dynamics of triphenylmethane dyes bonded to polyacrylamide gels before and after swelling was studied by a single-photon timing technique. Microviscosity in the gels after swelling was estimated to be 10-11 cP by examining the viscosity dependence of fluorescence dynamics of malachite green in various alcohols. The results were interpreted in terms of structured stiff water in a microcavity of the gels.

  1. Tissue characterization by time-resolved fluorescence spectroscopy of endogenous and exogenous fluorochromes: apparatus design and preliminary results

    NASA Astrophysics Data System (ADS)

    Glanzmann, Thomas M.; Ballini, Jean-Pierre; Jichlinski, Patrice; van den Bergh, Hubert; Wagnieres, Georges A.

    1996-12-01

    The biomedical use of an optical fiber-based spectro- temporal fluorometer that can endoscopically record the fluorescence decay of an entire spectrum without scanning is presented. The detector consists of a streak camera coupled to a spectrograph. A mode-locked argon ion pumped dye laser or a nitrogen laser-pumped dye laser are used as pulsed excitation light sources. We measured the fluorescence decays of endogenous fluorophores and of ALA-induced- protoporphyrin IX(PPIX) in an excised human bladder with a carcinoma in situ (CIS). Each autofluorescence decay can be decomposed in at least three exponential components for all tissue samples investigated if the excitation is at 425 nm. The decays of the autofluorescence of all normal sites of the human bladder are similar and they differ significantly from the decays measured on the CIS and the necrotic tissue. The fluorescence of the ALA-induced PPIX in the bladder is monoexponential with a lifetime of 15 (plus or minus 1) ns and this fluorescence lifetime does not change significantly between the normal urothelium and the CIS. A photoproduct of ALA-PPIX with a fluorescence maximum at 670 nm and a lifetime of 8 (plus or minus 1) ns was observed. The measurement of the decay of the autofluorescence allowed to correctly identify a normal tissue site that was classified as abnormal by the measurement of the ALA-PPIX fluorescence intensity.

  2. Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

    PubMed

    Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

    2007-01-15

    Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

  3. 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.

  4. Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy.

    PubMed

    Kollmann, Heiko; Becker, Simon F; Shirdel, Javid; Scholten, Alexander; Ostendorp, Anna; Lienau, Christoph; Koch, Karl-Wilhelm

    2012-06-15

    We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.

  5. Time-resolved fluorescence immunochromatographic assay developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin and zearalenone in maize and its products.

    PubMed

    Tang, Xiaoqian; Li, Peiwu; Zhang, Qi; Zhang, Zhaowei; Zhang, Wen; Jiang, Jun

    2017-09-13

    Aflatoxins and zearalenone (ZEN) are highly common mycotoxins in maize and maize-based products. This study aimed to report a time-resolved fluorescence immunochromatographic assay (TRFICA) developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin B1 (AFB1) and ZEN in maize and its products. A novel Eu/Tb(III) nanosphere with enhanced fluorescence was prepared as a label and conjugated to anti-idiotypic nanobody (AIdnb) and monoclonal antibody (mAb). On the basis of nanosphere-antibody conjugation, two patterns of competitive time-resolved strip methods (AIdnb-TRFICA and mAb-TRFICA) were established and compared. The half inhibition concentration of AIdnb-TRFICA was 0.46 and 0.86 ng  mL1 for AFB1 and ZEN, which was 18.3- and 20.3-fold more sensitive than that of mAb-TRFICA for AFB1 and ZEN, respectively. Under optimal conditions, AIdnb-TRFICA for dual mycotoxin was established and provided a quantitative relationship ranging from 0.13 to 4.54 ng  mL1 for AFB1 and 0.20 to 2.77 ng  mL1 for ZEN, with a detection limit of 0.05 and 0.07 ng  mL1 in the buffer solution, respectively. AIdnb-TRFICA showed good recoveries (72.6%-106.6%) in samples and was applied to detect dual mycotoxin in maize samples with satisfied results. To the best of our knowledge, it is the first report about time-resolved strip method based on AIdnbs for dual mycotoxin.

  6. Separation of indocyanine green boluses in the human brain and scalp based on time-resolved in-vivo fluorescence measurements

    NASA Astrophysics Data System (ADS)

    Jelzow, Alexander; Wabnitz, Heidrun; Obrig, Hellmuth; Macdonald, Rainer; Steinbrink, Jens

    2012-05-01

    Non-invasive detection of fluorescence from the optical tracer indocyanine green is feasible in the adult human brain when employing a time-domain technique with picosecond resolution. A fluorescence-based assessment may offer higher signal-to-noise ratio when compared to bolus tracking relying on changes in time-resolved diffuse reflectance. The essential challenge is to discriminate the fluorescence originating from the brain from contamination by extracerebral fluorescence and hence to reconstruct the bolus kinetics; however, a method to reliably perform the necessary separation is missing. We present a novel approach for the decomposition of the fluorescence contributions from the two tissue compartments. The corresponding sensitivity functions pertaining to the brain and to the extracerebral compartment are directly derived from the in-vivo measurement. This is achieved by assuming that during the initial and the late phase of bolus transit the fluorescence signal originates largely from one of the compartments. Solving the system of linear equations allows one to approximate time courses of a bolus for each compartment. We applied this method to repetitive measurements on two healthy subjects with an overall 34 boluses. A reconstruction of the bolus kinetics was possible in 62% of all cases.

  7. 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.

  8. Rapid and simple quantitation of methamphetamine by using a homogeneous time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer from europium to Cy5.

    PubMed

    Kimura, Hiroko; Matsumoto, Kazuko; Mukaida, Masahiro

    2005-01-01

    A rapid and simple homogeneous time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer from europium (Eu) to cyanine dye (Cy5) has been developed for the quantitation of methamphetamine. In this assay, Eu chelate was labeled to a conjugate of methamphetamine and bovine serum albumin (MA-BSA), as an energy donor, and Cy5 was labeled to anti-MA as an energy acceptor. The close proximity between the two labels in the immunocomplex permits energy transfer from the excited Eu(3+) donor. Therefore, by measuring the sensitized emission of Cy5 with the time-resolved assay, immunocomplex of MA-BSA and anti-MA can be measured in the homogeneous solution without separation steps within 30 min. By a competitive immunoassay, MA could be assayed in the range 0.1-1,000 ng/mL. The intra-assay variations were 5.4-14.8% at 5 different concentrations. When urine or serum samples were examined, the quenching of Eu fluorescence was observed, but the acceptor-to-donor ratio constantly depended upon the dilution of samples. Twenty urine samples were assayed, and the data showed a good correlation to those obtained by gas chromatography (r = 0.94). The homogeneous assay using Eu-Cy5 energy transfer is time-saving without any washing procedures and is suitable for screening drugs that are commonly abused.

  9. Time-resolved spectroscopic fluorescence imaging, transient absorption and vibrational spectroscopy of intact and photo-inhibited photosynthetic tissue.

    PubMed

    Lukins, Philip B; Rehman, Shakil; Stevens, Gregory B; George, Doaa

    2005-01-01

    Fluorescence, absorption and vibrational spectroscopic techniques were used to study spinach at the photosystem II (PS II), chloroplast and cellular levels and to determine the effects and mechanisms of ultraviolet-B (UV-B) photoinhibition of these structures. Two-photon fluorescence spectroscopic imaging of intact chloroplasts shows significant spatial variations in the component fluorescence spectra in the range 640-740 nm, indicating that the type and distribution of chlorophylls vary markedly with position in the chloroplast. The chlorophyll distributions and excitonic behaviour in chloroplasts and whole plant tissue were studied using picosecond time-gated fluorescence imaging, which also showed UV-induced kinetic changes that clearly indicate that UV-B induces both structural and excitonic uncoupling of chlorophylls within the light-harvesting complexes. Transient absorption measurements and low-frequency infrared and Raman spectroscopy show that the predominant sites of UV-B damage in PS II are at the oxygen-evolving centre (OEC) itself, as well as at specific locations near the OEC-binding sites.

  10. 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

    2012-11-07

    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.

  11. Excitation relaxation dynamics and energy transfer in fucoxanthin-chlorophyll a/c-protein complexes, probed by time-resolved fluorescence.

    PubMed

    Akimoto, Seiji; Teshigahara, Ayaka; Yokono, Makio; Mimuro, Mamoru; Nagao, Ryo; Tomo, Tatsuya

    2014-09-01

    In algae, light-harvesting complexes contain specific chlorophylls (Chls) and keto-carotenoids; Chl a, Chl c, and fucoxanthin (Fx) in diatoms and brown algae; Chl a, Chl c, and peridinin in photosynthetic dinoflagellates; and Chl a, Chl b, and siphonaxanthin in green algae. The Fx-Chl a/c-protein (FCP) complex from the diatom Chaetoceros gracilis contains Chl c1, Chl c2, and the keto-carotenoid, Fx, as antenna pigments, in addition to Chl a. In the present study, we investigated energy transfer in the FCP complex associated with photosystem II (FCPII) of C. gracilis. For these investigations, we analyzed time-resolved fluorescence spectra, fluorescence rise and decay curves, and time-resolved fluorescence anisotropy data. Chl a exhibited different energy forms with fluorescence peaks ranging from 677 nm to 688 nm. Fx transferred excitation energy to lower-energy Chl a with a time constant of 300fs. Chl c transferred excitation energy to Chl a with time constants of 500-600fs (intra-complex transfer), 600-700fs (intra-complex transfer), and 4-6ps (inter-complex transfer). The latter process made a greater contribution to total Chl c-to-Chl a transfer in intact cells of C. gracilis than in the isolated FCPII complexes. The lower-energy Chl a received excitation energy from Fx and transferred the energy to higher-energy Chl a. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy. Copyright © 2014. Published by Elsevier B.V.

  12. Lateral distribution of the transmembrane domain of influenza virus hemagglutinin revealed by time-resolved fluorescence imaging.

    PubMed

    Scolari, Silvia; Engel, Stephanie; Krebs, Nils; Plazzo, Anna Pia; De Almeida, Rodrigo F M; Prieto, Manuel; Veit, Michael; Herrmann, Andreas

    2009-06-05

    Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577-589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.

  13. 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.

  14. Time-Resolved Luminescence Spectroelectrochemistry at Screen-Printed Electrodes: Following the Redox-Dependent Fluorescence of [Ru(bpy)3](2).

    PubMed

    Martín-Yerga, Daniel; Pérez-Junquera, Alejandro; Hernández-Santos, David; Fanjul-Bolado, Pablo

    2017-09-25

    In this work, a compact instrument for time-resolved luminescence spectroelectrochemistry using low-cost disposable electrodes is reported. This instrument can be coupled with screen-printed electrodes via a specific cell and a reflection probe, which allows one to observe changes occurring at the electrode/solution interface. This approach allowed one to follow the fluorescence variation of electrofluorochromic species such as [Ru(bpy)3](2+) at screen-printed carbon electrodes. A strong correlation between the electrochemical processes and the fluorescence was found during potentiostatic or multipulsed amperometric measurements. A decrease of the fluorescence was observed when the [Ru(bpy)3](2+) was oxidized to [Ru(bpy)3](3+) and part of this fluorescence is recovered when [Ru(bpy)3](3+) was reduced to the initial species. Moreover, a significant increment of the fluorescence was found when the oxygen reduction reaction takes place, which also confirms its quenching effect. Finally, multipulsed amperometric detection was employed in order to obtain more information about the redox-dependent luminescence of [Ru(bpy)3](2+) finding a continuous quenching over time attributed to bleaching chlorine-based species.

  15. 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

  16. Liquid film characterization in horizontal, annular, two-phase, gas-liquid flow using time-resolved laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Farias, P. S. C.; Martins, F. J. W. A.; Sampaio, L. E. B.; Serfaty, R.; Azevedo, L. F. A.

    2012-03-01

    A non-intrusive optical technique was developed to provide time-resolved longitudinal and cross-sectional images of the liquid film in horizontal annular pipe flow of air and water, revealing the interfacial wave behavior. Quantitative information on the liquid film dynamics was extracted from the time-resolved images. The planar laser-induced fluorescence technique was utilized to allow for optical separation of the light emitted by the film from that scattered by the air-water interface. The visualization test section was fabricated from a tube presenting nearly the same refractive index as water, which allowed the visualization of the liquid film at regions very close to the pipe wall. Longitudinal images of the liquid film were captured using a high-frame-rate digital video camera synchronized with a high-repetition-rate laser. An image processing algorithm was developed to automatically detect the position of the air-water interface in each image frame. The thickness of the liquid film was measured at two axial stations in each processed image frame, providing time history records of the film thickness at two different positions. Wave frequency information was obtained by analyzing the time-dependent signals of film thickness for each of the two axial positions recorded. Wave velocities were measured by cross-correlating the amplitude signals from the two axial positions. For the film cross-section observations, two high-speed digital video cameras were used in a stereoscopic arrangement. Comparisons with results from different techniques available in literature indicate that the technique developed presents equivalent accuracy in measuring the liquid film properties. Time-resolved images of longitudinal and cross-section views of the film were recorded, which constitute valuable information provided by the technique implemented.

  17. Photophysical behavior of acridine with amines within the micellar microenvironment of SDS: a time-resolved fluorescence and laser flash photolysis study.

    PubMed

    Sarangi, Manas Kumar; Basu, Samita

    2011-10-06

    The photophysical behavior of acridine (Acr) shows a facilitated water assisted protonation equilibrium between its deprotonated (Acr* ∼ 3.4 ns) and protonated forms (AcrH(+)* ∼ 33 ns) within a confined environment of sodium dodecyl sulphate (SDS) micelles above the critical micellar concentration of 8 mM. The acidic interface of the micelles is capable of protonating Acr whereas deprotonated Acr is partitioned into the hydrophobic core. The time-resolved-area-normalized-emission spectra confirm the presence of both Acr* and AcrH(+)*, while time-resolved-emission spectra depict time evolution between them. Quenching of AcrH(+)* with triethylamine (TEA) results in a linear Stern-Volmer (S-V) plot, whereas non-linearity arises with N,N-dimethylaniline (DMA). Both steady-state and time-resolved quenching results with TEA are explained on the basis of excited state proton transfer (ESPT), however the reasons behind the quenching of excited Acr with DMA are proposed as ESPT followed by a photoinduced electron transfer. Partitioning of DMA at the interface makes it accessible for both Acr* and AcrH(+)* in hydrophobic and hydrophilic regions of micelles respectively. The rate of electron transfer at the interface is found to be slower compared to that in the hydrophobic core. Characterization of transient intermediates formed during ESPT and PET between Acr and amines by laser-flash photolysis also supports the observation obtained during fluorescence studies. The mode of interactions between Acr and amines inside micelles is controlled by the localization of the proton/electron donors and acceptors in different hydrophobic or hydrophilic regions of such nano-confined environments.

  18. Time-Resolved Fluorescence Spectroscopy and Imaging of DNA Labeled with DAPI and Hoechst 33342 Using Three-Photon Excitation

    PubMed Central

    Lakowicz, Joseph R.; Gryczynski, Ignacy; Malak, Henryk; Schrader, Martin; Engelhardt, Peter; Kano, Hiroski; Hell, Stefan W.

    1997-01-01

    We examined the fluorescence spectral properties of the DNA stains DAPI (4′,6-diamidino-2-phenylindole, hydrochloride) and Hoechst 33342 (bis-benzimide, or 2,5′-bi-1H-benzimidazole2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)) with two-photon (2hν) and three-photon (3hν) excitation using femtosecond pulses from a Ti:sapphire laser from 830 to 885 nm. The mode of excitation of DAPI bound to DNA changed from two-photon at 830 nm to three-photon at 885 nm. In contrast, Hoechst 33342 displayed only two-photon excitation from 830 to 885 nm. DAPI-DNA displayed the same emission spectra and decay times for 2hν and 3hν excitation. Hoechst 33342-DNA displayed the same intensity decay for excitation at 830 and 885 nm. Both probes displayed higher anisotropies for multiphoton excitation as compared to one-photon excitation with ultraviolet wavelengths, and DAPI-DNA displays a higher anisotropy for 3hν at 885 nm than for 2hν at 830 nm. We used 970-nm excitation of DAPI-stained chromosomes to obtain the first three-dimensional images with three-photon excitation. Three-photon excitation of DAPI-stained chromosomes at 970 nm was demonstrated by the power dependence in the fluorescence microscope. ImagesFIGURE 10FIGURE 11FIGURE 12 PMID:9017187

  19. 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.

  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.

    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.

  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

    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.

  2. Exactly soluble model of the time-resolved fluorescence return to thermal equilibrium in many-particle systems after excitation

    NASA Astrophysics Data System (ADS)

    Czachor, Andrzej

    2016-02-01

    In this paper we consider the assembly of weakly interacting identical particles, where the occupation of single-particle energy-levels at thermal equilibrium is governed by statistics. The analytic form of the inter-energy-level jump matrix is derived and analytic solution of the related eigen-problem is given. It allows one to demonstrate the nature of decline in time of the energy emission (fluorescence, recombination) of such many-level system after excitation in a relatively simple and unifying way - as a multi-exponential de-excitation. For the system of L energy levels the number of the de-excitation lifetimes is L-1. The lifetimes depend on the energy level spectrum as a whole. Two- and three-level systems are considered in detail. The impact of the energy level degeneracy on the lifetimes is discussed.

  3. 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.

  4. The equipment for time-resolved measurements of excitation-emission matrix of seawater fluorescence in natural conditions

    NASA Astrophysics Data System (ADS)

    Salyuk, Pavel A.; Nagorny, Ivan G.; Mayor, Alexander A.; Krikun, Vladimir A.; Shmirko, Konstantin A.

    2016-10-01

    Excitation-emission matrix (EEM) technique is widely used to study the properties of organic matter in seawater. Additional variations of the exciting radiation parameters (in addition to the wavelengths), such as duration and intensity, allow to get more spectral information about the object of research, and allow to identify some individual components or to study the dynamics of the molecular changes in the sample of analyzed liquid. One of the main limitations of the EEM is that usually necessary to carry out research in the laboratory on the specialized equipment. The paper describes the hardware system allowing carry out measurements in the field studies. The hardware system includes a set of light-emitted diodes (LEDs) working in the range from 245 to 600 nm, which produce sequential excitation fluorescence of the sample liquid, which is detected using a 32-channel photomultiplier tube (PMT). Number of LEDs can vary from 7 to 16. Through the use of multi-channel photomultiplier reached the required measurement sensitivity and efficiency measurement is less than 1 minute for full cycle. Minimum 4 channel in excitation range of 350-600 nm support the work of variable duration excitation pulses from microseconds to seconds. It makes possible to measure the fluorescence of the chlorophyll-a with closed or open reaction centers of phytoplankton cells. The use of multiple excitation radiation allows to activate various mechanisms of energy transfer in the photosynthetic apparatus of phytoplankton cells, improves measurement accuracy, reduces dependence on the variation in species composition and functional state of phytoplankton.

  5. Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy.

    PubMed

    Bannon, Catherine C; Campbell, Douglas A

    2017-01-01

    Diatoms are marine primary producers that sink in part due to the density of their silica frustules. Sinking of these phytoplankters is crucial for both the biological pump that sequesters carbon to the deep ocean and for the life strategy of the organism. Sinking rates have been previously measured through settling columns, or with fluorimeters or video microscopy arranged perpendicularly to the direction of sinking. These side-view techniques require large volumes of culture, specialized equipment and are difficult to scale up to multiple simultaneous measures for screening. We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll a fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; Thalassiosira pseudonana CCMP1335, chain-forming Skeletonema marinoi RO5A and Coscinodiscus radiatus CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d-1) using an RStudio script, SinkWORX. We thereby detected significant differences in sinking rates as larger diatom cells sank faster than smaller cells, and cultures in stationary phase sank faster than those in exponential phase. Our sinking rate estimates accord well with literature values from previously established methods. This well plate-based method can operate as a high throughput integrative phenotypic screen for factors that influence sinking rates including macromolecular allocations, nutrient availability or uptake rates, chain-length or cell size, degree of silification and progression through growth stages. Alternately the approach can be used to phenomically screen libraries of mutants.

  6. Homodimerization of Amyloid Precursor Protein at the Plasma Membrane: A homoFRET Study by Time-Resolved Fluorescence Anisotropy Imaging

    PubMed Central

    Devauges, Viviane; Marquer, Catherine; Lécart, Sandrine; Cossec, Jack-Christophe; Potier, Marie-Claude; Fort, Emmanuel; Suhling, Klaus; Lévêque-Fort, Sandrine

    2012-01-01

    Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer’s disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions. PMID:22973448

  7. Steady-state and time-resolved fluorescence of Esperase®: comparison with the X-ray structure in the region of the two tryptophans

    NASA Astrophysics Data System (ADS)

    Georgieva, D. N.; Nikolov, P.; Betzel, C.

    1998-08-01

    Fluorescence emission properties of the alkaline protease Esperase® have been investigated using steady-state and time-resolved fluorescence spectroscopy. The local polarity and solvent accessibility of the tryptophyl chromophores is characterized. Quenching studies demonstrated that Trp 6 and Trp 113 are 'buried' to acrylamide, iodide ions and caesium ions. An abnormally low tryptophan quantum yield was calculated showing that the emission of the two indole rings is significantly quenched by nearby side chains or peptide bonds. The fluorescence decay of PMS-Esperase® was well fitted by two exponentials with lifetimes of 2.7 and 0.35 ns. X-ray data for Esperase® (S. Klupsch, Ph.D. Thesis, University of Hamburg, Hamburg, Germany) in the region of the two tryptophans were used to explain the observed emission properties. Gln 182 and Asn 204 as well as Asn 117 and Met 119 are the most likely quenchers, respectively, of the Trp 6 and Trp 113 fluorescence. The two tryptophans in Esperase® are 'buried' in hydrophobic regions and are excellent intrinsic probes to study folding-unfolding reactions. Experiments in the presence and absence of added calcium ions demonstrated the stabilizing role of the Ca 2+-binding sites.

  8. Laguerre-based method for analysis of time-resolved fluorescence data: application to in-vivo characterization and diagnosis of atherosclerotic lesions

    PubMed Central

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

    2007-01-01

    We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability. PMID:16674179

  9. Tracking local conformational changes of ribonuclease A using picosecond time-resolved fluorescence of the six tyrosine residues.

    PubMed

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

    2007-06-15

    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 (tau(1) = 1.7 ns, tau(2) = 180 ps, and tau(3) = 30 ps) and preexponential coefficients (A(1) = 1, A(2) = 1, and A(3) = 4) at pH 7, 25 degrees 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 tau(1) to Tyr-76 (R = 12.8 A), tau(2) to Tyr-115 (R = 6.9 A), and tau(3) to Tyr-25, Tyr-73, Tyr-92, and Tyr-97 (all four at R = 5.5 +/- 0.3 A) at 23 degrees 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, T(m), obtained from Tyr-76 (T(m) = 59.2 degrees C) and Tyr-25 and Tyr-92 (T(m) = 58.2 degrees C) suggests a single unfolding event in this temperature range that affects all these regions similarly.

  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.

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

    PubMed

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

    2010-08-01

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

  13. Maximum entropy analysis of data simulations and practical aspects of time-resolved fluorescence measurements in the study of molecular interactions

    NASA Astrophysics Data System (ADS)

    Henry, Etienne; Deprez, Eric; Brochon, Jean-Claude

    2014-12-01

    Time-resolved fluorescence spectroscopy and microscopy is increasingly used to probe molecular interactions and dynamics events in vitro and in vivo. We point out some pitfalls to avoid in the data acquisition procedure using time correlated single photon counting. A good accuracy in fluorescence decay measurements is not only linked to the counts in the peak channel but also to the statistics at the end of the curve. A too narrow time interval between successive excitation leads to an overlap of decays, and that should be taken into account in data analysis. The counting rate in the peak channel of the excitation profile should be close to the one of the fluorescence decay. Many distributions of lifetimes can fit an incomplete or noisy data set to satisfying precisions corresponding to close values of χ2. The maximum entropy principle is appropriate to distinguish among these in a consistent way. It is also shown that encoding a prior knowledge about the system study dramatically improves the quality of the recovered distribution, particularly in case of a set of discrete components. Based on simulated noisy quantify maximum entropy method (MEM) data analysis, we propose a simple strategy for estimating the quality of information and inferences we can draw from experimental results.

  14. 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).

  15. 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

    2013-11-07

    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.

  16. An in vitro diagnosis of oral premalignant lesion using time-resolved fluorescence spectroscopy under UV excitation-a pilot study.

    PubMed

    Kanniyappan, Udayakumar; Prakasarao, Aruna; Dornadula, Koteeswaran; Singaravelu, Ganesan

    2016-06-01

    In spite of rapid advancement in cancer treatment, early diagnosis of cancer and medicable precursors are still the finest approach towards the assurance of patient lives and enhancement in the quality of their life. In this regard, the present study deals with the time-resolved fluorescence spectroscopy of normal and premalignant oral tissues under UV excitations (280nm and 310nm). The decay kinetics at 350nm emission of normal tissues exhibit higher fluorescence lifetime than that of premalignant tissues and subsequent statistical analysis shows that the data were statistically significant. Further, the decay kinetics at 450nm emission for normal and premalignant oral tissues was obtained. Subsequently, statistical analysis revealed that except fast component, rest of the component lifetimes and fractional amplitudes were not statistically significant. An attempt has also been made to explore the better statistical tool to discriminate premalignant tissues from normal ones at 350nm emission. Among stepwise linear discriminant analysis (SLDA) and receiver operator characteristics (ROC), the former discriminates premalignant from normal tissues with 86.7% specificity and 93.3% sensitivity. Hence, fluorescence lifetime spectroscopy at 350nm emission opens a new avenue for early detection of oral cancer.

  17. Vibrational Spectrum of an Excited State and Huang-Rhys Factors by Coherent Wave Packets in Time-Resolved Fluorescence Spectroscopy.

    PubMed

    Lee, Gyeongjin; Kim, Junwoo; Kim, So Young; Kim, Dong Eon; Joo, Taiha

    2017-01-10

    Coherent nuclear wave packet motions in an electronic excited state of a molecule are measured directly by time-resolved spontaneous fluorescence spectroscopy with an unprecedented time resolution by using two-photon absorption excitation and fluorescence upconversion by noncollinear sum frequency generation. With an estimated time resolution of approximately 25 fs, wave packet motions of vibrational modes up to 1600 cm(-1) are recorded for coumarin 153 in ethanol. Two-color transient absorption at 13 fs time resolution are measured to confirm the result. Vibrational displacements between the ground and excited states and Huang-Rhys factors (HRFs) are calculated by quantum mechanical methods and are compared with the experimental results. HRFs calculated by density functional theory (DFT) and time-dependent DFT reproduce the experiment adequately. This fluorescence-based method provides a unique and direct way to obtain the vibrational spectrum of a molecule in an electronic excited state and the HRFs, as well as the dynamics of excited states, and it might provide information on the structure of an excited state through the HRFs.

  18. 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.

  19. 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.

  20. Time-resolved fluorescence investigation of the human immunodeficiency virus type 1 nucleocapsid protein: influence of the binding of nucleic acids.

    PubMed Central

    Bombarda, E; Ababou, A; Vuilleumier, C; Gérard, D; Roques, B P; Piémont, E; Mély, Y

    1999-01-01

    Depending on the HIV-1 isolate, MN or BH10, the nucleocapsid protein, NCp7, corresponds to a 55- or 71-amino acid length product, respectively. The MN NCp7 contains a single Trp residue at position 37 in the distal zinc finger motif, and the BH10 NCp7 contains an additional Trp, at position 61 in the C-terminal chain. The time-resolved intensity decay parameters of the zinc-saturated BH10 NCp7 were determined and compared to those of single-Trp-containing derivatives. The fluorescence decay of BH10 NCp7 could be clearly represented as a linear combination (with respect to both lifetimes and fractional intensities) of the individual emitting Trp residues. This suggested the absence of interactions between the two Trp residues, a feature that was confirmed by molecular modeling and fluorescence energy transfer studies. In the presence of tRNAPhe, taken as a RNA model, the same conclusions hold true despite the large fluorescence decrease induced by the binding of tRNAPhe. Indeed, the fluorescence of Trp37 appears almost fully quenched, in keeping with a stacking of this residue with the bases of tRNAPhe. Despite the multiple binding sites in tRNAPhe, the large prevalence of ultrashort lifetimes, associated with the stacking of Trp37, suggests that this stacking constitutes a major feature in the binding process of NCp7 to nucleic acids. In contrast, Trp61 only stacked to a small extent with tRNAPhe. The behavior of this residue in the tRNAPhe-NCp7 complexes appeared to be rather heterogeneous, suggesting that it does not constitute a major determinant in the binding process. Finally, our data suggested that the binding of NCp7 proteins from the two HIV-1 strains to nonspecific nucleic acid sequences was largely similar. PMID:10049336

  1. Influence of substrates and MgADP on the time-resolved intrinsic fluorescence of phosphofructokinase from Escherichia coli. Correlation of tryptophan dynamics to coupling entropy.

    PubMed

    Johnson, J L; Reinhart, G D

    1994-03-08

    The influence of that MgADP and the substrate ligands MgATP and fructose 6-phosphate (Fru-6-P) have on the structure of E. coli phosphofructokinase (PFK) in the vicinity of the single tryptophan that exists in each subunit has been examined by employing both steady-state and time-resolved measurements of the tryptophan fluorescence. The accessibility of the tryptophan to iodide quenching is over 1 order of magnitude less than experienced by N-acetyltryptophanamide in solution but varies nonetheless with the state of ligation. Most, but not all, of these changes correlate with changes in the degree of local motion available to the tryptophan side chain as determined by steady-state and time-resolved polarization measurements. When the data obtained from differential polarization experiments are fit to a model in which the motion of the tryptophan side chain is able to move with high frequency within a cone of limited amplitude as part of an otherwise slowly tumbling spherical protein, it was found that ligands primarily affect the amplitude of the available local motion. By interpreting these effects with reference to the disproportionation equilibria which define the negative coupling free energy between MgADP and Fru-6-P and the positive coupling free energy between MgADP and MgATP, it is apparent that changes in the local motion amplitudes correlate with the sign of the component coupling entropy previously determined from van't Hoff analyses (Johnson & Reinhart, 1994).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. 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.

  3. Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers

    PubMed Central

    McDonald, S.L.R.; Maple, P.A.C.; Andrews, N.; Brown, K.E.; Ayres, K.L.; Scott, F.T.; Bassam, M. Al; Gershon, A.A.; Steinberg, S.P.; Breuer, J.

    2017-01-01

    Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥400mIU/mL and latter had levels <400 mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of >130 mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79–96), and 78% (95% CI 61–90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA. PMID:21192976

  4. Complex formation of neptunium(V) with 4-hydroxy-3-methoxybenzoic acid studied by time-resolved laser-induced fluorescence spectroscopy with ultra-short laser pulses.

    PubMed

    Vulpius, D; Geipel, G; Baraniak, L; Bernhard, G

    2006-03-01

    The complex formation of neptunium(V) with 4-hydroxy-3-methoxybenzoic acid (vanillic acid) was studied by time-resolved laser-induced fluorescence spectroscopy with ultra-short laser pulses using the fluorescence properties of 4-hydroxy-3-methoxybenzoic acid. A 2:1 complex of neptunium(V) with 4-hydroxy-3-methoxybenzoic acid was found. The stability constant of this complex was determined to be logbeta(210) = 7.33 +/- 0.10 at an ionic strength of 0.1 mol/l (NaClO(4)) and at 21 degrees C. The determination of the stability constant required an investigation of the excited-state proton transfer of 4-hydroxy-3-methoxybenzoic acid over the whole pH range. It was realized that 4-hydroxy-3-methoxybenzoic acid undergoes excited-state reactions only at pH values below 5. At pH values above 5 stability constants can be determined without kinetic calculation of the proton transfer.

  5. Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2.

    PubMed

    Wyhs, Nicolas; Walker, David; Giovinazzo, Hugh; Yegnasubramanian, Srinivasan; Nelson, William G

    2014-08-01

    Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z' factor = 0.58) emerged as a superior screening strategy compared with FP (Z' factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET-based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions. © 2014 Society for Laboratory Automation and Screening.

  6. Energy transfer in Anabaena variabilis filaments adapted to nitrogen-depleted and nitrogen-enriched conditions studied by time-resolved fluorescence.

    PubMed

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

    2017-02-16

    Nitrogen is among the most important nutritious elements for photosynthetic organisms such as plants, algae, and cyanobacteria. Therefore, nitrogen depletion severely compromises the growth, development, and photosynthesis of these organisms. To preserve their integrity under nitrogen-depleted conditions, filamentous nitrogen-fixing cyanobacteria reduce atmospheric nitrogen to ammonia, and self-adapt by regulating their light-harvesting and excitation energy-transfer processes. To investigate the changes in the primary processes of photosynthesis, we measured the steady-state absorption and fluorescence spectra and time-resolved fluorescence spectra (TRFS) of whole filaments of the nitrogen-fixing cyanobacterium Anabaena variabilis at 77 K. The filaments were grown in standard and nitrogen-free media for 6 months. The TRFS were measured with a picosecond time-correlated single photon counting system. Despite the phycobilisome degradation, the energy-transfer paths within phycobilisome and from phycobilisome to both photosystems were maintained. However, the energy transfer from photosystem II to photosystem I was suppressed and a specific red chlorophyll band appeared under the nitrogen-depleted condition.

  7. Time-resolved and Depth-dependent Photo-Degradation of Marine Dissolved Organic Matter Analyzed by Semi-continuous EEM Fluorescence Monitoring

    NASA Astrophysics Data System (ADS)

    Gonsior, M.; Timko, S.; Conte, M. H.; Schmitt-Kopplin, P.

    2016-02-01

    Ten liter water samples were collected at the Bermuda Atlantic Time Series Station (BATS) at 200 m intervals down to a maximum depth of 4530 m and solid-phase extracted. The methanol extracts were dried and re-dissolved in pure water and then used to determine the time-resolved photo-degradation of marine dissolved organic matter to be able to determine kinetic data. Excitation Emission Matrix (EEM) fluorescence spectra were recorded every 20 minutes using a custom-built flow-through photo-degradation system during 20 h of solar simulated light exposure. The resulting EEM spectra were modeled using Parallel Factor Analysis (PARAFAC) and results revealed reproducible and significant changes in the photo-degradation of marine FDOM originating from different depths. A five component model was fitted and the terrestrial-like components showed the expected high photo-reactivity, but surprisingly, the traditional marine-like peak showed slight photo-production in the surface layer, which might be the reason for its prevalence in the open ocean. Surface ocean waters were depleted in the highly photo-degradable components while protein-like fluorescent components were enriched, which was in agreement with previous studies. Ultrahigh resolution mass spectrometry confirmed unique aliphatic molecular ions in the Surface Ocean and hydrogen-deficient molecules at depth. Multivariate statistical analyses revealed strong correlations between unsaturated/aromatic molecular ions and depth, where aliphatic molecular ions were more prevalent in the Surface Ocean and aromatic molecular ions at depth. Strong correlations were also found between hydrogen-deficient molecular ions and the humic-like fluorescent components. The rapid photo-degradation of the deep-sea FDOM and the surface oceans relative depletion of aromatic molecular ions suggested that deep-ocean FDOM may be too photochemically labile to survive meridional overturning circulation.

  8. Development of filtration-based time-resolved fluorescence assay for the high-throughput screening of urotensin II receptor antagonist.

    PubMed

    Oh, Kwang-Seok; Lee, Sunghou; Lee, Byung Ho

    2011-10-01

    The time-resolved fluorescence (TRF) receptor binding assay has many advantages over the traditional radioligand binding assay in terms of sensitivity and reproducibility for the screening of receptor ligands. The TRF-based urotensin receptor (UT) binding assay with an automatic vacuum filtration system was developed and evaluated for the high-throughput screening of UT receptor antagonists. For this assay development, the human recombinant urotensin II (UII) was modified by labeling europium at its N-terminal position (Eu-UII) and used as a fluorescent tracer. The microsomal membrane fraction of UT receptor was prepared from HEK293 cells stably expressing the human UT receptor. The 50% inhibitory concentration (IC(50)) values of UII from competition binding assays with Eu-UII were 2.76 nM, which is very similar to that of fluorescence polarization (FP)-based UT receptor binding experiment (2.18 nM). Comparing with the FP-based receptor binding assay for UII (Z' factor, 0.36), the current TRF assay presented improved Z' factor (0.76) with a relatively higher signal-to-background ratio (1.5 and 2.1, respectively). The known high-affinity UT receptor antagonists, palosuran and SB657510, exhibited IC(50) values of 23.6 and 73.4 nM, respectively, which were consistent with the IC(50) values from FP-based receptor binding assay (30.6 and 78.7 nM, respectively). These results suggest that our filtration-based TRF UT receptor binding assay can achieve the desired sensitivity with higher reproducibility to adapt for the high-throughput screening of compound libraries.

  9. Time-resolved confocal fluorescence microscopy: novel technical features and applications for FLIM, FRET and FCS using a sophisticated data acquisition concept in TCSPC

    NASA Astrophysics Data System (ADS)

    Koberling, Felix; Krämer, Benedikt; Kapusta, Peter; Patting, Matthias; Wahl, Michael; Erdmann, Rainer

    2007-05-01

    In recent years time-resolved fluorescence measurement and analysis techniques became a standard in single molecule microscopy. However, considering the equipment and experimental implementation they are typically still an add-on and offer only limited possibilities to study the mutual dependencies with common intensity and spectral information. In contrast, we are using a specially designed instrument with an unrestricted photon data acquisition approach which allows to store spatial, temporal, spectral and intensity information in a generalized format preserving the full experimental information. This format allows us not only to easily study dependencies between various fluorescence parameters but also to use, for example, the photon arrival time for sorting and weighting the detected photons to improve the significance in common FCS and FRET analysis schemes. The power of this approach will be demonstrated for different techniques: In FCS experiments the concentration determination accuracy can be easily improved by a simple time-gated photon analysis to suppress the fast decaying background signal. A more detailed analysis of the arrival times allows even to separate FCS curves for species which differ in their fluorescence lifetime but, for example, cannot be distinguished spectrally. In multichromophoric systems like a photonic wire which undergoes unidirectional multistep FRET the lifetime information complements significantly the intensity based analysis and helps to assign the respective FRET partners. Moreover, together with pulsed excitation the time-correlated analysis enables directly to take advantage of alternating multi-colour laser excitation. This pulsed interleaved excitation (PIE) can be used to identify and rule out inactive FRET molecules which cause interfering artefacts in standard FRET efficiency analysis. We used a piezo scanner based confocal microscope with compact picosecond diode lasers as excitation sources. The timing performance

  10. Synthesis of a highly fluorescent beta-diketone-europium chelate and its utility in time-resolved fluoroimmunoassay of serum total thyroxine.

    PubMed

    Wu, Feng-Bo; Han, Shi-Quan; Zhang, Chao; He, You-Feng

    2002-11-15

    A new highly fluorescent beta-diketone-europium chelate was synthesized and employed as a tracer to develop a time-resolved fluoroimmunoassay (TRFIA) for detection of serum total thyroxine (T4). The tetradentate beta-diketone chelator, 1,10-bis(thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone (BTOT), was structurally composed of two units of thenoyltrifluoroacetone (TTA) derivatives but expressed fluorescence that was greatly enhanced, as compared to the original TTA molecules, in the presence of excess amount of Eu3+. The luminescence properties of the europium chelate of BTOT werestudied in aqueous solution. Chlorosulfonylation of BTOT afforded 1, 10-bis(5'-chlorosulfo-thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone (BCTOT), which could be coupled to proteins (i.e., streptavidin or the BSA-T4 conjugate) and used as a tracer for TRFIA. Although the BCTOT-Eu complex could be detected at a very low level (approximately 1.07 x 10(-12) mol/L) in buffered aqueous solution (50 mmoVLTris-HCl; pH, 8.0), the application of the chelate label in direct serum T4 TRFIA experienced a problem of matrix interference, which was probably caused by some unknown chelating components in the samples as a result of the fact that the fluorescence of the BCTOT-Eu chelate was prone to quenching or enhancement by some chelating reagents. To remove this problem, an indirect serum T4 TRFIA was proposed with the use of BCTOT-Eu-labeled streptavidin (SA) as signal generation reagent. The concentrations of T4 in 27 human serums were determined by indirect T4 TRFIA, and the assay results correlated well with those obtained by commercial Coming-CLIA (r = 0.955) and Wallac-DELFIA (r 0.965).

  11. Time-resolved nanosecond fluorescence lifetime imaging and picosecond infrared spectroscopy of combretastatin A-4 in solution and in cellular systems

    NASA Astrophysics Data System (ADS)

    Bisby, Roger H.; Botchway, Stanley W.; Greetham, Greg M.; Hadfield, John A.; McGown, Alan T.; Parker, Anthony W.; Scherer, Kathrin M.; Towrie, Mike

    2012-08-01

    Fluorescence lifetime images of intrinsic fluorescence obtained with two-photon excitation at 630 nm are shown following uptake of a series of E-combretastatins into live cells, including human umbilical vein endothelial cells (HUVECs) that are the target for the anticancer activity of combretastatins. Images show distribution of the compounds within the cell cytoplasm and in structures identified as lipid droplets by comparison with images obtained following Nile red staining of the same cells. The intracellular fluorescent lifetimes are generally longer than in fluid solution as a consequence of the high viscosity of the cellular environment. Following incubation, the intracellular concentrations of a fluorinated derivative of E-combretastatin A-4 in HUVECs are between two and three orders of magnitude higher than the concentration in the surrounding medium. Evidence is presented to indicate that at moderate laser powers (up to 6 mW), it is possible to isomerize up to 25% of the combretastatin within the femtolitre focal volume of the femtosecond laser beam. This suggests that it may be possible to activate the E-combretastatin (with low cellular toxicity) to the Z-isomer with high anticancer drug activity using two-photon irradiation. The isomerization of Z- and E-combretastatins by 266 nm irradiation has been probed by ultrafast time-resolved infrared spectroscopy. Results for the E-isomer show a rapid loss of excess vibrational energy in the excited state with a lifetime of 7 ps, followed by a slower process with a lifetime of 500 ps corresponding to the return to the ground state as also determined from the fluorescence lifetime. In contrast, the Z-isomer, whilst also appearing to undergo a rapid cooling of the initial excited state, has a much shorter overall excited state lifetime of 14 ps. DedicationThis paper is dedicated to the memory of Professor Christopher G Morgan (1949-2011). He was a valued colleague and friend at the University of Salford and made

  12. Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A.

    PubMed

    Blanc, Emilie; Wagner, Patrick; Plaisier, Fabrice; Schmitt, Martine; Durroux, Thierry; Bourguignon, Jean-Jacques; Partiseti, Michel; Dupuis, Elodie; Bihel, Frederic

    2015-09-01

    Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.

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

    PubMed Central

    Liu, Jikun; Mahtani, Prerna; Zhang, Panhe; Du, Bingchen; Ragupathy, Viswanath; Devadas, Krishnakumar

    2016-01-01

    Abstract 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

  14. 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

  15. 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

    2007-02-05

    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.

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

    PubMed

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

    2017-01-31

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

  17. Identification of Small Molecule Inhibitors of the Mitotic Kinase Haspin by High Throughput Screening using a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assay

    PubMed Central

    Patnaik, Debasis; Xian, Jun; Glicksman, Marcie A.; Cuny, Gregory D.; Stein, Ross L.; Higgins, Jonathan M.G.

    2008-01-01

    Summary Haspin/Gsg2 is a kinase that phosphorylates Histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin therefore is a novel target for development of anti-mitotic agents. We report the development of a high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for Haspin. Histone H3 peptide was used as a substrate, and a Europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at Km concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC50 determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant Histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of two unrelated kinases. EC50 values in cells were determined using a cell-based ELISA assay of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of Haspin that will have clear utility in basic research and possible use as starting points for development of anti-mitotic anticancer therapeutics. PMID:18978305

  18. Steady-state and time-resolved fluorescence investigation of 2-pyridone and 3-pyridone in solution and their specific binding to human serum albumin

    NASA Astrophysics Data System (ADS)

    Abou-Zied, Osama K.; Al-Shihi, Othman I. K.

    2008-02-01

    2-pyridone (2Py) and 3-pyridone (3Py) were examined in different solvents and their binding to human serum albumin (HSA) was studied using steady-state spectroscopy and time-resolved fluorescence. Solvation of 2Py and 3Py by water was examined in binary mixtures of 1,4-dioxane and water. Analysis of the absorption and fluorescence data reveals the solvation of the hydrogen bonding center in 2Py by one water molecule and in 3Py by three water molecules. A zwitterionic tautomer of 3Py is formed in water and shows distinct absorption peaks from the absorption of the neutral tautomer. Fluorescence of 3Py was observed in polar solvents only, whereas 2Py is fluorescent in polar and nonpolar solvents. The absorption and fluorescence spectra of 2Py in different solvents indicate less solute-solvent interaction in nonpolar solvents. This observation was confirmed by the measured longer fluorescence lifetime of 2Py in cyclohexane compared to that in water. The mechanism of binding of 2Py and 3Py as probe ligands to HSA was investigated by following the intensity change and lifetime of HSA fluorescence after excitation at 280 nm. The presence of 2Py and 3Py causes a reduction in the fluorescence intensity and lifetime of HSA. This observation indicates that subdomain IIA binding site (Sudlow site I) is the host of the probes and the reduction in the fluorescence of HSA is due to energy transfer from the Trp-214 residue to the probe in each case. The distance between Trp-214 and each of the probes was calculated using Förster theory for energy transfer to be 1.99 nm for HSA/2Py and 2.44 nm for HSA/3Py. The shorter distance in the former complex indicates more efficient energy transfer than in the latter. This was confirmed by estimating the quenching rate constant (k q) in each complex. k q was calculated to be 1.44 x 10 12 M -1s -1 for HSA/2Py and 3.45 x 10 11 M -1s -1 for HSA/3Py. The calculated distances and the k q values indicate a static quenching mechanism operative in

  19. Performance characteristics of a quantitative, standardised varicella zoster IgG time resolved fluorescence immunoassay (VZV TRFIA) for measuring antibody following natural infection.

    PubMed

    Chris Maple, P A; Gray, Jim; Brown, Kevin; Brown, David

    2009-04-01

    Infection by Varicella Zoster virus (VZV) during pregnancy has been associated with adverse foetal development and more severe disease in the mother. Accurate determination of VZV immunity in pregnant women exposed to VZV, with no history of chickenpox, guides therapeutic interventions. The accepted gold standard assay for the determination of immunity/protection against Varicella Zoster virus was for many years the fluorescent antibody to membrane antigen (FAMA) assay which is labour intensive and subjective. A validated alternative is the Merck glycoprotein EIA (Merck Sharp & Dohme Research Laboratories, West Point, PA, USA) which reports VZV IgG levels in enzyme units per ml (EU/ml) because an internal, non-international reference serum is used as calibrator. Comparison of different VZV IgG detection assays is hampered by a lack of an agreed cut-off in standardised units. A time resolved fluorescence immunoassay (TRFIA) for VZV IgG using British Standard VZV antibody has been developed and standardised. The limit of detection of VZV IgG by this assay was of the order 39-78mIU/ml. Following comparison with the Merck glycoprotein EIA and the application of the USA Advisory Committee on Immunization Practices recommended 5.0EU/ml cut-off the following standardised cut-offs in mIU/ml are proposed. A VZV TRFIA IgG cut-off of less than 100mIU/ml VZV IgG equates with susceptibility and an equivocal range of 100mIU/ml to less than 150mIU/ml is proposed. VZV IgG levels of 150mIU/ml, or greater, are indicative of natural infection at some time and the ability to mount a protective immune response is inferred.

  20. Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers.

    PubMed

    McDonald, S L R; Maple, P A C; Andrews, N; Brown, K E; Ayres, K L; Scott, F T; Al Bassam, M; Gershon, A A; Steinberg, S P; Breuer, J

    2011-03-01

    Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥ 60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥ 400 mIU/mL and latter had levels < 400 mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of > 130 mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79-96), and 78% (95% CI 61-90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. H-bonding vs non-H-bonding in 100% pyrene methacrylate comb polymers: self-assembly probed by time-resolved emission spectra and temperature dependent fluorescence.

    PubMed

    Kaushlendra, K; Asha, S K

    2014-05-08

    The differences in self-organization behavior in novel 100% pyrene labeled comb methacrylate polymers probed as a function of their varied origins of excimer formation are presented. The different structural variations in the polymers included the presence or absence of hydrogen bonding interactions in the form of urethane linkages, short or long alkyl spacer segments separating the pyrene units from the polymer backbone and linear versus kinked urethane linkage. The effect of variable concentration and temperature on the chemical shift of the NH proton of the urethane linkage was probed using (1)H NMR experiments conducted at temperatures varying from 25 to 70 °C at two different concentrations (2.5 and 25 mmol) in DMSO-d6 as solvent. The photophysical properties of the polymers in dilute DMF solutions were investigated by steady state emission, fluorescence decay studies, time-resolved emission spectra (TRES), and variable temperature emission studies. It was observed that the polymer poly(PBH) having a non-hydrogen-bondable ester linkage in the pendant chains formed an excimer completely via a static mechanism and the ground state aggregate species were not broken even at higher temperatures. The polymer poly(PIC) having a short hydrogen-bondable urethane linkage formed an excimer via a static as well as dynamic mechanism. The other hydrogen-bondable urethane methacrylate polymers having a linear linker poly(PHH) and kinked linker (PIHP) formed excimer mostly via a dynamic mechanism with a very small contribution from the static route. The TRES studies carried out for the polymers provided significant insight into the excimer formation mechanism in these polymers. The variable temperature fluorescence studies highlighted the differences in the H-bonded vs non-H-bonded polymer as a function of their excimer recovery upon cooling.

  2. A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry.

    PubMed

    Su, Jui-Lan; Fornwald, Jim; Rivers, Philip; Goldsworthy, Susan; Looney, Noeleen A; Hanvey, Jeff; Plumpton, Chris; Parham, Janet; Romanos, Michael; Kost, Thomas A; Kull, Frederick C

    2004-08-01

    A cell-based time-resolved fluorescence (celTRF) immunoassay is described for pre-screening antibodies to G protein-coupled receptor (GPCR) peptides that predicts suitability for immunohistochemistry (IHC). Rat GPCRs were expressed in Saos-2 human osteosarcoma cells via recombinant baculoviruses designed for mammalian cell expression, i.e., the transduced cells were used as a "screening lawn". The lawn was fixed and permeabilized similarly to IHC tissue. The celTRF, a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), employed Eu-labelled goat anti-rabbit IgG. It exhibited a broad dynamic range upon which enzyme-linked immunosorbant assay (ELISA)-positive affinity-purified anti-peptide antibody reagents were examined for specificity and potency. Over 150 anti-peptide reagents to 27 GPCRs were characterized. All celTRF-positive antibodies were found to be suitable for IHC, whereas ELISA alone did not predict IHC utility. Examples are illustrated with five rabbit anti-neuropeptide FF receptor 1 (NPFF1) antibodies, where a strong correlation between celTRF potency and IHC utility was observed in both applications. In contrast, two high anti-peptide ELISA titer but celTRF-negative antibodies failed to recognize the NPFF1 receptor in IHC. The celTRF assay was performed manually and in an automated fashion, in our case, using a Biomek FX station and Sami scheduling software. The celTRF is the first in vitro automated assay that offers confident pre-selection of antibodies for IHC and the versatility to accommodate the rapid screening of large numbers of GPCRs. The celTRF is readily applicable to other protein target classes.

  3. 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.

  4. Time-Resolved Fluorescent Resonance Energy Transfer Assay for Simple and Rapid Detection of Anti-Brucella Antibodies in Ruminant Serum Samples▿

    PubMed Central

    McGiven, John A.; Thompson, Iain J.; Commander, Nicola J.; Stack, Judy A.

    2009-01-01

    Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucella monoclonal antibody with a long-lifetime donor fluorophore and Brucella smooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n = 73) and uninfected (n = 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases. PMID:19656980

  5. 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

    2016-09-08

    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

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

    PubMed

    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.

  7. 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

  8. A comparison of the three isoforms of the light-harvesting complex II using transient absorption and time-resolved fluorescence measurements.

    PubMed

    Palacios, Miguel A; Standfuss, Joerg; Vengris, Mikas; van Oort, Bart F; van Stokkum, Ivo H M; Kühlbrandt, Werner; van Amerongen, Herbert; van Grondelle, Rienk

    2006-06-01

    In this article we report the characterization of the energy transfer process in the reconstituted isoforms of the plant light-harvesting complex II. Homotrimers of recombinant Lhcb1 and Lhcb2 and monomers of Lhcb3 were compared to native trimeric complexes. We used low-intensity femtosecond transient absorption (TA) and time-resolved fluorescence measurements at 77 K and at room temperature, respectively, to excite the complexes selectively in the chlorophyll b absorption band at 650 nm with 80 fs pulses and on the high-energy side of the chlorophyll a absorption band at 662 nm with 180 fs pulses. The subsequent kinetics was probed at 30-35 different wavelengths in the region from 635 to 700 nm. The rate constants for energy transfer were very similar, indicating that structurally the three isoforms are highly homologous and that probably none of them play a more significant role in light-harvesting and energy transfer. No signature has been found in the transient absorption measurements at 77 K for Lhcb3 which might suggest that this protein acts as a relative energy sink of the excitations in heterotrimers of Lhcb1/Lhcb2/Lhcb3. Minor differences in the amplitudes of some of the rate constants and in the absorption and fluorescence properties of some pigments were observed, which are ascribed to slight variations in the environment surrounding some of the chromophores depending on the isoform. The decay of the fluorescence was also similar for the three isoforms and multi-exponential, characterized by two major components in the ns regime and a minor one in the ps regime. In agreement with previous transient absorption measurements on native LHC II complexes, Chl b --> Chl a energy transfer exhibited very fast channels but at the same time a slow component (ps). The Chls absorbing at around 660 nm exhibited both fast energy transfer which we ascribe to transfer from 'red' Chl b towards 'red' Chl a and slow transfer from 'blue' Chl a towards 'red' Chl a. The

  9. Nuclear magnetic resonance, fluorescence correlation spectroscopy and time-resolved fluorescence anisotropy studies of intermolecular interactions in bis(1-methyl-1H-imidazol-3-ium-3-yl)dihydroborate bis(trifluoromethylsulfonyl)amide and its mixtures with various cosolvents

    NASA Astrophysics Data System (ADS)

    Sahu, Prabhat Kumar; Nanda, Raju; Seth, Sudipta; Ghosh, Arindam; Sarkar, Moloy

    2016-09-01

    Keeping in mind the potential usefulness of mixed ionic liquid (IL)-cosolvents systems in several industrial applications, intermolecular interactions between a borate-based IL, bis(1-methyl-1H-imidazol-3-ium-3-yl)dihydroborate bis(trifluoromethylsulfonyl)amide ([BIMIMDBA][TF2N]), and its binary mixtures with several molecular solvents has been investigated through NMR and fluorescence spectroscopy. Analysis of the 1H chemical shifts (δ/ppm) and translational diffusion coefficients (D) of the IL in different solvent mixtures demonstrate interplay of nonspecific (ion-dipole) and specific (hydrogen bonding) interactions in governing the properties of these mixtures. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy data provide evidence in favour of different IL-solvent interaction for different IL-cosolvent systems.

  10. A Time-Resolved Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of 14-3-3 Protein–Protein Interaction Inhibitors

    PubMed Central

    Fu, Robert W.; Lou, Bin; Zhao, Jing; Qui, Min; Khuri, Fadlo R.; Fu, Haian

    2013-01-01

    Abstract Protein–protein interaction networks mediate diverse biological processes by regulating various signaling hubs and clusters. 14-3-3 proteins, a family of phosphoserine/threonine-binding molecules, serve as major interaction hubs in eukaryotic cells and have emerged as promising therapeutic targets for various human diseases. In order to identify chemical probes for mechanistic studies and for potential therapeutic development, we have developed highly sensitive bioassays to monitor the interaction of 14-3-3 with a client protein. In this study, we describe a homogenous time-resolved fluorescence resonance energy transfer (TR-FRET) assay to detect the interaction of 14-3-3 with Bad, a proapoptotic member of the Bcl-2 family. Through a series of titration studies in which europium-labeled 14-3-3 serves as an FRET donor and a Dy647-labeled phosphorylated Bad, the peptide acts as an FRET acceptor, we have achieved a robust TR-FRET assay that is suitable for high-throughput screening (HTS) with an excellent signal-to-background ratio of >20 and Z′ values >0.7. This assay was further miniaturized to a 1,536-well format for ultra-HTS (uHTS), and exhibited a similar robust performance. The utility and performance of the assay for uHTS were validated by (i) known inhibitors, including peptide R18 and small molecule FOBISIN101, and (ii) screening of a 51,200 compound library. This simple and robust assay is generally applicable to detect the interaction of 14-3-3 with other client proteins. It provides a sensitive and easy-to-use tool to facilitate the discovery of 14-3-3 protein inhibitors as well as to study 14-3-3-mediated protein–protein interactions. PMID:23906346

  11. An investigation of dynamic surface tension, critical micelle concentration, and aggregation number of three nonionic surfactants using NMR, time-resolved fluorescence quenching, and maximum bubble pressure tensiometry.

    PubMed

    Kjellin, U R Mikael; Reimer, Johan; Hansson, Per

    2003-06-15

    Several physicochemical properties have been determined for N-dodecyllactobionamide (LABA), maltose 6'-O-dodecanoate (C12-maltose ester), and tetra(ethylene oxide) dodecyl amide (TEDAd). The increase in the flexibility of the sugar headgroup, enabling more possible molecular conformations, reduces the minimum area/molecule at the liquid-vapor interface obtained at the critical micelle concentration (cmc). The obtained cmc's were 0.35 mM (LABA), 0.3 mM (C12-maltose ester), and 0.5 mM (TEDAd). The monomer diffusion coefficient decreased with the molecular weight and increasing headgroup flexibility of the sugar headgroup, and values were in the range from 3.1 x 10(-10) to 3.6 x 10(-10) m2/s. The micelle diffusion coefficients (0.46 x 10(-10) to 0.68 x 10(-10) m2/s) indicated that the TEDAd micelles deviated most from spherical shape. The micelle aggregation numbers determined by time-resolved fluorescence quenching (TRFQ) were estimated to be 120+/-10 (LABA), 90+/-10 (C12-maltose ester), and 130+/-10 (TEDAd). The dynamic surface tension measurements show that the adsorption of TEDAd onto the liquid-vapor interface at short surface lifetimes is diffusion-limited, whereas an adsorption barrier is present for the sugar surfactants. The analysis of the dynamic surface tension data above the cmc shows that the rate of demicellization is faster for TEDAd than for the two sugar-based surfactants.

  12. Testing UK blood donors for exposure to human parvovirus 4 using a time-resolved fluorescence immunoassay to screen sera and Western blot to confirm reactive samples.

    PubMed

    Maple, Peter A C; Beard, Stuart; Parry, Ruth P; Brown, Kevin E

    2013-10-01

    Human parvovirus 4 (ParV4), a newly described member of the family Parvoviridae, like B19V, has been found in pooled plasma preparations. The extent, and significance, of ParV4 exposure in UK blood donors remain to be determined and reliable detection of ParV4 immunoglobulin (Ig)G, using validated methods, is needed. With ParV4 virus-like particles a ParV4 IgG time-resolved fluorescence immunoassay (TRFIA) was developed. There is no gold standard or reference assay for measuring ParV4 IgG and the utility of the TRFIA was first examined using a panel of sera from people who inject drugs (PWIDS)--a high-prevalence population for ParV4 infection. Western blotting was used to confirm the specificity of TRFIA-reactive sera. Two cohorts of UK blood donor sera comprising 452 sera collected in 1999 and 156 sera collected in 2009 were tested for ParV4 IgG. Additional testing for B19V IgG, hepatitis C virus antibodies (anti-HCV), and ParV4 DNA was also undertaken. The rate of ParV4 IgG seroprevalence in PWIDS was 20.7% and ParV4 IgG was positively associated with the presence of anti-HCV with 68.4% ParV4 IgG-positive sera testing anti-HCV-positive versus 17.1% ParV4 IgG-negative sera. Overall seropositivity for ParV4 IgG, in 608 UK blood donors was 4.76%. The ParV4 IgG seropositivity for sera collected in 1999 was 5.08%, compared to 3.84% for sera collected in 2009. No ParV4 IgG-positive blood donor sera had detectable ParV4 DNA. ParV4 IgG has been found in UK blood donors and this finding needs further investigation. © 2013 American Association of Blood Banks.

  13. Remarkable effects of solvent and substitution on the photo-dynamics of cytosine: a femtosecond broadband time-resolved fluorescence and transient absorption study.

    PubMed

    Ma, Chensheng; Cheng, Chopen Chan-Wut; Chan, Chris Tsz-Leung; Chan, Ruth Chau-Ting; Kwok, Wai-Ming

    2015-07-15

    Cytosine (Cyt) among all the nucleic acid bases features the most complex and least understood nonradiative deactivation, a process that is crucially important for its photostability. Herein, the excited state dynamics of Cyt and a series of its N1- and C5-derivatives, including the full set of Cyt nucleosides and nucleotides in DNA and RNA and the nucleosides of 5-methyl cytosine, 5-methylcytidine and 2'-deoxy-5-methylcytidine, have been investigated in water and in methanol employing femtosecond broadband time-resolved fluorescence coupled with fs transient absorption spectroscopy. The results reveal remarkable state-specific effects of the substitution and solvent in tuning distinctively the timescales and pathways of the nonradiative decays. For Cyt and the N1-derivatives, the nonradiative deactivations occur in a common two-state process through three channels, two from the light-absorbing ππ* state with respectively the sub-picosecond (∼0.2 ps) and the picosecond (∼1.5 ps) time constant, and the third is due to an optically dark nπ* state with the lifetime ranging from several to hundreds of picoseconds depending on solvents and substitutions. Compared to Cyt, the presence of the ribose or deoxyribose moiety at the N1 position of N1-derivatives facilitates the formation of the nπ* at the sub-picosecond timescale and at the same time increases its lifetime by ∼4-6 times in both water and methanol. In sharp contrast, the existence of the methyl group at the C5 position of the C5-derivatives eliminates completely the sub-picosecond ππ* channel and the channel due to the nπ*, but on the other hand slows down the decay of the ππ* state which after relaxation exhibits a single time constant of ∼4.1 to ∼7.6 ps depending on solvents. Varying the solvent from water to methanol accelerates only slightly the decay of the ππ* state in all the compounds; while for Cyt and its N1-derivatives, this change of solvent also retards strongly the n

  14. Group-selective antibodies based fluorescence immunoassay for monitoring opiate drugs.

    PubMed

    Gandhi, Sonu; Sharma, Prince; Capalash, Neena; Verma, R S; Suri, C Raman

    2008-09-01

    A novel carboxylic acid derivative of monoacetylmorphine (MAM-COOH) was synthesized and conjugated with bovine serum albumin (BSA) for generating polyclonal antibodies against the target molecule heroin and its major metabolites. The conjugate was characterized by fluorescence spectroscopy, polyacrylamide gel electrophoresis, and mass spectrometry to confirm the extent of haptenization of the carrier protein. A high titer (1:64,0000) of antibody was obtained by using the conjugate with an optimum protein/hapten molar ratio of 1:100. The generated antibody showed good binding affinity with heroin and its metabolites monoacetylmorphine (MAM) and morphine. The relative affinity constant (K (aff)) of the antibody was 3.1 x 10(7) l mol(-1), and the IC(50) values obtained for heroin, MAM, morphine, and codeine were 0.01, 0.013, 0.012, and 0.014 ng ml(-1), respectively. A fluorescence-based competitive inhibition immunoassay procedure was developed for the estimation of heroin and its major metabolites in standard and biofludic samples over a concentration range up to 0.01 ng ml(-1) with good signal reproducibility (p < 0.05). The method can be used as a convenient quantitative tool for the sensitive screening of major metabolites of heroin in biological samples.

  15. Time-resolved spectral studies of blue-green fluorescence of artichoke (Cynara cardunculus L. Var. Scolymus) leaves: identification of chlorogenic acid as one of the major fluorophores and age-mediated changes.

    PubMed

    Morales, Fermín; Cartelat, Aurélie; Alvarez-Fernández, Ana; Moya, Ismael; Cerovic, Zoran G

    2005-12-14

    Synchrotron radiation and the time-correlated single-photon counting technique were used to investigate the spectral and time-resolved characteristics of blue-green fluorescence (BGF) of artichoke leaves. Leaves emitted BGF under ultraviolet (UV) excitation; the abaxial side was much more fluorescent than the adaxial side, and in both cases, the youngest leaves were much more fluorescent than the oldest ones. The BGF of artichoke leaves was dominated by the presence of hydroxycinnamic acids. A decrease in the percentage of BGF attributable to the very short kinetic component (from 42 to 20%), in the shape of the BGF excitation spectra, and chlorogenic acid concentrations indicate that there is a loss of hydroxycinnamic acid with leaf age. Studies on excitation, emission, and synchronized fluorescence spectra of leaves and trichomes and chlorogenic acid contents indicate that chlorogenic acid is one of the main blue-green fluorophores in artichoke leaves. Results of the present study indicate that 20-42% (i.e., the very short kinetic component) of the overall BGF is emitted by chlorogenic acid. Time-resolved BGF measurements could be a means to extract information on chlorogenic acid fluorescence from the overall leaf BGF.

  16. Prediction of Complement-Binding Capacity of HLA Antibodies Based on Mean Fluorescence Intensity.

    PubMed

    Zhao, J; Fu, Y X; Yang, T; Shen, Z Y; Wu, C L

    2016-01-01

    Human leukocyte antigen (HLA) antibodies estimated by Luminex single-antigen beads, especially those that fix complement, are associated with antibody-mediated rejection and graft failure. However, the relationship between HLA antibody strength and complement-binding ability is controversial. Serum samples of 31 sensitized renal patients waiting for renal transplantation were retrospectively analyzed by IgG-Luminex to identify HLA antibodies and in parallel by C1q-Luminex to determine the complement binding of HLA antibodies. The percentage of HLA class I antibodies binding with C1q was lower than that of HLA class II antibodies (43.2% vs. 51.3%, P = .006). The mean fluorescence intensities (MFI) of IgG-Luminex correlated with the MFI of C1q-Luminex for the same antibodies (Spearman correlation; class I, r = 0.665, P < .01; class II, r = 0.761, P < .01). Receiver operating characteristic (ROC) curve analysis showed that the MFIs of HLA antibodies by IgG-Luminex predicted their C1q-binding abilities (area under the curve [AUC] class I = 0.917; AUC class II = 0.927). Using MFI cutoff values of 8238 and 6754 in IgG-Luminex for HLA class I and class II antibodies, respectively, the sensitivity and specificity for C1q binding were 82.4% and 87.4% for class I antibodies and 90.9% and 82% for class II antibodies. The MFI of HLA antibodies by IgG-Luminex predicts the complement-binding capability to a certain extent before transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. The dependence of the ultrafast relaxation kinetics of the S(2) and S(1) states in beta-carotene homologs and lycopene on conjugation length studied by femtosecond time-resolved absorption and Kerr-gate fluorescence spectroscopies.

    PubMed

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

    2009-06-07

    The ultrafast relaxation kinetics of all-trans-beta-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 S(2)(1(1)B(u) (+)) state were precisely determined by the time-resolved fluorescence. The kinetics of the optically forbidden S(1)(2(1)A(g) (-)) state were observed by the time-resolved absorption measurements. The dependence of the S(1) 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 S(2) 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 S(2) relaxation rates can be explained by the presence of an intermediate state (here called the S(x) state) located between the S(2) and S(1) 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 S(2)-->S(x)-->S(1)-->S(0). 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.

  18. 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.

  19. Influence of heterogeneity of confined water on photophysical behavior of acridine with amines: a time-resolved fluorescence and laser flash photolysis study.

    PubMed

    Sarangi, Manas Kumar; Dey, Debarati; Basu, Samita

    2011-01-20

    The photophysical behavior of acridine (Acr) shows facilitated water-assisted protonation equilibrium between its deprotonted (Acr* ∼ 10 ns) and protonated forms (AcrH(+*) ∼ 28 ns) within confined region of ordered water molecules inside AOT/H(2)O/n-heptane reverse micelles (RMs). The time-resolved-area-normalized-emission spectra confirm both Acr* and AcrH(+*), while time-resolved-emission spectra depict time evolution between them. Quenching of AcrH(+*) with N,N-dimethylaniline (DMA) is a purely diffusion-controlled bimolecular quenching with linear Stern-Volmer (S-V) plot, while nonlinearity arises with triethylamine (TEA) that forms ground state complex with AcrH(+) (AcrH(+)··H(2)O··TEA) indicating both static and dynamic quenching. Transient intermediates, DMA(•+) and AcrH(•) infer photoinduced electron transfer from DMA to Acr, while those from AcrH(+)··H(2)O··TEA complex suggest water mediated excited-state proton transfer (ESPT) between AcrH(+) and TEA. The ESPT becomes faster in larger RMs due to enhanced mobility of hydronium ions in AcrH(+)··H(2)O··TEA, which reduces in smaller RMs as water becomes much more constrained owing to stronger complexation by excess confinement.

  20. Time-Resolved Measurements in Optoelectronic Microbioanalysis

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory; Kossakovski, Dmitri

    2003-01-01

    A report presents discussion of time-resolved measurements in optoelectronic microbioanalysis. Proposed microbioanalytical laboratory-on-a-chip devices for detection of microbes and toxic chemicals would include optoelectronic sensors and associated electronic circuits that would look for fluorescence or phosphorescence signatures of multiple hazardous biomolecules in order to detect which ones were present in a given situation. The emphasis in the instant report is on gating an active-pixel sensor in the time domain, instead of filtering light in the wavelength domain, to prevent the sensor from responding to a laser pulse used to excite fluorescence or phosphorescence while enabling the sensor to respond to the decaying fluorescence or phosphorescence signal that follows the laser pulse. The active-pixel sensor would be turned on after the laser pulse and would be used to either integrate the fluorescence or phosphorescence signal over several lifetimes and many excitation pulses or else take time-resolved measurements of the fluorescence or phosphorescence. The report also discusses issues of multiplexing and of using time-resolved measurements of fluorophores with known different fluorescence lifetimes to distinguish among them.

  1. Complexity of Lipid Domains and Rafts in Giant Unilamellar Vesicles Revealed by Combining Imaging and Microscopic and Macroscopic Time-Resolved Fluorescence

    PubMed Central

    de Almeida, Rodrigo F. M.; Borst, JanWillem; Fedorov, Alexander; Prieto, Manuel; Visser, Antonie J. W. G.

    2007-01-01

    The application of fluorescence lifetime imaging microscopy to study gel/fluid and raftlike lipid domains in giant unilamellar vesicles (GUVs) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine/cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid-ordered (lo)/liquid-disordered (ld) phase separation were clearly visualized upon two-photon excitation. Fluorescence intensity decays in different regions of a GUV were also obtained with the microscope in fixed laser-beam configuration. The ensemble behavior of the system was studied by obtaining fluorescence intensity decays of Rhod-DOPE in nongiant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of lo raftlike phase, based on fluorescence lifetime imaging microscopy histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of three lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the lo phase is formed in the binary dipalmitoylphosphatidylcholine/cholesterol but not in the dioleoylphosphatidylcholine/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex substructure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed. PMID:17449668

  2. Detailed Study of BSA Adsorption on Micro- and Nanocrystalline Diamond/β-SiC Composite Gradient Films by Time-Resolved Fluorescence Microscopy.

    PubMed

    Handschuh-Wang, Stephan; Wang, Tao; Druzhinin, Sergey I; Wesner, Daniel; Jiang, Xin; Schönherr, Holger

    2017-01-24

    The adsorption of bovine serum albumin (BSA) on micro- and nanocrystalline diamond/β-SiC composite films synthesized using the hot filament chemical vapor deposition (HFCVD) technique has been investigated by confocal fluorescence lifetime imaging microscopy. BSA labeled with fluorescein isothiocyanate (FITC) was employed as a probe. The BSA(FITC) conjugate was found to preferentially adsorb on both O-/OH-terminated microcrystalline and nanocrystalline diamond compared to the OH-terminated β-SiC, resulting in an increasing amount of BSA adsorbed to the gradient surfaces with an increasing diamond/β-SiC ratio. The different strength of adsorption (>30 times for diamond with a grain size of 570 nm) coincides with different surface energy parameters and differing conformational changes upon adsorption. Fluorescence data of the adsorbed BSA(FITC) on the gradient film with different diamond coverage show a four-exponential decay with decay times of 3.71, 2.54, 0.66, and 0.13 ns for a grain size of 570 nm. The different decay times are attributed to the fluorescence of thiourea fluorescein residuals of linked FITC distributed in BSA with different dye-dye and dye-surface distances. The longest decay time was found to correlate linearly with the diamond grain size. The fluorescence of BSA(FITC) undergoes external dynamic fluorescence quenching on the diamond surface by H- and/or sp(2)-defects and/or by amorphous carbon or graphite phases. An acceleration of the internal fluorescence concentration quenching in BSA(FITC) because of structural changes of albumin due to adsorption, is concluded to be a secondary contributor. These results suggest that the micro- and nanocrystalline diamond/β-SiC composite gradient films can be utilized to spatially control protein adsorption and diamond crystallite size, which facilitates systematic studies at these interesting (bio)interfaces.

  3. Detection of radiation-induced brain necrosis in live rats using label-free time-resolved fluorescence spectroscopy (TRFS) (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hartl, Brad A.; Ma, Htet S. W.; Sridharan, Shamira; Hansen, Katherine; Klich, Melanie; Perks, Julian; Kent, Michael; Kim, Kyoungmi; Fragoso, Ruben; Marcu, Laura

    2017-02-01

    Differentiating radiation-induced necrosis from recurrent tumor in the brain remains a significant challenge to the neurosurgeon. Clinical imaging modalities are not able to reliably discriminate the two tissue types, making biopsy location selection and surgical management difficult. Label-free fluorescence lifetime techniques have previously been shown to be able to delineate human brain tumor from healthy tissues. Thus, fluorescence lifetime techniques represent a potential means to discriminate the two tissues in real-time during surgery. This study aims to characterize the endogenous fluorescence lifetime signatures from radiation induced brain necrosis in a tumor-free rat model. Fischer rats received a single fraction of 60 Gy of radiation to the right hemisphere using a linear accelerator. Animals underwent a terminal live surgery after gross necrosis had developed, as verified with MRI. During surgery, healthy and necrotic brain tissue was measured with a fiber optic needle connected to a multispectral fluorescence lifetime system. Measurements of the necrotic tissue showed a 48% decrease in intensity and 20% increase in lifetimes relative to healthy tissue. Using a support vector machine classifier and leave-one-out validation technique, the necrotic tissue was correctly classified with 94% sensitivity and 97% specificity. Spectral contribution analysis also confirmed that the primary source of fluorescence contrast lies within the redox and bound-unbound population shifts of nicotinamide adenine dinucleotide. A clinical trial is presently underway to measure these tissue types in humans. These results show for the first time that radiation-induced necrotic tissue in the brain contains significantly different metabolic signatures that are detectable with label-free fluorescence lifetime techniques.

  4. Nonphotochemical quenching of excitation energy in photosystem II. A picosecond time-resolved study of the low yield of chlorophyll a fluorescence induced by single-turnover flash in isolated spinach thylakoids.

    PubMed

    Vasil'ev, S; Bruce, D

    1998-08-04

    Chlorophyll a fluorescence emission is widely used as a noninvasive measure of a number of parameters related to photosynthetic efficiency in oxygenic photosynthetic organisms. The most important component for the estimation of photochemistry is the relative increase in fluorescence yield between dark-adapted samples which have a maximal capacity for photochemistry and a minimal fluorescence yield (F0) and light-saturated samples where photochemistry is saturated and fluorescence yield is maximal (Fm). However, when photosynthesis is saturated with a short (less than 50 micro(s)) flash of light, which induces only one photochemical turnover of photosystem II, the maximal fluorescence yield is significantly lower (Fsat) than when saturation is achieved with a millisecond duration multiturnover flash (Fm). To investigate the origins of the difference in fluorescence yield between these two conditions, our time-resolved fluorescence apparatus was modified to allow collection of picosecond time-resolved decay kinetics over a short time window immediately following a saturating single-turnover flash (Fsat) as well as after a multiturnover saturating pulse (Fm). Our data were analyzed with a global kinetic model based on an exciton radical pair equilibrium model for photosystem II. The difference between Fm and Fsat was modeled well by changing only the rate constant for quenching of excitation energy in the antenna of photosystem II. An antenna-based origin for the quenching was verified experimentally by the observation that addition of the antenna quencher 5-hydroxy-1,4-naphthoquinone to thylakoids under Fm conditions resulted in decay kinetics and modeled kinetic parameters very similar to those observed under Fsat conditions in the absence of added quinone. Our data strongly support the origin of low fluorescence yield at Fsat to be an antenna-based nonphotochemical quenching of excitation energy in photosystem II which has not usually been considered explicitly in

  5. Multimode Surface Functional Group Determination: Combining Steady-State and Time-Resolved Fluorescence with X-ray Photoelectron Spectroscopy and Absorption Measurements for Absolute Quantification.

    PubMed

    Fischer, Tobias; Dietrich, Paul M; Unger, Wolfgang E S; Rurack, Knut

    2016-01-19

    The quantitative determination of surface functional groups is approached in a straightforward laboratory-based method with high reliability. The application of a multimode BODIPY-type fluorescence, photometry, and X-ray photoelectron spectroscopy (XPS) label allows estimation of the labeling ratio, i.e., the ratio of functional groups carrying a label after reaction, from the elemental ratios of nitrogen and fluorine. The amount of label on the surface is quantified with UV/vis spectrophotometry based on the molar absorption coefficient as molecular property. The investigated surfaces with varying density are prepared by codeposition of 3-(aminopropyl)triethoxysilane (APTES) and cyanoethyltriethoxysilane (CETES) from vapor. These surfaces show high functional group densities that result in significant fluorescence quenching of surface-bound labels. Since alternative quantification of the label on the surface is available through XPS and photometry, a novel method to quantitatively account for fluorescence quenching based on fluorescence lifetime (τ) measurements is shown. Due to the complex distribution of τ on high-density surfaces, the stretched exponential (or Kohlrausch) function is required to determine representative mean lifetimes. The approach is extended to a commercial Rhodamine B isothiocyanate (RITC) label, clearly revealing the problems that arise from such charged labels used in conjunction with silane surfaces.

  6. 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

  7. Electronic energy migration on different time scales: concentration dependence of the time-resolved anisotropy and fluorescence quenching of Lumogen Red in poly(methyl methacrylate).

    PubMed

    Colby, Kathryn A; Burdett, Jonathan J; Frisbee, Robert F; Zhu, Lingyan; Dillon, Robert J; Bardeen, Christopher J

    2010-03-18

    Electronic energy transfer plays an important role in many types of organic electronic devices. Forster-type theories of exciton diffusion provide a way to calculate diffusion constants and lengths, but their applicability to amorphous polymer systems must be evaluated. In this paper, the perylenediimide dye Lumogen Red in a poly(methyl methacrylate) host matrix is used to test theories of exciton motion over Lumogen Red concentrations (C(LR)'s) ranging from 1 x 10(-4) to 5 x 10(-2) M. Two experimental quantities are measured. First, time-resolved anisotropy decays in films containing only Lumogen Red provide an estimate of the initial energy transfer rate from the photoexcited molecule. Second, the Lumogen Red lifetime decays in mixed systems where the dyes Malachite Green and Rhodamine 700 act as energy acceptors are measured to estimate the diffusive quenching of the exciton. From the anisotropy measurements, it is found that theory accurately predicts both the C(LR)(-2) concentration dependence of the polarization decay time tau(pol), as well as its magnitude to within 30%. The theory also predicts that the diffusive quenching rate is proportional to C(LR)(alpha), where alpha ranges between 1.00 and 1.33. Experimentally, it is found that alpha = 1.1 +/- 0.2 when Malachite Green is used as an acceptor, and alpha = 1.2 +/- 0.2 when Rhodamine 700 is the acceptor. On the basis of the theory that correctly describes the anisotropy data, the exciton diffusion constant is projected to be 4-9 nm(2)/ns. By use of several different analysis methods for the quenching data, the experimental diffusion constant is found to be in the range of 0.32-1.20 nm(2)/ns. Thus the theory successfully describes the early time anisotropy data but fails to quantitatively describe the quenching experiments which are sensitive to motion on longer time scales. The data are consistent with the idea that orientational and energetic disorder leads to a time-dependent exciton migration rate

  8. Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation

    PubMed Central

    1995-01-01

    The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature- dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of

  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. Time-Resolved Fluorescence Anisotropy of Bicyclo[1.1.1]pentane/Tolane-Based Molecular Rods Included in Tris(o-phenylenedioxy)cyclotriphosphazene (TPP).

    PubMed

    Cipolloni, Marco; Kaleta, Jiří; Mašát, Milan; Dron, Paul I; Shen, Yongqiang; Zhao, Ke; Rogers, Charles T; Shoemaker, Richard K; Michl, Josef

    2015-04-23

    We examine the fluorescence anisotropy of rod-shaped guests held inside the channels of tris(o-phenylenedioxy)cyclotriphosphazene (TPP) host nanocrystals, characterized by powder X-ray diffraction and solid state NMR spectroscopy. We address two issues: (i) are light polarization measurements on an aqueous colloidal solution of TPP nanocrystals meaningful, or is depolarization by scattering excessive? (ii) Can measurements of the rotational mobility of the included guests be performed at low enough loading levels to suppress depolarization by intercrystallite energy transfer? We find that meaningful measurements are possible and demonstrate that the long axis of molecular rods included in TPP channels performs negligible vibrational motion.

  11. A study of the time-resolved fluorescence spectrum and red edge effect of ANF in a room-temperature ionic liquid.

    PubMed

    Hu, Zhonghan; Margulis, Claudio J

    2006-06-15

    In a recent article, we have analyzed using molecular dynamics simulations the steady-state red edge effect (REE) observed by Samanta and co-workers when the fluorescent probe 2-amino-7-nitrofluorene (ANF) is photoexcited at different wavelengths in 1-butyl-3-methylimidazolium ([BMIM+]) hexafluorophosphate ([PF6-]). In this letter, we predict the time- and wavelength-dependent emission spectra of ANF in the same ionic solvent. From the analysis of our simulated data, we are able to derive an approximate time scale for reorganization of the solvent around the solute probe. The effect that slow varying local liquid environments have on the overall time-dependent signal is also discussed.

  12. Time-Resolved Fluorescence Anisotropy of Bicyclo[1.1.1]pentane/Tolane-Based Molecular Rods Included in Tris(o-phenylenedioxy)cyclotriphosphazene (TPP)

    PubMed Central

    2015-01-01

    We examine the fluorescence anisotropy of rod-shaped guests held inside the channels of tris(o-phenylenedioxy)cyclotriphosphazene (TPP) host nanocrystals, characterized by powder X-ray diffraction and solid state NMR spectroscopy. We address two issues: (i) are light polarization measurements on an aqueous colloidal solution of TPP nanocrystals meaningful, or is depolarization by scattering excessive? (ii) Can measurements of the rotational mobility of the included guests be performed at low enough loading levels to suppress depolarization by intercrystallite energy transfer? We find that meaningful measurements are possible and demonstrate that the long axis of molecular rods included in TPP channels performs negligible vibrational motion. PMID:25937858

  13. Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells. Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer.

    PubMed

    Gazi, Lucien; López-Giménez, Juan F; Rüdiger, Martin P; Strange, Philip G

    2003-10-01

    Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of approximately 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R-(-)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands.

  14. Dynamic Solvent Control of a Reaction in Ionic Deep Eutectic Solvents: Time-Resolved Fluorescence Measurements of Reactive and Nonreactive Dynamics in (Choline Chloride + Urea) Melts.

    PubMed

    Das, Anuradha; Biswas, Ranjit

    2015-08-06

    Dynamic fluorescence anisotropy and Stokes shift measurements of [f choline chloride + (1 - f) urea)] deep eutectic solvents at f = 0.33 and 0.40 have been carried out using a dipolar solute, coumarin 153 (C153), in the temperature range 298 ≤ T ≤ 333 K. Subsequently, measured time-dependent solvent response is utilized to investigate the dynamic solvent control on the measured rates of photoexcited intramolecular charge transfer (ICT) reactions of two molecules, 4-(1-azetidinyl)benzonitrile (P4C) and 4-(1-pyrrolidinyl)benzonitrile (P5C), occurring in these media. Measured average reaction time scales (⟨τ(rxn)⟩) exhibit the following dependence on average solvation times scales (⟨τ(s)⟩): ⟨τ(rxn)⟩ ∝ ⟨τ(s)⟩(α) with α = 0.5 and 0.35 for P4C and P5C, respectively. Such a strong dynamic solvent control of ⟨τ(rxn)⟩, particularly for P4C, is different from earlier observations with these ICT molecules in conventional molecular solvents. Excitation wavelength-dependent fluorescence emissions of C153 and trans-2-[4-(dimethylamino)styryl]-benzothiazole (DMASBT), which differ widely in average fluorescence lifetimes (⟨τ(life)⟩), suggest the presence of substantial spatial heterogeneity in these systems. Dynamic heterogeneity is reflected via the following fractional viscosity (η) dependences of ⟨τ(s)⟩ and ⟨τ(r)⟩ (⟨τ(r)⟩ being solute's average rotation time): ⟨τx⟩ ∝ (η/T)(p) with 0.7 ≤ p ≤ 0.9. Different correlations between ⟨τ(s)⟩ and ⟨τ(r)⟩ emerge at different temperature regimes, indicating variable frictional coupling at low and high temperatures. Estimated dynamic Stokes shifts in these media vary between ∼1200 and ∼1600 cm(-1), more than 50% of which possess a time scale much faster than the temporal resolution (∼75 ps) employed in these measurements. Estimated activation energy for η is closer to that for ⟨τ(r)⟩ than that for ⟨τ(s)⟩, suggesting ⟨τ(s)⟩ being more decoupled

  15. Comparison of microenvironments of aqueous sodium dodecyl sulfate micelles in the presence of inorganic and organic salts: a time-resolved fluorescence anisotropy approach.

    PubMed

    Dutt, G B

    2005-11-08

    Microenvironments of aqueous sodium dodecyl sulfate (SDS) micelles was examined in the presence of additives such as sodium chloride and p-toluidine hydrochloride (PTHC) by monitoring the fluorescence anisotropy decays of two hydrophobic probes, 2,5-dimethyl-1,4-dioxo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DMDPP) and coumarin 6 (C6). It has been well-established that SDS micelles undergo a sphere-to-rod transition and that their mean hydrodynamic radius increases from 19 to 100 A upon the addition of 0.0-0.7 M NaCl at 298 K. A similar size and shape transition is induced by PTHC at concentrations that are 20 times lower compared to that of NaCl. This study was undertaken to find out how the microviscosity of the micelles is influenced under these circumstances. It was noticed that the microviscosity of the SDS/NaCl system increased by approximately 45%, whereas there was a less than 10% variation in the microviscosity of the SDS/PTHC system. The large increase in the microviscosity of the former system with salt concentration has been rationalized on the basis of the high concentration of sodium ions in the headgroup region of the micelles and their ability to strongly coordinate with the water present in this region, which decreases the mobility of the probe molecules.

  16. Time-resolved vibrational spectroscopy

    SciTech Connect

    Tokmakoff, Andrei; Champion, Paul; Heilweil, Edwin J.; Nelson, Keith A.; Ziegler, Larry

    2009-05-14

    This document contains the Proceedings from the 14th International Conference on Time-Resolved Vibrational Spectroscopy, which was held in Meredith, NH from May 9-14, 2009. The study of molecular dynamics in chemical reaction and biological processes using time-resolved spectroscopy plays an important role in our understanding of energy conversion, storage, and utilization problems. Fundamental studies of chemical reactivity, molecular rearrangements, and charge transport are broadly supported by the DOE's Office of Science because of their role in the development of alternative energy sources, the understanding of biological energy conversion processes, the efficient utilization of existing energy resources, and the mitigation of reactive intermediates in radiation chemistry. In addition, time-resolved spectroscopy is central to all fiveof DOE's grand challenges for fundamental energy science. The Time-Resolved Vibrational Spectroscopy conference is organized biennially to bring the leaders in this field from around the globe together with young scientists to discuss the most recent scientific and technological advances. The latest technology in ultrafast infrared, Raman, and terahertz spectroscopy and the scientific advances that these methods enable were covered. Particular emphasis was placed on new experimental methods used to probe molecular dynamics in liquids, solids, interfaces, nanostructured materials, and biomolecules.

  17. Rate constant for the reaction of OH with methyl iodide, a re-determination by flash photolysis of water vapour and time resolved resonance fluorescence of OH

    NASA Astrophysics Data System (ADS)

    Zhang, Shaoliang; Strekowski, Rafal; Zetzsch, Cornelius

    2010-05-01

    Methyl iodide is a major source gas for atmospheric iodine, and it is mainly emitted from the ocean. Aqueous-phase reactions, such as hydrolysis and exchange reactions with chloride control its emissions to the atmosphere, where its lifetime is limited to less than a week, mainly by photolysis. A minor contribution to the loss processes in the troposphere is the gas-phase reaction with OH radicals, that has been investigated by several authors. On the other hand, this reaction turned out to be uncertain in spite of interest in nuclear safety after the International Phebus Fission Product programme, initiated in 1988. Some of the most important observed phenomena with regard to the chemistry of iodine were not predicted, clearly showing the need for carrying out rate constant determinations for the reactions of I2 and CH3I with OH, which is a major oxidant product from the air radiolysis under accident conditions. We have measured the rate constant for the reaction OH + CH3I - H2O + CH2I in He at 260 mbar in the temperature range from 298 to 362 K. OH radicals were produced by flash photolysis of H2O in the vacuum-UV at wavelengths > 115 nm using a Xe flash lamp with a MgF2 window. Time profiles of OH radicals are monitored by resonance fluorescence of the A2 Σ - X2 Π transition at 308 nm, induced by the emission from a microwave discharge of a flow of He and H2O, a few Torr each. The signal is monitored by photon counting and multichannel scaling, collecting the counts from 50 flashes each, obtaind by pulsed photolysis of various mixtures of H2O and CH3I under slow-flow conditions. Decays of OH in the presence of CH3I are observed to be exponential, and the decay rates are found to be linearly dependent on the concentration of CH3I. Rate constants, k ± 2σ (in units of 10-14 cm3 s-1) of 4.14±0.20, 6.33±0.68, 7.31±1.18 and 8.24±1.60 at 298, 326, 352 and 362 K, respectively, are obtained from linear regressions and lead to an Arrhenius expression of k = 1.5

  18. Time resolved fluorescence and energy transfer analysis of Nd(3+)-Yb (3+)-Er (3+) triply-doped Ba-Al-metaphosphate glasses for an eye safe emission (1.54 microm).

    PubMed

    Sontakke, Atul D; Biswas, Kaushik; Mandal, Ashis K; Annapurna, Kalyandurg

    2010-01-01

    This paper reports on the preparation and systematic analysis of energy transfer mechanisms in Nd(3+)-Yb(3+)-Er(3+) co-doped new series of barium-alumino-metaphosphate glasses. The time resolved fluorescence of Nd(3+) in triply doped Ba-Al-metaphosphate glasses have revealed that, Yb(3+) ions could function as quite efficient bridge for an energy transfer between Nd(3+) and Er(3+) ions. As a result, a fourfold emission enhancement at 1.54 mum of Er(3+) ions has been achieved through an excitation of (4)F(5/2) level of Nd(3+) at 806 nm for the glass having 3 mol% Yb(3+) with an energy transfer efficiency reaching up to 94%. Decay of donor (Nd(3+)) ion fluorescence has been analyzed based on theoretical models such as direct energy transfer model (Inokuti-Hirayama) and migration assisted energy transfer models (Burshtein's hopping and Yokota-Tanimoto's diffusion). The corresponding energy transfer parameters have been evaluated and discussed. Primarily, electrostatic dipole-dipole (s approximately 6) interactions are found to be responsible for the occurrence of energy transfer process in theses glasses.

  19. Binding of 7-methoxy-4-(aminomethyl)-coumarin to wild-type and W128F mutant cytochrome P450 2D6 studied by time-resolved fluorescence spectroscopy

    PubMed Central

    Stortelder, Aike; Keizers, Peter H. J.; Oostenbrink, Chris; De Graaf, Chris; De Kruijf, Petra; Vermeulen, Nico P. E.; Gooijer, Cees; Commandeur, Jan N. M.; Van Der Zwan, Gert

    2005-01-01

    Enzyme structure and dynamics may play a main role in substrate binding and the subsequent steps in the CYP (cytochrome P450) catalytic cycle. In the present study, changes in the structure of human CYP2D6 upon binding of the substrate are studied using steady-state and time-resolved fluorescence methods, focusing not only on the emission of the tryptophan residues, but also on emission of the substrate. As a substrate, MAMC [7-methoxy-4-(aminomethyl)-coumarin] was selected, a compound exhibiting native fluorescence. As well as the wild-type, the W128F (Trp128→Phe) mutant of CYP2D6 was studied. After binding, a variety of energy transfer possibilities exist, and molecular dynamics simulations were performed to calculate distances and relative orientations of donors and acceptors. Energy transfer from Trp128 to haem appeared to be important; its emission was related to the shortest of the three average tryptophan fluorescence lifetimes observed for CYP2D6. MAMC to haem energy transfer was very efficient as well: when bound in the active site, the emission of MAMC was fully quenched. Steady-state anisotropy revealed that besides the MAMC in the active site, another 2.4% of MAMC was bound outside of the active site to wild-type CYP2D6. The tryptophan residues in CYP2D6 appeared to be less accessible for the external quenchers iodide and acrylamide in presence of MAMC, indicating a tightening of the enzyme structure upon substrate binding. However, the changes in the overall enzyme structure were not very large, since the emission characteristics of the enzyme were not very different in the presence of MAMC. PMID:16190863

  20. Engineering out motion: a surface disulfide bond alters the mobility of tryptophan 22 in cytochrome b5 as probed by time-resolved fluorescence and 1H NMR experiments.

    PubMed

    Storch, E M; Grinstead, J S; Campbell, A P; Daggett, V; Atkins, W M

    1999-04-20

    In the accompanying paper [Storch et al. (1999) Biochemistry 38, 5054-5064] equilibrium denaturation studies and molecular dynamics (MD) simulations were used to investigate localized dynamics on the surface of cytochrome b5 (cyt b5) that result in the formation of a cleft. In those studies, an S18C:R47C disulfide mutant was engineered to inhibit cleft mobility. Temperature- and urea-induced denaturation studies revealed significant differences in Trp 22 fluorescence between the wild-type and mutant proteins. On the basis of the results, it was proposed that wild type populates a conformational ensemble that is unavailable to the disulfide mutant and is mediated by cleft mobility. As a result, the solvent accessibility of Trp 22 is decreased in S18C:R47C, suggesting that the local environment of this residue is less mobile due to the constraining effects of the disulfide on cleft dynamics. To further probe the structural effects on the local environment of Trp 22 caused by inhibition of cleft formation, we report here the results of steady-state and time-resolved fluorescence quenching, differential phase/modulation fluorescence anisotropy, and 1H NMR studies. In Trp fluorescence experiments, the Stern-Volmer quenching constant increases in wild type versus the oxidized disulfide mutant with increasing temperature. At 50 degrees C, KSV is nearly 1.5-fold greater in wild type compared to the oxidized disulfide mutant. In the reduced disulfide mutant, KSV was the same as wild type. The bimolecular collisional quenching constant, kq, for acrylamide quenching of Trp 22 increases 2.7-fold for wild type and only 1.8-fold for S18C:R47C, upon increasing the temperature from 25 to 50 degrees C. The time-resolved anisotropy decay at 25 degrees C was fit to a double-exponential decay for both the wild type and S18C:R47C. Both proteins exhibited a minor contribution from a low-amplitude fast decay, consistent with local motion of Trp 22. This component was more prevalent in

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

    PubMed

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

    2017-02-20

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

  2. Dynamics of solvent and rotational relaxation of coumarin 153 in room-temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate confined in Brij-35 micelles: a picosecond time-resolved fluorescence spectroscopic study.

    PubMed

    Chakraborty, Anjan; Seth, Debabrata; Chakrabarty, Debdeep; Setua, Palash; Sarkar, Nilmoni

    2005-12-15

    The dynamics of solvent and rotational relaxation of Coumarin 153 (C-153) in ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]) and in the ionic liquid confined in Brij-35 micellar aggregates have been investigated using steady-state and time-resolved fluorescence spectroscopy. We observed slower dynamics in the presence of micellar aggregates as compared to the pure IL. However, the slowing down in the solvation time on going from neat IL to IL-confined micelles is much smaller compared to that on going from water to water-confined micellar aggregates. The increase in solvation and rotational time in micelles is attributed to the increase in viscosity of the medium. The slow component is assumed to be dependent on the viscosity of the solution and involves large-scale rearrangement of the anions and cations while fast component is assumed to originate from the initial response of the anions during excitation. The slow component increases due to the increase in the viscosity of the medium and increase in fast component is probably due to the hydrogen bonding between the anions and polar headgroup of the surfactant. The dynamics of solvent relaxation was affected to a small extent due to the micelle formation.

  3. Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145 --> Met145 mutant AGT: comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy.

    PubMed Central

    Federwisch, M; Hassiepen, U; Bender, K; Dewor, M; Rajewsky, M F; Wollmer, A

    1997-01-01

    Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 degrees C but remained monomeric at 4 degrees C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8. 4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The alpha-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its alpha-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered. PMID:9164873

  4. Multiplexed Salivary Protein Profiling for Patients with Respiratory Diseases using Fiber-Optic Bundles and Fluorescent Antibody-Based Microarrays

    PubMed Central

    Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R.

    2013-01-01

    Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. Based on a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin 8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin 1 beta (IL-1β) salivary biomarkers in the sub-picomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p<0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis. PMID:23972398

  5. A Direct Interaction between the Sigma-1 Receptor and the hERG Voltage-gated K+ Channel Revealed by Atomic Force Microscopy and Homogeneous Time-resolved Fluorescence (HTRF®)*

    PubMed Central

    Balasuriya, Dilshan; D'Sa, Lauren; Talker, Ronel; Dupuis, Elodie; Maurin, Fabrice; Martin, Patrick; Borgese, Franck; Soriani, Olivier; Edwardson, J. Michael

    2014-01-01

    The sigma-1 receptor is an endoplasmic reticulum chaperone protein, widely expressed in central and peripheral tissues, which can translocate to the plasma membrane and modulate the function of various ion channels. The human ether-à-go-go-related gene encodes hERG, a cardiac voltage-gated K+ channel that is abnormally expressed in many human cancers and is known to interact functionally with the sigma-1 receptor. Our aim was to investigate the nature of the interaction between the sigma-1 receptor and hERG. We show that the two proteins can be co-isolated from a detergent extract of stably transfected HEK-293 cells, consistent with a direct interaction between them. Atomic force microscopy imaging of the isolated protein confirmed the direct binding of the sigma-1 receptor to hERG monomers, dimers, and tetramers. hERG dimers and tetramers became both singly and doubly decorated by sigma-1 receptors; however, hERG monomers were only singly decorated. The distribution of angles between pairs of sigma-1 receptors bound to hERG tetramers had two peaks, at ∼90 and ∼180° in a ratio of ∼2:1, indicating that the sigma-1 receptor interacts with hERG with 4-fold symmetry. Homogeneous time-resolved fluorescence (HTRF®) allowed the detection of the interaction between the sigma-1 receptor and hERG within the plane of the plasma membrane. This interaction was resistant to sigma ligands, but was decreased in response to cholesterol depletion of the membrane. We suggest that the sigma-1 receptor may bind to hERG in the endoplasmic reticulum, aiding its assembly and trafficking to the plasma membrane. PMID:25266722

  6. Development of time-resolved fluorescent based [EU]-GTP binding assay for selection of human Histamine 3 receptor antagonists/inverse agonist: a potential target for Alzheimer's treatment.

    PubMed

    Singh, Jitendra K; Maniyar, Reema C; Shirsath, Vikas S

    2012-04-01

    The histamine H3 receptor is an attractive G protein-coupled receptor drug target that regulates neurotransmission in the central nervous system and plays a crucial role in cognitive and homeostatic functions. This receptor exhibits molecular, pharmacological, and functional heterogeneity that affects the preclinical development of effective antagonists. The range of assay technologies like radio isotope based [35S] GTPγS binding assay, luminescent based reporter gene assay (In-direct cAMP measurement) for binding and signaling have been developed in High Throughput Screening (HTS) laboratories for the identification of hit or lead compounds acting on H3 receptor. The [35S] GTPγS binding assay still remains a useful and a simple technique to demonstrate receptor activation and is one of the few functional, cell-free assays that has set the standards in the field of research. However, its radioactive nature imposes clear limitations to its use in regular laboratory practice and in high-throughput experimentation. Herein, we have developed and optimized a membrane based non-radioactive assay using a europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence technology. The characterization of H3 agonist or antagonist with HTRF platform has revealed a rank order potency (pEC50 & P K B) comparable to that from isotopic functional studies measured by liquid scintillation counter (LSC). Lastly, the Eu-GTP binding assay has been found to be highly robust (Z' factor 0.84) with high percentage over basal counts. This assay can be utilized as a component of cascade for the screening of H3 receptor ligands.

  7. Time-resolved Raman spectroscopy for in situ planetary mineralogy.

    PubMed

    Blacksberg, Jordana; Rossman, George R; Gleckler, Anthony

    2010-09-10

    Planetary mineralogy can be revealed through a variety of remote sensing and in situ investigations that precede any plans for eventual sample return. We briefly review those techniques and focus on the capabilities for on-surface in situ examination of Mars, Venus, the Moon, asteroids, and other bodies. Over the past decade, Raman spectroscopy has continued to develop as a prime candidate for the next generation of in situ planetary instruments, as it provides definitive structural and compositional information of minerals in their natural geological context. Traditional continuous-wave Raman spectroscopy using a green laser suffers from fluorescence interference, which can be large (sometimes saturating the detector), particularly in altered minerals, which are of the greatest geophysical interest. Taking advantage of the fact that fluorescence occurs at a later time than the instantaneous Raman signal, we have developed a time-resolved Raman spectrometer that uses a streak camera and pulsed miniature microchip laser to provide picosecond time resolution. Our ability to observe the complete time evolution of Raman and fluorescence spectra in minerals makes this technique ideal for exploration of diverse planetary environments, some of which are expected to contain strong, if not overwhelming, fluorescence signatures. We discuss performance capability and present time-resolved pulsed Raman spectra collected from several highly fluorescent and Mars-relevant minerals. In particular, we have found that conventional Raman spectra from fine grained clays, sulfates, and phosphates exhibited large fluorescent signatures, but high quality spectra could be obtained using our time-resolved approach.

  8. Time-Resolved Photoluminescence and Photovoltaics

    SciTech Connect

    Metzger, W. K.; Ahrenkiel, R. K.; Dippo, P.; Geisz, J.; Wanlass, M. W.; Kurtz, S.

    2005-01-01

    The time-resolved photoluminescence (TRPL) technique and its ability to characterize recombination in bulk photovoltaic semiconductor materials are reviewed. Results from a variety of materials and a few recent studies are summarized and compared.

  9. Time-resolved transillumination and optical tomography

    NASA Astrophysics Data System (ADS)

    de Haller, Emmanuel B.

    1996-01-01

    In response to an invitation by the editor-in-chief, I would like to present the current status of time-domain imaging. With exciting new photon diffusion techniques being developed in the frequency domain and promising optical coherence tomography, time-resolved transillumination is in constant evolution and the subject of passionate discussions during the numerous conferences dedicated to this subject. The purpose of time-resolved optical tomography is to provide noninvasive, high-resolution imaging of the interior of living bodies by the use of nonionizing radiation. Moreover, the use of visible to near-infrared wavelength yields metabolic information. Breast cancer screening is the primary potential application for time-resolved imaging. Neurology and tissue characterization are also possible fields of applications. Time- resolved transillumination and optical tomography should not only improve diagnoses, but the welfare of the patient. As no overview of this technique has yet been presented to my knowledge, this paper briefly describes the various methods enabling time-resolved transillumination and optical tomography. The advantages and disadvantages of these methods, as well as the clinical challenges they face are discussed. Although an analytic and computable model of light transport through tissues is essential for a meaningful interpretation of the transillumination process, this paper will not dwell on the mathematics of photon propagation.

  10. Detection of colorectal cancer using time-resolved autofluorescence spectrometer

    NASA Astrophysics Data System (ADS)

    Fu, Sheng; Kwek, Leong-Chuan; Chia, Teck-Chee; Lim, Chu-Sing; Tang, Choong-Leong; Ang, Wuan-Suan; Zhou, Miao-Chang; Loke, Po-Ling

    2006-04-01

    As we know Quantum mechanics is a mathematical theory that can describe the behavior of objects that are at microscopic level. Time-resolved autofluorescence spectrometer monitors events that occur during the lifetime of the excited state. This time ranges from a few picoseconds to hundreds of nanoseconds. That is an extremely important advance as it allows environmental parameters to be monitored in a spatially defined manner in the specimen under study. This technique is based on the application of Quantum Mechanics. This principle is applied in our project as we are trying to use different fluorescence spectra to detect biological molecules commonly found in cancerous colorectal tissue and thereby differentiate the cancerous and non-cancerous colorectal polyps more accurately and specifically. In this paper, we use Fluorescence Lifetime Spectrometer (Edinburgh Instruments FL920) to measure decay time of autofluorescence of colorectal cancerous and normal tissue sample. All specimens are from Department of Colorectal Surgery, Singapore General Hospital. The tissues are placed in the time-resolved autofluorescence instrument, which records and calculates the decay time of the autofluorescence in the tissue sample at the excitation and emission wavelengths pre-determined from a conventional spectrometer. By studying the decay time,τ, etc. for cancerous and normal tissue, we aim to present time-resolved autofluorescence as a feasible technique for earlier detection of malignant colorectal tissues. By using this concept, we try to contribute an algorithm even an application tool for real time early diagnosis of colorectal cancer for clinical services.

  11. Time resolved thermal lens in edible oils

    NASA Astrophysics Data System (ADS)

    Albuquerque, T. A. S.; Pedreira, P. R. B.; Medina, A. N.; Pereira, J. R. D.; Bento, A. C.; Baesso, M. L.

    2003-01-01

    In this work time resolved thermal lens spectrometry is applied to investigate the optical properties of the following edible oils: soya, sunflower, canola, and corn oils. The experiments were performed at room temperature using the mode mismatched thermal lens configuration. The results showed that when the time resolved procedure is adopted the technique can be applied to investigate the photosensitivity of edible oils. Soya oil presented a stronger photochemical reaction as compared to the other investigated samples. This observation may be relevant for future studies evaluating edible oils storage conditions and also may contribute to a better understanding of the physical and chemical properties of this important foodstuff.

  12. Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by Aβ(1-40) and Aβ(1-42)

    SciTech Connect

    Lindberg, David J.; Wranne, Moa S.; Gilbert Gatty, Mélina; Westerlund, Fredrik; Esbjörner, Elin K.

    2015-03-06

    Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation. - Highlights: • ThT emission is more intense with Aβ(1-40) fibrils than with Aβ(1-42) fibrils. • Aβ(1-42) fibrils induce longer ThT fluorescence lifetimes and higher quantum yield. • ThT emission intensity in Aβ fibril samples reports on fibril morphology. • The ThT fluorescence lifetime is a characteristic feature of each A

  13. Comparison of the rate constants for energy transfer in the light-harvesting protein, C-phycocyanin, calculated from Foerster`s theory and experimentally measured by time-resolved fluorescence spectroscopy

    SciTech Connect

    Debreczeny, Martin Paul

    1994-05-01

    We have measured and assigned rate constants for energy transfer between chromophores in the light-harvesting protein C-phycocyanin (PC), in the monomeric and trimeric aggregation states, isolated from Synechococcus sp. PCC 7002. In order to compare the measured rate constants with those predicted by Fdrster`s theory of inductive resonance in the weak coupling limit, we have experimentally resolved several properties of the three chromophore types ({beta}{sub 155} {alpha}{sub 84}, {beta}{sub 84}) found in PC monomers, including absorption and fluorescence spectra, extinction coefficients, fluorescence quantum yields, and fluorescence lifetimes. The cpcB/C155S mutant, whose PC is missing the {beta}{sub 155} chromophore, was, useful in effecting the resolution of the chromophore properties and in assigning the experimentally observed rate constants for energy transfer to specific pathways.

  14. Effect of PpIX photoproducts formation on pO2 measurement by time-resolved delayed fluorescence spectroscopy of PpIX in solution and in vivo.

    PubMed

    Huntosova, Veronika; Gerelli, Emmanuel; Zellweger, Matthieu; Wagnières, Georges

    2016-11-01

    The measurement of Protoporphyrin IX delayed fluorescence lifetime is a minimally invasive method for monitoring the levels of oxygen in cells and tissues. The excitation of Protoporphyrin IX during this measurement can lead to the formation of photoproducts in vitro and in vivo. The influence of their luminescence on the measured Protoporphyrin IX delayed fluorescence lifetimes was studied in solution and in vivo on the Chick's chorioallantoic membrane (CAM) model under various oxygen enriched air conditions (0mmHg, 37mmHg and 155mmHg). The presence of photoproducts disturbs such measurements since the delayed fluorescence emission of some of them spectrally overlaps with that of Protoporphyrin IX. One possible way to avoid this obstacle is to detect Protoporphyrin IX's delayed fluorescence lifetime in a very specific spectral range (620-640nm). Another possibility is to excite Protoporphyrin IX with light doses much lower than 10J/cm(2), quite possibly as low as a fraction 1J/cm(2) at 405nm. This leads to an increased accuracy of pO2 detection. Furthermore, this method allows combination of diagnosis and therapy in one step. This helps to improve detection systems and real-time identification of tissue respiration, which is tuned for the detection of PpIX luminescence and not its photoproducts.

  15. Comparison of the performance of the LIAISON VZV-IgG and VIDAS automated enzyme linked fluorescent immunoassays with reference to a VZV-IgG time-resolved fluorescence immunoassay and implications of choice of cut-off for LIAISON assay.

    PubMed

    Maple, P A C; Rathod, P; Smit, E; Gray, J; Brown, D; Boxall, E H

    2009-01-01

    Determination of Varicella Zoster virus (VZV) immune status in pregnant women without history of chickenpox is important in identifying those who genuinely need VZV immune globulin prophylaxis following significant exposure to chickenpox or shingles. Immune status testing requires highly sensitive and specific immunoassays for timely and accurate results. To compare the performance of DiaSorin LIAISON and Biomerieux VIDAS VZV-IgG assays with reference to a VZV-IgG time-resolved fluorescence immunoassay (TRFIA). A panel of sera collected from 65 pregnant contacts of VZV and 62 individuals tested for VZV immunity was tested in all three assays. Dose-response curves were generated using International Standards W1044 and 90/690. Sensitivity and specificity of VIDAS compared to VZV-TRFIA was 54.5% and 97.9% respectively and for LIAISON compared to VZV-TRFIA was 67% and 100% respectively. Both assays correlated well with TRFIA with R2 correlation coefficients of 0.79 and 0.76 respectively. Dose-response curves showed both Standards behaved in a similar manner in each assay. For VIDAS, the test cut-off value of 0.9 correlated with 275-280mIU/ml and for LIAISON a cut-off value of 150mIU/ml correlated with 208-219mIU/ml. By dose-response data and in comparison with TRFIA, LIAISON is more sensitive and specific than VIDAS.

  16. Time resolved astronomy with the SALT

    NASA Astrophysics Data System (ADS)

    Buckley, D. A. H.; Crawford, S.; Gulbis, A. A. S.; McPhate, J.; Nordsieck, K. H.; Potter, S. B.; O'Donoghue, D.; Siegmund, O. H. W.; Schellart, P.; Spark, M.; Welsh, B. Y.; Zietsman, E.

    2010-07-01

    While time resolved astronomical observations are not new, the extension of such studies to sub-second time resolution is and has resulted in the opening of a new observational frontier, High Time Resolution Astronomy (HTRA). HTRA studies are well suited to objects like compact binary stars (CVs and X-ray binaries) and pulsars, while asteroseismology of pulsating stars, occultations, transits and the study of transients, will all benefit from such HTRA studies. HTRA has been a SALT science driver from the outset and the first-light instruments, namely the UV-VIS imager, SALTICAM, and the multi-purpose Robert Stobie Spectrograph (RSS), both have high time resolution modes. These are described, together with some observational examples. We also discuss the commissioning observations with the photon counting Berkeley Visible Image Tube camera (BVIT) on SALT. Finally we describe the software tools, developed in Python, to reduce SALT time resolved observations.

  17. Time-resolved photoemission using attosecond streaking

    NASA Astrophysics Data System (ADS)

    Nagele, S.; Pazourek, R.; Wais, M.; Wachter, G.; Burgdörfer, J.

    2014-04-01

    We theoretically study time-resolved photoemission in atoms as probed by attosecond streaking. We review recent advances in the study of the photoelectric efect in the time domain and show that the experimentally accessible time shifts can be decomposed into distinct contributions that stem from the feld-free photoionization process itself and from probe-field induced corrections. We perform accurate quantum-mechanical as well as classical simulations of attosecond streaking for efective one-electron systems and determine all relevant contributions to the time delay with attosecond precision. In particular, we investigate the properties and limitations of attosecond streaking for the transition from short-ranged potentials (photodetachment) to long-ranged Coulomb potentials (photoionization). As an example for a more complex system, we study time-resolved photoionization for endohedral fullerenes A@C60 and discuss how streaking time shifts are modifed due to the interaction of the C60 cage with the probing infrared streaking field.

  18. The differences in short- and long-term varicella-zoster virus (VZV) immunoglobulin G levels following varicella vaccination of healthcare workers measured by VZV fluorescent-antibody-to-membrane-antigen assay (FAMA), VZV time-resolved fluorescence immunoassay and a VZV purified glycoprotein enzyme immunoassay.

    PubMed

    Maple, P A C; Haedicke, J; Quinlivan, M; Steinberg, S P; Gershon, A A; Brown, K E; Breuer, J

    2016-08-01

    Healthcare workers (HCWs) reporting no history of varicella frequently receive varicella vaccination (vOka) if they test varicella-zoster virus (VZV) immunoglobulin G (IgG) negative. In this study, the utilities of VZV-IgG time-resolved fluorescence immunoassay (VZV-TRFIA) and a commercial VZV-IgG purified glycoprotein enzyme immunoassay (gpEIA) currently used in England for confirming VZV immunity have been compared to the fluorescent-antibody-to-membrane-antigen assay (FAMA). A total of 110 HCWs received two doses of vOka vaccine spaced 6 weeks apart and sera collected pre-vaccination (n = 100), at 6 weeks post-completion of vaccination (n = 86) and at 12-18 months follow-up (n = 73) were analysed. Pre-vaccination, by FAMA, 61·0% sera were VZV IgG negative, and compared to FAMA the sensitivities of VZV-TRFIA and gpEIA were 74·4% [95% confidence interval (CI) 57·9-87·0] and 46·2% (95% CI 30·1-62·8), respectively. Post-completion of vaccination the seroconversion rate by FAMA was 93·7% compared to rates of 95·8% and 70·8% determined by VZV-TRFIA and gpEIA, respectively. At 12-18 months follow-up seropositivity rates by FAMA, VZV-TRFIA and gpEIA were 78·1%, 74·0% and 47·9%, respectively. Compared to FAMA the sensitivities of VZV-TRFIA and gpEIA for measuring VZV IgG following vaccination were 96·4% (95% CI 91·7-98·8) and 74·6% (95% CI 66·5-81·6), respectively. Using both FAMA and VZV-TRFIA to identify healthy adult VZV susceptibles and measure seroconversion showed that vOka vaccination of HCWs is highly immunogenic.

  19. Time-resolved optical spectroscopy of wood.

    PubMed

    D'Andrea, C; Farina, A; Comelli, D; Pifferi, A; Taroni, P; Valentini, G; Cubeddu, R; Zoia, L; Orlandi, M; Kienle, A

    2008-05-01

    We have proposed and experimentally demonstrated that picosecond time-resolved optical spectroscopy in the visible/near-infrared (NIR) region (700-1040 nm) is a useful technique for noninvasive characterization of wood. This technique has been demonstrated on both softwood and hardwood samples treated in different ways simulating the aging process suffered by waterlogged woods. In all the cases, alterations of absorption and scattering spectra were observed, revealing changes of chemical and structural composition.

  20. Time-resolved inverse Raman spectroscopy.

    PubMed

    Rahn, L A

    1982-02-01

    A technique for obtaining sensitive, highly reproducible, time-resolved inverse Raman measurements is reported. Experimental results are presented for the nitrogen vibrational Q branch at a pressure of 10 atm. For these measurements the signal, normalized to the pump-laser energy, exhibits fluctuations of 1.4% (rms) about the average of 500 measurements; these deviations are within a factor of 2 of the quantum noise limit.

  1. Time-resolved multiphoton imaging of basal cell carcinoma

    NASA Astrophysics Data System (ADS)

    Cicchi, R.; Sestini, S.; De Giorgi, V.; Stambouli, D.; Carli, P.; Massi, D.; Pavone, F. S.

    2007-02-01

    We investigated human cutaneous basal cell carcinoma ex-vivo samples by combined time resolved two photon intrinsic fluorescence and second harmonic generation microscopy. Morphological and spectroscopic differences were found between malignant skin and corresponding healthy skin tissues. In comparison with normal healthy skin, cancer tissue showed a different morphology and a mean fluorescence lifetime distribution slightly shifted towards higher values. Topical application of delta-aminolevulinic acid to the lesion four hours before excision resulted in an enhancement of the fluorescence signal arising from malignant tissue, due to the accumulation of protoporphyrines inside tumor cells. Contrast enhancement was prevalent at tumor borders by both two photon fluorescence microscopy and fluorescence lifetime imaging. Fluorescence-based images showed a good correlation with conventional histopathological analysis, thereby supporting the diagnostic accuracy of this novel method. Combined morphological and lifetime analysis in the study of ex-vivo skin samples discriminated benign from malignant tissues, thus offering a reliable, non-invasive tool for the in-vivo analysis of inflammatory and neoplastic skin lesions.

  2. Structural kinetics of myosin by transient time-resolved FRET

    PubMed Central

    Nesmelov, Yuri E.; Agafonov, Roman V.; Negrashov, Igor V.; Blakely, Sarah E.; Titus, Margaret A.; Thomas, David D.

    2011-01-01

    For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)2FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)2FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)2FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism. PMID:21245357

  3. Time Resolved Studies Of Adsorbed Species

    NASA Astrophysics Data System (ADS)

    Howard, J.; Nicol, J. M.

    1985-12-01

    A time-resolved Fourier transform IR study of ethyne adsorbed on ZnNaA zeolite yields results very different from those reported for related systems. Initially two species (A and B) are formed by the interaction of C2H2 with the cations. Whereas species A (π-bonded C2H2) was found to be removed immediately on evacuation, species B (probably Zn-acetylide) was not fully removed after 60 mins evacuation. In the presence of the gas phase, bands due to Species A decreased slowly in intensity as new bands due to adsorbed ethanal were observed.

  4. Time Resolved Deposition Measurements in NSTX

    SciTech Connect

    C.H. Skinner; H. Kugel; A.L. Roquemore; J. Hogan; W.R. Wampler; the NSTX team

    2004-08-03

    Time-resolved measurements of deposition in current tokamaks are crucial to gain a predictive understanding of deposition with a view to mitigating tritium retention and deposition on diagnostic mirrors expected in next-step devices. Two quartz crystal microbalances have been installed on NSTX at a location 0.77m outside the last closed flux surface. This configuration mimics a typical diagnostic window or mirror. The deposits were analyzed ex-situ and found to be dominantly carbon, oxygen, and deuterium. A rear facing quartz crystal recorded deposition of lower sticking probability molecules at 10% of the rate of the front facing one. Time resolved measurements over a 4-week period with 497 discharges, recorded 29.2 {micro}g/cm{sup 2} of deposition, however surprisingly, 15.9 {micro}g/cm{sup 2} of material loss occurred at 7 discharges. The net deposited mass of 13.3 {micro}g/cm{sup 2} matched the mass of 13.5 {micro}g/cm{sup 2} measured independently by ion beam analysis. Monte Carlo modeling suggests that transient processes are likely to dominate the deposition.

  5. Time-resolved RNA SHAPE chemistry.

    PubMed

    Mortimer, Stefanie A; Weeks, Kevin M

    2008-12-03

    Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry yields quantitative RNA secondary and tertiary structure information at single nucleotide resolution. SHAPE takes advantage of the discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl group is modulated by local nucleotide flexibility in the RNA backbone. Flexible nucleotides are reactive toward hydroxyl-selective electrophiles, whereas constrained nucleotides are unreactive. Initial versions of SHAPE chemistry, which employ isatoic anhydride derivatives that react on the minute time scale, are emerging as the ideal technology for monitoring equilibrium structures of RNA in a wide variety of biological environments. Here, we extend SHAPE chemistry to a benzoyl cyanide scaffold to make possible facile time-resolved kinetic studies of RNA in approximately 1 s snapshots. We then use SHAPE chemistry to follow the time-dependent folding of an RNase P specificity domain RNA. Tertiary interactions form in two distinct steps with local tertiary contacts forming an order of magnitude faster than long-range interactions. Rate-determining tertiary folding requires minutes despite that no non-native interactions must be disrupted to form the native structure. Instead, overall folding is limited by simultaneous formation of interactions approximately 55 A distant in the RNA. Time-resolved SHAPE holds broad potential for understanding structural biogenesis and the conformational interconversions essential to the functions of complex RNA molecules at single nucleotide resolution.

  6. Time-resolved resonance Raman spectroscopy: exploring reactive intermediates.

    PubMed

    Sahoo, Sangram Keshari; Umapathy, Siva; Parker, Anthony W

    2011-10-01

    The study of reaction mechanisms involves systematic investigations of the correlation between structure, reactivity, and time. The challenge is to be able to observe the chemical changes undergone by reactants as they change into products via one or several intermediates such as electronic excited states (singlet and triplet), radicals, radical ions, carbocations, carbanions, carbenes, nitrenes, nitrinium ions, etc. The vast array of intermediates and timescales means there is no single "do-it-all" technique. The simultaneous advances in contemporary time-resolved Raman spectroscopic techniques and computational methods have done much towards visualizing molecular fingerprint snapshots of the reactive intermediates in the microsecond to femtosecond time domain. Raman spectroscopy and its sensitive counterpart resonance Raman spectroscopy have been well proven as means for determining molecular structure, chemical bonding, reactivity, and dynamics of short-lived intermediates in solution phase and are advantageous in comparison to commonly used time-resolved absorption and emission spectroscopy. Today time-resolved Raman spectroscopy is a mature technique; its development owes much to the advent of pulsed tunable lasers, highly efficient spectrometers, and high speed, highly sensitive multichannel detectors able to collect a complete spectrum. This review article will provide a brief chronological development of the experimental setup and demonstrate how experimentalists have conquered numerous challenges to obtain background-free (removing fluorescence), intense, and highly spectrally resolved Raman spectra in the nanosecond to microsecond (ns-μs) and picosecond (ps) time domains and, perhaps surprisingly, laid the foundations for new techniques such as spatially offset Raman spectroscopy. © 2011 Society for Applied Spectroscopy

  7. An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

    USDA-ARS?s Scientific Manuscript database

    A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

  8. Time-resolved Temperature Measurements in SSPX

    SciTech Connect

    Ludington, A R; Hill, D N; McLean, H S; Moller, J; Wood, R D

    2006-08-14

    We seek to measure time-resolved electron temperatures in the SSPX plasma using soft X-rays from free-free Bremsstrahlung radiation. To increase sensitivity to changes in temperature over the range 100-300 eV, we use two photodiode detectors sensitive to different soft X-ray energies. The detectors, one with a Zr/C coating and the other with a Ti/Pd coating, view the plasma along a common line of sight tangential to the magnetic axis of the spheromak, where the electron temperature is a maximum. The comparison of the signals, over a similar volume of plasma, should be a stronger function of temperature than a single detector in the range of Te< 300 eV. The success of using photodiodes to detect changing temperatures along a chord will make the case for designing an array of the detectors, which could provide a time changing temperature profile over a larger portion of the plasma.

  9. Time resolved ion beam induced charge collection

    SciTech Connect

    SEXTON,FREDERICK W.; WALSH,DAVID S.; DOYLE,BARNEY L.; DODD,PAUL E.

    2000-04-01

    Under this effort, a new method for studying the single event upset (SEU) in microelectronics has been developed and demonstrated. Called TRIBICC, for Time Resolved Ion Beam Induced Charge Collection, this technique measures the transient charge-collection waveform from a single heavy-ion strike with a {minus}.03db bandwidth of 5 GHz. Bandwidth can be expanded up to 15 GHz (with 5 ps sampling windows) by using an FFT-based off-line waveform renormalization technique developed at Sandia. The theoretical time resolution of the digitized waveform is 24 ps with data re-normalization and 70 ps without re-normalization. To preserve the high bandwidth from IC to the digitizing oscilloscope, individual test structures are assembled in custom high-frequency fixtures. A leading-edge digitized waveform is stored with the corresponding ion beam position at each point in a two-dimensional raster scan. The resulting data cube contains a spatial charge distribution map of up to 4,096 traces of charge (Q) collected as a function of time. These two dimensional traces of Q(t) can cover a period as short as 5 ns with up to 1,024 points per trace. This tool overcomes limitations observed in previous multi-shot techniques due to the displacement damage effects of multiple ion strikes that changed the signal of interest during its measurement. This system is the first demonstration of a single-ion transient measurement capability coupled with spatial mapping of fast transients.

  10. Time-resolved local strain tracking microscopy for cell mechanics

    NASA Astrophysics Data System (ADS)

    Aydin, O.; Aksoy, B.; Akalin, O. B.; Bayraktar, H.; Alaca, B. E.

    2016-02-01

    A uniaxial cell stretching technique to measure time-resolved local substrate strain while simultaneously imaging adherent cells is presented. The experimental setup comprises a uniaxial stretcher platform compatible with inverted microscopy and transparent elastomer samples with embedded fluorescent beads. This integration enables the acquisition of real-time spatiotemporal data, which is then processed using a single-particle tracking algorithm to track the positions of fluorescent beads for the subsequent computation of local strain. The present local strain tracking method is demonstrated using polydimethylsiloxane (PDMS) samples of rectangular and dogbone geometries. The comparison of experimental results and finite element simulations for the two sample geometries illustrates the capability of the present system to accurately quantify local deformation even when the strain distribution is non-uniform over the sample. For a regular dogbone sample, the experimentally obtained value of local strain at the center of the sample is 77%, while the average strain calculated using the applied cross-head displacement is 48%. This observation indicates that considerable errors may arise when cross-head measurement is utilized to estimate strain in the case of non-uniform sample geometry. Finally, the compatibility of the proposed platform with biological samples is tested using a unibody PDMS sample with a well to contain cells and culture media. HeLa S3 cells are plated on collagen-coated samples and cell adhesion and proliferation are observed. Samples with adherent cells are then stretched to demonstrate simultaneous cell imaging and tracking of embedded fluorescent beads.

  11. Time Resolved Microfluorescence In Biomedical Diagnosis

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert

    1985-12-01

    A measuring system combining subnanosecond laser-induced fluorescence with microscopic signal detection was installed and used for diverse projects in the biomedical and environmental fields. These projects range from tumor diagnosis and enzymatic analysis to measurements of the activity of methanogenic bacteria, which affect biogas production and waste water cleaning. The advantages of this method and its practical applicability are discussed.

  12. Time-Resolved Microfluorescence In Biomedical Diagnosis

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert

    1985-02-01

    A measuring system combining subnanosecond laser-induced fluorescence with microscopic signal detection was installed and used for diverse projects in the biomedical and environmental field. These projects are ranging from tumor diagnosis and enzymatic analysis to measurements of the activity of methanogenic bacteria which effect biogas production and waste water cleaning. The advantages of this method and its practical applicability are discussed.

  13. Time-resolved spectral imaging: better photon economy, higher accuracy

    NASA Astrophysics Data System (ADS)

    Fereidouni, Farzad; Reitsma, Keimpe; Blab, Gerhard A.; Gerritsen, Hans C.

    2015-03-01

    Lifetime and spectral imaging are complementary techniques that offer a non-invasive solution for monitoring metabolic processes, identifying biochemical compounds, and characterizing their interactions in biological tissues, among other tasks. Newly developed instruments that perform time-resolved spectral imaging can provide even more information and reach higher sensitivity than either modality alone. Here we report a multispectral lifetime imaging system based on a field-programmable gate array (FPGA), capable of operating at high photon count rates (12 MHz) per spectral detection channel, and with time resolution of 200 ps. We performed error analyses to investigate the effect of gate width and spectral-channel width on the accuracy of estimated lifetimes and spectral widths. Temporal and spectral phasors were used for analysis of recorded data, and we demonstrated blind un-mixing of the fluorescent components using information from both modalities. Fractional intensities, spectra, and decay curves of components were extracted without need for prior information. We further tested this approach with fluorescently doubly-labeled DNA, and demonstrated its suitability for accurately estimating FRET efficiency in the presence of either non-interacting or interacting donor molecules.

  14. Time-resolved and polarised microspectroscopy of thin films of bio- and nanomaterials

    NASA Astrophysics Data System (ADS)

    Soleimaninejad, Hamid; Hong, Yuning; Smith, Trevor A.

    2016-09-01

    We report on the use of time-resolved and polarised evanescent wave-induced fluorescence anisotropy measurements to probe molecular photophysics, motion and energy migration of fluorescent species in close proximity to a silica/film interface. In particular we show that the fluorescence decay and anisotropy of common fluorophores varies as a function of the plane of the fluorophore with respect to the interface, the distance from the interface, and as a function of position (using polarised EW imaging). We have applied time-resolved and polarised EW-induced fluorescence microspectroscopic measurements to dyes, thin polymer nanoparticle films and cells on silica surfaces, probing the variation in the photophysical dynamics within the films.

  15. Monoclonal antibody-based fluorescence polarization immunoassay for high throughput screening of furaltadone and its metabolite AMOZ in animal feeds and tissues.

    PubMed

    Xu, Zhen-Lin; Zhang, Shi-Wei; Sun, Yuan-Ming; Shen, Yu-Dong; Lei, Hong-Tao; Jiang, Yue-Ming; Eremin, Sergei A; Yang, Jin-Yi; Wang, Hong

    2013-07-01

    A simple and fast homogeneous fluorescent polarization immunoassay (FPIA) was developed for the determination of furaltadone and its metabolite 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ). Monoclonal antibody with high cross-reactivity to furaltadone and the nitrophenyl derivative of AMOZ (NPAMOZ) were produced against a novel immunogen and the effects of several synthesized tracers on FPIA sensitivity studied. The proposed FPIA, using an optimum antibody and tracer pair, had an IC₅₀ of 4.3 µg L⁻¹ and limit of detection at 0.6 µg L⁻¹ for furaltadone, and 2.7 µgL⁻¹ and 0.3 µg L⁻¹ for NPAMOZ. Recoveries of furaltadone from animal feeds by FPIA ranged from 79.6 to 87.7%, while recoveries of AMOZ from animal tissues ranged from 72.9 to 83.1%. Good correlation (R>0.99) between the results of this FPIA and a standard analytical method was obtained. The FPIA does not require separation or washing steps and the total time required for equilibrium of the antibody-tracer interaction is only 10 min. These results indicated that the proposed FPIA offers great potential and utility for the high throughput screening of furaltadone residues in animal feed and its metabolite AMOZ residues in animal tissues.

  16. Biochemical Applications Of Frequency-Domain Fluorometry; Determination Of Time-Resolved Anisotropies And Emission Spectra

    NASA Astrophysics Data System (ADS)

    Lakowicz, Joseph R.; Gryczynski, Ignazy; Cherek, Henryh; Laczko, Gabor; Joshi, Nanda

    1987-01-01

    Measurements of time-resolved fluorescence are often used for studies of biological macromolecules. Such measurements are usually performed in the time-domain, by measurement of the time-dependent emission following pulsed excitation. It has recently become possible to measure the frequency-response of the emission to intensity modulated light, over a wide range of modulation frequencies. We used frequency-domain fluorometers which operates from 1 to 220 MHz, and more recently to 2000 MHz. The frequency-domain data provide excellent resolution of time-dependent spectral parameters. It is now possible to resolve closely spaced fluorescence lifetimes, to determine multi-exponential decays of anisotropy and to determine time-resolved emission spectra of samples which display time-dependent spectral shifts. In this article we show representative results on tryptophan fluorescence from proteins and for protein-bound fluorophores.

  17. Time-resolved spectral analysis of Radachlorin luminescence in water

    NASA Astrophysics Data System (ADS)

    Belik, V. P.; Gadzhiev, I. M.; Semenova, I. V.; Vasyutinskii, O. S.

    2017-05-01

    We report results of spectral- and time-resolved study of Radachlorin photosensitizer luminescence in water in the spectral range of 950-1350nm and for determination of the photosensitizer triplet state and the singlet oxygen lifetimes responsible for singlet oxygen generation and degradation. At any wavelength within the explored spectral range the luminescence decay contained two major contributions: a fast decay at the ns time scale and a slow evolution at the μs time scale. The fast decay was attributed to electric dipole fluorescence transitions in photosensitizer molecules and the slow evolution to intercombination phosphorescence transitions in singlet oxygen and photosensitizer molecules. Relatively high-amplitude ns peak observed at all wavelengths suggests that singlet oxygen monitoring with spectral isolation methods alone, without additional temporal resolution can be controversial. In the applied experimental conditions the total phosphorescence signal at any wavelength contained a contribution from the photosensitizer triplet state decay, while at 1274nm the singlet oxygen phosphorescence dominated. The results obtained can be used for optimization of the methods of singlet oxygen monitoring and imaging.

  18. Time-resolved spectral analysis of Radachlorin luminescence in water.

    PubMed

    Belik, V P; Gadzhiev, I M; Semenova, I V; Vasyutinskii, O S

    2017-05-05

    We report results of spectral- and time-resolved study of Radachlorin photosensitizer luminescence in water in the spectral range of 950-1350nm and for determination of the photosensitizer triplet state and the singlet oxygen lifetimes responsible for singlet oxygen generation and degradation. At any wavelength within the explored spectral range the luminescence decay contained two major contributions: a fast decay at the ns time scale and a slow evolution at the μs time scale. The fast decay was attributed to electric dipole fluorescence transitions in photosensitizer molecules and the slow evolution to intercombination phosphorescence transitions in singlet oxygen and photosensitizer molecules. Relatively high-amplitude ns peak observed at all wavelengths suggests that singlet oxygen monitoring with spectral isolation methods alone, without additional temporal resolution can be controversial. In the applied experimental conditions the total phosphorescence signal at any wavelength contained a contribution from the photosensitizer triplet state decay, while at 1274nm the singlet oxygen phosphorescence dominated. The results obtained can be used for optimization of the methods of singlet oxygen monitoring and imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Time-resolved pulsed EPR: microwave and optical detection

    SciTech Connect

    Trifunac, A.D.; Smith, J.P.

    1981-01-01

    Time-resolved pulsed EPR spectrometers are described. EPR spectra, kinetic profiles, and relaxation studies are used to illustrate some capabilities of the pulsed EPR experiment. Optical detection of time-resolved EPR spectra of radical ion pairs is used to study radical-ion recombination kinetics, recombination pathways, and the structure of radical anions and cations. 17 figures.

  20. Ultrafast time-resolved vibrational spectroscopies of carotenoids in photosynthesis.

    PubMed

    Hashimoto, Hideki; Sugisaki, Mitsuru; Yoshizawa, Masayuki

    2015-01-01

    This review discusses the application of time-resolved vibrational spectroscopies to the studies of carotenoids in photosynthesis. The focus is on the ultrafast time regime and the study of photophysics and photochemistry of carotenoids by femtosecond time-resolved stimulated Raman and four-wave mixing spectroscopies. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

  1. Cervical cancer detection by time-resolved spectra of blood components

    NASA Astrophysics Data System (ADS)

    Kalaivani, Rudran; Masilamani, Vadivel; AlSalhi, Mohamad Saleh; Devanesan, Sandhanasamy; Ramamurthy, P.; Palled, Siddanna R.; Ganesh, K. M.

    2014-05-01

    Fluorescence spectral techniques are very sensitive, and hence they are gaining importance in cancer detection. The biomarkers indicative of cancer could be identified and quantified by spectral or time domain fluorescence spectroscopy. The results of an investigation of time-resolved spectra of cellular components of blood obtained from cervical cancer patients and normal controls are given. The cancer indicative biomarker in this paper is porphyrin; it has a fluorescence decay time of 60% more in samples of cancer patients than those of normal controls. Based on such measurements, a randomized set comprising samples from cancer patients and controls (N=27 in total) could be classified with sensitivity (92%) and specificity (86%).

  2. PREFACE: Time-resolved scanning tunnelling microscopy Time-resolved scanning tunnelling microscopy

    NASA Astrophysics Data System (ADS)

    Zandvliet, Harold J. W.; Lin, Nian

    2010-07-01

    out the potential landscape of the system (often a molecule or an atom) under study [4, 5]. However, the dynamical processes might also be induced by the tunnelling process itself [6, 7]. In the field of molecular science, excited single molecule experiments have been especially performed [8]. As a nice example, we refer to the work of Sykes' group [9] on thioether molecular rotors. In addition, several groups explore the possibility of combining time-resolved scanning tunnelling microscopy with optical techniques [10, 11]. Although the majority of studies that have been performed so far focus on rather simple systems under nearly ideal and well-defined conditions, we anticipate that time-resolved scanning tunnelling microscopy can also be applied in other research areas, such as biology and soft condensed matter, where the experimental conditions are often less ideal. We hope that readers will enjoy this collection of papers and that it will trigger them to further explore the possibilities of this simple, but powerful technique. References [1] Besenbacher F, Laegsgaard E and Stengaard I 2005 Mater. Today 8 26 [2] van Houselt A and Zandvliet H J W 2010 Rev. Mod. Phys. 82 1593 [3] Tringides M C and Hupalo M 2010 J. Phys.: Condens. Matter 22 264002 [4] Ronci F, Colonna S, Cricenti A and Le Lay G 2010 J. Phys.: Condens. Matter 22 264003 [5] van Houselt A, Poelsema B and Zandvliet H J W 2010 J. Phys.: Condens. Matter 22 264004 [6] Sprodowski C, Mehlhorn M and Morgenstern K 2010 J. Phys.: Condens. Matter 22 264005 [7] Saedi A, Poelsema B and Zandvliet H J W 2010 J. Phys.: Condens. Matter 22 264007 [8] Sloan P A 2010 J. Phys.: Condens. Matter 22 264001 [9] Jewell A D, Tierney H L, Baber A E, Iski E V, Laha M M and Sykes E C H 2010 J. Phys.: Condens. Matter 22 264006 [10] Riedel D 2010 J. Phys.: Condens. Matter 22 264009 [11] Terada Y, Yoshida S, Takeuchi O and Shigekawa H 2010 J. Phys.: Condens. Matter 22 264008

  3. Thymine Dimer Formation probed by Time-Resolved Vibrational Spectroscopy

    NASA Astrophysics Data System (ADS)

    Schreier, Wolfgang J.; Schrader, Tobias E.; Roller, Florian O.; Gilch, Peter; Zinth, Wolfgang; Kohler, Bern

    Cyclobutane pyrimidine dimers are the major photoproducts formed when DNA is exposed to UV light. Femtosecond time-resolved vibrational spectroscopy reveals that thymine dimers are formed in thymidine oligonucleotides in an ultrafast photoreaction.

  4. High-speed detector for time-resolved diffraction studies

    PubMed Central

    Singh, Bipin; Miller, Stuart R.; Bhandari, Harish B.; Graceffa, Rita; Irving, Thomas C.; Nagarkar, Vivek V.

    2013-01-01

    There are a growing number of high brightness synchrotron sources that require high-frame-rate detectors to provide the time-scales required for performing time-resolved diffraction experiments. We report on the development of a very high frame rate CMOS X-ray detector for time-resolved muscle diffraction and time-resolved solution scattering experiments. The detector is based on a low-afterglow scintillator, provides a megapixel resolution with frame rates of up to 120,000 frames per second, an effective pixel size of 64 µm, and can be adapted for various X-ray energies. The paper describes the detector design and initial results of time-resolved diffraction experiments on a synchrotron beamline. PMID:24489595

  5. Direct time-resolved spectroscopic investigation of intramolecular hydrogen atom transfer of deoxyblebbistatin

    NASA Astrophysics Data System (ADS)

    Li, Ming-De; Zhu, Ruixue; Lee Phillips, David

    2017-09-01

    The photophysics and photochemistry of deoxyblebbistatin was investigated using femtosecond time-resolved transient absorption spectroscopy. An ultrafast intramolecular hydrogen atom transfer (IHAT) appears to take place via the first singlet excited state of deoxyblebbistatin within 8 ps. Absorption and fluorescence photochemical results indicate the IHAT process leads to mainly conversion of deoxyblebbistatin into an enol form final product which was observed and characterized by resonance Raman spectroscopy.

  6. Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

    PubMed Central

    Krishna, Katla Sai; Biswas, Sanchita; Navin, Chelliah V.; Yamane, Dawit G.; Miller, Jeffrey T.; Kumar, Challa S.S.R.

    2013-01-01

    Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst. PMID:24327099

  7. The ULTRA Laser System—For Time-Resolved Spectroscopy

    NASA Astrophysics Data System (ADS)

    Parker, Anthony

    2010-08-01

    Time-resolved vibrational spectroscopy (TRVS) has many unique features and capabilities for elucidating the structural changes of transient species across the fs to ms time frame. The recently developed 10 KHz, 10W ULTRA laser system represents the very latest technology. ULTRA's performance and capabilities will be described for the time resolved infrared, 2D infrared and femtosecond stimulated Raman spectroscopy techniques with examples taken from organometallic intermediates, organic excited states and DNA tertiary structure.

  8. Seventh international conference on time-resolved vibrational spectroscopy

    SciTech Connect

    Dyer, R.B.; Martinez, M.A.D.; Shreve, A.; Woodruff, W.H.

    1997-04-01

    The International Conference on Time-Resolved Vibrational Spectroscopy (TRVS) is widely recognized as the major international forum for the discussion of advances in this rapidly growing field. The 1995 conference was the seventh in a series that began at Lake Placid, New York, 1982. Santa Fe, New Mexico, was the site of the Seventh International Conference on Time-Resolved Vibrational Spectroscopy, held from June 11 to 16, 1995. TRVS-7 was attended by 157 participants from 16 countries and 85 institutions, and research ranging across the full breadth of the field of time-resolved vibrational spectroscopy was presented. Advances in both experimental capabilities for time-resolved vibrational measurements and in theoretical descriptions of time-resolved vibrational methods continue to occur, and several sessions of the conference were devoted to discussion of these advances and the associated new directions in TRVS. Continuing the interdisciplinary tradition of the TRVS meetings, applications of time-resolved vibrational methods to problems in physics, biology, materials science, and chemistry comprised a large portion of the papers presented at the conference.

  9. Comparison of a commercial Varicella Zoster glycoprotein IgG enzyme immunoassay with a reference time resolved fluorescence immunoassay (VZV TRFIA) for measuring VZV IgG in sera from pregnant women, sera sent for confirmatory testing and pre and post vOka vaccination sera from healthcare workers.

    PubMed

    Maple, P A C; Breuer, J; Quinlivan, M; Kafatos, G; Brown, K E

    2012-03-01

    Recently, a commercial, standardised VZV IgG glycoprotein EIA, Binding Site VaccZyme™VZV glycoprotein IgG low level EIA (VaccZyme™EIA) has become available. The VaccZyme™EIA is more robust and user friendly than the reference VZV time-resolved fluorescence immunoassay (VZV TRFIA). To assess the usefulness of the VaccZyme™EIA in the diagnostic laboratory by comparing VZV IgG levels generated by both assays on serum panels representing, non-vaccinated, and vOka vaccinated populations. Sera from non-vaccinated individuals were tested; 248 from pregnant women, 117 from various patient groups referred to the Virus Reference Department for confirmatory VZV IgG testing and 102 from healthcare workers enrolled in a study (ROVE) of antibody/IgG response to vOka. From the ROVE study, 282 post vaccination sera were tested; 108 and 101 collected at six weeks post first and second doses of vOka, respectively, and 73 collected at 18 month follow-up. Sensitivities and specificities (equivocals treated as negatives) of the VaccZyme™EIA for sera from pregnant women were 97.8% (95% CI: [94.6%, 99.4%]) and 96.8% (95% CI: [89.0%, 99.6%]), respectively, and for sera referred for confirmatory testing were 81.2% (95% CI: [71.2%, 88.8%]) and 96.9% (95% CI: [83.8%, 99.9%]), respectively, and for ROVE baseline sera were 54.2% (95% CI: [32.8%, 74.4%]) and 100% (95% CI: [95.4%, 100.0%]), respectively. For the post vOka serum panels sensitivities of the VaccZyme™EIA ranged from 65.3% (95% CI: [50.4%, 78.3%]) to 80.4% (95% CI: [71.1%, 87.8%]). Specificities were all 100%. Correlation with VZV TRFIA was high and agreement varied between the serum panels tested. VaccZyme™EIA is recommended for detecting VZV IgG in sera from non-vaccinated populations; however, caution is advised when measuring post vOka VZV IgG levels. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Latent fingerprint and trace explosives detection by photoluminescence and time-resolved imaging

    NASA Astrophysics Data System (ADS)

    Bouldin, Kimberly Kay

    Latent fingerprint detection by photoluminescence is a well-developed field. Many development techniques exist and are currently being employed in forensic laboratories to detect fingerprints by making them luminescent. However, in forensic science, time-resolved imaging techniques, designed to suppress background fluorescence that interferes with fingerprint detectability, are to date not used outside of the research laboratory, and the chemistry necessary to use time-resolved imaging for fingerprint detection is somewhat limited. For this reason, the first section of this dissertation deals with fingerprint detection methods that have direct application to time-resolved imaging techniques. Trace explosive detection field methods based on chemical reactions have until recently utilized only colorimetric products. To increase the sensitivity of such detection, a field explosive test kit which produces a product that is both colorimetric and luminescent is studied. Detection sensitivity can be gained by taking advantage of the luminescence of these products, something that has not been done to date. When the appropriate chemistry is chosen for explosive detection, time-resolved imaging techniques may again be applicable. This dissertation thus looks at possibilities of taking trace explosives detection to this next level.

  11. Time-resolved experiments in the frequency domain using synchrotron radiation (invited)

    SciTech Connect

    De Stasio, G. ); Giusti, A.M.; Parasassi, T.; Ravagnan, G. ); Sapora, O. )

    1992-01-01

    PLASTIQUE is the only synchrotron radiation beam line in the world that performs time-resolved fluorescence experiments in frequency domain. These experiments are extremely valuable sources of information on the structure and the dynamics of molecules. This technique measures fluorescence lifetimes with picosecond resolution in the near UV spectral range. Such accurate measurements are rendered possible by taking phase and modulation data, and by the advantages of the cross-correlation technique. A successful experiment demonstrated the radiation damage induced by low doses of radiation on rabbit blood cell membranes.

  12. Fibre optic time-resolved spectroscopy using CMOS-SPAD arrays

    NASA Astrophysics Data System (ADS)

    Ehrlich, K.; Kufcsák, A.; Krstajić, N.; Henderson, R. K.; Thomson, R. R.; Tanner, M. G.

    2017-02-01

    In vivo fibre optic fluorescence-based sensing is the use of synthesised fluorophores which interrogate the local environment via variation in their fluorescence emission, addressed through an optic fibre. However, the emission intensity is influenced by intrinsic factors such as photobleaching, quantitative factors like concentration dependency and background signals from autofluorescence of tissue and the delivery optical fibre. Many of these problems can be addressed by using time-resolved spectroscopy which measures variations in the fluorescent lifetime. We present a versatile fibre-based time-resolved spectrograph based on a CMOS SPAD line sensor capable of acquiring time and spectral resolved fluorescent lifetime data in a single measurement exploiting the time-correlated single photon counting (TCSPC) technique. It is shown that these TCSPC histograms enable the differentiation between autofluorescence of tissue and synthesized fluorophores, as well as the removal of unwanted fibre background through post-processed time-gating. As a proof-of-principle application the pH- dependent changes in fluorescent lifetime of 5-carboxyuorescein (FAM) are measured.

  13. Time-Resolved Autofluorescence Imaging of Human Donor Retina Tissue from Donors with Significant Extramacular Drusen

    PubMed Central

    Schweitzer, Dietrich; Gaillard, Elizabeth R.; Dillon, James; Mullins, Robert F.; Russell, Stephen; Hoffmann, Birgit; Peters, Sven; Hammer, Martin; Biskup, Christoph

    2012-01-01

    Purpose. Time and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen. Methods. Tissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The repetition rate was 76 MHz and the pulse width was 170 femtoseconds (fs). The time-resolved autofluorescence was recorded simultaneously in 16 spectral channels (445–605 nm) and bi-exponentially fitted. Results. RPE can be discriminated clearly from Bruch's membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected. Conclusions. The demonstrated differentiation of fluorescent structures by their fluorescence decay time is important for interpretation of in vivo measurements by the new fluorescence lifetime imaging (FLIM) ophthalmoscopy on healthy subjects as well as on patients. PMID:22511622

  14. Protein-ligand interactions probed by time-resolved crystallography

    SciTech Connect

    Schmidt, M.; Ihee, H.; Pahl, R.; Srajer, V.

    2005-03-09

    Time-resolved (TR) crystallography is a unique method for determining the structures of intermediates in biomolecular reactions. The technique reached its mature stage with the development of the powerful third-generation synchrotron X-ray sources, and the advances in data processing and analysis of time-resolved Laue crystallographic data. A time resolution of 100 ps has been achieved and relatively small structural changes can be detected even from only partial reaction initiation. The remaining challenge facing the application of this technique to a broad range of biological systems is to find an efficient and rapid, system-specific method for the reaction initiation in the crystal. Other frontiers for the technique involve the continued improvement in time resolution and further advances in methods for determining intermediate structures and reaction mechanisms. The time-resolved technique, combined with trapping methods and computational approaches, holds the promise for a complete structure-based description of biomolecular reactions.

  15. A time-resolved image sensor for tubeless streak cameras

    NASA Astrophysics Data System (ADS)

    Yasutomi, Keita; Han, SangMan; Seo, Min-Woong; Takasawa, Taishi; Kagawa, Keiichiro; Kawahito, Shoji

    2014-03-01

    This paper presents a time-resolved CMOS image sensor with draining-only modulation (DOM) pixels for tube-less streak cameras. Although the conventional streak camera has high time resolution, the device requires high voltage and bulky system due to the structure with a vacuum tube. The proposed time-resolved imager with a simple optics realize a streak camera without any vacuum tubes. The proposed image sensor has DOM pixels, a delay-based pulse generator, and a readout circuitry. The delay-based pulse generator in combination with an in-pixel logic allows us to create and to provide a short gating clock to the pixel array. A prototype time-resolved CMOS image sensor with the proposed pixel is designed and implemented using 0.11um CMOS image sensor technology. The image array has 30(Vertical) x 128(Memory length) pixels with the pixel pitch of 22.4um. .

  16. Time-Resolved X-Ray Crystallography of Heme Proteins

    SciTech Connect

    Srajer, Vukica; Royer, Jr., William E.

    2008-04-29

    Heme proteins, with their natural photosensitivity, are excellent systems for the application of time-resolved crystallographic methods. Ligand dissociation can be readily initiated by a short laser pulse with global structural changes probed at the atomic level by X-rays in real time. Third-generation synchrotrons provide 100-ps X-ray pulses of sufficient intensity for monitoring very fast processes. Successful application of such time-resolved crystallographic experiments requires that the structural changes being monitored are compatible with the crystal lattice. These techniques have recently permitted observing for the first time allosteric transitions in real time for a cooperative dimeric hemoglobin.

  17. Time-resolved x-ray crystallography of heme proteins

    PubMed Central

    Royer, William E.

    2012-01-01

    Heme proteins, with their natural photosensitivity, are excellent systems for the application of time-resolved crystallographic methods. Ligand dissociation can be readily initiated by a short laser pulse with global structural changes probed at the atomic level by X-rays in real time. Third generation synchrotrons provide 100ps X-ray pulses of sufficient intensity for monitoring very fast processes. Successful application of such time-resolved crystallographic experiments requires that the structural changes being monitored are compatible with the crystal lattice. These techniques have permitted observing allosteric transitions in real time for a cooperative dimeric hemoglobin. PMID:18433638

  18. Time-resolved photon emission from layered turbid media

    NASA Astrophysics Data System (ADS)

    Hielscher, Andreas H.; Liu, Hanli; Chance, Britton; Tittel, Frank K.; Jacques, Steven L.

    1996-02-01

    We present numerical and experimental results of time-resolved emission profiles from various layered turbid media. Numerical solutions determined by time-resolved Monte Carlo simulations are compared with measurements on layered-tissue phantoms made from gelatin. In particular, we show that in certain cases the effects of the upper layers can be eliminated. As a practical example, these results are used to analyze in vivo measurements on the human head. This demonstrates the influence of skin, skull, and meninges on the determination of the blood oxygenation in the brain.

  19. Time-resolved photon emission from layered turbid media

    SciTech Connect

    Hielscher, A.H.; Liu, H.; Chance, B.; Tittel, F.K.; Jacques, S.L.

    1996-02-01

    We present numerical and experimental results of time-resolved emission profiles from various layered turbid media. Numerical solutions determined by time-resolved Monte Carlo simulations are compared with measurements on layered-tissue phantoms made from gelatin. In particular, we show that in certain cases the effects of the upper layers can be eliminated. As a practical example, these results are used to analyze {ital in} {ital vivo} measurements on the human head. This demonstrates the influence of skin, skull, and meninges on the determination of the blood oxygenation in the brain. {copyright} {ital 1996 Optical Society of America.}

  20. Alteration of time-resolved autofluorescence properties of rat aorta, induced by diabetes mellitus

    NASA Astrophysics Data System (ADS)

    Uherek, M.; Uličná, O.; Vančová, O.; Muchová, J.; Ďuračková, Z.; Šikurová, L.; Chorvát, D.

    2016-10-01

    Changes in autofluorescence properties of isolated rat aorta, induced by diabetes mellitus, were detected using time-resolved fluorescence spectroscopy with pulsed ultraviolet (UV) laser excitation. We demonstrated that time-resolved spectroscopy was able to detect changes in aorta tissues related to diabetes and unambiguously discriminate diabetic (τ 1 0.63  ±  0.05 ns, τ 2 3.66  ±  0.10 ns) samples from the control (τ 1 0.76  ±  0.03 ns, τ 2 4.48  ±  0.15 ns) group. We also report changes in the ratio of relative amplitudes of the two lifetime component in aorta tissue during diabetes, most likely related to the pseudohypoxic state with altered NADH homeostasis.

  1. Time-Resolved Photodetachment Anisotropy: Gas-Phase Rotational and Vibrational Dynamics of the Fluorescein Anion.

    PubMed

    Horke, Daniel A; Chatterley, Adam S; Bull, James N; Verlet, Jan R R

    2015-01-02

    The photoelectron signal of the singly deprotonated fluorescein anion is found to be highly dependent on the relative polarization between pump and probe pulses, and time-resolved photodetachment anisotropy (TR-PA) is developed as a probe of the rotational dynamics of the chromophore. The total photoelectron signal shows both rotational and vibrational wavepacket dynamics, and we demonstrate how TR-PA can readily disentangle these dynamical processes. TR-PA in fluorescein presents specific opportunities for its development as a probe for rotational dynamics in large biomolecules as fluorescein derivatives are commonly incorporated in complex biomolecules and have been used extensively in time-resolved fluorescence anisotropy measurements, to which TR-PA is a gas-phase analogue.

  2. Time-Resolved Emission Spectroscopy of Field Reversed Configuration Thruster

    DTIC Science & Technology

    2016-08-31

    Field Reversed Configuration (FRC) thrusters are candidates for next generation high -powered electric propulsion (EP) • Advantages over competing...16468 Introduction • Field Reversed Configuration (FRC) thrusters are candidates for next generation high -powered electric propulsion (EP) • Advantages...Briefing Charts 3. DATES COVERED (From - To) 06 September 2016 - 01 November 2016 4. TITLE AND SUBTITLE Time-Resolved Emission Spectroscopy of Field

  3. Time-resolved imaging of cavitation effects during laser lithotripsy

    NASA Astrophysics Data System (ADS)

    Siano, Salvatore; Pini, Roberto; Salimbeni, Renzo; Vannini, Matteo

    1995-01-01

    We devised a diagnostic technique based on a pump-and-probe scheme that provided time- resolved imaging of photofragmentation effects during laser lithotripsy. The evolution of the cavitation bubble induced on kidney stone samples by underwater irradiation with a XeCl excimer laser is presented and analyzed.

  4. Time-resolved Förster-resonance-energy-transfer DNA assay on an active CMOS microarray

    PubMed Central

    Schwartz, David Eric; Gong, Ping; Shepard, Kenneth L.

    2008-01-01

    We present an active oligonucleotide microarray platform for time-resolved Förster resonance energy transfer (TR-FRET) assays. In these assays, immobilized probe is labeled with a donor fluorophore and analyte target is labeled with a fluorescence quencher. Changes in the fluorescence decay lifetime of the donor are measured to determine the extent of hybridization. In this work, we demonstrate that TR-FRET assays have reduced sensitivity to variances in probe surface density compared with standard fluorescence-based microarray assays. Use of an active array substrate, fabricated in a standard complementary metal-oxide-semiconductor (CMOS) process, provides the additional benefits of reduced system complexity and cost. The array consists of 4096 independent single-photon avalanche diode (SPAD) pixel sites and features on-chip time-to-digital conversion. We demonstrate the functionality of our system by measuring a DNA target concentration series using TR-FRET with semiconductor quantum dot donors. PMID:18515059

  5. The analysis of time-resolved optical waveguide absorption spectroscopy based on positive matrix factorization.

    PubMed

    Liu, Ping; Li, Zhu; Li, Bo; Shi, Guolong; Li, Minqiang; Yu, Daoyang; Liu, Jinhuai

    2013-08-01

    Time-resolved optical waveguide absorption spectroscopy (OWAS) makes use of an evanescent field to detect the polarized absorption spectra of sub-monomolecular adlayers. This technique is suitable for the investigation of kinetics at the solid/liquid interface of dyes, pigments, fluorescent molecules, quantum dots, metallic nanoparticles, and proteins with chromophores. In this work, we demonstrate the application of positive matrix factorization (PMF) to analyze time-resolved OWAS for the first time. Meanwhile, PCA is researched to compare with PMF. The absorption/desorption kinetics of Rhodamine 6G (R6G) onto a hydrophilic glass surface and the dynamic process of Meisenheimer complex between Cysteine and TNT are selected as samples to verify experimental system and analytical methods. The results are shown that time-resolved OWAS can well record the absorption/desorption of R6G onto a hydrophilic glass surface and the dynamic formation process of Meisenheimer complexes. The feature of OWAS extracted by PMF is dynamic and consistent with the results analyzed by the traditional function of time/wavelength-absorbance. Moreover, PMF prevents the negative factors from occurring, avoids contradicting physical reality, and makes factors more easily interpretable. Therefore, we believe that PMF will provide a valuable analysis route to allow processing of increasingly large and complex data sets.

  6. Time-resolved rotational spectroscopy of para-difluorobenzene·Ar

    NASA Astrophysics Data System (ADS)

    Weichert, A.; Riehn, C.; Matylitsky, V. V.; Jarzeba, W.; Brutschy, B.

    2002-07-01

    We report on time-resolved rotational spectroscopy experiments of the cluster para-difluorobenzene·Ar ( pDFB·Ar) by picosecond laser pulses in a supersonic expansion. Rotational coherences of pDFB·Ar are generated by resonant electronic excitation and probed by time-resolved fluorescence depletion spectroscopy and time-resolved photoionization ((1+1') PPI) spectroscopy. The former allows the determination of both ground and excited state rotational constants, whereas the latter technique enables the separate study of the excited state with the benefit of mass-selective detection. Since pDFB·Ar represents a near symmetric oblate rotor, persistent J-type transients with tJ≈ n/2( A+ B) could be measured. From their analysis, (A″+B″)=2234.9±2 MHz and (A'+B')=2237.9±2 MHz were obtained. A structural investigation, based on data of the pDFB monomer, is presented resulting in a pDFB·Ar center-of-mass distance of both moieties of R z=3.543±0.017 Å with a change of ΔR z=-0.057±0.009 Å upon electronic excitation. These results are compared to data of former frequency-resolved experiments and ab initio computations.

  7. New time-resolved micro-photoluminescence spectroscopy of natural and synthetic analogue minerals

    NASA Astrophysics Data System (ADS)

    Panczer, G.; Ollier, N.; Champagnon, B.; Gaft, M.

    2003-04-01

    Minerals as well as geomaterials often present light emissions under UV or visible excitations. This property called photoluminescence is due to low concentration impurities such as the rare earths, the transition elements and the lanthanides. The induced color is used for ore prospection but only spectroscopic analyses indicate the nature of the emitted centers. However natural samples contained numerous luminescent centers simultaneously and with regular steady-state measurements (such as in cathodoluminescence) all the emissions are often over lapping. In order to record the contributions of each separate center, it is possible to use time-resolved measurements based on the decay time of the emissions and using pulsed laser excitation. Some characteristic examples will be presented on apatites, zircons as well as gemstones. Geomaterials present as well micro scale heterogeneities (growth zoning, inclusions, devitrification, microphases...). Precise identification and optical effects of such heterogeneities have to be taken into account. To reach the microscale using photo luminescence studies, a microscope has be modified to allowed pulsed laser injection (from UV to visible), beam focus with micro scale resolution on the sample (<10 μm), as well as time resolved collection of micro fluorescence. Such equipment allows now undertaking time-resolved measurements of microphases. Applications on geomaterials will be presented.

  8. Use of time-resolved spectroscopy as a method to monitor carotenoids present in tomato extract obtained using ultrasound treatment.

    PubMed

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

    2016-01-01

    Compounds exhibiting antioxidant activity have received much interest in the food industry because of their potential health benefits. Carotenoids such as lycopene, which in the human diet mainly derives from tomatoes (Solanum lycopersicum), have attracted much attention in this aspect and the study of their extraction, processing and storage procedures is of importance. Optical techniques potentially offer advantageous non-invasive and specific methods to monitor them. To obtain both fluorescence and Raman information to ascertain if ultrasound assisted extraction from tomato pulp has a detrimental effect on lycopene. Use of time-resolved fluorescence spectroscopy to monitor carotenoids in a hexane extract obtained from tomato pulp with application of ultrasound treatment (583 kHz). The resultant spectra were a combination of scattering and fluorescence. Because of their different timescales, decay associated spectra could be used to separate fluorescence and Raman information. This simultaneous acquisition of two complementary techniques was coupled with a very high time-resolution fluorescence lifetime measurement of the lycopene. Spectroscopic data showed the presence of phytofluene and chlorophyll in addition to lycopene in the tomato extract. The time-resolved spectral measurement containing both fluorescence and Raman data, coupled with high resolution time-resolved measurements, where a lifetime of ~5 ps was attributed to lycopene, indicated lycopene appeared unaltered by ultrasound treatment. Detrimental changes were, however, observed in both chlorophyll and phytofluene contributions. Extracted lycopene appeared unaffected by ultrasound treatment, while other constituents (chlorophyll and phytofluene) were degraded. Copyright © 2015 John Wiley & Sons, Ltd.

  9. Planetary Surface Exploration Using Time-Resolved Laser Spectroscopy on Rovers and Landers

    NASA Astrophysics Data System (ADS)

    Blacksberg, Jordana; Alerstam, Erik; Maruyama, Yuki; Charbon, Edoardo; Rossman, George

    2013-04-01

    Planetary surface exploration using laser spectroscopy has become increasingly relevant as these techniques become a reality on Mars surface missions. The ChemCam instrument onboard the Curiosity rover is currently using laser induced breakdown spectroscopy (LIBS) on a mast-mounted platform to measure elemental composition of target rocks. The RLS Raman Spectrometer is included on the payload for the ExoMars mission to be launched in 2018 and will identify minerals and organics on the Martian surface. We present a next-generation instrument that builds on these widely used techniques to provide a means for performing both Raman spectroscopy and LIBS in conjunction with microscopic imaging. Microscopic Raman spectroscopy with a laser spot size smaller than the grains of interest can provide surface mapping of mineralogy while preserving morphology. A very small laser spot size (~ 1 µm) is often necessary to identify minor phases that are often of greater interest than the matrix phases. In addition to the difficulties that can be posed by fine-grained material, fluorescence interference from the very same material is often problematic. This is particularly true for many of the minerals of interest that form in environments of aqueous alteration and can be highly fluorescent. We use time-resolved laser spectroscopy to eliminate fluorescence interference that can often make it difficult or impossible to obtain Raman spectra. As an added benefit, we have found that with small changes in operating parameters we can include microscopic LIBS using the same hardware. This new technique relies on sub-ns, high rep-rate lasers with relatively low pulse energy and compact solid state detectors with sub-ns time resolution. The detector technology that makes this instrument possible is a newly developed Single-Photon Avalanche Diode (SPAD) sensor array based on Complementary Metal-Oxide Semiconductor (CMOS) technology. The use of this solid state time-resolved detector offers a

  10. Radiative lifetime measurements of some Gd I levels by time-resolved laser spectroscopy

    NASA Astrophysics Data System (ADS)

    Shang, Xue; Zhou, Chunxiao; Dai, Zhenwen

    2017-04-01

    Natural radiative lifetimes for 27 excited levels of Gd I in the energy range from 28215.140 to 43963.900 cm-1 were measured using time-resolved laser-induced fluorescence (TR-LIF) technique in an atom beam produced by laser-induced plasma. All the lifetimes obtained in this paper range from 8.4 to 833 ns with the uncertainties within ten percent. A comparison with a few previously reported values was performed and good agreement between them was achieved. To our best knowledge, 18 lifetimes of Gd I are reported for the first time.

  11. Exploiting time-resolved magnetic field effects for determining radical ion reaction rates

    NASA Astrophysics Data System (ADS)

    Bessmertnykh, A. O.; Borovkov, V. I.; Bagryansky, V. A.; Molin, Yu N.

    2016-07-01

    The capabilities of the method of time-resolved magnetic field effect in determining the rates of charge transfer reactions between radical ions and molecules on a nanosecond time scale have been investigated. The approach relies on the electron spin coherence in radical pair's partners generated by ionizing radiation. The spin evolution of the pair is sensitive to the reaction since the latter results in changing magnetic interactions of the unpaired electron. This process can be monitored by magnetic-field-sensitive fluorescence from an irradiated sample that is illustrated using reactions involving alkane radical cations. The accuracy and limitations of the approach are discussed.

  12. Time resolved optical tomography of the human forearm

    NASA Astrophysics Data System (ADS)

    Hillman, Elizabeth M. C.; Hebden, Jeremy C.; Schweiger, Martin; Dehghani, Hamid; Schmidt, Florian E. W.; Delpy, David T.; Arridge, Simon R.

    2001-04-01

    A 32-channel time-resolved optical imaging instrument has been developed principally to study functional parameters of the new-born infant brain. As a prelude to studies on infants, the device and image reconstruction methodology have been evaluated on the adult human forearm. Cross-sectional images were generated using time-resolved measurements of transmitted light at two wavelengths. All data were acquired using a fully automated computer-controlled protocol. Images representing the internal scattering and absorbing properties of the arm are presented, as well as images that reveal physiological changes during a simple finger flexion exercise. The results presented in this paper represent the first simultaneous tomographic reconstruction of the internal scattering and absorbing properties of a clinical subject using purely temporal data, with additional co-registered difference images showing repeatable absorption changes at two wavelengths in response to exercise.

  13. Time resolved optical tomography of the human forearm.

    PubMed

    Hillman, E M; Hebden, J C; Schweiger, M; Dehghani, H; Schmidt, F E; Delpy, D T; Arridge, S R

    2001-04-01

    A 32-channel time-resolved optical imaging instrument has been developed principally to study functional parameters of the new-born infant brain. As a prelude to studies on infants, the device and image reconstruction methodology have been evaluated on the adult human forearm. Cross-sectional images were generated using time-resolved measurements of transmitted light at two wavelengths. All data were acquired using a fully automated computer-controlled protocol. Images representing the internal scattering and absorbing properties of the arm are presented, as well as images that reveal physiological changes during a simple finger flexion exercise. The results presented in this paper represent the first simultaneous tomographic reconstruction of the internal scattering and absorbing properties of a clinical subject using purely temporal data, with additional co-registered difference images showing repeatable absorption changes at two wavelengths in response to exercise.

  14. Time-Resolved Rayleigh Scattering Measurements in Hot Gas Flows

    NASA Technical Reports Server (NTRS)

    Mielke, Amy F.; Elam, Kristie A.; Sung, Chih-Jen

    2008-01-01

    A molecular Rayleigh scattering technique is developed to measure time-resolved gas velocity, temperature, and density in unseeded gas flows at sampling rates up to 32 kHz. A high power continuous-wave laser beam is focused at a point in an air flow field and Rayleigh scattered light is collected and fiber-optically transmitted to the spectral analysis and detection equipment. The spectrum of the light, which contains information about the temperature and velocity of the flow, is analyzed using a Fabry-Perot interferometer. Photomultipler tubes operated in the photon counting mode allow high frequency sampling of the circular interference pattern to provide time-resolved flow property measurements. Mean and rms velocity and temperature fluctuation measurements in both an electrically-heated jet facility with a 10-mm diameter nozzle and also in a hydrogen-combustor heated jet facility with a 50.8-mm diameter nozzle at NASA Glenn Research Center are presented.

  15. Time-resolved studies of individual molecular rotors.

    PubMed

    Jewell, April D; Tierney, Heather L; Baber, Ashleigh E; Iski, Erin V; Laha, Michael M; Sykes, E Charles H

    2010-07-07

    Thioether molecular rotors show great promise as nanoscale models for exploring the fundamental limits of thermally and electrically driven molecular rotation. By using time-resolved measurements which increase the time resolution of the scanning tunneling microscope we were able to record the dynamics of individual thioether molecular rotors as a function of surface structure, rotor chemistry, thermal energy and electrical excitation. Our results demonstrate that the local surface structure can have a dramatic influence on the energy landscape that the molecular rotors experience. In terms of rotor structure, altering the length of the rotor's alkyl tails allowed the origin of the barrier to rotation to be more fully understood. Finally, time-resolved measurement of electrically excited rotation revealed that vibrational excitation of a C-H bond in the rotor's alkyl tail is an efficient channel with which to excite rotation, and that the excitation is a one-electron process. © 2010 IOP Publishing Ltd

  16. Time resolved Schlieren imaging of DBD actuator flow fields

    NASA Astrophysics Data System (ADS)

    Nourgostar, Cyrus; Oksuz, Lutfi; Hershkowitz, Noah

    2009-10-01

    Schlieren imaging methods measure the first derivative of density in the direction of a knife-edge spatial filter. It has been used extensively in aerodynamic research to visualize the structure of flow fields. With a single barrier planer dielectric barrier discharge (DBD) actuator, Schlieren images clearly show the absence of significant vertical air flow normal to the surface, and no more than few millimeters thick induced boundary layer flow. A gated intensified CCD camera along with a Schlieren system can not only visualize the flow field induced by the actuator, but also temporarily resolve the images of the flow and plasma field. Our time resolved images with triangular applied voltage waveforms indicate that several separate discharge regimes occur during positive and negative going half cycles of single and double barrier DBD actuators. Time resolved Schlieren imaging of both single and double barrier DBDs with different applied waveforms, discharge parameters and electrode geometries reveal important information on the induced flow structure.

  17. Benchtop time-resolved magneto-optical Kerr magnetometer.

    PubMed

    Barman, Anjan; Kimura, T; Otani, Y; Fukuma, Y; Akahane, K; Meguro, S

    2008-12-01

    We present here the construction and application of a compact benchtop time-resolved Kerr magnetometer to measure the magnetization precession in magnetic thin films and lithographically patterned elements. As opposed to very expensive femtosecond lasers this system is built upon a picosecond pulsed injection diode laser and electronic pulse and delay generators. The precession is triggered by the electronic pulses of controlled duration and shape, which is launched onto the sample by a microstrip line. We used polarized optical pulses synchronous to the electronic pulses to measure the magneto-optical Kerr rotation. The system is integrated in a conventional upright microscope configuration with separate illumination, imaging, and magneto-optical probe paths. The system offers high stability, relative ease of alignment, sample changing, and a long range of time delay. We demonstrate the measurements of time-resolved dynamics of a Permalloy microwire and microdot using this system, which showed dynamics at two different time scales.

  18. Time-resolved single dopant charge dynamics in silicon

    NASA Astrophysics Data System (ADS)

    Rashidi, Mohammad; Burgess, Jacob A. J.; Taucer, Marco; Achal, Roshan; Pitters, Jason L.; Loth, Sebastian; Wolkow, Robert A.

    2016-10-01

    As the ultimate miniaturization of semiconductor devices approaches, it is imperative that the effects of single dopants be clarified. Beyond providing insight into functions and limitations of conventional devices, such information enables identification of new device concepts. Investigating single dopants requires sub-nanometre spatial resolution, making scanning tunnelling microscopy an ideal tool. However, dopant dynamics involve processes occurring at nanosecond timescales, posing a significant challenge to experiment. Here we use time-resolved scanning tunnelling microscopy and spectroscopy to probe and study transport through a dangling bond on silicon before the system relaxes or adjusts to accommodate an applied electric field. Atomically resolved, electronic pump-probe scanning tunnelling microscopy permits unprecedented, quantitative measurement of time-resolved single dopant ionization dynamics. Tunnelling through the surface dangling bond makes measurement of a signal that would otherwise be too weak to detect feasible. Distinct ionization and neutralization rates of a single dopant are measured and the physical process controlling those are identified.

  19. Time resolved optical spectra from MIG welding arc ignitions

    SciTech Connect

    Eriksen, P.

    1985-03-01

    Optical radiation from MIG (GMAW) welding arc ignitions has been measured with a rapid scan spectrometer. The time resolved spectral measurements reveal a substantial overshoot of ultraviolet radiation during the ignition phase of a 200 A aluminum arc. Calculations which follow the ACGIH guidelines show that, at a welding current of 300 A, the unprotected eye at a distance of 0.5 m may suffer a flash after the reception of radiation from only one ignition.

  20. The RATIO method for time-resolved Laue crystallography

    PubMed Central

    Coppens, Philip; Pitak, Mateusz; Gembicky, Milan; Messerschmidt, Marc; Scheins, Stephan; Benedict, Jason; Adachi, Shin-ichi; Sato, Tokushi; Nozawa, Shunsuke; Ichiyanagi, Kohei; Chollet, Matthieu; Koshihara, Shin-ya

    2009-01-01

    A RATIO method for analysis of intensity changes in time-resolved pump–probe Laue diffraction experiments is described. The method eliminates the need for scaling the data with a wavelength curve representing the spectral distribution of the source and removes the effect of possible anisotropic absorption. It does not require relative scaling of series of frames and removes errors due to all but very short term fluctuations in the synchrotron beam. PMID:19240334

  1. Ultrafast Time-Resolved Electron Diffraction with Megavolt Electron Beams

    SciTech Connect

    Hastings, J.B.; Rudakov, F.M.; Dowell, D.H.; Schmerge, J.F.; Cardoza, J.D.; Castro, J.M.; Gierman, S.M.; Loos, H.; Weber, P.M.; /Brown U.

    2006-10-24

    An rf photocathode electron gun is used as an electron source for ultrafast time-resolved pump-probe electron diffraction. We observed single-shot diffraction patterns from a 160 nm Al foil using the 5.4 MeV electron beam from the Gun Test Facility at the Stanford Linear Accelerator. Excellent agreement with simulations suggests that single-shot diffraction experiments with a time resolution approaching 100 fs are possible.

  2. Studies of Minerals, Organic and Biogenic Materials through Time-Resolved Raman Spectroscopy

    NASA Technical Reports Server (NTRS)

    Garcia, Christopher S.; Abedin, M. Nurul; Ismail, Syed; Sharma, Shiv K.; Misra, Anupam K.; Nyugen, Trac; Elsayed-Ali, hani

    2009-01-01

    A compact remote Raman spectroscopy system was developed at NASA Langley Research center and was previously demonstrated for its ability to identify chemical composition of various rocks and minerals. In this study, the Raman sensor was utilized to perform time-resolved Raman studies of various samples such as minerals and rocks, Azalea leaves and a few fossil samples. The Raman sensor utilizes a pulsed 532 nm Nd:YAG laser as excitation source, a 4-inch telescope to collect the Raman-scattered signal from a sample several meters away, a spectrograph equipped with a holographic grating, and a gated intensified CCD (ICCD) camera system. Time resolved Raman measurements were carried out by varying the gate delay with fixed short gate width of the ICCD camera, allowing measurement of both Raman signals and fluorescence signals. Rocks and mineral samples were characterized including marble, which contain CaCO3. Analysis of the results reveals the short (approx.10-13 s) lifetime of the Raman process, and shows that Raman spectra of some mineral samples contain fluorescence emission due to organic impurities. Also analyzed were a green (pristine) and a yellow (decayed) sample of Gardenia leaves. It was observed that the fluorescence signals from the green and yellow leaf samples showed stronger signals compared to the Raman lines. Moreover, it was also observed that the fluorescence of the green leaf was more intense and had a shorter lifetime than that of the yellow leaf. For the fossil samples, Raman shifted lines could not be observed due the presence of very strong short-lived fluorescence.

  3. Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6.

    PubMed

    Karvinen, Jarkko; Elomaa, Annika; Mäkinen, Maija Liisa; Hakala, Harri; Mukkala, Veli Matti; Peuralahti, Jari; Hurskainen, Pertti; Hovinen, Jari; Hemmilä, Ilkka

    2004-02-15

    Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.

  4. Relationship between time-resolved and non-time-resolved Beer-Lambert law in turbid media.

    PubMed

    Nomura, Y; Hazeki, O; Tamura, M

    1997-06-01

    The time-resolved Beer-Lambert law proposed for oxygen monitoring using pulsed light was extended to the non-time-resolved case in a scattered medium such as living tissues with continuous illumination. The time-resolved Beer-Lambert law was valid for the phantom model and living tissues in the visible and near-infrared regions. The absolute concentration and oxygen saturation of haemoglobin in rat brain and thigh muscle could be determined. The temporal profile of rat brain was reproduced by Monte Carlo simulation. When the temporal profiles of rat brain under different oxygenation states were integrated with time, the absorbance difference was linearly related to changes in the absorption coefficient. When the simulated profiles were integrated, there was a linear relationship within the absorption coefficient which was predicted for fractional inspiratory oxygen concentration from 10 to 100% and, in the case beyond the range of the absorption coefficient, the deviation from linearity was slight. We concluded that an optical pathlength which is independent of changes in the absorption coefficient is a good approximation for near-infrared oxygen monitoring.

  5. Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase

    PubMed Central

    Ibrahim, Ziad; Martel, Anne; Moulin, Martine; Kim, Henry S.; Härtlein, Michael; Franzetti, Bruno; Gabel, Frank

    2017-01-01

    We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale. PMID:28102317

  6. Application of spectral unmixing in multi-wavelength time-resolved spectroscopy

    NASA Astrophysics Data System (ADS)

    Chorvat, D., Jr.; Mateasik, A.; Kirchnerova, J.; Chorvatova, A.

    2007-09-01

    We present a new approach for analysis of multi-wavelength time-resolved spectroscopy data, based on sequential spectral unmixing. Principal component analysis was used to identify the number and spectral profiles of the main components of intrinsic flavin signal in multi-wavelength time-resolved fluorescence recordings from isolated living cardiac myocytes. To determine these components, natural variations in the cardiomyocyte autofluorescence spectra were induced by modulators of mitochondrial metabolism and respiration. Using aforementioned approach we have identified two main components of intrinsic flavin emission in cardiac myocytes. The first component show emission maximum at 486-504 nm and mean lifetime of 1.2 nanoseconds, the second component with peak at 522 nm has two-exponential decay with fluorescence lifetimes of 0.3 and 3.1 nanoseconds. Comparison of gathered new results to our previous studies of flavins in vitro and in cardiac cells clearly points to the fact that the estimated spectral components correspond to flavin adenine dinucleotide (FAD) bound to enzyme(s) of mitochondrial metabolic chain, and to free FAD, respectively.

  7. Lucas-Kanade fluid trajectories for time-resolved PIV

    NASA Astrophysics Data System (ADS)

    Yegavian, Robin; Leclaire, Benjamin; Champagnat, Frédéric; Illoul, Cédric; Losfeld, Gilles

    2016-08-01

    We introduce a new method for estimating fluid trajectories in time-resolved PIV. It relies on a Lucas-Kanade paradigm and consists in a simple and direct extension of a two-frame estimation with FOLKI-PIV (Champagnat et al 2011 Exp. Fluids 50 1169-82). The so-called Lucas-Kanade Fluid Trajectories (LKFT) are assumed to be polynomial in time, and are found as the minimizer of a global functional, in which displacements are sought so as to match the intensities of a series of images pairs in the sequence, in the least-squares sense. All pairs involve the central image, similar to other recent time-resolved approaches (FTC (Lynch and Scarano 2013 Meas. Sci. Technol. 24 035305) and FTEE (Jeon et al 2014 Exp. Fluids 55 1-16)). As switching from a two-frame to a time-resolved objective simply amounts to adding terms in a functional, no significant additional algorithmic element is required. Similar to FOLKI-PIV the method is very well suited for GPU acceleration, which is an important feature as computational complexity increases with the image sequence size. Tests on synthetic data exhibiting peak-locking show that increasing the image sequence size strongly reduces both associated bias and random error, and that LKFT has a remaining total error comparable to that of FTEE on this case. Results on case B of the third PIV challenge (Stanislas et al 2008 Exp. Fluids 45 27-71) also show its ability to drastically reduce the error in situations with low signal-to-noise ratio. These results are finally confirmed on experimental images acquired in the near-field of a low Reynolds number jet. Strong reductions in peak-locking, spatial and temporal noise compared to two-frame estimation are also observed, on the displacement components themselves, as well as on spatial or temporal derivatives, such as vorticity and material acceleration.

  8. Time resolved spectral behavior of bright BATSE precursors

    NASA Astrophysics Data System (ADS)

    Burlon, D.; Ghirlanda, G.; Ghisellini, G.; Greiner, J.; Celotti, A.

    2009-10-01

    Aims: Gamma ray bursts (GRBs) are sometimes preceded by dimmer emission episodes, called “precursors”, whose nature is still a puzzle: they could either have the same origin as the main emission episode or they could be due to another mechanism. We investigate if precursors have some spectral distinctive feature with respect to the main GRB episodes. Methods: To this aim we compare the spectral evolution of the precursor with that of the main GRB event. We also study if and how the spectral parameters, and in particular the peak of the ν Fν spectrum of time resolved spectra, correlates with the flux. This allows us to test if the spectra of the precursor and of the main event belong to the same correlation (if any). We searched GRBs with precursor activity in the complete sample of 2704 bursts detected by BATSE finding that 12% of GRBs have one or more precursors. Among these we considered the bursts with time resolved spectral analysis performed by Kaneko et al. ( 2006, ApJS, 166, 298), selecting those having at least two time resolved spectra for the precursor. Results: We find that precursors and main events have very similar spectral properties. The spectral evolution within precursors has similar trends as the spectral evolution observed in the subsequent peaks. Also the typical spectral parameters of the precursors are similar to those of the main GRB events. Moreover, in several cases we find that within the precursors the peak energy of the spectrum is correlated with the flux similarly to what happens in the main GRB event. This strongly favors models in which the precursor is due to the same fireball physics of the main emission episodes. Figures 8 to 41 are only available in electronic form at http://www.aanda.org

  9. Spectral and time-resolved measurements of marine oil pollution by YAG laser fluorosensor

    NASA Astrophysics Data System (ADS)

    Yamagishi, Susumu; Hitomi, Kazuo; Yamanouchi, Hiroshi

    1998-08-01

    This paper describes a compact imaging lidar system capable of detecting fluorescence of substances excited by the third harmonic generator of the YAG laser using CCD camera with gated image intensifier. The system mounted on a small airplane or ships will provide the spreading image of oil spills and classification of substances for clean-up operations. From image data of the water Raman scatters, we present a method to measure the extinction coefficients for the oil film thickness measurement. As the reference to interpret the data obtained in the field, the time-resolved fluorescence characteristics of fuel oils and industrial chemical substances were measured with a streak scope in the laboratory within the wavelength of 350 to 575 nm and with decay time up to 500 ns.

  10. A modified stopped-flow apparatus for time-resolved protein phosphorescence

    NASA Astrophysics Data System (ADS)

    Strambini, Giovanni B.; Puntoni, Alessandro; Gonnelli, Margherita

    1997-12-01

    A type of commercial apparatus was modified and integrated in order to implement the detection of time-resolved protein phosphorescence in the stopped-flow technique. Laser excitation, photomultiplier protection from the intense fluorescence pulse, fluorescence integration, and data acquisition are all synchronized by a trigger module that takes over standard computer control of the stopped-flow apparatus. A detailed protocol is given for effective deoxygenation of the sample and flow lines and for avoiding contamination of the solutions by quenching impurities. The performance of the apparatus was tested by comparing the phosphorescence decay kinetics of the protein horse liver alcohol dehydrogenase in the stopped-flow apparatus and in a standard phosphorimeter. The time resolution of phosphorescence detection in the stopped-flow apparatus is 10 ms and the sensitivity in terms of chromophores concentration is about 0.1 μM.

  11. Time-Resolved Conformational Dynamics in Hydrocarbon Chains

    SciTech Connect

    Minitti, Michael P.; Weber, Peter M.

    2007-06-22

    Internal rotation about carbon-carbon bonds allows N,N-dimethyl-2-butanamine (DM2BA) and N,N-dimethyl-3-hexanamine (DM3HA) to assume multiple conformeric structures. We explore the equilibrium composition and dynamics between such conformeric structures using Rydberg fingerprint spectroscopy. Time constants for conformeric interconversion of DM2BA (at 1.79 eV of internal energy) are 19 and 66 ps, and for DM3HA (1.78 eV) 23 and 41 ps. For the first time, a time-resolved and quantitative view of conformational dynamics of flexible hydrocarbon molecules at high temperatures is revealed.

  12. Picosecond time-resolved measurements of dense plasma line shifts

    DOE PAGES

    Stillman, C. R.; Nilson, P. M.; Ivancic, S. T.; ...

    2017-06-13

    Picosecond time-resolved x-ray spectroscopy is used to measure the spectral line shift of the 1s2p–1s2 transition in He-like Al ions as a function of the instantaneous plasma conditions. The plasma temperature and density are inferred from the Al Heα complex using a nonlocal-thermodynamic-equilibrium atomic physics model. The experimental spectra show a linearly increasing red shift for electron densities of 1 to 5 × 1023 cm–3. Furthermore, the measured line shifts are broadly consistent with a generalized analytic line-shift model based on calculations of a self-consistent field ion sphere model.

  13. Time-resolved SERS for characterizing extracellular vesicles

    NASA Astrophysics Data System (ADS)

    Rojalin, Tatu; Saari, Heikki; Somersalo, Petter; Laitinen, Saara; Turunen, Mikko; Viitala, Tapani; Wachsmann-Hogiu, Sebastian; Smith, Zachary J.; Yliperttula, Marjo

    2017-02-01

    The aim of this work is to develop a platform for characterizing extracellular vesicles (EV) by using gold-polymer nanopillar SERS arrays simultaneously circumventing the photoluminescence-related disadvantages of Raman with a time-resolved approach. EVs are rich of biochemical information reporting of, for example, diseased state of the biological system. Currently, straightforward, label-free and fast EV characterization methods with low sample consumption are warranted. In this study, SERS spectra of red blood cell and platelet derived EVs were successfully measured and their biochemical contents analyzed using multivariate data analysis techniques. The developed platform could be conveniently used for EV analytics in general.

  14. A compact electron gun for time-resolved electron diffraction

    SciTech Connect

    Robinson, Matthew S.; Lane, Paul D.; Wann, Derek A.

    2015-01-15

    A novel compact time-resolved electron diffractometer has been built with the primary goal of studying the ultrafast molecular dynamics of photoexcited gas-phase molecules. Here, we discuss the design of the electron gun, which is triggered by a Ti:Sapphire laser, before detailing a series of calibration experiments relating to the electron-beam properties. As a further test of the apparatus, initial diffraction patterns have been collected for thin, polycrystalline platinum samples, which have been shown to match theoretical patterns. The data collected demonstrate the focusing effects of the magnetic lens on the electron beam, and how this relates to the spatial resolution of the diffraction pattern.

  15. Time Resolved Phonon Spectroscopy, Version 1.0

    SciTech Connect

    Goett, Johnny; Zhu, Brian

    2016-12-22

    TRPS code was developed for the project "Time Resolved Phonon Spectroscopy". Routines contained in this piece of software were specially created to model phonon generation and tracking within materials that interact with ionizing radiation, particularly applicable to the modeling of cryogenic radiation detectors for dark matter and neutrino research. These routines were created to link seamlessly with the open source Geant4 framework for the modeling of radiation transport in matter, with the explicit intent of open sourcing them for eventual integration into that code base.

  16. Time resolved structural dynamics of butadiyne-linked porphyrin dimers

    PubMed Central

    Camargo, Franco V. A.; Hall, Christopher R.; Anderson, Harry L.; Meech, Stephen R.; Heisler, Ismael A.

    2016-01-01

    In this work, the timescales and mechanisms associated with the structural dynamics of butadiyne-linked porphyrin dimers are investigated through time resolved narrowband pump/broadband probe transient absorption spectroscopy. Our results confirm previous findings that the broadening is partly due to a distribution of structures with different (dihedral) angular conformations. Comparison of measurements with excitations on the red and blue sides of the Q-band unravel the ground and excited state conformational re-equilibration timescales. Further comparison to a planarized dimer, through the addition of a ligand, provides conclusive evidence for the twisting motion performed by the porphyrin dimer in solution. PMID:26798839

  17. Sensitive, time-resolved, broadband spectroscopy of single transient processes

    NASA Astrophysics Data System (ADS)

    Fjodorow, Peter; Baev, Ivan; Hellmig, Ortwin; Sengstock, Klaus; Baev, Valery M.

    2015-09-01

    Intracavity absorption spectroscopy with a broadband Er3+-doped fiber laser is applied to time-resolved measurements of transient gain and absorption in electrically excited Xe and Kr plasmas. The achieved time resolution for broadband spectral recording of a single process is 25 µs. For pulsed-periodic processes, the time resolution is limited by the laser pulse duration, which is set here to 3 µs. This pulse duration also predefines the effective absorption path length, which amounts to 900 m. The presented technique can be applied to multicomponent analysis of single transient processes such as shock tube experiments, pulse detonation engines, or explosives.

  18. Time resolved cryogenic cooling analysis of the Cornell Injector Cryomodule

    NASA Astrophysics Data System (ADS)

    Eichhorn, R.; Markham, S.; Smith, E.; Quigley, P.

    2015-12-01

    Managing parallel cryogenic flows has become a key challenge in designing efficient and smart cryo-modules for particle accelerators. In analysing the heating dynamics of the Cornell high current injector module a computational tool has been set-up allowing time resolved analysis and optimization. We describe the computational methods and data sets we have used, report the results and compare them to measured data from the module being in good agreement. Mitigation strategies developed on basis of this model have helped us in pushing the operational limitations.

  19. Femtosecond time-resolved electronic relaxation dynamics in tetrathiafulvalene

    SciTech Connect

    Staedter, D.; Polizzi, L.; Thiré, N.; Mairesse, Y.; Mayer, P.; Blanchet, V.

    2015-05-21

    In the present paper, the ultrafast electronic relaxation of tetrathiafulvalene (TTF) initiated around 4 eV is studied by femtosecond time-resolved velocity-map imaging. The goal is to investigate the broad double structure observed in the absorption spectrum at this energy. By monitoring the transients of the parent cation and its fragments and by varying the pump and the probe wavelengths, two internal conversions and intramolecular vibrational relaxation are detected both on the order of a few hundred of femtoseconds. Photoelectron images permit the assignment of a dark electronic state involved in the relaxation. In addition, the formation of the dimer of TTF has been observed.

  20. Coherent state statistics from time-resolved photon counting

    NASA Astrophysics Data System (ADS)

    Ravi, Harish; Prabhakar, Anil

    2011-01-01

    Time resolved photon counting was used to separate the different photon states emitted from a strongly attenuated laser source. We first describe a method to quantify the efficiency of our gated avalanche photo-detector, by relying on known Poissonian statistics. The detector was then optimized under different temperature and bias voltage conditions using the noise equivalent power as a metric. Finally, coherent pulses are sent into a ring cavity, such that the tapped output from the cavity forms a series of time multiplexed pulses, which then yield the photon counting statistics. We observed good agreement between theoretical estimates and experimental observations, to as low as 0.01% probability of detection.

  1. Picosecond time-resolved measurements of dense plasma line shifts

    NASA Astrophysics Data System (ADS)

    Stillman, C. R.; Nilson, P. M.; Ivancic, S. T.; Golovkin, I. E.; Mileham, C.; Begishev, I. A.; Froula, D. H.

    2017-06-01

    Picosecond time-resolved x-ray spectroscopy is used to measure the spectral line shift of the 1 s 2 p -1 s2 transition in He-like Al ions as a function of the instantaneous plasma conditions. The plasma temperature and density are inferred from the Al H eα complex using a nonlocal-thermodynamic-equilibrium atomic physics model. The experimental spectra show a linearly increasing redshift for electron densities of 1 -5 ×1023c m-3 . The measured line shifts are broadly consistent with a generalized analytic line-shift model based on calculations of a self-consistent field ion-sphere model.

  2. Experimental Time Resolved Electron Beam Temperature Measurements Using Bremsstrahlung Diagnostics

    SciTech Connect

    Menge, P.R.; Maenchen, J.E.; Mazarakis, M.G.; Rosenthal, S.E.

    1999-06-25

    Electron beam temperature, {beta}{perpendicular} (= v{perpendicular}/v), is important to control for the development of high dose flash radiographic bremsstrahlung sources. At high voltage (> 5 MV) increasing electron beam temperature has a serious deleterious effect on dose production. The average and time resolved behavior of beam temperature was measured during radiographic experiments on the HERMES III accelerator (10 MV, 50 kA, 70 ns). A linear array of thermoluminescent dosimeters (TLDs) were used to estimate the time integrated average of beam temperature. On and off-axis photoconducting diamond (PCD) detectors were used to measure the time resolved bremsstrahlung dose rate, which is dependent on beam energy and temperature. The beam temperature can be determined by correlating PCD response with accelerator voltage and current and also by analyzing the ratio of PCD amplitudes on and off axis. This ratio is insensitive to voltage and current and thus, is more reliable than utilizing absolute dose rate. The data is unfolded using comparisons with Monte Carlo simulations to obtain absolute beam temperatures. The data taken on HERMES III show abrupt increases in {beta}{perpendicular} midway through the pulse indicating rapid onset of beam instability.

  3. Time-resolved videothermography at above-frame-rate frequencies

    NASA Astrophysics Data System (ADS)

    Shepard, Steven M.; Sass, David T.; Imirowicz, Thomas P.; Meng, A.

    1991-03-01

    Temporal aliasing is a problem common all video imaging systems. IR video systems are particularly susceptible because of the relatively low (30 Hz) RS-170 frame rate. We have developed a system which compensates for the effects of aliasing, and allows images of repetitive thermal phenomena at frequencies up to 4 kHz to be acquired. The system utilizes an unmodified commercial imager (Inframetrics IR 600) and a microcomputer. Data is acquired selectively using a 'line-by-line' scheme which compares the motion of the camera's horizontal scanning mirror to a reference signal correlated to the event of interest. Only those horizontal lines that have a user-specified phase relationship to the reference signal are acquired. The resulting images have an effective integration time of 125 microseconds. This ability to acquire 'time-resolved' IR images allows investigation of phenomena not possible with a conventional RS-170 IR imaging system. For example, a conventional thermal image of an integrated circuit operating at several kilohertz yields no temporal information whatsoever. By comparison, we have acquired time-resolved images which show the AC heating and cooling of the circuit, so that we may identify AC hot spots. The system has also been applied to a running internal combustion engine. The imager views the piston head through an IR transparent (ZnS) window. Acquired images clearly show the motion of the flame front through the combustion chamber, and the subsequent heating and cooling of the piston head.

  4. Time-resolved luminescent lateral flow assay technology.

    PubMed

    Song, Xuedong; Knotts, Michael

    2008-09-26

    We here report a detection technology that integrates highly sensitive time-resolved luminescence technique into lateral flow assay platform to achieve excellent detection performance with low cost. We have developed very bright, surface-functionalized and mono-dispersed phosphorescent nanoparticles of long lifetime under ambient conditions. The phosphorescent nanoparticles have been used to conjugate with monoclonal antibody for C-reactive protein (CRP), an inflammatory biomarker. Lateral flow immunoassay devices have been developed using the conjugate for highly sensitive detection of CRP. The CRP assay can achieve a detection sensitivity of <0.2 ngmL(-1) in serum with a linear response from 0.2 to 200 ngmL(-1) CRP. We have also developed a low cost time-resolved luminescence reader for the lateral flow immunoassay (LFIA) devices. The reader does not use expensive band pass filter and still provide very low detection background and high detection sensitivity on solid substrates such as nitrocellulose membranes. The reader can detect less than 2.5 ng phosphorescent particles captured on a nitrocellulose membrane strip with more than three orders of magnitude linear detection dynamic range. The technology should find a number of applications, ranging from clinical diagnostics, detection of chemical and biological warfare agents, to food and environmental monitoring.

  5. Time-resolved quasielastic neutron scattering studies of native photosystems.

    PubMed

    Pieper, Jörg

    2010-01-01

    The internal molecular dynamics of proteins plays an important role in a number of functional processes in native photosystems. Prominent examples include the photocycle of bacteriorhodopsin and electron transfer in the reaction center of plant photosystem II. In this regard, the recently developed technique of time-resolved quasielastic neutron scattering with laser excitation opens up new perspectives for the study of protein/membrane dynamics in specific functional states of even complex systems. The first direct observation of a functionally modulated protein dynamics has just recently been reported for the model system bacteriorhodopsin (Pieper et al., Phys. Rev. Lett. 100, 2008, 228103.), where a transient softening of the protein was observed on a timescale of approximately 1 ms along with the large-scale structural change in the M-intermediate of bacteriorhodopsin. In contrast, photosystem II membrane fragments with inhibited electron transfer show a suppression of protein dynamics approximately 160 mus after the actinic laser flash (Pieper and Renger, Biochemistry 48, 2009, 6111). This effect may reflect aggregation-like conformational changes capable of dissipation of excess excitation energy to prevent photodamage in the absence of Q(A)-->Q(B) electron transfer. These findings indicate that proteins exhibit a remarkable flexibility to accommodate different functional processes. This contribution will discuss methodical aspects, challenges, and recent applications of laser-excited, time-resolved quasielastic neutron scattering.

  6. Time Resolved FTIR Analysis of Tailpipe Exhaust for Several Automobiles

    NASA Astrophysics Data System (ADS)

    White, Allen R.; Allen, James; Devasher, Rebecca B.

    2011-06-01

    The automotive catalytic converter reduces or eliminates the emission of various chemical species (e.g. CO, hydrocarbons, etc.) that are the products of combustion from automobile exhaust. However, these units are only effective once they have reached operating temperature. The design and placement of catalytic converters has changed in order to reduce both the quantity of emissions and the time that is required for the converter to be effective. In order to compare the effectiveness of catalytic converters, time-resolved measurements were performed on several vehicles, including a 2010 Toyota Prius, a 2010 Honda Fit, a 1994 Honda Civic, and a 1967 Oldsmobile 442 (which is not equipped with a catalytic converter but is used as a baseline). The newer vehicles demonstrate bot a reduced overall level of CO and hydrocarbon emissions but are also effective more quickly than older units. The time-resolved emissions will be discussed along with the impact of catalytic converter design and location on the measured emissions.

  7. Time-resolved multispectral imaging of combustion reaction

    NASA Astrophysics Data System (ADS)

    Huot, Alexandrine; Gagnon, Marc-André; Jahjah, Karl-Alexandre; Tremblay, Pierre; Savary, Simon; Farley, Vincent; Lagueux, Philippe; Guyot, Éric; Chamberland, Martin; Marcotte, Fréderick

    2015-05-01

    Thermal infrared imaging is a field of science that evolves rapidly. Scientists have used for years the simplest tool: thermal broadband cameras. This allows to perform target characterization in both the longwave (LWIR) and midwave (MWIR) infrared spectral range. Infrared thermal imaging is used for a wide range of applications, especially in the combustion domain. For example, it can be used to follow combustion reactions, in order to characterize the injection and the ignition in a combustion chamber or even to observe gases produced by a flare or smokestack. Most combustion gases such as carbon dioxide (CO2) selectively absorb/emit infrared radiation at discrete energies, i.e. over a very narrow spectral range. Therefore, temperatures derived from broadband imaging are not reliable without prior knowledge about spectral emissivity. This information is not directly available from broadband images. However, spectral information is available using spectral filters. In this work, combustion analysis was carried out using Telops MS-IR MW camera which allows multispectral imaging at a high frame rate. A motorized filter wheel allowing synchronized acquisitions on eight (8) different channels was used to provide time-resolved multispectral imaging of combustion products of a candle in which black powder has been burnt to create a burst. It was then possible to estimate the temperature by modeling spectral profile derived from information obtained with the different spectral filters. Comparison with temperatures obtained using conventional broadband imaging illustrates the benefits of time-resolved multispectral imaging for the characterization of combustion processes.

  8. Time-resolved multispectral imaging of combustion reactions

    NASA Astrophysics Data System (ADS)

    Huot, Alexandrine; Gagnon, Marc-André; Jahjah, Karl-Alexandre; Tremblay, Pierre; Savary, Simon; Farley, Vincent; Lagueux, Philippe; Guyot, Éric; Chamberland, Martin; Marcotte, Frédérick

    2015-10-01

    Thermal infrared imaging is a field of science that evolves rapidly. Scientists have used for years the simplest tool: thermal broadband cameras. These allow to perform target characterization in both the longwave (LWIR) and midwave (MWIR) infrared spectral range. Infrared thermal imaging is used for a wide range of applications, especially in the combustion domain. For example, it can be used to follow combustion reactions, in order to characterize the injection and the ignition in a combustion chamber or even to observe gases produced by a flare or smokestack. Most combustion gases, such as carbon dioxide (CO2), selectively absorb/emit infrared radiation at discrete energies, i.e. over a very narrow spectral range. Therefore, temperatures derived from broadband imaging are not reliable without prior knowledge of spectral emissivity. This information is not directly available from broadband images. However, spectral information is available using spectral filters. In this work, combustion analysis was carried out using a Telops MS-IR MW camera, which allows multispectral imaging at a high frame rate. A motorized filter wheel allowing synchronized acquisitions on eight (8) different channels was used to provide time-resolved multispectral imaging of combustion products of a candle in which black powder has been burnt to create a burst. It was then possible to estimate the temperature by modeling spectral profiles derived from information obtained with the different spectral filters. Comparison with temperatures obtained using conventional broadband imaging illustrates the benefits of time-resolved multispectral imaging for the characterization of combustion processes.

  9. Time-resolved single dopant charge dynamics in silicon

    PubMed Central

    Rashidi, Mohammad; Burgess, Jacob A. J.; Taucer, Marco; Achal, Roshan; Pitters, Jason L.; Loth, Sebastian; Wolkow, Robert A.

    2016-01-01

    As the ultimate miniaturization of semiconductor devices approaches, it is imperative that the effects of single dopants be clarified. Beyond providing insight into functions and limitations of conventional devices, such information enables identification of new device concepts. Investigating single dopants requires sub-nanometre spatial resolution, making scanning tunnelling microscopy an ideal tool. However, dopant dynamics involve processes occurring at nanosecond timescales, posing a significant challenge to experiment. Here we use time-resolved scanning tunnelling microscopy and spectroscopy to probe and study transport through a dangling bond on silicon before the system relaxes or adjusts to accommodate an applied electric field. Atomically resolved, electronic pump-probe scanning tunnelling microscopy permits unprecedented, quantitative measurement of time-resolved single dopant ionization dynamics. Tunnelling through the surface dangling bond makes measurement of a signal that would otherwise be too weak to detect feasible. Distinct ionization and neutralization rates of a single dopant are measured and the physical process controlling those are identified. PMID:27782125

  10. Formulation for Time-resolved Aerodynamic Damping in Dynamic Stall

    NASA Astrophysics Data System (ADS)

    Corke, Thomas; Bowles, Patrick; Coleman, Dusty; Thomas, Flint

    2012-11-01

    A new Hilbert transform formulation of the equation of motion for a pitching airfoil in a uniform stream yields a time resolved aerodynamic damping factor, Ξ (t) = (√{ (Cm2 (t) +C m2 } /αmax) sinψ (t) , where Cm (t) is the instantaneous pitch moment coefficient, and C m (t) is the Hilbert transform of Cm (t) , αmax is the pitching amplitude, and ψ (t) is the time-resolved phase difference between the aerodynamic pitch moment and the instantaneous angle of attack. A Ξ (t) < 0 indicates unstable pressure loading that can be considered a necessary condition to excite stall flutter in an elastic airfoil. This will be illustrated in experiments with conditions producing ``light'' dynamic stall for a range of Mach numbers from 0.3-0.6. These reveal large negative excursions of Ξ (t) during the pitch-up portion of the cycle that correlates with the formation and convection of the dynamic stall vortex. The fact that the cycle-integrated damping coefficient is positive in all these cases underscores how the traditional diagnostic masks much of the physics that underlies the destabilizing effect of the dynamic stall process. This new insight can explain instances of transient limit-cycle growth of helicopter rotor vibrations. Supported by Bell Helicopter.

  11. CCD time-resolved photometry of faint cataclysmic variables. II

    SciTech Connect

    Szkody, P.; Howell, S.B.; Mateo, M.; Kreidl, T.J. Planetary Science Institute, Tucson, AZ Mount Wilson and Las Campanas Observatories, Pasadena, CA Lowell Observatory, Flagstaff, AZ )

    1989-10-01

    Time-resolved optical broad-band light curves obtained from differential photometry on sequential CCD frames of the known or suspected cataclysmic variable FO And, EH Aqr, WX Cet, XX Cet, AL Com, V503 Cyg, AH Eri, CP Eri, IR Gem, RW UMi, PG0134+070, and US 3215 are presented. The analysis of the light curves with coverage of greater than 2 hrs shows repeatable periodicity in five objects. PG0134+070 exhibits eclipses of 1.3-1.8 mag depth with a period of 313 min. V503 Cyg has a 0.7-1.0 mag peak-to-peak modulation with a period of 109 min. IR Gem shows a large modulation at the orbital period of 99 min, and comparison with previous data indicates that this modulation may have an amplitude dependent on outburst phase. AH Eri reveals a 0.1-0.3 mag modulation, at a period of 42 min. Better time-resolved data on AL Com confirm the 0.4-mag variation reported by Howell and Szkody (1988) at a period of 42 min. These latter two short periods likely indicate magnetic systems. There is also some evidence of periodicity in RW UMi and WX Cet which must be confirmed with further data. 25 refs.

  12. Nonadiabatic and Time-Resolved Photoelectron Spectroscopy for Molecular Systems.

    PubMed

    Flick, Johannes; Appel, Heiko; Rubio, Angel

    2014-04-08

    We quantify the nonadiabatic contributions to the vibronic sidebands of equilibrium and explicitly time-resolved nonequilibrium photoelectron spectra for a vibronic model system of trans-polyacetylene. Using exact diagonalization, we directly evaluate the sum-over-states expressions for the linear-response photocurrent. We show that spurious peaks appear in the Born-Oppenheimer approximation for the vibronic spectral function, which are not present in the exact spectral function of the system. The effect can be traced back to the factorized nature of the Born-Oppenheimer initial and final photoemission states and also persists when either only initial or final states are replaced by correlated vibronic states. Only when correlated initial and final vibronic states are taken into account are the spurious spectral weights of the Born-Oppenheimer approximation suppressed. In the nonequilibrium case, we illustrate for an initial Franck-Condon excitation and an explicit pump-pulse excitation how the vibronic wavepacket motion of the system can be traced in the time-resolved photoelectron spectra as a function of the pump-probe delay.

  13. Time-resolved spectroscopy of the Mercury 6 3P1 state

    NASA Technical Reports Server (NTRS)

    Halstead, J. A.; Reeves, R. R.

    1981-01-01

    The time-resolved fluorescence was observed from the Hg 6 3P1 state under the influence of the earth's magnetic field and with applied fields of up to 14 G. Modulation of the fluorescence decay signal was observed as a function of both time and space and can be interpreted in terms of a classical precession of the excited atom about the magnetic field or as quantum beats resulting from interference between coherently populated Zeeman sublevels. This modulation was studied for each of the five resolvable components of the hyperfine structure separately. The fluorescence from the even isotopes was determined to be almost completely modulated while the fluorescence from the odd isotopes was only partially modulated. The frequency of modulation of the fluorescence from the mercury-202 isotope was observed as a function of the applied magnetic field and a value for the Lande factor of 1.46 + or - 0.03 was obtained. This is within experimental error of the accepted value of 1.486. In addition, the frequency of modulation as a function of applied magnetic field was determined for each of the three resolvable components with more than one contributing isotopic hyperfine line. An investigation of the effect of radiation trapping on the degree modulation was also made.

  14. Ultrafast time-resolved spectroscopy of the light-harvesting complex 2 (LH2) from the photosynthetic bacterium Thermochromatium tepidum

    SciTech Connect

    Niedzwiedzki, Dariusz M.; Fuciman, Marcel; Kobayashi, Masayuki; Frank, Harry A.; Blankenship, Robert E.

    2011-10-08

    The light-harvesting complex 2 from the thermophilic purple bacterium Thermochromatium tepidum was purified and studied by steady-state absorption and fluorescence, sub-nanosecond-time-resolved fluorescence and femtosecond time-resolved transient absorption spectroscopy. The measurements were performed at room temperature and at 10 K. The combination of both ultrafast and steady-state optical spectroscopy methods at ambient and cryogenic temperatures allowed the detailed study of carotenoid (Car)-to-bacteriochlorophyll (BChl) as well BChl-to-BChl excitation energy transfer in the complex. The studies show that the dominant Cars rhodopin (N = 11) and spirilloxanthin (N = 13) do not play a significant role as supportive energy donors for BChl a. This is related with their photophysical properties regulated by long π-electron conjugation. On the other hand, such properties favor some of the Cars, particularly spirilloxanthin (N = 13) to play the role of the direct quencher of the excited singlet state of BChl.

  15. Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs.

    PubMed

    Massey, Melissa; Ancona, Mario G; Medintz, Igor L; Algar, W Russ

    2015-12-01

    Nucleic acid hybridization probes are sought after for numerous assay and imaging applications. These probes are often limited by the properties of fluorescent dyes, prompting the development of new probes where dyes are paired with novel or nontraditional luminescent materials. Luminescent terbium complexes are an example of such a material, and these complexes offer several unique spectroscopic advantages. Here, we demonstrate two nonstem-loop designs for light-up nucleic acid hybridization beacons that utilize time-resolved Förster resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved emission from the dye provides an analytical signal. Both designs are based on probe oligonucleotides that are labeled at their opposite termini with Tb and a fluorescent reporter dye. In one design, a probe is partially blocked with a quencher dye-labeled oligonucleotide, and target hybridization is signaled through toehold-mediated strand displacement and loss of a competitive FRET pathway. In the other design, the intrinsic folding properties of an unblocked probe are utilized in combination with a temporal mechanism for signaling target hybridization. This temporal mechanism is based on a recently elucidated "sweet spot" for TR-FRET measurements and exploits distance control over FRET efficiencies to shift the Tb lifetime within or outside the time-gated detection window for measurements. Both the blocked and unblocked beacons offer nanomolar (femtomole) detection limits, response times on the order of minutes, multiplexing through the use of different reporter dyes, and detection in complex matrices such as serum and blood. The blocked beacons offer better mismatch selectivity, whereas the unblocked beacons are simpler in design. The temporal mechanism of signaling utilized with the unblocked beacons also plays a significant role with the blocked beacons and represents a new and effective

  16. Spectral characteristics of time resolved magnonic spin Seebeck effect

    SciTech Connect

    Etesami, S. R.; Chotorlishvili, L.; Berakdar, J.

    2015-09-28

    Spin Seebeck effect (SSE) holds promise for new spintronic devices with low-energy consumption. The underlying physics, essential for a further progress, is yet to be fully clarified. This study of the time resolved longitudinal SSE in the magnetic insulator yttrium iron garnet concludes that a substantial contribution to the spin current stems from small wave-vector subthermal exchange magnons. Our finding is in line with the recent experiment by S. R. Boona and J. P. Heremans [Phys. Rev. B 90, 064421 (2014)]. Technically, the spin-current dynamics is treated based on the Landau-Lifshitz-Gilbert equation also including magnons back-action on thermal bath, while the formation of the time dependent thermal gradient is described self-consistently via the heat equation coupled to the magnetization dynamics.

  17. Time-resolved thermal transport in compositionally modulated metal films

    SciTech Connect

    Clemens, B.M.; Eesley, G.L.; Paddock, C.A.

    1988-01-15

    We report on investigations of one-dimensional thermal transport in compositionally modulated metal films produced with a systematic variation in atomic lattice mismatch. In the case of Ni-Cu, Ni-Mo, Ni-Ti, and Ni-Zr, we observe the relative effects of interfacial disorder on thermal diffusion. Our observations demonstrate the thermal impedance of a single metal-metal interface and indicate that thermal diffusion in a bilayer film is strongly influenced by the interface between contacting metal pairs. This study is made possible by picosecond time-resolved thermoreflectance measurements which probe thermal transport perpendicular to the film plane. This technique can impact on our understanding of electron scattering and transport across metallic boundaries, and it provides a means of inferring electrical transport properties.

  18. Photon-Counting Arrays for Time-Resolved Imaging

    PubMed Central

    Antolovic, I. Michel; Burri, Samuel; Hoebe, Ron A.; Maruyama, Yuki; Bruschini, Claudio; Charbon, Edoardo

    2016-01-01

    The paper presents a camera comprising 512 × 128 pixels capable of single-photon detection and gating with a maximum frame rate of 156 kfps. The photon capture is performed through a gated single-photon avalanche diode that generates a digital pulse upon photon detection and through a digital one-bit counter. Gray levels are obtained through multiple counting and accumulation, while time-resolved imaging is achieved through a 4-ns gating window controlled with subnanosecond accuracy by a field-programmable gate array. The sensor, which is equipped with microlenses to enhance its effective fill factor, was electro-optically characterized in terms of sensitivity and uniformity. Several examples of capture of fast events are shown to demonstrate the suitability of the approach. PMID:27367697

  19. Multidimensional Time-Resolved Spectroscopy of Vibrational Coherence in Biopolyenes

    NASA Astrophysics Data System (ADS)

    Buckup, Tiago; Motzkus, Marcus

    2014-04-01

    Multidimensional femtosecond time-resolved vibrational coherence spectroscopy allows one to investigate the evolution of vibrational coherence in electronic excited states. Methods such as pump-degenerate four-wave mixing and pump-impulsive vibrational spectroscopy combine an initial ultrashort laser pulse with a nonlinear probing sequence to reinduce vibrational coherence exclusively in the excited states. By carefully exploiting specific electronic resonances, one can detect vibrational coherence from 0 cm-1 to over 2,000 cm-1 and map its evolution. This review focuses on the observation and mapping of high-frequency vibrational coherence for all-trans biological polyenes such as β-carotene, lycopene, retinal, and retinal Schiff base. We discuss the role of molecular symmetry in vibrational coherence activity in the S1 electronic state and the interplay of coupling between electronic states and vibrational coherence.

  20. Time-resolved polarization study of anisotropy in bacteriorhodopsin

    SciTech Connect

    Wan, Chaozhi; Qian, Jun; Johnson, C.K. )

    1990-11-01

    Time-resolved polarization spectroscopy is sensitive to the orientational dynamics of chromophores, and as a result it can be applied to study internal motion in restrictive environments. This paper describes the application of polarization spectroscopy to the photoactive protein bacteriorhodopsin on time scales from picoseconds to hundreds of microseconds. Anisotropy persists in both the ground-state bacteriorhodopsin population and in the photocycle intermediates due to a population bottleneck in the bacteriorhodopsin photocycle. The time dependence of the polarization signal expected for a sequence of intermediates is described and is shown to be sensitive both to population kinetics and to internal motion. The observed time dependence of the polarization signal reveals internal motion in the purple membrane fragments on the time scale of the K {yields} L transition ({approximately} 1 {mu}s). The results are consistent with the known kinetics of the bacteriorhodopsin photocycle. Evidence is also presented and discussed for a decay component on a time scale of < 50 ps.

  1. Revealing Deactivation Pathways Hidden in Time-Resolved Photoelectron Spectra

    NASA Astrophysics Data System (ADS)

    Ruckenbauer, Matthias; Mai, Sebastian; Marquetand, Philipp; González, Leticia

    2016-10-01

    Time-resolved photoelectron spectroscopy is commonly employed with the intention to monitor electronic excited-state dynamics occurring in a neutral molecule. With the help of theory, we show that when excited-state processes occur on similar time scales the different relaxation pathways are completely obscured in the total photoionization signal recorded in the experiment. Using non-adiabatic molecular dynamics and Dyson norms, we calculate the photoionization signal of cytosine and disentangle the transient contributions originating from the different deactivation pathways of its tautomers. In the simulations, the total signal from the relevant keto and enol tautomers can be decomposed into contributions either from the neutral electronic state populations or from the distinct mechanistic pathways across the multiple potential surfaces. The lifetimes corresponding to these contributions cannot be extracted from the experiment, thereby illustrating that new experimental setups are necessary to unravel the intricate non-adiabatic pathways occurring in polyatomic molecules after irradiation by light.

  2. Nonselective and polarization effects in time-resolved optogalvanic spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhechev, D.; Steflekova, V.

    2016-02-01

    Three interfering effects in optogalvanic (OG) spectroscopy are identified in a hollow cathode discharge (HCD) - OG detector. The laser beam is found to generate two nonselective processes, namely photoelectron emission (PE) from the cathode surface with a sub-breakdown bias applied, and nonresonant space ionization. The convolution of these galvanic contributions was determined experimentally as an instrumental function and a deconvolution procedure to determine the actual OG signal was developed. Specific plasma conductance is detected dependent on the polarization of the laser beam irradiating. Linearly/circularly polarized light beam is found to induce OG signals differ in amplitude (and their shape parameters in the time-resolved OG signals (TROGS)). The phenomena coherence and specific conductance are found to be in causal relationship. The additional conductance due to coherent states of atoms manifests itself as an intrinsic instrumental property of OG detector.

  3. FXR LIA Optimization - Time-resolved OTR Emittance Measurement

    SciTech Connect

    Jacob, J; Ong, M; Wargo, P; LeSage, G

    2005-07-21

    The Flash X-Ray Radiography (FXR) facility at Lawrence Livermore National Laboratory utilizes a high current, long pulse linear induction accelerator to produce high doses of x-ray radiation. Accurate characterization of the transverse beam emittance is required in order to facilitate accelerator modeling and tuning efforts and, ultimately, to optimize the final focus spot size, yielding higher resolution radiographs. In addition to conventional magnet scan, pepper-pot, and multiple screen techniques, optical transition radiation (OTR) has been proven as a useful emittance measurement diagnostic and is particularly well suited to the FXR accelerator. We shall discuss the time-resolved emittance characterization of an induction linac electron beam using OTR, and we will present our experimental apparatus and analysis software. We shall also develop the theoretical background of beam emittance and transition radiation.

  4. Examining Electron-Boson Coupling Using Time-Resolved Spectroscopy

    SciTech Connect

    Sentef, Michael; Kemper, Alexander F.; Moritz, Brian; Freericks, James K.; Shen, Zhi-Xun; Devereaux, Thomas P.

    2013-12-26

    Nonequilibrium pump-probe time-domain spectroscopies can become an important tool to disentangle degrees of freedom whose coupling leads to broad structures in the frequency domain. Here, using the time-resolved solution of a model photoexcited electron-phonon system, we show that the relaxational dynamics are directly governed by the equilibrium self-energy so that the phonon frequency sets a window for “slow” versus “fast” recovery. The overall temporal structure of this relaxation spectroscopy allows for a reliable and quantitative extraction of the electron-phonon coupling strength without requiring an effective temperature model or making strong assumptions about the underlying bare electronic band dispersion.

  5. Towards time-resolved serial crystallography in a microfluidic device

    PubMed Central

    Pawate, Ashtamurthy S.; Šrajer, Vukica; Schieferstein, Jeremy; Guha, Sudipto; Henning, Robert; Kosheleva, Irina; Schmidt, Marius; Ren, Zhong; Kenis, Paul J. A.; Perry, Sarah L.

    2015-01-01

    Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR1/pRE46Q and pR2/pRCW states at 10 µs and the pB1 intermediate at 1 ms. The strategies presented here have tremendous potential for extension to chemical triggering methods for reaction initiation and for extension to dynamic, multivariable analyses. PMID:26144226

  6. Fast Time-Resolved Aerosol Collector: Proof of Concept

    SciTech Connect

    Yu, Xiao-Ying; Cowin, James P.; Iedema, Martin J.; Ali, Hashim M.

    2010-10-12

    Atmospheric particles can be collected in the field on substrates for subsequent laboratory analysis via chemically sensitive single particle methods such as scanning electron microscopy with energy dispersed x-ray analysis. With moving substrates one can achieve time resolution of seconds to minutes. We demonstrate how to increase the time resolution to a few milliseconds to provide real-time information in this paper. Our fast time-resolved aerosol collector (“Fast-TRAC”) microscopically observes the particle collection on substrate and record an on-line video. Particle arrivals are resolved to within a single frame (17 to 4 ms in this setup), and the spatial locations are matched to the subsequent single particle analysis. This approach provides real-time information on particle size and number concentration. Applications are expected in airborne studies of cloud microstructure, pollution plumes, and long term site monitoring.

  7. Spectral characteristics of time resolved magnonic spin Seebeck effect

    NASA Astrophysics Data System (ADS)

    Etesami, S. R.; Chotorlishvili, L.; Berakdar, J.

    2015-09-01

    Spin Seebeck effect (SSE) holds promise for new spintronic devices with low-energy consumption. The underlying physics, essential for a further progress, is yet to be fully clarified. This study of the time resolved longitudinal SSE in the magnetic insulator yttrium iron garnet concludes that a substantial contribution to the spin current stems from small wave-vector subthermal exchange magnons. Our finding is in line with the recent experiment by S. R. Boona and J. P. Heremans [Phys. Rev. B 90, 064421 (2014)]. Technically, the spin-current dynamics is treated based on the Landau-Lifshitz-Gilbert equation also including magnons back-action on thermal bath, while the formation of the time dependent thermal gradient is described self-consistently via the heat equation coupled to the magnetization dynamics.

  8. Revealing Deactivation Pathways Hidden in Time-Resolved Photoelectron Spectra

    PubMed Central

    Ruckenbauer, Matthias; Mai, Sebastian; Marquetand, Philipp; González, Leticia

    2016-01-01

    Time-resolved photoelectron spectroscopy is commonly employed with the intention to monitor electronic excited-state dynamics occurring in a neutral molecule. With the help of theory, we show that when excited-state processes occur on similar time scales the different relaxation pathways are completely obscured in the total photoionization signal recorded in the experiment. Using non-adiabatic molecular dynamics and Dyson norms, we calculate the photoionization signal of cytosine and disentangle the transient contributions originating from the different deactivation pathways of its tautomers. In the simulations, the total signal from the relevant keto and enol tautomers can be decomposed into contributions either from the neutral electronic state populations or from the distinct mechanistic pathways across the multiple potential surfaces. The lifetimes corresponding to these contributions cannot be extracted from the experiment, thereby illustrating that new experimental setups are necessary to unravel the intricate non-adiabatic pathways occurring in polyatomic molecules after irradiation by light. PMID:27762396

  9. Time-resolved measurements of equilibrium profiles in MST

    NASA Astrophysics Data System (ADS)

    Deng, B. H.; Brower, D. L.; Ding, W. X.; Yates, T. F.; Anderson, J. K.; Caspary, K.; McCollam, K. J.; Prager, S. C.; Reusch, J. A.; Sarff, J. S.; Craig, D.

    2007-11-01

    Based on the high-speed, three-wave, far-infrared polarimeter-interferometer measurement of Bpol profiles and external coil measurements of Btave and Btw, a new method is developed to derive Btor and other equilibrium profiles (J// and q) with high time resolution. Using Faraday's law, the inductive electric field (E//) profile is also deduced from the temporal derivatives of the time-resolved magnetic field profiles. The derived B(0) values have excellent agreement with direct measurements using a Motional Stark Effect (MSE) diagnostic. Evolution of equilibrium profiles during single sawtooth events in MST, both the slow linear ramp and crash phases, are presented. Profile scaling with plasma current Ip and reversal parameter F is also explored. MHD stability is tested from the spatial gradients of the J// and q profiles, and correlation with fluctuation mode amplitude is investigated. Future improvements to equilibrium reconstruction are expected by measuring Btor(r,t) directly via Cotton-Mouton interferometry.

  10. Time-resolved phase-sensitive second harmonic generation spectroscopy

    NASA Astrophysics Data System (ADS)

    Nowakowski, Paweł J.; Woods, David A.; Bain, Colin D.; Verlet, Jan R. R.

    2015-02-01

    A methodology based on time-resolved, phase-sensitive second harmonic generation (SHG) for probing the excited state dynamics of species at interfaces is presented. It is based on an interference measurement between the SHG from the sample and a local oscillator generated at a reference together with a lock-in measurement to remove the large constant offset from the interference. The technique is characterized by measuring the phase and excited state dynamics of the dye malachite green at the water/air interface. The key attributes of the technique are that the observed signal is directly proportional to sample concentration, in contrast to the quadratic dependence from non-phase sensitive SHG, and that the real and imaginary parts of the 2nd order non-linear susceptibility can be determined independently. We show that the method is highly sensitive and can provide high quality excited state dynamics in short data acquisition times.

  11. Time-resolve study of the photooxygenation of 3-hydroxyflavone

    SciTech Connect

    Studer, S.L.; Brewer, W.E.; Martinez, M.L.; Chou, Pi-Tai )

    1989-09-13

    3-hydroxyflavones are prototype molecules for studying the dynamics of the proton-transfer reaction in the excited state as well as in the ground state. Their lasing action has been reported with a high efficiency of laser gain. Unfortunately, a major obstruction to the practical application of 3-hydroxy-flavones as laser dyes is due to a photochemical reaction possibly involving molecular oxygen. In this communication we report a direct time-resolved study of the photooxygenation of 3-hydroxyflavone (3HF) in the normal state as well as in the tau-tomer state. Our results not only provide a detailed mechanism of the photooxygenation reaction but also give indirect evidence to support the role of the triplet state in the reverse proton-transfer reaction.

  12. Time-resolved spectroscopy of low-dimensional semiconductor structures

    NASA Astrophysics Data System (ADS)

    Murphy, Joseph R.

    This dissertation is a survey of ultrafast time-resolved optical measurements conducted on a variety of low-dimensional semiconductor systems to further the understanding of the dynamic behavior in the following systems: ZnMnTe/ZnSe quantum dots, ZnTe/ZnMnSe quantum dots, InGaAs quantum wells, CdMnSe colloidal quantum dots, multi-shell CdSe/CdMnS/CdS colloidal nanoplatelets, and graphene and graphene-related solutions and films. Using time-resolved photoluminescence to study epitaxially-grown ZnTe and ZnMnTe quantum dots in corresponding ZnMnSe and ZnSe matrices, the location dependence of manganese ions in respect to magnetic polaron formation is shown. The structure with manganese ions located in the matrix exhibited magnetic polaron behavior consistent with previous literature, whereas the structure with the magnetic ions located within the quantum dots exhibited unconventional magnetic polaron properties. These properties, including temperature and magnetic field insensitivity, were explained through the use of a model that predicted an increased internal magnetic field due to a decreased effective volume of the magnetic polaron and a higher effective temperature due to laser heating. Magneto-time-resolved photoluminescence measurements on a system of colloidal CdMnSe quantum dots show that the magnetic polaron properties differ significantly from the epitaxially grown quantum dots. First the timescales at which the magnetic polaron forms and the polarization saturates are different by more than an order of magnitude, and second, the magnetic polaron energy exhibited step-like behavior as the strength of the externally applied magnetic field is increased. The field dependent MP formation energy that is observed experimentally is explained as due to the breaking of the antiferromagnetic coupling of Mn dimers within the QDs. This model is further verified by the observation of quantized behavior in the Zeeman energy splitting. Through the use of magneto

  13. Towards time-resolved serial crystallography in a microfluidic device.

    PubMed

    Pawate, Ashtamurthy S; Šrajer, Vukica; Schieferstein, Jeremy; Guha, Sudipto; Henning, Robert; Kosheleva, Irina; Schmidt, Marius; Ren, Zhong; Kenis, Paul J A; Perry, Sarah L

    2015-07-01

    Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR(1)/pR(E46Q) and pR(2)/pR(CW) states at 10 µs and the pB1 intermediate at 1 ms. The strategies presented here have tremendous potential for extension to chemical triggering methods for reaction initiation and for extension to dynamic, multivariable analyses.

  14. Femtosecond time-resolved ERE-CARS of PM650

    NASA Astrophysics Data System (ADS)

    He, Ping; Fan, Rongwei; Chen, Deying; Li, Xiaohui; Xia, Yuanqin; Yu, Xin; Wang, Jialing; Jiang, Yugang

    2012-07-01

    We utilize femtosecond time-resolved electronic resonance-enhanced coherent anti-Stokes Raman scattering (ERE-CARS) to investigate new information for pyrromethene 650 (PM650) dye molecules. For this purpose, the vibrational properties of PM650 are registered in diluted solutions of several organisms. We observe a strong Raman vibrational mode with a wavenumber difference of about 48 cm- 1 in its organic solutions. This may be linked to the intramolecular electron transfer (ICT) process from the aromatic ring to the cyano group in PM650. The influence of the solvent effects on the vibrational dynamics of PM650 is also investigated. The vibrational properties of PM650 dye molecules diluted in polar organic solutions are light solvent-dependent.

  15. Time-Resolved Infrared (TRIR) Studies of Oxycarbonylnitrenes.

    PubMed

    Chavez, Tyler A; Liu, Yonglin; Toscano, John P

    2016-08-05

    N-Ethyloxycarbonyl-S,S-dibenzothiphene sulfilimine and N-t-butyloxycarbonyl-S,S-dibenzothiphene sulfilimine have been utilized as precursors to ethoxycarbonylnitrene and t-butyloxycarbonylnitrene. B3LYP/6-31G(d) calculations predict triplet ground states for both oxycarbonylnitrenes, albeit by small margins. Triplet ethoxycarbonylnitrene and triplet t-butyloxycarbonylnitrene have been observed following photolysis of these sulfilimine precursors by time-resolved infrared (TRIR) spectroscopy. Kinetic studies show that ethoxycarbonylnitrene reacts with solvents such as acetonitrile and cyclohexane, while t-butyloxycarbonylnitrene undergoes an intramolecular insertion reaction to produce 5,5-dimethyl oxazolidinone. Product analysis following photolysis of N-t-butyloxycarbonyl-S,S-dibenzothiphene sulfilimine confirms that the oxazolidinone is the major product with an estimated yield of 90%. The products from these two nitrenes are derived from the corresponding singlet nitrene, either directly or via thermal repopulation of the singlet from the lower-energy triplet nitrene.

  16. Time-resolved, dual heterodyne phase collection transient grating spectroscopy

    NASA Astrophysics Data System (ADS)

    Dennett, Cody A.; Short, Michael P.

    2017-05-01

    The application of optical heterodyne detection for transient grating spectroscopy (TGS) using a fixed, binary phase mask often relies on taking the difference between signals captured at multiple heterodyne phases. To date, this has been accomplished by manually controlling the heterodyne phase between measurements with an optical flat. In this letter, an optical configuration is presented which allows for collection of TGS measurements at two heterodyne phases concurrently through the use of two independently phase controlled interrogation paths. This arrangement allows for complete, heterodyne amplified TGS measurements to be made in a manner not constrained by a mechanical actuation time. Measurements are instead constrained only by the desired signal-to-noise ratio. A temporal resolution of between 1 and 10 s, demonstrated here on single crystal metallic samples, will allow TGS experiments to be used as an in-situ, time-resolved monitoring technique for many material processing applications.

  17. Theoretical Studies of Time-Resolved Photoelectron Spectra of IBr^-

    NASA Astrophysics Data System (ADS)

    Horvath, Samantha; McCoy, Anne B.; Pitzer, Russell M.

    2009-06-01

    In the present study, we examine the time-resolved photoelectron spectra of IBr^-. In the photodetachment studies performed by Sanov and co-workers and Lineberger and co-workers, the anionic species, prepared in its electronic ground state (^2Σ_{1/2}^+), is excited to either its \\Atilde' (^2Π_{3/2}) or \\Btilde (^2Σ_{1/2}^+) excited state, before electron photodetachment and dissociation on the \\Ctilde (^1Π_1) or higher-lying excited states of the neutral species, respectively. In this work, we use the electronic structure program Columbus to calculate the six lowest electronic states of IBr^- and the ten lowest Σ states of IBr at the MR-SO-CISD/aug-cc-pVDZ level of theory/basis, using relativistic core potentials for I and Br. Experimentally determined electronic states of IBr are also used. Vibrational eigenstates for these electronic states are calculated in a discrete variable representation, and propagation of the thermally populated \\Xtilde-state vibrational wave functions on either the \\Atilde' or \\Btilde electronic states of the anion is performed using a Lanczos scheme. We then take time-dependent overlaps between these propagated states and the vibrational eigenstates of the neutral surface. Results for IBr^- show good agreement with the experimental time-resolved spectra. Extensions to IBr^-(CO_2)_n (n < 2) will also be discussed. R. Mabbs, K. Pichugin, and A. Sanov, J. Chem. Phys., 2005 122, 174305 Leonid Sheps, Elisa M. Miller, and W. C. Lineberger (private communication) E. Wrede, S. Laubach, S. Schulenburg, A. Brown, E. R. Wouters, A. J. Orr-Ewing, and M. N. R. Ashfold, J. Chem. Phys., 2001, 114(6), 2629. D. T. Colbert and W. H. Miller, J. Chem. Phys., 1992, 96(3), 1982

  18. Toward picosecond time-resolved X-ray absorption studies of interfacial photochemistry

    NASA Astrophysics Data System (ADS)

    Gessner, Oliver; Mahl, Johannes; Neppl, Stefan

    2016-05-01

    We report on the progress toward developing a novel picosecond time-resolved transient X-ray absorption spectroscopy (TRXAS) capability for time-domain studies of interfacial photochemistry. The technique is based on the combination of a high repetition rate picosecond laser system with a time-resolved X-ray fluorescent yield setup that may be used for the study of radiation sensitive materials and X-ray spectroscopy compatible photoelectrochemical (PEC) cells. The mobile system is currently deployed at the Advanced Light Source (ALS) and may be used in all operating modes (two-bunch and multi-bunch) of the synchrotron. The use of a time-stamping technique enables the simultaneous recording of TRXAS spectra with delays between the exciting laser pulses and the probing X-ray pulses spanning picosecond to nanosecond temporal scales. First results are discussed that demonstrate the viability of the method to study photoinduced dynamics in transition metal-oxide semiconductor (SC) samples under high vacuum conditions and at SC-liquid electrolyte interfaces during photoelectrochemical water splitting. Opportunities and challenges are outlined to capture crucial short-lived intermediates of photochemical processes with the technique. This work was supported by the Department of Energy Office of Science Early Career Research Program.

  19. Efficiency estimates and practical aspects of an optical Kerr gate for time-resolved luminescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Dmitruk, I.; Shynkarenko, Ye; Dmytruk, A.; Aleksiuk, D.; Kadan, V.; Korenyuk, P.; Zubrilin, N.; Blonskiy, I.

    2016-12-01

    We report experience of assembling an optical Kerr gate setup at the Femtosecond Laser Center for collective use at the Institute of Physics of the National Academy of Sciences of Ukraine. This offers an inexpensive solution to the problem of time-resolved luminescence spectroscopy. Practical aspects of its design and alignment are discussed and its main characteristics are evaluated. Theoretical analysis and numerical estimates are performed to evaluate the efficiency and the response time of an optical Kerr gate setup for fluorescence spectroscopy with subpicosecond time resolution. The theoretically calculated efficiency is compared with the experimentally measured one of ~12% for Crown 5 glass and ~2% for fused silica. Other characteristics of the Kerr gate are analyzed and ways to improve them are discussed. A method of compensation for the refractive index dispersion in a Kerr gate medium is suggested. Examples of the application of the optical Kerr gate setup for measurements of the time-resolved luminescence of Astra Phloxine and Coumarin 30 dyes and both linear and nonlinear chirp parameters of a supercontinuum are presented.

  20. Time-Resolved Luminescence Nanothermometry with Nitrogen-Vacancy Centers in Nanodiamonds.

    PubMed

    Tzeng, Yan-Kai; Tsai, Pei-Chang; Liu, Hsiou-Yuan; Chen, Oliver Y; Hsu, Hsiang; Yee, Fu-Goul; Chang, Ming-Shien; Chang, Huan-Cheng

    2015-06-10

    Measuring temperature in nanoscale spatial resolution either at or far from equilibrium is of importance in many scientific and technological applications. Although negatively charged nitrogen-vacancy (NV(-)) centers in diamond have recently emerged as a promising nanometric temperature sensor, the technique has been applied only under steady state conditions so far. Here, we present a three-point sampling method that allows real-time monitoring of the temperature changes over ±100 K and a pump-probe-type experiment that enables the study of nanoscale heat transfer with a temporal resolution of better than 10 μs. The utility of the time-resolved luminescence nanothermometry was demonstrated with 100 nm fluorescent nanodiamonds spin-coated on a glass substrate and submerged in gold nanorod solution heated by a near-infrared laser, and the validity of the measurements was verified with finite-element numerical simulations. The combined theoretical and experimental approaches will be useful to implement time-resolved temperature sensing in laser processing of materials and even for devices in operation at the nanometer scale.

  1. Time-Resolved Fluorometry Based Sandwich Hybridisation Assay for HLA-DQA1 Typing

    PubMed Central

    Sjöroos, Minna; Ilonen, Jorma; Reijonen, Helena; Lövgren, Timo

    1998-01-01

    A microtitration plate based time-resolved fluorescence (TRF) hybridisation assay was developed for HLA typing utilising biotinylated sequence-specific catching probes and europium (Eu) labelled gene locus-specific detection probe to allow time-resolved fluorometer reading of the reaction. In an application for HLA-DQA typing a 228 base pair long region of the polymorphic exon 2 of DQA1 gene was amplified and the denatured PCR product distributed into streptavidin-coated microtitration wells together with the detection probe and one of the catching probes. After incubation and washes, the enhancement solution was added and specific hybridisation signal detected by measuring the emitted light. A series of 100 isolated genomic DNA samples were studied using biotinylated probes specific for DQA1*01, *0101/0104, *0103/0201/0601, *0201, *03, *0401/0601, *05 and *0502 alleles with results demonstrating the capacity of the test to detect aimed alleles. A series of whole blood spot samples were also studied and the results confirmed the applicability of this modification of the test. PMID:9706458

  2. Frequency domain approach for time-resolved pump-probe microscopy using intensity modulated laser diodes

    NASA Astrophysics Data System (ADS)

    Miyazaki, J.; Kawasumi, K.; Kobayashi, T.

    2014-09-01

    We present a scheme for time-resolved pump-probe microscopy using intensity modulated laser diodes. The modulation frequencies of the pump and probe beams are varied up to 500 MHz with fixed frequency detuning typically set at 15 kHz. The frequency response of the pump-probe signal is detected using a lock-in amplifier referenced at the beat frequency. This frequency domain method is capable of characterizing the nanosecond to picosecond relaxation dynamics of sample species without the use of a high speed detector or a high frequency lock-in amplifier. Furthermore, as the pump-probe signal is based on the nonlinear interaction between the two laser beams and the sample, our scheme provides better spatial resolution than the conventional diffraction-limited optical microscopes. Time-resolved pump-probe imaging of fluorescence beads and aggregates of quantum dots demonstrates that this method is useful for the microscopic analysis of optoelectronic devices. The system is implemented using compact and low-cost laser diodes, and thus has a broad range of applications in the fields of photochemistry, optical physics, and biological imaging.

  3. Electron-transfer acceleration investigated by time resolved infrared spectroscopy.

    PubMed

    Vlček, Antonín; Kvapilová, Hana; Towrie, Michael; Záliš, Stanislav

    2015-03-17

    Ultrafast electron transfer (ET) processes are important primary steps in natural and artificial photosynthesis, as well as in molecular electronic/photonic devices. In biological systems, ET often occurs surprisingly fast over long distances of several tens of angströms. Laser-pulse irradiation is conveniently used to generate strongly oxidizing (or reducing) excited states whose reactions are then studied by time-resolved spectroscopic techniques. While photoluminescence decay and UV-vis absorption supply precise kinetics data, time-resolved infrared absorption (TRIR) and Raman-based spectroscopies have the advantage of providing additional structural information and monitoring vibrational energy flows and dissipation, as well as medium relaxation, that accompany ultrafast ET. We will discuss three cases of photoinduced ET involving the Re(I)(CO)3(N,N) moiety (N,N = polypyridine) that occur much faster than would be expected from ET theories. [Re(4-N-methylpyridinium-pyridine)(CO)3(N,N)](2+) represents a case of excited-state picosecond ET between two different ligands that remains ultrafast even in slow-relaxing solvents, beating the adiabatic limit. This is caused by vibrational/solvational excitation of the precursor state and participation of high-frequency quantum modes in barrier crossing. The case of Re-tryptophan assemblies demonstrates that excited-state Trp → *Re(II) ET is accelerated from nanoseconds to picoseconds when the Re(I)(CO)3(N,N) chromophore is appended to a protein, close to a tryptophan residue. TRIR in combination with DFT calculations and structural studies reveals an interaction between the N,N ligand and the tryptophan indole. It results in partial electronic delocalization in the precursor excited state and likely contributes to the ultrafast ET rate. Long-lived vibrational/solvational excitation of the protein Re(I)(CO)3(N,N)···Trp moiety, documented by dynamic IR band shifts, could be another accelerating factor. The last

  4. New method for measuring time-resolved spectra of lanthanide emission using square-wave excitation

    SciTech Connect

    Qin, Feng; Zhao, Hua; Cai, Wei; Duan, Qianqian; Zhang, Zhiguo; Cao, Wenwu

    2013-11-15

    A method using modulated continuous wave (CW) visible laser to measure time-resolved fluorescence spectra of trivalent rare-earth ions has been developed. Electro-optic modulator was used to modulate the CW pumping laser with a rise time of 2 μs. CW Nd{sup 3+} lasers were used as examples to present the method. Upconversion dynamic process of Ho{sup 3+} was studied utilizing a 532 nm CW laser. Quantum cutting dynamic process from Tb{sup 3+} to Yb{sup 3+} was analyzed by a 473 nm CW laser. This method can be applied to any CW laser such as He-Ne laser, Ar{sup +} laser, Kr{sup +} laser, Ti:sapphire laser, etc.

  5. New method for measuring time-resolved spectra of lanthanide emission using square-wave excitation.

    PubMed

    Qin, Feng; Zhao, Hua; Duan, Qianqian; Cai, Wei; Zhang, Zhiguo; Cao, Wenwu

    2013-11-01

    A method using modulated continuous wave (CW) visible laser to measure time-resolved fluorescence spectra of trivalent rare-earth ions has been developed. Electro-optic modulator was used to modulate the CW pumping laser with a rise time of 2 μs. CW Nd(3+) lasers were used as examples to present the method. Upconversion dynamic process of Ho(3+) was studied utilizing a 532 nm CW laser. Quantum cutting dynamic process from Tb(3+) to Yb(3+) was analyzed by a 473 nm CW laser. This method can be applied to any CW laser such as He-Ne laser, Ar(+) laser, Kr(+) laser, Ti:sapphire laser, etc.

  6. Time-resolved laser studies on the proton pump mechanism of bacteriorhodopsin

    SciTech Connect

    El-Sayed, M.A.

    1990-01-01

    During the past three years, a number of studies have been made to examine the different properties of bacteriorhodopsin and the effect of the different perturbations on its photocycle. A number of spectroscopic tools were used in these studies: (1) flash optical spectroscopy; (2) tryptophan fluorescence intensity, spectrum and decay; (3) time-resolved resonance Raman spectroscopy on the femtosecond time scale; (4) circular and magnetic circular dichroism; and (5) differential scanning calorimetry. The different perturbations used include: (1) pH changes, (2) temperature changes, (3) removing metal cations, (4) removing the lipids, (5) adding anesthetic drugs (dibucane.HCl) to bacteriorhodopsin, and (6) mutagenic substitution of some amino acids. Some of the important conclusions of this work are summarized.

  7. New method for measuring time-resolved spectra of lanthanide emission using square-wave excitation

    NASA Astrophysics Data System (ADS)

    Qin, Feng; Zhao, Hua; Duan, Qianqian; Cai, Wei; Zhang, Zhiguo; Cao, Wenwu

    2013-11-01

    A method using modulated continuous wave (CW) visible laser to measure time-resolved fluorescence spectra of trivalent rare-earth ions has been developed. Electro-optic modulator was used to modulate the CW pumping laser with a rise time of 2 μs. CW Nd3+ lasers were used as examples to present the method. Upconversion dynamic process of Ho3+ was studied utilizing a 532 nm CW laser. Quantum cutting dynamic process from Tb3+ to Yb3+ was analyzed by a 473 nm CW laser. This method can be applied to any CW laser such as He-Ne laser, Ar+ laser, Kr+ laser, Ti:sapphire laser, etc.

  8. Time-resolved analysis and visualization of dynamic processes in living cells

    PubMed Central

    Tvaruskó, W.; Bentele, M.; Misteli, T.; Rudolf, R.; Kaether, C.; Spector, D. L.; Gerdes, H. H.; Eils, R.

    1999-01-01

    Recent development of in vivo microscopy techniques, including green fluorescent proteins, has allowed the visualization of a wide range of dynamic processes in living cells. For quantitative and visual interpretation of such processes, new concepts for time-resolved image analysis and continuous time–space visualization are required. Here, we describe a versatile and fully automated approach consisting of four techniques, namely highly sensitive object detection, fuzzy logic-based dynamic object tracking, computer graphical visualization, and measurement in time–space. Systematic model simulations were performed to evaluate the reliability of the automated object detection and tracking method. To demonstrate potential applications, the method was applied to the analysis of secretory membrane traffic and the functional dynamics of nuclear compartments enriched in pre-mRNA splicing factors. PMID:10393928

  9. Time-resolved optical sensing of oligonucleotide hybridization via Au colloidal nanoparticles.

    PubMed

    Liu, Gang L; Rodriguez, Victoria B; Lee, Luke P

    2005-11-01

    Au nanoparticles have distinctive absorption spectra whose peak position or particle plasmon resonance wavelength is highly sensitive to molecule adsorption on their surfaces. Spherical Au nanoparticles are surface-modified by amino-functionalized self-assembly-monolayer and used as optical probes in the fluorescence-label-free spectroscopic detection of sub-nanomole oligonucleotides. Time-resolved studies of the immobilization and hybridization of oligonucleotides on the surface of Au nanoparticles were carried out. By measuring peak shift of absorption spectra of the Au colloidal nanoparticles over time, the samples of 15 nM 20 mer target and mismatched oligonucleotides are distinguished by their different influences on the particle plasmon resonance wavelength. The approach presented in this paper extends the application of Au nanoparticles as the optical probe in oligonucleotide recognitions without prior sample labeling.

  10. Time-resolved neutron imaging at ANTARES cold neutron beamline

    NASA Astrophysics Data System (ADS)

    Tremsin, A. S.; Dangendorf, V.; Tittelmeier, K.; Schillinger, B.; Schulz, M.; Lerche, M.; Feller, W. B.

    2015-07-01

    In non-destructive evaluation with X-rays light elements embedded in dense, heavy (or high-Z) matrices show little contrast and their structural details can hardly be revealed. Neutron radiography, on the other hand, provides a solution for those cases, in particular for hydrogenous materials, owing to the large neutron scattering cross section of hydrogen and uncorrelated dependency of neutron cross section on the atomic number. The majority of neutron imaging experiments at the present time is conducted with static objects mainly due to the limited flux intensity of neutron beamline facilities and sometimes due to the limitations of the detectors. However, some applications require the studies of dynamic phenomena and can now be conducted at several high intensity beamlines such as the recently rebuilt ANTARES beam line at the FRM-II reactor. In this paper we demonstrate the capabilities of time resolved imaging for repetitive processes, where different phases of the process can be imaged simultaneously and integrated over multiple cycles. A fast MCP/Timepix neutron counting detector was used to image the water distribution within a model steam engine operating at 10 Hz frequency. Within <10 minutes integration the amount of water was measured as a function of cycle time with a sub-mm spatial resolution, thereby demonstrating the capabilities of time-resolved neutron radiography for the future applications. The neutron spectrum of the ANTARES beamline as well as transmission spectra of a Fe sample were also measured with the Time Of Flight (TOF) technique in combination with a high resolution beam chopper. The energy resolution of our setup was found to be ~ 0.8% at 5 meV and ~ 1.7% at 25 meV. The background level (most likely gammas and epithermal/fast neutrons) of the ANTARES beamline was also measured in our experiments and found to be on the scale of 3% when no filters are installed in the beam. Online supplementary data available from stacks.iop.org/jinst/10

  11. Time-Resolved Measurements of Carbon Nanotube and Nanohorn Growth

    NASA Astrophysics Data System (ADS)

    Geohegan, David

    2005-11-01

    Mechanisms for carbon nanotube growth have been investigated for both laser vaporization (LV) and chemical vapor deposition (CVD) synthesis techniques through the use of time-resolved, in situ laser-based diagnostics for the measurement of absolute growth rates. Optimization of both the production of loose single-wall carbon nanotubes (SWNTs) by LV and the sustained growth of mm-long, vertically-aligned carbon nanotube arrays (VANTAs) by CVD are described. For SWNT growth by laser co-vaporization of carbon and trace metal catalysts at high (1200 C) temperatures, nanotubes are found to grow at ˜ 1--5 microns/second to lengths of only several microns, as determined by gated-ICCD imaging and laser spectroscopy of the plume of ejected material. Efforts to scale the LV production of SWNTs utilizing an industrial Nd:YAG laser (600 W average power, 1-500 Hz repetition rate, 0.5-10ms pulse width) are described. In addition to vaporizing material at much higher rates, the high-power laser irradiation provides sufficient plasma plume density and temperature to enable the growth of novel single-wall carbon nanohorn (SWNH) structures without the need for metal catalysts in the target. Applications of these SWNH structures as metal catalyst supports will be discussed. Through the application of time-resolved reflectivity and direct imaging, CVD growth of VANTAs from hydrocarbon gases at sustained rates of 0.2 -- 0.5 microns/second have been directly measured over millimeters of length at lower (˜ 700 C) temperatures. Now, through a new laser-CVD setup at the ALPS (Advanced Laser Processing and Synthesis) facility at ORNL, high-power laser heating is being employed for the fast and position-controlled growth of carbon nanotubes on substrates. In situ fast optical pyrometry is employed to record the rapid thermal processing of metal-catalyst-prepared substrates to investigate the nucleation and early growth behavior of CVD-grown nanotubes. New nanotube growth and tunable Raman

  12. Fielding of a time-resolved tomographic diagnostic

    SciTech Connect

    Daniel Frayer, Brian Cox, Wendi Dreesen, Douglas Johnson, Morris Kaufman

    2008-03-01

    A diagnostic instrument has been developed for the acquisition of high-speed time-resolved images at the Dual-Axis Radiographic Hydrodynamic Test (DARHT) Facility at Los Alamos National Laboratory. The instrument was developed in order to create time histories of the electron beam. Four discrete optical subsystems view Cerenkov light generated at an X-ray target inside of a vacuum envelope. Each system employs cylindrical optics to image light in one direction and collapse light in the orthogonal direction. Each of the four systems images and collapses in unique axes, thereby capturing unique information. Light along the imaging axis is relayed via optical fiber to streak cameras. A computer is used to reconstruct the original image from the four optically collapsed images. Due to DARHT’s adverse environment, the instrument can be operated remotely to adjust optical parameters and contains a subsystem for remote calibration. The instrument was deployed and calibrated, and has been used to capture and reconstruct images. Matters of alignment, calibration, control, resolution, adverse conditions and maintenance will be discussed.

  13. Chopper system for time resolved experiments with synchrotron radiation.

    PubMed

    Cammarata, Marco; Eybert, Laurent; Ewald, Friederike; Reichenbach, Wolfgang; Wulff, Michael; Anfinrud, Philip; Schotte, Friedrich; Plech, Anton; Kong, Qingyu; Lorenc, Maciej; Lindenau, Bernd; Räbiger, Jürgen; Polachowski, Stephan

    2009-01-01

    A chopper system for time resolved pump-probe experiments with x-ray beams from a synchrotron is described. The system has three parts: a water-cooled heatload chopper, a high-speed chopper, and a millisecond shutter. The chopper system, which is installed in beamline ID09B at the European Synchrotron Radiation Facility, provides short x-ray pulses for pump-probe experiments with ultrafast lasers. The chopper system can produce x-ray pulses as short as 200 ns in a continuous beam and repeat at frequencies from 0 to 3 kHz. For bunch filling patterns of the synchrotron with pulse separations greater than 100 ns, the high-speed chopper can isolate single 100 ps x-ray pulses that are used for the highest time resolution. A new rotor in the high-speed chopper is presented with a single pulse (100 ps) and long pulse (10 micros) option. In white beam experiments, the heatload of the (noncooled) high-speed chopper is lowered by a heatload chopper, which absorbs 95% of the incoming power without affecting the pulses selected by the high speed chopper.

  14. Time-Resolved Hard X-Ray Spectrometer

    SciTech Connect

    Kenneth Moya; Ian McKennaa; Thomas Keenana; Michael Cuneob

    2007-03-01

    Wired array studies are being conducted at the SNL Z accelerator to maximize the x-ray generation for inertial confinement fusion targets and high energy density physics experiments. An integral component of these studies is the characterization of the time-resolved spectral content of the x-rays. Due to potential spatial anisotropy in the emitted radiation, it is also critical to diagnose the time-evolved spectral content in a space-resolved manner. To accomplish these two measurement goals, we developed an x-ray spectrometer using a set of high-speed detectors (silicon PIN diodes) with a collimated field-of-view that converged on a 1-cm-diameter spot at the pinch axis. Spectral discrimination is achieved by placing high Z absorbers in front of these detectors. We built two spectrometers to permit simultaneous different angular views of the emitted radiation. Spectral data have been acquired from recent Z shots for the radial and polar views. UNSPEC1 has been adapted to analyze and unfold the measured data to reconstruct the x-ray spectrum. The unfold operator code, UFO2, is being adapted for a more comprehensive spectral unfolding treatment.

  15. Time resolved spectroscopic NMR imaging using hyperpolarized 129Xe

    NASA Astrophysics Data System (ADS)

    Han, S.; Kühn, H.; Häsing, F. W.; Münnemann, K.; Blümich, B.; Appelt, S.

    2004-04-01

    We have visualized the melting and dissolution processes of xenon (Xe) ice into different solvents using the methods of nuclear magnetic resonance (NMR) spectroscopy, imaging, and time resolved spectroscopic imaging by means of hyperpolarized 129Xe. Starting from the initial condition of a hyperpolarized solid Xe layer frozen on top of an ethanol (ethanol/water) ice block we measured the Xe phase transitions as a function of time and temperature. In the pure ethanol sample, pieces of Xe ice first fall through the viscous ethanol to the bottom of the sample tube and then form a thin layer of liquid Xe/ethanol. The xenon atoms are trapped in this liquid layer up to room temperature and keep their magnetization over a time period of 11 min. In the ethanol/water mixture (80 vol%/20%), most of the polarized Xe liquid first stays on top of the ethanol/water ice block and then starts to penetrate into the pores and cracks of the ethanol/water ice block. In the final stage, nearly all the Xe polarization is in the gas phase above the liquid and trapped inside the pores. NMR spectra of homogeneous samples of pure ethanol containing thermally polarized Xe and the spectroscopic images of the melting process show that very high concentrations of hyperpolarized Xe (about half of the density of liquid Xe) can be stored or delivered in pure ethanol.

  16. Femtosecond time-resolved MeV electron diffraction

    DOE PAGES

    Zhu, Pengfei; Zhu, Y.; Hidaka, Y.; ...

    2015-06-02

    We report the experimental demonstration of femtosecond electron diffraction using high-brightness MeV electron beams. High-quality, single-shot electron diffraction patterns for both polycrystalline aluminum and single-crystal 1T-TaS2 are obtained utilizing a 5 fC (~3 × 104 electrons) pulse of electrons at 2.8 MeV. The high quality of the electron diffraction patterns confirms that electron beam has a normalized emittance of ~50 nm rad. The transverse and longitudinal coherence length is ~11 and ~2.5 nm, respectively. The timing jitter between the pump laser and probe electron beam was found to be ~100 fs (rms). The temporal resolution is demonstrated by observing themore » evolution of Bragg and superlattice peaks of 1T-TaS2 following an 800 nm optical pump and was found to be 130 fs. Lastly, our results demonstrate the advantages of MeV electrons, including large elastic differential scattering cross-section and access to high-order reflections, and the feasibility of ultimately realizing below 10 fs time-resolved electron diffraction.« less

  17. Time resolved EUV spectra from Zpinching capillary discharge plasma

    NASA Astrophysics Data System (ADS)

    Jancarek, Alexandr; Nevrkla, Michal; Nawaz, Fahad

    2015-09-01

    We developed symmetrically charged driver to obtain high voltage, high current Z-pinching capillary discharge. Plasma is created by up to 70 kA, 29 ns risetime current pulse passing through a 5 mm inner diameter, 224 mm long capillary filled with gas to initial pressure in the range of 1 kPa. Due to the low inductance design of the driver, the pinch is observable directly from the measured current curve. Time-integrated and time-resolved spectra of discharge plasma radiation are recorded together with the capillary current and analyzed. The most encouraging spectra were captured in the wavelength range 8.3 ÷ 14 nm. This spectral region contains nitrogen Balmer series lines including potentially lasing NVII 2 - 3 transition. Spectral lines are identified in the NIST database using the FLY kinetic code. The line of 13.38 nm wavelength, transition NVII 2 - 3, was observed in gated, and also in time-integrated spectra for currents >60 kA. This work has been supported by the Ministry of Education, Youth and Sports of the Czech Republic grants LG13029.

  18. Time-resolved infrared spectroscopic techniques as applied to channelrhodopsin

    PubMed Central

    Ritter, Eglof; Puskar, Ljiljana; Bartl, Franz J.; Aziz, Emad F.; Hegemann, Peter; Schade, Ulrich

    2015-01-01

    Among optogenetic tools, channelrhodopsins, the light gated ion channels of the plasma membrane from green algae, play the most important role. Properties like channel selectivity, timing parameters or color can be influenced by the exchange of selected amino acids. Although widely used, in the field of neurosciences for example, there is still little known about their photocycles and the mechanism of ion channel gating and conductance. One of the preferred methods for these studies is infrared spectroscopy since it allows observation of proteins and their function at a molecular level and in near-native environment. The absorption of a photon in channelrhodopsin leads to retinal isomerization within femtoseconds, the conductive states are reached in the microsecond time scale and the return into the fully dark-adapted state may take more than minutes. To be able to cover all these time regimes, a range of different spectroscopical approaches are necessary. This mini-review focuses on time-resolved applications of the infrared technique to study channelrhodopsins and other light triggered proteins. We will discuss the approaches with respect to their suitability to the investigation of channelrhodopsin and related proteins. PMID:26217670

  19. Femtosecond time-resolved MeV electron diffraction

    SciTech Connect

    Zhu, Pengfei; Zhu, Y.; Hidaka, Y.; Wu, L.; Cao, J.; Berger, H.; Geck, J.; Kraus, R.; Pjerov, S.; Shen, Y.; Tobey, R. I.; Hill, J. P.; Wang, X. J.

    2015-06-02

    We report the experimental demonstration of femtosecond electron diffraction using high-brightness MeV electron beams. High-quality, single-shot electron diffraction patterns for both polycrystalline aluminum and single-crystal 1T-TaS2 are obtained utilizing a 5 fC (~3 × 104 electrons) pulse of electrons at 2.8 MeV. The high quality of the electron diffraction patterns confirms that electron beam has a normalized emittance of ~50 nm rad. The transverse and longitudinal coherence length is ~11 and ~2.5 nm, respectively. The timing jitter between the pump laser and probe electron beam was found to be ~100 fs (rms). The temporal resolution is demonstrated by observing the evolution of Bragg and superlattice peaks of 1T-TaS2 following an 800 nm optical pump and was found to be 130 fs. Lastly, our results demonstrate the advantages of MeV electrons, including large elastic differential scattering cross-section and access to high-order reflections, and the feasibility of ultimately realizing below 10 fs time-resolved electron diffraction.

  20. Fielding of a Time-Resolved Tomographic Diagnostic

    SciTech Connect

    Daniel Frayer, Brian Cox, Wendi Dreesen, Douglas Johnson, Mike Jones, Morris Kaufman

    2008-09-11

    A diagnostic instrument has been developed for the acquisition of high-speed time-resolved images at the Dual-Axis Radiographic Hydrodynamic Test (DARHT) Facility at Los Alamos National Laboratory. The instrument was developed in order to create time histories of the electron beam. Four discrete optical subsystems view Cerenkov light generated at an x-ray target inside of a vacuum envelope. Each system employs cylindrical optics to image light in one direction and collapse light in the orthogonal direction. Each of the four systems images and collapses in unique axes, thereby capturing unique information. Light along the imaging axis is relayed via optical fiber to streak cameras. A computer is used to reconstruct the original image from the four optically collapsed images. Due to DARHT’s adverse environment, the instrument can be operated remotely to adjust optical parameters and contains a subsystem for remote calibration. The instrument was deployed and calibrated, and has been used to capture and reconstruct images. Matters of alignment, calibration, control, resolution, and adverse conditions will be discussed.

  1. Time-resolved neurite mechanics by thermal fluctuation assessments.

    PubMed

    Gárate, Fernanda; Betz, Timo; Pertusa, María; Bernal, Roberto

    2015-12-30

    In the absence of simple noninvasive measurements, the knowledge of temporal and spatial variations of axons mechanics remains scarce. By extending thermal fluctuation spectroscopy (TFS) to long protrusions, we determine the transverse amplitude thermal fluctuation spectra that allow direct and simultaneous access to three key mechanics parameters: axial tension, bending flexural rigidity and plasma membrane tension. To test our model, we use PC12 cell protrusions-a well-know biophysical model of axons-in order to simplify the biological system under scope. For instance, axial and plasma membrane tension are found in the range of nano Newton and tens of pico Newtons per micron respectively. Furthermore, our results shows that the TFS technique is capable to distinguish quasi-identical protrusions. Another advantage of our approach is the time resolved nature of the measurements. Indeed, in the case of long term experiments on PC12 protrusions, TFS has revealed large temporal, correlated variations of the protrusion mechanics, displaying extraordinary feedback control over the axial tension in order to maintain a constant tension value.

  2. Time-resolved pump-probe experiments at the LCLS

    SciTech Connect

    Glownia, James; Cryan, J.; Andreasson, J.; Belkacem, A.; Berrah, N.; Blaga, C.L.; Bostedt, C.; Bozek, J.; DiMauro, L.F.; Fang, L.; Frisch, J.; Gessner, O.; Guhr, M.; Hajdu, J.; Hertlein, M.P.; Hoener, M.; Huang, G.; Kornilov, O.; Marangos, J.P.; March, A.M.; McFarland, B.K.; /SLAC /Stanford U., Phys. Dept. /SLAC /IRAMIS, Saclay /Stanford U., Phys. Dept. /Georgia Tech /Argonne /Kansas State U. /SLAC /Stanford U., Phys. Dept. /SLAC /Stanford U., Appl. Phys. Dept. /Stanford U., Appl. Phys. Dept. /SLAC /LBNL /Argonne /SLAC /SLAC /Stanford U., Appl. Phys. Dept. /Stanford U., Phys. Dept.

    2011-08-12

    The first time-resolved x-ray/optical pump-probe experiments at the SLAC Linac Coherent Light Source (LCLS) used a combination of feedback methods and post-analysis binning techniques to synchronize an ultrafast optical laser to the linac-based x-ray laser. Transient molecular nitrogen alignment revival features were resolved in time-dependent x-ray-induced fragmentation spectra. These alignment features were used to find the temporal overlap of the pump and probe pulses. The strong-field dissociation of x-ray generated quasi-bound molecular dications was used to establish the residual timing jitter. This analysis shows that the relative arrival time of the Ti:Sapphire laser and the x-ray pulses had a distribution with a standard deviation of approximately 120 fs. The largest contribution to the jitter noise spectrum was the locking of the laser oscillator to the reference RF of the accelerator, which suggests that simple technical improvements could reduce the jitter to better than 50 fs.

  3. Time-resolved pump-probe experiments at the LCLS.

    PubMed

    Glownia, James M; Cryan, J; Andreasson, J; Belkacem, A; Berrah, N; Blaga, C I; Bostedt, C; Bozek, J; DiMauro, L F; Fang, L; Frisch, J; Gessner, O; Gühr, M; Hajdu, J; Hertlein, M P; Hoener, M; Huang, G; Kornilov, O; Marangos, J P; March, A M; McFarland, B K; Merdji, H; Petrovic, V S; Raman, C; Ray, D; Reis, D A; Trigo, M; White, J L; White, W; Wilcox, R; Young, L; Coffee, R N; Bucksbaum, P H

    2010-08-16

    The first time-resolved x-ray/optical pump-probe experiments at the SLAC Linac Coherent Light Source (LCLS) used a combination of feedback methods and post-analysis binning techniques to synchronize an ultrafast optical laser to the linac-based x-ray laser. Transient molecular nitrogen alignment revival features were resolved in time-dependent x-ray-induced fragmentation spectra. These alignment features were used to find the temporal overlap of the pump and probe pulses. The strong-field dissociation of x-ray generated quasi-bound molecular dications was used to establish the residual timing jitter. This analysis shows that the relative arrival time of the Ti:Sapphire laser and the x-ray pulses had a distribution with a standard deviation of approximately 120 fs. The largest contribution to the jitter noise spectrum was the locking of the laser oscillator to the reference RF of the accelerator, which suggests that simple techni