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Sample records for fluorescence correlation spectroscopic

  1. The spectroscopic basis of Fluorescence Triple Correlation Spectroscopy

    PubMed Central

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

    2012-01-01

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

  2. Fluorescence spectroscopic detection of early injury-induced atherosclerosis

    NASA Astrophysics Data System (ADS)

    Lucas, Alexandra; Perk, Masis; Wen, Yue; Smith, Carol

    1992-08-01

    Laser-induced fluorescence spectroscopy has been used for the detection of advanced atherosclerotic lesions. Angioplasty balloon-mediated injury was examined spectroscopically in order to assess the sensitivity of fluorescence spectroscopy for detection of early atherosclerosis. Abdominal aortic balloon angioplasty was performed via femoral artery cutdown in nine White Leghorn roosters (five normal, four atherogenic diet). Roosters were sacrificed at 1, 2, 4, 8, and 12 week intervals. Fluorescence emission spectra (n equals 114) were recorded from each aortic section (XeCl excimer laser, 308 nm, 1.5 - 2.0 mJ/pulse, 5 Hz). Changes in normalized fluorescence emission intensity were correlated with selected sections of histology. All balloon-injured segments showed intimal fibrous proliferation. For intimal thickness measuring > 70 (mu) , fluorescence emission intensity was decreased at 440 - 460 nm (p < 0.0005). Lesions complicated by thrombus also had lower fluorescence emission at 425 - 450 nm when compared to histologically normal aorta (p < 0.009). In injured segments high cholesterol diet resulted in lower recorded fluorescence emission at 440 - 460 nm (p < 0.001) associated with the increase in intimal thickness. Spectra from uninjured elastic aorta (aortic arch and thoracic aorta) had greater fluorescence intensity at 380 - 445 nm than muscular (abdominal) aorta (p < 0.01), therefore, only spectra from injured and uninjured segments of corresponding areas of the aorta were compared. The conclusion is: (1) Early intimal proliferative changes after angioplasty can be detected by fluorescence spectroscopy. (2) Spectra from elastic thoracic aorta differ significantly from the spectra of muscular abdominal aorta.

  3. Fluorescence spectroscopic studies of DNA dynamics

    SciTech Connect

    Scalettar, B.A.

    1987-04-01

    Random solvent induced motions of DNA are manifest as nanosecond torsional oscillations of the helix backbone, nanosecond through millisecond bending deformations and overall rotational and translational diffusion of the polymer. Fluorescence spectroscopy is used to study this spectrum of DNA motions while ethidium monoazide was covalently bounded. The steady state fluorescence depolarization data indicate that the covalent monoazide/DNA complex exhibits internal motions characterized by an average angular amplitude of 26 degrees confirming reports of fast torsional oscillations in noncovalent ethidium bromide/DNA systems. Data obtained by use of a new polarized photobleaching recovery technique (FPR) reflect both the rotational dynamics of the polymer and the reversible photochemistry of the dye. To isolate the reorientational motion of the DNA, the FPR experiments were ran in two modes that differ only in the polarization of the bleaching light. A quotient function constructed from the data obtained in these two modes monitors only the rotational component of the FPR recovery. In specific applications those bending deformations of long DNA molecules that have characteristic relaxation times on the order of 100 microseconds have been resolved. A fluorescence correlation technique that relates fluctuations in particle number to center-of-mass motion was used to measure translational diffusion on coefficients of the plasmid PBR322 and a short oligomeric DNA. A theory that describes angular correlation in systems exhibiting cyclic, biologically directed reorientation and random Brownian rotation is developed.

  4. Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

    NASA Astrophysics Data System (ADS)

    Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

    2005-04-01

    This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

  5. Correlation fluorescence method of amine detection

    NASA Astrophysics Data System (ADS)

    Myslitsky, Valentin F.; Tkachuk, Svetlana S.; Rudeichuk, Volodimir M.; Strinadko, Miroslav T.; Slyotov, Mikhail M.; Strinadko, Marina M.

    1997-12-01

    The amines fluorescence spectra stimulated by UV laser radiation are investigated in this paper. The fluorescence is stimulated by the coherent laser beam with the wavelength 0.337 micrometers . At the sufficient energy of laser stimulation the narrow peaks of the fluorescence spectra are detected besides the wide maximum. The relationship between the fluorescence intensity and the concentration of amines solutions are investigated. The fluorescence intensity temporal dependence on wavelength 0.363 micrometers of the norepinephrine solution preliminarily radiated by UV laser with wavelength 0.337 micrometers was found. The computer stimulated and experimental investigations of adrenaline and norepinephrine mixtures fluorescence spectra were done. The correlation fluorescent method of amines detection is proposed.

  6. Photon correlation system for fluorescence lifetime measurements

    NASA Astrophysics Data System (ADS)

    Morgan, C. G.; Murray, J. G.; Mitchell, A. C.

    1995-07-01

    The construction and testing of a dual-channel photon correlator is reported for the frequency domain imaging of fluorescence lifetimes using photon-counting detection. A light source modulated at radio frequency excites fluorescence, which is detected using an imaging single-photon detector. After discrimination, single-photon events are processed in parallel by the correlation circuit, the purpose of which is to allow both the mean phase delay and the demodulation of fluorescence to be calculated relative to a reference signal derived from the modulated excitation source. Outputs from the correlator are integrated in a computer, resulting in accumulation of images which have been statistically filtered by sine and cosine transforms, and which can be manipulated within the computer to generate a resultant image where contrast depends on fluorescence lifetime rather than fluorescence intensity.

  7. Fluorescence Spectroscopic Properties of Normal and Abnormal Biomedical Materials

    NASA Astrophysics Data System (ADS)

    Pradhan, Asima

    Steady state and time-resolved optical spectroscopy and native fluorescence is used to study the physical and optical properties occurring in diseased and non-diseased biological human tissue, in particular, cancer of the human breast, artery and the dynamics of a photosensitizer useful in photodynamic therapy. The main focus of the research is on the optical properties of cancer and atherosclerotic tissues as compared to their normal counterparts using the different luminescence based spectroscopic techniques such as steady state fluorescence, time-resolved fluorescence, excitation spectroscopy and phosphorescence. The excitation and steady-state spectroscopic fluorescence using visible excitation wavelength displays a difference between normal and malignant tissues. This difference is attributed to absorption of the emission by hemoglobin in normal tissues. This method using 488nm fails to distinguish neoplastic tissue such as benign tissues and tumors from malignant tumors. The time-resolved fluorescence at visible, near -uv and uv excitation wavelengths display non-exponential profiles which are significantly different for malignant tumors as compared to non-malignant tissues only with uv excitation. The differences observed with visible and near-uv excitation wavelengths are not as significant. The non-exponential profiles are interpreted as due to a combination of fluorophores along with the action of non-radiative processes. Low temperature luminescence studies confirm the occurrence of non-radiative decay processes while temporal studies of various relevant biomolecules indicate the probable fluorophores responsible for the observed signal in tissues. Phosphorescence from human tissues have been observed for the first time and lifetimes of a few hundred nanoseconds are measured for malignant and benign tissues. Time-resolved fluorescence studies of normal artery and atherosclerotic plaque have shown that a combination of two excitation wavelengths can

  8. Spectroscopic Analysis of Today's Compact Fluorescent Light Bulbs

    NASA Astrophysics Data System (ADS)

    Pluhar, Edward

    2012-03-01

    In today's consumer market, there are many different light bulbs that claim to produce `natural' light. In my research, I both quantitatively and qualitatively analyzed this claim. First, utilizing a spectroscope, I compared the spectra emitted by different brands and types of compact fluorescent light (CFL) bulbs to the spectra emitted by the Sun. Once the bulbs were quantitatively analyzed, I proceeded to qualitatively analyze them by exposing subjects to the different bulbs. The subjects were asked to rate the quality of color in different pictures illuminated by each type of CFL. From these tests, I was able to determine the ``best'' CFL bulbs, and conclude whether the health risks associated with CFL bulbs outweigh the cost savings, longevity of the bulbs, and/or quality of light benefits.

  9. Fluorescent-Spectroscopic Research of in Vivo Tissues Pathological Conditions

    NASA Astrophysics Data System (ADS)

    Giraev, K. M.; Ashurbekov, N. A.; Medzhidov, R. T.

    The steady-state spectra of autofluorescence and the reflection coefficient on the excitation wavelength of some stomach tissues in vivo with various pathological conditions (surface gastritis, displasia, cancer) are measured under excitation by the nitrogen laser irradiation (λex=337.1 nm). The contour expansion of obtained fluorescence spectra into contributions of components is conducted by the Gaussian-Lorentzian curves method. It is shown that at least 7 groups of fluorophores forming a total luminescence spectrum can be distinguished during the development of displasia and tumor processes. The correlation of intensities of flavins and NAD(P)·H fluorescence is determined and the degree of respiratory activity of cells for the functional condition considered is estimated. The evaluations of the fluorescence quantum yield of the tissue's researched are given.

  10. Dual-focus fluorescence correlation spectroscopy.

    PubMed

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

    2013-01-01

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

  11. Position-sensitive scanning fluorescence correlation spectroscopy.

    PubMed

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

    2005-08-01

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

  12. Synchronous fluorescence spectroscopic study of solvatochromic curcumin dye

    NASA Astrophysics Data System (ADS)

    Patra, Digambara; Barakat, Christelle

    2011-09-01

    Curcumin, the main yellow bioactive component of turmeric, has recently acquired attention by chemists due its wide range of potential biological applications as an antioxidant, an anti-inflammatory, and an anti-carcinogenic agent. This molecule fluoresces weakly and poorly soluble in water. In this detailed study of curcumin in thirteen different solvents, both the absorption and fluorescence spectra of curcumin was found to be broad, however, a narrower and simple synchronous fluorescence spectrum of curcumin was obtained at Δ λ = 10-20 nm. Lippert-Mataga plot of curcumin in different solvents illustrated two sets of linearity which is consistent with the plot of Stokes' shift vs. the ET30. When Stokes's shift in wavenumber scale was replaced by synchronous fluorescence maximum in nanometer scale, the solvent polarity dependency measured by λSFSmax vs. Lippert-Mataga plot or ET30 values offered similar trends as measured via Stokes' shift for protic and aprotic solvents for curcumin. Better linear correlation of λSFSmax vs. π* scale of solvent polarity was found compared to λabsmax or λemmax or Stokes' shift measurements. In Stokes' shift measurement both absorption/excitation as well as emission (fluorescence) spectra are required to compute the Stokes' shift in wavenumber scale, but measurement could be done in a very fast and simple way by taking a single scan of SFS avoiding calculation and obtain information about polarity of the solvent. Curcumin decay properties in all the solvents could be fitted well to a double-exponential decay function.

  13. Fluorescence correlation spectroscopy in semiadhesive wall proximity.

    PubMed

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

    2011-11-01

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

  14. Dual-wavelength excitation to reduce background fluorescence for fluorescence spectroscopic quantitation of erythrocyte zinc protoporphyrin-IX and protoporphyrin-IX from whole blood and oral mucosa

    NASA Astrophysics Data System (ADS)

    Hennig, Georg; Vogeser, Michael; Holdt, Lesca M.; Homann, Christian; Großmann, Michael; Stepp, Herbert; Gruber, Christian; Erdogan, Ilknur; Hasmüller, Stephan; Hasbargen, Uwe; Brittenham, Gary M.

    2014-02-01

    Erythrocyte zinc protoporphyrin-IX (ZnPP) and protoporphyrin-IX (PPIX) accumulate in a variety of disorders that restrict or disrupt the biosynthesis of heme, including iron deficiency and various porphyrias. We describe a reagent-free spectroscopic method based on dual-wavelength excitation that can measure simultaneously both ZnPP and PPIX fluorescence from unwashed whole blood while virtually eliminating background fluorescence. We further aim to quantify ZnPP and PPIX non-invasively from the intact oral mucosa using dual-wavelength excitation to reduce the strong tissue background fluorescence while retaining the faint porphyrin fluorescence signal originating from erythrocytes. Fluorescence spectroscopic measurements were made on 35 diluted EDTA blood samples using a custom front-face fluorometer. The difference spectrum between fluorescence at 425 nm and 407 nm excitation effectively eliminated background autofluorescence while retaining the characteristic porphyrin peaks. These peaks were evaluated quantitatively and the results compared to a reference HPLC-kit method. A modified instrument using a single 1000 μm fiber for light delivery and detection was used to record fluorescence spectra from oral mucosa. For blood measurements, the ZnPP and PPIX fluorescence intensities from the difference spectra correlated well with the reference method (ZnPP: Spearman's rho rs = 0.943, p < 0.0001; PPIX: rs = 0.959, p < 0.0001). In difference spectra from oral mucosa, background fluorescence was reduced significantly, while porphyrin signals remained observable. The dual-wavelength excitation method evaluates quantitatively the ZnPP/heme and PPIX/heme ratios from unwashed whole blood, simplifying clinical laboratory measurements. The difference technique reduces the background fluorescence from measurements on oral mucosa, allowing for future non-invasive quantitation of erythrocyte ZnPP and PPIX.

  15. 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

  16. 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.

  17. Fluorescence spectroscopic detection of virus-induced atherosclerosis

    NASA Astrophysics Data System (ADS)

    Yan, Wei-dong; Perk, Masis; Nation, Patric N.; Power, Robert F.; Liu, Liying; Jiang, Xiuyan; Lucas, Alexandra

    1994-07-01

    Laser-induced fluorescence (LF) has been developed as a diagnostic tool for the detection of atherosclerosis. We have examined the use of LF for the identification of accelerated atherosclerotic plaque growth induced by Marek's Disease Virus (MDV) infection in White Leghorn rooster chicks (R) as well as plaque regression after treatment. Twenty-eight newborn R were infected with 12,000 cfu of MDV. Twelve parallel control R had saline injection. LF spectra were recorded from the arteries in vitro with a CeramOptec laser angioplasty catheter during 308 nm XeCl excimer laser excitation. Significant differences were detected at 440 to 475, 525, 550, 600, and 650 nm in MDV-R (p<0.05). In a subsequent study, 60 R were infected with 5,000 cfu of MDV, and were then treated with either Pravastatin (PRV) or placebo at 3 months post infection. These PRV-R were followed for 6 months to detect changes in atherosclerotic plaque development. PRV reduced intimal proliferation produced by MDV infection on histological examination (PRV-R 128.0+/- 44.0 micrometers , placebo-R 412.2+/- 91.5 micrometers , pequals0.007). MDV infected, PRV treated R were examined for LF changes that correlated with decreased atherosclerosis. There was an associated significant increase in LF intensity in PRV-R at 405 to 425 nm (p<0.001). In conclusion, LF can detect intimal proliferation in virus- induced atherosclerosis and atherosclerotic plaque regression after PRV therapy.

  18. Fluorescence spectroscopic analysis on interaction of fleroxacin with pepsin.

    PubMed

    Lian, Shuqin; Wang, Guirong; Zhou, Liping; Yang, Dongzhi

    2013-01-01

    The interaction between fleroxacin (FLX) and pepsin was investigated by spectrofluorimetry. The effects of FLX on pepsin showed that the microenvironment of tryptophan residues and molecular conformation of pepsin were changed based on fluorescence quenching and synchronous fluorescence spectroscopy in combination with three-dimensional fluorescence spectroscopy. Static quenching was suggested and it was proved that the fluorescence quenching of pepsin by FLX was related to the formation of a new complex and a non-radiation energy transfer. The quenching constants KSV , binding constants K and binding sites n were calculated at different temperatures. The molecular interaction distance (r = 6.71) and energy transfer efficiency (E = 0.216) between pepsin and FLX were obtained according to the Forster mechanism of non-radiation energy transfer. Hydrophobic and electrostatic interaction played a major role in FLX-pepsin association. In addition, the hydrophobic interaction and binding free energy were further tested by molecular modeling study.

  19. Single molecule spectroscopic characterization of a far-red fluorescent protein (HcRed) from the Anthozoa coral Heteractis crispa

    NASA Astrophysics Data System (ADS)

    Cotlet, Mircea; Habuchi, Satoshi; Whitier, Jennifer E.; Werner, James H.; De Schryver, Frans C.; Hofkens, Johan; Goodwin, Peter M.

    2006-02-01

    We report on the photophysical properties of a far-red intrinsic fluorescent protein by means of single molecule and ensemble spectroscopic methods. The green fluorescent protein (GFP) from Aequorea victoria is a popular fluorescent marker with genetically encoded fluorescence and which can be fused to any biological structure without affecting its function. GFP and its variants provide emission colors from blue to yellowish green. Red intrinsic fluorescent proteins from Anthozoa species represent a recent addition to the emission color palette provided by GFPs. Red intrinsic fluorescent markers are on high demand in protein-protein interaction studies based on fluorescence-resonance energy transfer or in multicolor tracking studies or in cellular investigations where autofluorescence possesses a problem. Here we address the photophysical properties of a far-red fluorescent protein (HcRed), a mutant engineered from a chromoprotein cloned from the sea anemone Heteractis crispa, by using a combination of ensemble and single molecule spectroscopic methods. We show evidence for the presence of HcRed protein as an oligomer and for incomplete maturation of its chromophore. Incomplete maturation results in the presence of an immature (yellow) species absorbing/fluorescing at 490/530-nm. This yellow chromophore is involved in a fast resonance-energy transfer with the mature (purple) chromophore. The mature chromophore of HcRed is found to adopt two conformations, a Transoriented form absorbing and 565-nm and non-fluorescent in solution and a Cis-oriented form absorbing at 590-nm and emitting at 645-nm. These two forms co-exist in solution in thermal equilibrium. Excitation-power dependence fluorescence correlation spectroscopy of HcRed shows evidence for singlet-triplet transitions in the microseconds time scale and for cis-trans isomerization occurring in a time scale of tens of microseconds. Single molecule fluorescence data recorded from immobilized HcRed proteins, all

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

    PubMed Central

    Tian, Yu; Martinez, Michelle M.

    2011-01-01

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

  1. Fluorescence spectroscopic study on the interaction of resveratrol with lipoxygenase

    NASA Astrophysics Data System (ADS)

    Pinto, María del Carmen; Duque, Antonio Luis; Macías, Pedro

    2010-09-01

    The interaction of lipoxygenase with (E)-resveratrol was investigated by fluorescence spectroscopy. The data obtained revealed that the quenching of intrinsic fluorescence of lipoxygenase is produced by the formation of a complex lipoxygenase-(E)-resveratrol. From the value obtained for the binding constant, according to the Stern-Volmer modified equation, was deduced the existence of static quenching mechanism and, as consequence, the existence of a strong interaction between (E)-resveratrol and lipoxygenase. The values obtained for the thermodynamic parameter Δ H (-3.58 kJ mol -1) and Δ S (87.97 J mol -1K -1) suggested the participation of hydrophobic interactions and hydrogen bonds in the stabilization of the complex ligand-protein. From the static quenching we determined that only exist one independent binding site. Based on the Förster energy transfer theory, the distance between the acceptor ((E)-resveratrol) and the donor (Trp residues of lipoxygenase) was calculated to be 3.42 nm. Finally, based on the information obtained from the evaluation of synchronous and three-dimensional fluorescence spectroscopy, we deduced that the interaction of (E)-resveratrol with lipoxygenase produces micro-environmental and conformational alterations of protein in the binding region.

  2. Correlating the chemical and spectroscopic characteristics of natural organic matter with the photodegradation of sulfamerazine.

    PubMed

    Batista, Ana Paula S; Teixeira, Antonio Carlos S C; Cooper, William J; Cottrell, Barbara A

    2016-04-15

    The role of aquatic natural organic matter (NOM) in the removal of contaminants of emerging concern has been widely studied. Sulfamerazine (SMR), a sulfonamide antibiotic detected in aquatic environments, is implicated in environmental toxicity and may contribute to the resistance of bacteria to antibiotics. In aquatic systems sulfonamides may undergo direct photodegradation, and, indirect photodegradation through the generation of reactive species. Because some forms of NOM inhibit the photodegradation there is an increasing interest in correlating the spectroscopic parameters of NOM as potential indicators of its degradation in natural waters. Under the conditions used in this study, SMR hydrolysis was shown to be negligible; however, direct photolysis is a significant in most of the solutions studied. Photodegradation was investigated using standard solutions of NOM: Suwannee River natural organic matter (SRNOM), Suwannee River humic acid (SRHA), Suwannee River fulvic acid (SRFA), and Aldrich humic acid (AHA). The steady-state concentrations and formation rates of the reactive species and the SMR degradation rate constants (k1) were correlated with NOM spectroscopic parameters determined using UV-vis absorption, excitation-emission matrix (EEM) fluorescence spectroscopy, and proton nuclear magnetic resonance ((1)H NMR). SMR degradation rate constants (k1) were correlated with steady-state concentrations of NOM triplet-excited state ([(3)NOM(∗)]ss) and the corresponding formation rates ((3)NOM*) for SRNOM, SRHA, and AHA. The efficiency of SMR degradation was highest in AHA solution and was inhibited in solutions of SRFA. The steady-state concentrations of singlet oxygen ([(1)O2]ss) and the SMR degradation rate constants with singlet oxygen (k1O2) were linearly correlated with the total fluorescence and inversely correlated with the carbohydrate/protein content ((1)H NMR) for all forms of NOM. The total fluorescence and EEMs Peak A were confirmed as indicators

  3. Fluorescence probes of spectroscopic and dynamical aspects of molecular photoionization

    NASA Astrophysics Data System (ADS)

    Poliakoff, Erwin D.

    1988-11-01

    Studies were made of vibrationally resolved aspects of shape resonant excitation in the photoionization of N(2)0. This experiment was performed by generating dispersed fluorescence spectra from electronically excited photoions. These results are the first vibrationally resolved results on a polyatomic shape resonance. In vibrationally resolved measurements, different internuclear configurations are probed by sampling alternative vibrational levels of the ion. As a result, the continuum electron behavior can be mapped out most clearly, and the qualitative aspects of the electron ejection can be understood clearly. A central motivation for studying polyatomic shape resonances is that alternative vibrational modes may be explored, revealing facets that are nonexistent for diatomic systems, which are the only systems that have been characterized previously.

  4. Space-resolved fluorescence spectroscopic measurements with an optical fiber probe

    NASA Astrophysics Data System (ADS)

    Li, Enbang; Qiu, Hialin

    2008-12-01

    By monitoring of the emitted signal from a sample while varying the excitation wavelength, emission wavelength or both of them, fluorescence spectroscopy has become a powerful diagnostic technology. Fluorescence spectrometers can be used to measure and record the fluorescence spectra of a given sample, and have been successfully applied in different areas including biology, biochemistry, chemistry, medicine, environmental science, material science, food industry, and pharmaceutical industry. In order to increase the flexibility and applicability of conventional fluorescence spectrometers, we design an optic fiber probe for conducting the UV/Vis excitation light to a sample under study, and for collecting the fluorescence produced by the sample. Different excitation/emission fiber bundle arrangements have been fabricated and their performances have been evaluated and compared. Fiber adaptors which can be used for different commercial fluorescence spectrometers are also developed. In order to achieve space-resolved fluorescence spectroscopic measurements, we connect the fiber probe to a microscope which is mounted on a 3D traverse stage. Experiments and measurement results using the space-resolved fiber optic fluorescence spectrometer are presented in this paper.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2007-04-28

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

  7. Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

    PubMed Central

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

    2012-01-01

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

  8. Nonlinear spectroscopic theory of displaced harmonic oscillators with differing curvatures: a correlation function approach.

    PubMed

    Fidler, Andrew F; Engel, Gregory S

    2013-10-03

    We present a theory for a bath model in which we approximate the adiabatic nuclear potential surfaces on the ground and excited electronic states by displaced harmonic oscillators that differ in curvature. Calculations of the linear and third-order optical response functions employ an effective short-time approximation coupled with the cumulant expansion. In general, all orders of correlation contribute to the optical response, indicating that the solvation process cannot be described as Gaussian within the model. Calculations of the linear absorption and fluorescence spectra resulting from the theory reveal a stronger temperature dependence of the Stokes shift along with a general asymmetry between absorption and fluorescence line shapes, resulting purely from the difference in the phonon side band. We discuss strategies for controlling spectral tuning and energy-transfer dynamics through the manipulation of the excited-state and ground-state curvature. Calculations of the nonlinear response also provide insights into the dynamics of the system-bath interactions and reveal that multidimensional spectroscopies are sensitive to a difference in curvature between the ground- and excited-state adiabatic surfaces. This extension allows for the elucidation of short-time dynamics of dephasing that are accessible in nonlinear spectroscopic methods.

  9. Correlated blinking of fluorescent emitters mediated by single plasmons

    NASA Astrophysics Data System (ADS)

    Bouchet, D.; Lhuillier, E.; Ithurria, S.; Gulinatti, A.; Rech, I.; Carminati, R.; De Wilde, Y.; Krachmalnicoff, V.

    2017-03-01

    We observe time-correlated emission between a single CdSe/CdS/ZnS quantum dot exhibiting single-photon statistics and a fluorescent nanobead located micrometers apart. This is accomplished by coupling both emitters to a silver nanowire. Single plasmons are created on the latter from the quantum dot, and transfer energy to excite in turn the fluorescent nanobead. We demonstrate that the molecules inside the bead show the same blinking behavior as the quantum dot.

  10. [Studies on the oxidation of tyrosine induced by hydroxyl radical with fluorescence spectroscopic method].

    PubMed

    Sun, Yan-hui; Wang, Wei-long; Wu, Lin-sheng; Jia, Xiao-li

    2011-07-01

    Dityrosine is a marker of tyrosine oxidation. To study effecting factors of hydroxyl radical on tyrosine oxidation, synchronous fluorescence spectra with two dimensional correlation was used. The results showed that the peak position and intensity of dityrosine changed while pH value varied. In the system of tyrosine oxidation, with the increment of tyrosine concentration, the concentration of dityrosine decreased. With the increment of hydrogen peroxide concentration, the concentration of dityrosine increased. The oxidation reaction was prone to taking place in acid conditions while difficult to develop in basic conditions. With the development of oxidation reaction, the fluorescence intensity of dityrosine increased and then decreased. Two dimentional correlation synchronous fluorescence spectra showed that the variation in the intensity at 292 nm preceded that of 281, 300 and 374 nm. Thus, fluorescence spectroscopy was simple and easy for studying tyrosine oxidation induced by hydroxyl radical.

  11. Fluorescence, spectroscopic and NLO properties of green tea extract in deoxyribonucleic acid

    NASA Astrophysics Data System (ADS)

    Manea, Ana-Maria; Rau, Ileana; Kajzar, Francois; Meghea, Aurelia

    2013-11-01

    Natural, purely biological deoxyribonucleic acid (DNA)-green tea extract (GTE) complexes at different concentrations were prepared and characterized for their spectroscopic, fluorescent, linear and nonlinear optical properties. The complexes can be processed into good optical quality thin films by solution casting. They fluoresce when excited in UV absorption band, with a significantly larger quantum yield for the DNA-GTE complex than for a pure GTE solution. The thin film refractive indices were determined by Fabry-Perot (FP) interference patterns. The third-order nonlinear optical (NLO) properties of thin films were determined by the optical third-harmonic generation technique at 1064.2 nm fundamental wavelength. The phase of THG susceptibility was determined from the concentration variation of THG susceptibility. It reveals presence of a two-photon resonance with a band lying in the optical gap.

  12. Spectroscopic parameters of the cuticle and ethanol extracts of the fluorescent cave isopod Mesoniscusgraniger (Isopoda, Oniscidea).

    PubMed

    Giurginca, Andrei; Šustr, Vladimír; Tajovský, Karel; Giurginca, Maria; Matei, Iulia

    2015-01-01

    The body surface of the terrestrial isopod Mesoniscusgraniger (Frivaldsky, 1863) showed blue autofluorescence under UV light (330-385 nm), using epifluorescence microscopy and also in living individuals under a UV lamp with excitation light of 365 nm. Some morphological cuticular structures expressed a more intense autofluorescence than other body parts. For this reason, only the cuticle was analyzed. The parameters of autofluorescence were investigated using spectroscopic methods (molecular spectroscopy in infrared, ultraviolet-visible, fluorescence, and X-ray fluorescence spectroscopy) in samples of two subspecies of Mesoniscusgraniger preserved in ethanol. Samples excited by UV light (from 350 to 380 nm) emitted blue light of wavelengths 419, 420, 441, 470 and 505 nm (solid phase) and 420, 435 and 463 (ethanol extract). The results showed that the autofluorescence observed from living individuals may be due to some β-carboline or coumarin derivatives, some crosslinking structures, dityrosine, or due to other compounds showing similar excitation-emission characteristics.

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

    PubMed

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

    2013-06-07

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

  14. 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.

  15. A combined Raman-fluorescence spectroscopic probe for tissue diagnostics applications

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

    We designed and developed two different optical fibre probes for combined Raman and fluorescence spectroscopic measurements on human tissues. The experimental setup combines fluorescence spectroscopy and Raman spectroscopy in a multimodal approach. Two laser diodes, respectively emitting in the UV (378 nm) and in the visible (445 nm), were used for fluorescence spectroscopy. An additional laser diode emitting in the NIR (785 nm) was used for Raman spectroscopy. Laser light was delivered to the tissue under examination through a multimode optical fibre located in the centre of the fibre bundle probe. The surrounding 24 optical fibres were used for collection of the signal of interest and for delivering light to a common detection unit. Both fluorescence and Raman spectra were acquired on a cooled CCD camera, connected to a spectrograph. The device was successfully used for diagnosing melanocytic lesions in a good agreement with common routine histology. Additional measurements were performed on other human tissue samples, such as colon tissue and brain tissue in order to test the capability of the device for diagnosing a broader range of tissue lesions and malignancies. The system has the potential to improve diagnostic capabilities on a broad range of tissues and to be used for endoscopic inspections in the near future.

  16. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  17. Correlated quadratures of resonance fluorescence and the generalized uncertainty relation

    NASA Technical Reports Server (NTRS)

    Arnoldus, Henk F.; George, Thomas F.; Gross, Rolf W. F.

    1994-01-01

    Resonance fluorescence from a two-state atom has been predicted to exhibit quadrature squeezing below the Heisenberg uncertainty limit, provided that the optical parameters (Rabi frequency, detuning, laser linewidth, etc.) are chosen carefully. When the correlation between two quadratures of the radiation field does not vanish, however, the Heisenberg limit for quantum fluctuations might be an unrealistic lower bound. A generalized uncertainty relation, due to Schroedinger, takes into account the possible correlation between the quadrature components of the radiation, and it suggests a modified definition of squeezing. We show that the coherence between the two levels of a laser-driven atom is responsible for the correlation between the quadrature components of the emitted fluorescence, and that the Schrodinger uncertainty limit increases monotonically with the coherence. On the other hand, the fluctuations in the quadrature field diminish with an increasing coherence, and can disappear completely when the coherence reaches 1/2, provided that certain phase relations hold.

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

    PubMed

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

    2004-11-01

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

  19. Diffusivity of asphaltene molecules by fluorescence correlation spectroscopy.

    PubMed

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

    2006-07-06

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

  20. Fluorescence correlation spectroscopy: Diagnostics for sparse molecules

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2009-10-12

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

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

    PubMed

    Winterflood, Christian M; Seeger, Stefan

    2016-05-01

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

  3. Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

    PubMed Central

    Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

    2013-01-01

    Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

  4. Principles and applications of fluorescence lifetime correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Beranová, Lenka; Humpolícková, Jana; Hof, Martin

    2009-05-01

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

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

    PubMed Central

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

    2005-01-01

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

  6. A cryogenic fluorescence spectroscopic study of uranyl carbonate, phosphate, and oxyhydroxide minerals

    SciTech Connect

    Wang, Zheming; Zachara, John M.; Liu, Chongxuan; Gassman, Paul L.; Felmy, Andrew R.; Clark, Sue B.

    2008-11-03

    In this work we have applied liquid-helium temperature (LHeT) time-resolved laser-induced fluorescence spectroscopy (TRLIF) to characterize a series of natural and synthetic minerals of uranium carbonate, phosphate and oxyhydroxides including rutherfordine, zellerite, liebigite, phosphuranylite, meta-autunite, meta-torbernite, uranyl phosphate, sodium-uranyl-phosphate, bequerelite, clarkeite, curite, schoepite and compregnacite, and compared their spectral characteristics among these minerals as well as our previously published data on uranyl silicates. For the carbonate minerals, the fluorescence spectra depend on the stoichiometry of the mineral. For the phosphate minerals the fluorescence spectra closely resemble each other despite the differences in their composition and structure. For all uranium oxyhydroxides, the fluorescence spectra are largely red-shifted as compared with those of the uranium carbonates and phosphates and their vibronic bands are broadened and less resolved. The much enhanced spectra resolution at LHeT allows more accurate calculation of the O=U=O symmetrical stretch frequency, ν1, corresponding to the average spacing of the vibronic peaks of the fluorescence spectra and the spectral origin as reflected by the position of the first vibronic band. It was found that both the average ν1 and λ1 values correlate well with the average basicity of the inorganic anion.

  7. In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish

    NASA Astrophysics Data System (ADS)

    Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

    2014-03-01

    Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

  8. First fluorescence spectroscopic investigation of Am(III) complexation with an organic carboxylic ligand, pyromellitic acid

    NASA Astrophysics Data System (ADS)

    Barkleit, Astrid; Geipel, Gerhard; Acker, Margret; Taut, Steffen; Bernhard, Gert

    2011-01-01

    For the first time Am(III) complexation with a small organic ligand could be identified and characterized with time-resolved laser-induced fluorescence spectroscopy (TRLFS) at room temperature and trace metal concentration. With pyromellitic acid (1,2,4,5-benzene-tetracarboxylic acid, BTC) as ligand spectroscopic characteristics for the Am-BTC complex system were determined at pH 5.0, an ionic strength of 0.1 M (NaClO 4) and room temperature. The fluorescence lifetimes were determined to be 23.2 ± 2.2 ns for Am 3+(aq) and 27.2 ± 1.2 ns for the Am-BTC 1:1 complex; the emission maximum for the 5D 1- 7F 1 transition is 691 nm for both species. The complex stability constant for the Am-BTC 1:1 complex was calculated to be log β110 = 5.42 ± 0.16.

  9. First fluorescence spectroscopic investigation of Am(III) complexation with an organic carboxylic ligand, pyromellitic acid.

    PubMed

    Barkleit, Astrid; Geipel, Gerhard; Acker, Margret; Taut, Steffen; Bernhard, Gert

    2011-01-01

    For the first time Am(III) complexation with a small organic ligand could be identified and characterized with time-resolved laser-induced fluorescence spectroscopy (TRLFS) at room temperature and trace metal concentration. With pyromellitic acid (1,2,4,5-benzene-tetracarboxylic acid, BTC) as ligand spectroscopic characteristics for the Am-BTC complex system were determined at pH 5.0, an ionic strength of 0.1 M (NaClO4) and room temperature. The fluorescence lifetimes were determined to be 23.2±2.2 ns for Am3+(aq) and 27.2±1.2 ns for the Am-BTC 1:1 complex; the emission maximum for the 5D1-(7)F1 transition is 691 nm for both species. The complex stability constant for the Am-BTC 1:1 complex was calculated to be logβ110=5.42±0.16.

  10. Fluorescence spectroscopic studies of tyrosine environment and ligand binding of plant calmodulin

    NASA Astrophysics Data System (ADS)

    Sanyal, Gautam; Thompson, Faith; Puett, David

    1990-05-01

    Recent studies in our laboratories have focused on using tyrosine (Tyr) fluorescence of calmodulin (CaM) and tryptophan (Trp) fluorescence of CaM-bound peptdies as intrinsic probes of structure and interactions of this Ca2+ regulatory protein. Plant CaM contains a single Tyr (Tyr.-l38) and vertebrate CaM contains two (Tyr-99 and Tyr-.l38). Neither protein contains Trp. The fluorescence properties of Tyr-138 of wheat-germ CaM is sensitive to conformational changes induced by perturbations such as Ca2+ ligation or depletion, and pH changes. Effects of these perturbations on quantum yield, lifetime and dynamic quenching of Tyr-l38 fluorescence are reported. We have also studied binding of amphiphilic peptides to wheat-germ CaM. A comparison of wheat CaM induced changes in the fluorescence properties of a single Trp of these peptides with those induced by bovine testes CaM indicate general similarities of the peptide binding surfaces of plant and mammalian CaMs. Frequency domain measurements of decay of intensity and anisotropy have suggested some orientational freedom and local motion of the Trp residue of CaM-bound peptide, independent of the overall protein motion, even when the Trp is expected to be buried in the doubly apolar protein-peptide interface. Calmodulin (CaM) is a ubiquitous calcium binding protein which is believed to regulate several different enzymes in diverse cells (Klee et al., 1982). Much of the structural work to date has been carried out on mammalian CaM. However, CaM has also been isolated from plant and invertebrate sources, and a high degree of sequence homology with vertebrate CaM has been found. The amino acid sequence of wheat germ CaM shows eleven substitutions, two insertions and one deletion compared with the 148.-residue bovine brain CaM (Toda et al., 1985). Specific differences with mammalian CaM at two sites make plant CaM attractive for fluorescence spectroscopic studies. These are: (1) The presence of a single tyrosine residue (Tyr

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

    NASA Astrophysics Data System (ADS)

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

    2007-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

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

    PubMed

    Xi, Xu Guang; Deprez, Eric

    2010-07-01

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

  14. Correlated fluorescence blinking in two-dimensional semiconductor heterostructures

    NASA Astrophysics Data System (ADS)

    Xu, Weigao; Liu, Weiwei; Schmidt, Jan F.; Zhao, Weijie; Lu, Xin; Raab, Timo; Diederichs, Carole; Gao, Weibo; Seletskiy, Denis V.; Xiong, Qihua

    2016-12-01

    ‘Blinking’, or ‘fluorescence intermittency’, refers to a random switching between ‘ON’ (bright) and ‘OFF’ (dark) states of an emitter; it has been studied widely in zero-dimensional quantum dots and molecules, and scarcely in one-dimensional systems. A generally accepted mechanism for blinking in quantum dots involves random switching between neutral and charged states (or is accompanied by fluctuations in charge-carrier traps), which substantially alters the dynamics of radiative and non-radiative decay. Here, we uncover a new type of blinking effect in vertically stacked, two-dimensional semiconductor heterostructures, which consist of two distinct monolayers of transition metal dichalcogenides (TMDs) that are weakly coupled by van der Waals forces. Unlike zero-dimensional or one-dimensional systems, two-dimensional TMD heterostructures show a correlated blinking effect, comprising randomly switching bright, neutral and dark states. Fluorescence cross-correlation spectroscopy analyses show that a bright state occurring in one monolayer will simultaneously lead to a dark state in the other monolayer, owing to an intermittent interlayer carrier-transfer process. Our findings suggest that bilayer van der Waals heterostructures provide unique platforms for the study of charge-transfer dynamics and non-equilibrium-state physics, and could see application as correlated light emitters in quantum technology.

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

    PubMed Central

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

    2015-01-01

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

  16. Preliminary investigation of intrinsic UV fluorescence spectroscopic changes associated with proteolytic digestion of bovine articular cartilage

    NASA Astrophysics Data System (ADS)

    Lewis, William; Padilla-Martinez, Juan-Pablo; Ortega-Martinez, Antonio; Franco, Walfre

    2016-03-01

    Degradation and destruction of articular cartilage is the etiology of osteoarthritis (OA), an entity second only to cardiovascular disease as a cause of disability in the United States. Joint mechanics and cartilage biochemistry are believed to play a role in OA; an optical tool to detect structural and chemical changes in articular cartilage might offer benefit for its early detection and treatment. The objective of the present study was to identify the spectral changes in intrinsic ultraviolet (UV) fluorescence of cartilage that occur after proteolytic digestion of cartilage. Bovine articular cartilage samples were incubated in varying concentrations of collagenase ranging from 10ug/mL up to 5mg/mL for 18 hours at 37°C, a model of OA. Pre- and post-incubation measurements were taken of the UV excitation-emission spectrum of each cartilage sample. Mechanical tests were performed to determine the pre- and post-digestion force/displacement ratio associated with indentation of each sample. Spectral changes in intrinsic cartilage fluorescence and stiffness of the cartilage were associated with proteolytic digestion. In particular, changes in the relative intensity of fluorescence peaks associated with pentosidine crosslinks (330 nm excitation, 390 nm emission) and tryptophan (290 nm excitation, 340 nm emission) were found to correlate with different degrees of cartilage digestion and cartilage stiffness. In principle, it may be possible to use UV fluorescence spectral data for early detection of damage to articular cartilage, and as a surrogate measure for cartilage stiffness.

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

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

    PubMed

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

    2013-04-01

    Fluorescence Correlation Spectroscopy (FCS) is widely used to quantitate reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes.

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

    PubMed Central

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

    2013-01-01

    Fluorescence Correlation Spectroscopy (FCS) is widely used to quantitate reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes. PMID:23525193

  20. Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Liang, Lifang; Xing, Da; Chen, Tongshen; Pei, Yihui

    2007-11-01

    Fluorescence correlation spectroscopy (FCS) is a new kind of real-time, high-speed and single-molecule technique. It is used to detect the kinetic characteristics of fluorescent dye such as diffusion coefficient in the aqueous solution. Combined with confocal microscope optics, it has been now widely applied in cell biological research. Through a time correlation analysis of spontaneous intensity fluctuations, this technique with EGFP as a probe is capable of determining viscosity of fluids according to Stokes-Einstein equation. Nucleoplasmic viscosity is an important physical parameter to quantify the rheological characteristics of the nucleoplasm. Investigation on nucleoplasmic viscosity plays an important role in further understanding intranuclear environment. In this paper, FCS is introduced to noninvasively investigate nucleoplasmic viscosity of living cells. The results show that nucleoplasmic viscosity of lung adenocarcinoma (ASTC-a-1) cells is 2.55+/-0.61 cP and nucleoplasmic viscosity is larger than cytoplasmic viscosity at 37 °C (pH 7.4). In addition, significant changes in nucleoplasmic viscosity are detected by FCS when cells are exposed to hyper or hypotonic medium. Our study suggests that FCS can be used to detect the kinetic characteristics of biomolecules in living cells and thus helps to investigate the dynamic changes of the microenvironment in the cell.

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

    PubMed

    Piskorz, Tomasz K; Ochab-Marcinek, Anna

    2014-05-08

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

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

    PubMed

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

    2009-02-01

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

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

    PubMed Central

    Thompson, N L; Axelrod, D

    1983-01-01

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

  4. Spectroscopic Evidence of Anthropogenic Compounds Extraction from Polymers by Fluorescent Dissolved Organic Matter in Natural Water

    NASA Astrophysics Data System (ADS)

    Miranda, M.; Trojzuck, A.; Voss, D.; Gassmann, S.; Zielinski, O.

    2016-04-01

    FDOM is one of the most important carriers of anthropogenic compounds in natural waters. It can combine with environmental contaminants and polymers to form diverse chemical structures. To this end, here a microfluidic chip was designed for the analysis of these changes in fluorescent dissolved organic matter (FDOM) fingerprints due to thermal treatment and varying time intervals of exposure. Excitation Emission Matrix Spectroscopy (EEMS) approach was utilized to detect and identify the inherent compounds in sampled FDOM. Strong direct correlations were founded, Spearman rank correlation values (ρ = 0.85 at α = 0.1, n = 4) and linear correlation R2 = 0.8359 were noted between thermal treatment pattern 2 and fluorescence intensity of samples. Materials, acrylic based glue and cyclic olefin copolymer (COC) polymer, used to design the microfluidic sensor were determined to possess unique spectral features in the ultraviolet to green spectrum using EEMS. The study therefore provides an insight on methods to identify contaminants in natural waters. This underlines the potential of optical sensors providing measurements at fast intervals, enabling environmental monitoring.

  5. Fluorescent-spectroscopic and imaging methods of investigations for diagnostics of head and neck tumors and control of PDT

    NASA Astrophysics Data System (ADS)

    Edinak, N. J.; Shental, Victor V.; Komov, D. V.; Vacoulovskaia, E. G.; Tabolinovskaia, T. D.; Abdullin, N. A.; Pustynsky, I.; Chatikchine, V. H.; Loschenov, Victor B.; Meerovich, Gennadii A.; Stratonnikov, Alexander A.; Linkov, Kirill G.; Agafonov, Vladimir I.; Zuravleva, V.; Lukjanets, Eugeny A.

    1996-01-01

    Methodics of PDT control and fluorescent-spectroscopic diagnostic of head and neck tumors and mammary gland cancer (nodular) with the use of Kr, He-Ne and semiconductor lasers and photosensitizer (PS) -- Al phtalocyanin (Photosense) are discussed. The results show that applied diagnostic methods permit us not only to identify the topology and malignancy of a tumor but also to correct PDT process directly during irradiation.

  6. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    SciTech Connect

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

    2014-08-14

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

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

    NASA Astrophysics Data System (ADS)

    He, Hai-Tao; Marguet, Didier

    2011-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

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

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

    SciTech Connect

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

    2011-01-01

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

  10. Interpretation of fluorescence correlation spectra of biopolymer solutions.

    PubMed

    Phillies, George D J

    2016-05-01

    Fluorescence correlation spectroscopy (FCS) is regularly used to study diffusion in non-dilute "crowded" biopolymer solutions, including the interior of living cells. For fluorophores in dilute solution, the relationship between the FCS spectrum G(t) and the diffusion coefficient D is well-established. However, the dilute-solution relationship between G(t) and D has sometimes been used to interpret FCS spectra of fluorophores in non-dilute solutions. Unfortunately, the relationship used to interpret FCS spectra in dilute solutions relies on an assumption that is not always correct in non-dilute solutions. This paper obtains the correct form for interpreting FCS spectra of non-dilute solutions, writing G(t) in terms of the statistical properties of the fluorophore motions. Approaches for applying this form are discussed.

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

    PubMed

    Elson, Elliot L

    2004-01-01

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

  12. Correlation Between Bulk Material Defects and Spectroscopic Response in Cadmium Zinc Telluride Detectors

    NASA Technical Reports Server (NTRS)

    Parker, Bradford H.; Stahle, C. M.; Barthelmy, S. D.; Parsons, A. M.; Tueller, J.; VanSant, J. T.; Munoz, B. F.; Snodgrass, S. J.; Mullinix, R. E.

    1999-01-01

    One of the critical challenges for large area cadmium zinc telluride (CdZnTe) detector arrays is obtaining material capable of uniform imaging and spectroscopic response. Two complementary nondestructive techniques for characterizing bulk CdZnTe have been developed to identify material with a uniform response. The first technique, infrared transmission imaging, allows for rapid visualization of bulk defects. The second technique, x-ray spectral mapping, provides a map of the material spectroscopic response when it is configured as a planar detector. The two techniques have been used to develop a correlation between bulk defect type and detector performance. The correlation allows for the use of infrared imaging to rapidly develop wafer mining maps. The mining of material free of detrimental defects has the potential to dramatically increase the yield and quality of large area CdZnTe detector arrays.

  13. 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.

  14. Fluorescence Correlation Spectroscopy and Nonlinear Stochastic Reaction-Diffusion

    SciTech Connect

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

    2014-05-30

    The currently existing theory of fluorescence correlation spectroscopy (FCS) is based on the linear fluctuation theory originally developed by Einstein, Onsager, Lax, and others as a phenomenological approach to equilibrium fluctuations in bulk solutions. For mesoscopic reaction-diffusion systems with nonlinear chemical reactions among a small number of molecules, a situation often encountered in single-cell biochemistry, it is expected that FCS time correlation functions of a reaction-diffusion system can deviate from the classic results of Elson and Magde [Biopolymers (1974) 13:1-27]. We first discuss this nonlinear effect for reaction systems without diffusion. For nonlinear stochastic reaction-diffusion systems there are no closed solutions; therefore, stochastic Monte-Carlo simulations are carried out. We show that the deviation is small for a simple bimolecular reaction; the most significant deviations occur when the number of molecules is small and of the same order. Extending Delbrück-Gillespie’s theory for stochastic nonlinear reactions with rapidly stirring to reaction-diffusion systems provides a mesoscopic model for chemical and biochemical reactions at nanometric and mesoscopic level such as a single biological cell.

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

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

    NASA Astrophysics Data System (ADS)

    Widengren, Jerker; Mets, Ülo; Rigler, Rudolf

    1999-12-01

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

  17. Synthesis of a fluorescent 7-methylguanosine analog and a fluorescence spectroscopic study of its reaction with wheatgerm cap binding proteins.

    PubMed Central

    Ren, J; Goss, D J

    1996-01-01

    In the initiation of protein synthesis, the mRNA 5'-terminal 7-methylguanosine cap structure and several recognition proteins play a pivotal role. For the study of this cap binding reaction, one approach is to use fluorescence spectroscopy. A ribose diol-modified fluorescent cap analog, anthraniloyl-m7GTP (Ant-m7GTP), was designed and synthesized for this purpose. This fluorescent cap analog was found to have a high quantum yield, resistance to photobleaching and avoided overlap of excitation and emission wavelengths with those of proteins. The binding of Ant-m7GTP with wheatgerm initiation factors elF-4F and elF-(iso)4F was determined. The fluorescent cap analog and m7GTP had similar interactions with both cap binding proteins. Fluorescence quenching experiments showed that the microenvironment of Ant-m7GTP when bound to protein was hydrophobic. PMID:8836193

  18. Echo planar correlated spectroscopic imaging: implementation and pilot evaluation in human calf in vivo.

    PubMed

    Lipnick, Scott; Verma, Gaurav; Ramadan, Saadallah; Furuyama, Jon; Thomas, M Albert

    2010-10-01

    Exploiting the speed benefits of echo-planar imaging (EPI), the echo-planar spectroscopic imaging (EPSI) sequence facilitates recording of one spectral and two to three spatial dimensions faster than the conventional magnetic resonance spectroscopic imaging (MRSI). A novel four dimensional (4D) echo-planar correlated spectroscopic imaging (EP-COSI) was implemented on a whole body 3 T MRI scanner combining two spectral with two spatial encodings. Similar to EPSI, the EP-COSI sequence used a bipolar spatial read-out train facilitating simultaneous spatial and spectral encoding, and the conventional phase and spectral encodings for the other spatial and indirect spectral dimensions, respectively. Multiple 2D correlated spectroscopy (COSY) spectra were recorded over the spatially resolved volume of interest (VOI) localized by a train of three slice-selective radiofrequency (RF) pulses (90°-180°-90°). After the initial optimization using phantom solutions, the EP-COSI data were recorded from the lower leg of eight healthy volunteers including one endurance trained volunteer. Pilot results showed acceptable spatial and spectral quality achievable using the EP-COSI sequence. There was a detectable separation of cross peaks arising from the skeletal muscle intramyocellular lipids (IMCLs) and extramyocellular lipids (EMCLs) saturated and unsaturated pools. Residual dipolar interaction between the N-methylene and N-methyl protons of creatine/phosphocreatine (Cr/PCr) was also observed in the tibialis anterior region.

  19. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    DOE PAGES

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

    2014-08-14

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

  20. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Tsekouras, Konstantinos

    2015-03-01

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

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

    PubMed

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

    2010-02-01

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

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

    SciTech Connect

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

    2011-01-01

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

  3. The Intermediate Scattering Function in Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Guerra, Rodrigo; Andrews, Ballard; Sen, Pabitra

    2006-03-01

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

  4. Binding of caffeine with caffeic acid and chlorogenic acid using fluorescence quenching, UV/vis and FTIR spectroscopic techniques.

    PubMed

    Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

    2016-03-01

    The interactions of caffeine (CF) with chlorogenic acid (CGA) and caffeic acid (CFA) were investigated by fluorescence quenching, UV/vis and Fourier transform infrared (FTIR) spectroscopic techniques. The results of the study indicated that the fluorescence quenching between caffeine and hydroxycinnamic acids could be rationalized in terms of static quenching or the formation of non-fluorescent CF-CFA and CF-CGA complexes. From fluorescence quenching spectral analysis, the quenching constant (KSV), quenching rate constant (kq), number of binding sites (n), thermodynamic properties and conformational changes of the interaction were determined. The quenching constants (KSV) between CF and CGA, CFA are 1.84 × 10(4) and 1.04 × 10(4) L/mol at 298 K and their binding site n is ~ 1. Thermodynamic parameters determined using the Van't Hoff equation indicated that hydrogen bonds and van der Waal's forces have a major role in the reaction of caffeine with caffeic acid and chlorogenic acid. The 3D fluorescence, UV/vis and FTIR spectra also showed that the binding of CF with CFA and CGA induces conformational changes in CFA and CGA.

  5. [Vermicomposting of different organic materials and three-dimensional excitation emission matrix fluorescence spectroscopic characterization of their dissolved organic matter].

    PubMed

    Yang, Wei; Wang, Dong-sheng; Liu, Man-qiang; Hu, Feng; Li, Hui-xin; Huang, Zhong-yang; Chang, Yi-jun; Jiao, Jia-guo

    2015-10-01

    In this experiment, different proportions of the cattle manure, tea-leaf, herb and mushroom residues, were used as food for earthworm (Eisenia fetida) to study the growth of the earth-worm. Then the characteristics and transformation of nutrient content and three-dimensional excitation emission matrix fluorescence (3DEEM) of dissolved organic matter (DOM) during vermistabilization were investigated by means of chemical and spectroscopic methods. The result showed that the mixture of different ratios of cattle manure with herb residue, and cattle manure with tea-leaf were conducive to the growth of earthworm, while the materials compounded with mushroom residue inhibited the growth of earthworm. With the increasing time of verimcomposting, the pH in vermicompost tended to be circumneutral and weakly acidic, and there were increases in electrical conductivity, and the contents of total nitrogen, total phosphorus, available nitrogen, and available phosphorus, while the total potassium and available potassium increased first and then decreased, and the organic matter content decreased. 3DEEM and fluorescence regional integration results indicated that, the fluorescence of protein-like fluorescence peaks declined significantly, while the intensity of humic-like fluorescence peak increased significantly in DOM. Vermicomposting process might change the compositions of DOM with elevated concentrations of humic acid and fulvic acid in the organics. In all, this study suggested the suitability of 3DEEM for monitoring the organics transformation and assessing the maturity in the vermicomposting.

  6. Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy

    PubMed Central

    Paez Segala, Maria G.; Sun, Mei G.; Shtengel, Gleb; Viswanathan, Sarada; Baird, Michelle A.; Macklin, John J.; Patel, Ronak; Allen, John R.; Howe, Elizabeth S.; Piszczek, Grzegorz; Hess, Harald F.; Davidson, Michael W.; Wang, Yalin; Looger, Loren L.

    2014-01-01

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they cease to function following heavy fixation, hindering advanced applications such as correlative light and electron microscopy. Here we report engineered variants of the photoconvertible Eos fluorescent protein that function normally in heavily fixed (0.5–1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy. PMID:25581799

  7. Hybrid plasmonic platforms based on silica-encapsulated gold nanorods as effective spectroscopic enhancers for Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Gabudean, A. M.; Biro, D.; Astilean, S.

    2012-12-01

    Surface-enhanced Raman scattering (SERS) nano-tags are of increasing interest in biomedical research as viable alternatives to bio-imaging techniques based on semiconductor quantum dots or fluorescent molecules. In this work, we fabricate silica-coated gold nanorods (AuNRs) encoded with two molecular labels to operate as highly effective spectroscopic nano-tags in near-infrared SERS (NIR-SERS) and surface-enhanced resonance Raman scattering combined with metal-enhanced fluorescence (SERRS-MEF), respectively. Specifically, a non-fluorescent molecule with strong affinity for a gold surface (para-aminothiophenol, p-ATP) and a common dye (Nile Blue, NB) with lower affinity have been successfully tested as NIR-SERS nano-tags under laser excitation at 785 nm. Moreover, as a result of designing AuNRs with a plasmon resonance band overlapping the electronic absorption band of the encoded NB molecule, a dual SERRS and MEF performance has been devised under resonant excitation at 633 nm. We explain this result by considering a partial desorption of NB molecules from the metal surface and their trapping into the silica shell at favorable distances to avoid quenching and enhance the fluorescence signal. Finally, we prove that the silica shell prevents the desorption or chemical transformation of p-ATP into p,p‧-dimercaptoazobenzene species, as previously noticed, thus providing a highly stable SERRS signal, which is crucial for imaging applications.

  8. A combined fluorescence spectroscopic and electrochemical approach for the study of thioredoxins.

    PubMed

    Voicescu, Mariana; Rother, Dagmar; Bardischewsky, Frank; Friedrich, Cornelius G; Hellwig, Petra

    2011-01-11

    A new way to study the electrochemical properties of proteins by coupling front-face fluorescence spectroscopy with an optically transparent thin-layer electrochemical cell is presented. First, the approach was examined on the basis of the redox-dependent conformational changes in tryptophans in cytochrome c, and its redox potential was successfully determined. Second, an electrochemically induced fluorescence analysis of periplasmic thiol-disulfide oxidoreductases SoxS and SoxW was performed. SoxS is essential for maintaining chemotrophic sulfur oxidation of Paracoccus pantotrophus active in vivo, while SoxW is not essential. According to the potentiometric redox titration of tryptophan fluorescence, the midpoint potential of SoxS was -342 ± 8 mV versus the standard hydrogen electrode (SHE') and that of SoxW was -256 ± 10 mV versus the SHE'. The fluorescence properties of the thioredoxins are presented and discussed together with the intrinsic fluorescence contribution of the tyrosines.

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

    NASA Astrophysics Data System (ADS)

    Punj, Deep; Rigneault, Hervé; Wenger, Jérôme

    2014-05-01

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

  10. Macromolecular competition titration method accessing thermodynamics of the unmodified macromolecule-ligand interactions through spectroscopic titrations of fluorescent analogs.

    PubMed

    Bujalowski, Wlodzimierz; Jezewska, Maria J

    2011-01-01

    Analysis of thermodynamically rigorous binding isotherms provides fundamental information about the energetics of the ligand-macromolecule interactions and often an invaluable insight about the structure of the formed complexes. The Macromolecular Competition Titration (MCT) method enables one to quantitatively obtain interaction parameters of protein-nucleic acid interactions, which may not be available by other methods, particularly for the unmodified long polymer lattices and specific nucleic acid substrates, if the binding is not accompanied by adequate spectroscopic signal changes. The method can be applied using different fluorescent nucleic acids or fluorophores, although the etheno-derivatives of nucleic acid are especially suitable as they are relatively easy to prepare, have significant blue fluorescence, their excitation band lies far from the protein absorption spectrum, and the modification eliminates the possibility of base pairing with other nucleic acids. The MCT method is not limited to the specific size of the reference nucleic acid. Particularly, a simple analysis of the competition titration experiments is described in which the fluorescent, short fragment of nucleic acid, spanning the exact site-size of the protein-nucleic acid complex, and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. Although the MCT method is predominantly discussed as applied to studying protein-nucleic acid interactions, it can generally be applied to any ligand-macromolecule system by monitoring the association reaction using the spectroscopic signal originating from the reference macromolecule in the presence of the competing macromolecule, whose interaction parameters with the ligand are to be determined.

  11. Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues.

    PubMed Central

    Reshetnyak, Y K; Koshevnik, Y; Burstein, E A

    2001-01-01

    In our previous paper (Reshetnyak, Ya. K., and E. A. Burstein. 2001. Biophys. J. 81:1710-1734) we confirmed the existence of five statistically discrete classes of emitting tryptophan fluorophores in proteins. The differences in fluorescence properties of tryptophan residues of these five classes reflect differences in interactions of excited states of tryptophan fluorophores with their microenvironment in proteins. Here we present a system of describing physical and structural parameters of microenvironments of tryptophan residues based on analysis of atomic crystal structures of proteins. The application of multidimensional statistical methods of cluster and discriminant analyses for the set of microenvironment parameters of 137 tryptophan residues of 48 proteins with known three-dimensional structures allowed us to 1) demonstrate the discrete nature of ensembles of structural parameters of tryptophan residues in proteins; 2) assign spectral components obtained after decomposition of tryptophan fluorescence spectra to individual tryptophan residues; 3) find a correlation between spectroscopic and physico-structural features of the microenvironment; and 4) reveal differences in structural and physical parameters of the microenvironment of tryptophan residues belonging to various spectral classes. PMID:11509384

  12. Spectroscopic Ellipsometry and Fluorescence Study of Thermochromism in an Ultrathin Poly(diacetylene) Film: Reversibility and Transition Kinetics

    SciTech Connect

    CARPICK,R.W.; MAYER,THOMAS M.; SASAKI,DARRYL Y.; BURNS,ALAN R.

    2000-01-18

    We have investigated the thermochromic transition of an ultrathin poly(diacetylene) film. The Langmuir film is composed of three layers of polymerized 10,12-pentacosadiynoic acid [CH{sub 3}(CH{sub 2}){sub 11}C{triple_bond}CC{triple_bond}C(CH{sub 2}){sub 8}COOH] (poly-PCDA) organized into crystalline domains on a silicon substrate. Spectroscopic ellipsometry and fluorescence intensity measurements are obtained with in-situ temperature control. Poly-PCDA films exhibit a reversible thermal transition between the initial blue form and an intermediate ''purple'' form that exists only at elevated temperature (between 303-333 K), followed by an irreversible transition to the red form after annealing above 320 K. We propose that the purple form is thermally distorted blue poly-PCDA, and may represent a transitional configuration in the irreversible conversion to red. This hypothesis is supported by the appearance of unique features in the absorption spectra for each form as derived from the ellipsometry measurements. Significant fluorescence emission occurs only with the red form, and is reduced at elevated temperatures while the absorption remains unchanged. Reduced emission is likely related to thermal fluctuations of the hydrocarbon side chains. Time-resolved fluorescence measurements of the irreversible transition have been performed. Using a first-order kinetic analysis of these measurements we deduce an energy barrier of 17.6 {+-} 1.1 kcal mol{sup -1} between the blue and red forms.

  13. Study of diffusion in polymer solutions and networks by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Chehreghanianzabi, Yasaman

    Diffusion in polymer solutions and networks is a topic of vast importance in many fields related to medical devices, tissue engineering, and drug delivery. Understanding diffusion in such environments is also essential for describing molecular transport through biological systems such as cells and tissues. Fluorescence correlation spectroscopy (FCS) is single molecule spectroscopic technique that measures the fluctuations of fluorescent probes in a defined confocal volume and correlates them in time to give information on diffusion times, concentrations, and interactions as well as indirectly, on macromolecular structure or conformation. In the first project we used diffusivity data obtained by FCS to develop a novel homogenization theory model to accurately predict solute diffusivity in polymer solutions. We focused on a setting where diffusivity was hindered by obstruction only. By choosing experimental conditions that satisfied the model assumptions, we were able to validate the homogenization theory model. While testing diffusivity in various polymer solutions, we also observed an unexpected phenomenon--a dramatic decrease in diffusivity of small fluorophores in dilute solutions of polyethylene glycol (PEG), which led to the second project. Here, we determined that the rapid drop was due to a complexation between the PEG and the fluorophore. We also determined that this complexation was highly specific and could be attributed to hydrogel bonding between the ether oxygen of PEG and the carboxylic hydrogen of the fluorophore. We then transitioned to a more complex hydrogel network environment, namely fluorophore diffusivity in various alginate hydrogels--varied by concentration and modifications with a cell adhesive ligand. Importantly, we were able to determine that while the fluorophore diffusivity was hindered due to electrostatic interactions, it was the same irrespective of the alginate concentration or modifications. The last part of this thesis was focused

  14. Fluorescence spectroscopic characterization of dissolved organic matter fractions in soils in soil aquifer treatment.

    PubMed

    Xue, Shuang; Zhao, Qingliang; Wei, Liangliang; Song, Youtao; Tie, Mei

    2013-06-01

    This work investigated the effect of soil aquifer treatment (SAT) operation on the fluorescence characteristics of dissolved organic matter (DOM) fractions in soils through laboratory-scale soil columns with a 2-year operation. The resin adsorption technique (with XAD-8 and XAD-4 resins) was employed to characterize the dissolved organic matter in soils into five fractions, i.e., hydrophobic acid (HPO-A), hydrophobic neutral (HPO-N), transphilic acid (TPI-A), transphilic neutral (TPI-N), and hydrophilic fraction (HPI). The synchronous fluorescence spectra revealed the presence of soluble microbial byproduct- and humic acid-like components and polycyclic aromatic compounds in DOM in soils, and SAT operation resulted in the enrichment of these fluorescent materials in all DOM fractions in the surface soil (0-12.5 cm). More importantly, the quantitative method of fluorescence regional integration was used in the analysis of excitation-emission matrix (EEM) spectra of DOM fractions in soils. The cumulative EEM volume (Φ T, n ) results showed that SAT operation led to the enrichment of more fluorescent components in HPO-A and TPI-A, as well as the dominance of less fluorescent components in HPO-N, TPI-N, and HPI in the bottom soil (75-150 cm). Total Φ T, n values, which were calculated as [Formula: see text], suggested an accumulation of fluorescent organic matter in the upper 75 cm of soil as a consequence of SAT operation. The distribution of volumetric fluorescence among five regions (i.e., P i, n ) results revealed that SAT caused the increased content of humic-like fluorophores as well as the decreased content of protein-like fluorophores in both HPO-A and TPI-A in soils.

  15. Availability of fluorescence spectroscopic in the accompaniment of formation of corneal cross-linking

    NASA Astrophysics Data System (ADS)

    Costa, M. M.; Kurachi, C.; Bagnato, V. S.; Faria e Sousa, S. J.; Ventura, L.

    2010-02-01

    The corneal cross-linking is a method that associates riboflavin and ultraviolet light to induce a larger mechanical resistance at cornea. This method has been used for the treatment of Keratoconus. Since cross-linking is recent as treatment, there is a need to verify the effectiveness of the method. Therefore, the viability of the fluorescence spectroscopy technique to follow the cross-linking formation at cornea was studied. Corneas were divided in two measuring procedures: M1 (cornea + riboflavin), and M2 (cornea + riboflavina + light irradiation, 365nm). For fluorescence measurements, a spectrofluorimeter was used, where several wavelengths were selected (between 320nm and 370nm) for cornea excitation. Several fluorescence spectra were collected, at 10 min-interval, during 60 min. Spectra allowed one to observe two very well defined bands of fluorescence: the first one at 400nm (collagen), and the second one at 520nm (riboflavin). After spectra analyses, a decrease of collagen fluorescence was observed for both groups. For riboflavin, on the other hand, there was a fluorescence increase for M1, and a decrease for M2. Thus, it is possible to conclude that it this technique is sensitive for the detection of tissue structural changes during cross-linking treatment, encouraging subsequent studies on quantification of cross-linking promotion in tissue.

  16. A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy

    SciTech Connect

    Gong, S.; Labanca, I.; Rech, I.; Ghioni, M.

    2014-10-15

    Fluorescence correlation spectroscopy (FCS) is a well-established technique to study binding interactions or the diffusion of fluorescently labeled biomolecules in vitro and in vivo. Fast FCS experiments require parallel data acquisition and analysis which can be achieved by exploiting a multi-channel Single Photon Avalanche Diode (SPAD) array and a corresponding multi-input correlator. This paper reports a 32-channel FPGA based correlator able to perform 32 auto/cross-correlations simultaneously over a lag-time ranging from 10 ns up to 150 ms. The correlator is included in a 32 × 1 SPAD array module, providing a compact and flexible instrument for high throughput FCS experiments. However, some inherent features of SPAD arrays, namely afterpulsing and optical crosstalk effects, may introduce distortions in the measurement of auto- and cross-correlation functions. We investigated these limitations to assess their impact on the module and evaluate possible workarounds.

  17. Optical caries diagnostics: comparison of laser spectroscopic PNC method with method of laser integral fluorescence

    NASA Astrophysics Data System (ADS)

    Masychev, Victor I.

    2000-11-01

    In this research we present the results of approbation of two methods of optical caries diagnostics: PNC-spectral diagnostics and caries detection by laser integral fluorescence. The research was conducted in a dental clinic. PNC-method analyses parameters of probing laser radiation and PNC-spectrums of stimulated secondary radiations: backscattering and endogenous fluorescence of caries-involved bacterias. He-Ne-laser ((lambda) =632,8 nm, 1-2mW) was used as a source of probing (stimulated) radiation. For registration of signals, received from intact and pathological teeth PDA-detector was applied. PNC-spectrums were processed by special algorithms, and were displayed on PC monitor. The method of laser integral fluorescence was used for comparison. In this case integral power of fluorescence of human teeth was measured. As a source of probing (stimulated) radiation diode lasers ((lambda) =655 nm, 0.1 mW and 630nm, 1mW) and He-Ne laser were applied. For registration of signals Si-photodetector was used. Integral power was shown in a digital indicator. Advantages and disadvantages of these methods are described in this research. It is disclosed that the method of laser integral power of fluorescence has the following characteristics: simplicity of construction and schema-technical decisions. However the method of PNC-spectral diagnostics are characterized by considerably more sensitivity in diagnostics of initial caries and capability to differentiate pathologies of various stages (for example, calculus/initial caries). Estimation of spectral characteristics of PNC-signals allows eliminating a number of drawbacks, which are character for detection by method of laser integral fluorescence (for instance, detection of fluorescent fillings, plagues, calculus, discolorations generally, amalgam, gold fillings as if it were caries.

  18. Fluorescent bovine serum albumin interacting with the antitussive quencher dextromethorphan: a spectroscopic insight.

    PubMed

    Durgannavar, Amar K; Patgar, Manjanath B; Nandibewoor, Sharanappa T; Chimatadar, Shivamurti A

    2016-05-01

    The interaction of dextromethorphan hydrobromide (DXM) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV-vis absorption, synchronous fluorescence spectra (SFS), 3D fluorescence spectra, Fourier transform infrared (FTIR) spectroscopy and circular dichroism under simulated physiological conditions. DXM effectively quenched the intrinsic fluorescence of BSA. Values of the binding constant, K(A), are 7.159 × 10(3), 9.398 × 10(3) and 16.101 × 10(3)  L/mol; the number of binding sites, n, and the corresponding thermodynamic parameters ΔG°, ΔH° and ΔS° between DXM and BSA were calculated at different temperatures. The interaction between DXM and BSA occurs through dynamic quenching and the effect of DXM on the conformation of BSA was analyzed using SFS. The average binding distance, r, between the donor (BSA) and acceptor (DXM) was determined based on Förster's theory. The results of fluorescence spectra, UV-vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of DXM.

  19. NEW CORRECTION PROCEDURE FOR X-RAY SPECTROSCOPIC FLUORESCENCE DATA: SIMULATIONS AND EXPERIMENT.

    SciTech Connect

    ABLETT, J.M.; WOICIK, J.C.; KAO, C.C.

    2004-08-02

    X-ray fluorescence spectroscopy is a widely used method for determining the electronic configuration and local structure of dilute species with high sensitivity. In the dilute limit, and for thin films, the X-ray fluorescence signal is directly proportional to the atomic sub-shell absorption coefficient. However, for concentrated samples, the well-documented self-absorption effect often leads to the severe suppression of XANES (X-ray Absorption Near-Edge Structure) and EXAFS (Extended X-ray Absorption Fine-Structure) amplitudes. Thus to recover the real value of the sub-shell absorption coefficient, it is important to apply correction procedures to the measured fluorescence spectra. In this paper, we describe a new straightforward method to correct for self-absorption effects (the difference in the measured fluorescence signal compared to that of the true sub-shell photoabsorption coefficient) in XANES and EXAFS fluorescence measurements. Using a variety of sample and detector configurations, this method is used to extract the sub-shell absorption coefficient on elemental nickel and thick single-crystals of Gd{sub 3}Ga{sub 5}O{sub 12} and LaAlO{sub 3}.

  20. New Correction Procedure For X-ray Spectroscopic Fluorescence Data: Simulations and Experiment

    SciTech Connect

    Ablett,J.; Woicik, J.; Kao, C.

    2005-01-01

    X-ray fluorescence spectroscopy is a widely used method for determining the electronic configuration and local structure of dilute species with high sensitivity. In the dilute limit, and for thin films, the X-ray fluorescence signal is directly proportional to the atomic sub-shell absorption coefficient. However, for concentrated samples, the well-documented self-absorption effect often leads to the severe suppression of XANES (X-ray Absorption Near-Edge Structure) and EXAFS (Extended X-ray Absorption Fine-Structure) amplitudes. Thus to recover the real value of the sub-shell absorption coefficient, it is important to apply correction procedures to the measured fluorescence spectra. In this paper, we describe a new straightforward method to correct for self-absorption effects (the difference in the measured fluorescence signal compared to that of the true sub-shell photoabsorption coefficient) in XANES and EXAFS fluorescence measurements. Using a variety of sample and detector configurations, this method is used to extract the sub-shell absorption coefficient on elemental nickel and thick single-crystals of Gd{sub 3}Ga{sub 5}O{sub 12} and LaAlO{sub 3}.

  1. Spectroscopic ellipsometry and fluorescence study of thermochromism in an ultrathin poly(diacetylene) film: Reversibility and transition kinetics

    SciTech Connect

    Carpick, R.W.; Mayer, T.M.; Sasaki, D.Y.; Burns, A.R.

    2000-05-16

    The authors have investigated the thermochromic transition of an ultrathin poly(diacetylene)film. The Langmuir film is composed of three layers of polymerized 10,12-pentacosadiynoic acid [Ch{sub 3}(CH{sub 2}){sub 11}C{triple_bond}CC{triple_bond}C(CH{sub 2}){sub 8}COOH] (poly-PCDA) organized into crystalline domains on a silicon substrate. Spectroscopic ellipsometry and fluorescence intensity measurements are obtained with in situ temperature control. Poly-PCDA films exhibit a reversible thermal transition between the initial blue form and an intermediate purple form that exists only at elevated temperature (between 303 and 333 K), followed by an irreversible transition to the red form after annealing above 320 K. The authors propose that the purple form is thermally distorted blue poly-PCDA and may represent a transitional configuration in the irreversible conversion to red. This hypothesis is supported by the appearance of unique features in the absorption spectra for each form as derived from the ellipsometry measurements. Significant fluorescence emission occurs only with the red form and is reduced at elevated temperatures while the absorption remains unchanged. Reduced emission is likely related to thermal fluctuations of the hydrocarbon side chains. Time-resolved fluorescence is likely related to thermal fluctuations of the hydrocarbon side chains. Time-resolved fluorescence measurements of the irreversible transition have been performed. Using a first-order kinetic analysis of these measurements, the authors deduce an energy barrier of 17.6 {+-} 1.1 kcal mol{sup {minus}1} between the blue and red forms.

  2. Vibrational nano-spectroscopic imaging correlating structure with intermolecular coupling and dynamics.

    PubMed

    Pollard, Benjamin; Muller, Eric A; Hinrichs, Karsten; Raschke, Markus B

    2014-04-11

    Molecular self-assembly, the function of biomembranes and the performance of organic solar cells rely on nanoscale molecular interactions. Understanding and control of such materials have been impeded by difficulties in imaging their properties with the desired nanometre spatial resolution, attomolar sensitivity and intermolecular spectroscopic specificity. Here we implement vibrational scattering-scanning near-field optical microscopy with high spectral precision to investigate the structure-function relationship in nano-phase separated block copolymers. A vibrational resonance is used as a sensitive reporter of the local chemical environment and we image, with few nanometre spatial resolution and 0.2 cm(-1) spectral precision, solvatochromic Stark shifts and line broadening correlated with molecular-scale morphologies. We discriminate local variations in electric fields between nano-domains with quantitative agreement with dielectric continuum models. This ability to directly resolve nanoscale morphology and associated intermolecular interactions can form a basis for the systematic control of functionality in multicomponent soft matter systems.

  3. Correlation of spectroscopic parameters with ligand basicity for uranyl bis(hexafluoroacetylacetonate) adducts

    SciTech Connect

    Bray, R.G.; Kramer, G.M.

    1983-06-22

    The infrared transition frequencies (vapor and solution phases) of the uranyl and hexafluoroacetylacetonate (hfacac) moieties, as well as /sup 13/C and /sup 1/H NMR shifts, correlate linearly with the relative basicity of the neutral bases (B) for 15 UO/sub 2/(hfacac)/sub 2/ adducts. Solvation effects and relative entropy changes appear to be minimal for the base-exchange equilibrium, suggesting that the observed shifts in thes easily measurable spectroscopic properties predominantly reflect the Lewis acid-base relative bond strengths. We interpret the observed shifts in terms of electronic structure perturbations of both the uranyl and hfacac moieties arising from changes in neutral base (L-M) bonding. 6 figures, 2 tables.

  4. Dynamics-function correlation in Cu, Zn superoxide dismutase: a spectroscopic and molecular dynamics simulation study.

    PubMed Central

    Falconi, M; Stroppolo, M E; Cioni, P; Strambini, G; Sergi, A; Ferrario, M; Desideri, A

    2001-01-01

    A single mutation (Val29-->Gly) at the subunit interface of a Cu, Zn superoxide dismutase dimer leads to a twofold increase in the second order catalytic rate, when compared to the native enzyme, without causing any modification of the structure or the electric field distribution. To check the role of dynamic processes in this catalytic enhancement, the flexibility of the dimeric protein at the subunit interface region has been probed by the phosphorescence and fluorescence properties of the unique tryptophan residue. Multiple spectroscopic data indicate that Trp83 experiences a very similar, and relatively hydrophobic, environment in both wild-type and mutant protein, whereas its mobility is distinctly more restrained in the latter. Molecular dynamics simulation confirms this result, and provides, at the molecular level, details of the dynamic change felt by tryptophan. Moreover, the simulation shows that the loops surrounding the active site are more flexible in the mutant than in the native enzyme, making the copper more accessible to the incoming substrate, and being thus responsible for the catalytic rate enhancement. Evidence for increased, dynamic copper accessibility also comes from faster copper removal in the mutant by a metal chelator. These results indicate that differences in dynamic, rather than structural, features of the two enzymes are responsible for the observed functional change. PMID:11371434

  5. Energy calibration and gain correction of pixelated spectroscopic x-ray detectors using correlation optimised warping

    NASA Astrophysics Data System (ADS)

    Egan, C. K.; Scuffham, J. W.; Veale, M. C.; Wilson, M. D.; Seller, P.; Cernik, R. J.

    2017-01-01

    We describe the implementation of a reliable, robust and flexible gain correction and energy calibration algorithm for pixelated spectroscopic x-ray detectors. This algorithm uses a data processing method known as correlation optimised warping which aligns shifted datasets by means of a segmental linear stretching and compression of the spectral data in order to best correlate with a reference spectrum. We found the algorithm to be very robust against low-count spectroscopy, and was reliable in a range of different spectroscopic applications. Analysis of the integrated spectrum over all pixels for a Cerium K-alpha x-ray emission (at 34.72 keV) yielded a peak width of 2.45 keV before alignment and 1.11 keV after alignment. This compares favourably with the best in class pixel peak width of 0.76 keV and the mean peak width for all pixels of 1.00 keV. We also found the algorithm to be more user friendly than other peak-search algorithms because there is less external input. A key advantage of this algorithm is that it requires no prior knowledge of the input spectral characteristics, shape or quality of the data. This therefore lends itself to being useful for in-line processing and potentially removes the need for a separate calibration standard (e.g. a radioactive source). This algorithm can be used for any system that simultaneously collects large numbers of spectral data—including multi-element detectors.

  6. Steady state fluorescence spectroscopic characterization of normal and diabetic urine at selective excitation wavelength 280 nm

    NASA Astrophysics Data System (ADS)

    Kesavan, Anjana; Pachaiappan, Rekha; Aruna, Prakasa Rao; Ganesan, Singaravelu

    2016-03-01

    Urine is considered diagnostically important for tits native fluorophores and they vary in their distribution, concentration and physiochemical properties, depending upon the metabolic condition of the subject. In this study, we have made an attempt, to characterize the urine of normal subject and diabetic patients under medication by native fluorescence spectroscopy at 280 nm excitation. Further, the fluorescence data were analyzed employing the multivariate statistical method linear discriminant analysis (LDA) using leave one out cross validation method. The results were promising in discriminating diabetic urine from that of normal urine. This study in future may be extended to check the feasibility in ruling out the coexisting disorders such as cancer.

  7. Stochastic Micro-Pattern for Automated Correlative Fluorescence - Scanning Electron Microscopy

    PubMed Central

    Begemann, Isabell; Viplav, Abhiyan; Rasch, Christiane; Galic, Milos

    2015-01-01

    Studies of cellular surface features gain from correlative approaches, where live cell information acquired by fluorescence light microscopy is complemented by ultrastructural information from scanning electron micrographs. Current approaches to spatially align fluorescence images with scanning electron micrographs are technically challenging and often cost or time-intensive. Relying exclusively on open-source software and equipment available in a standard lab, we have developed a method for rapid, software-assisted alignment of fluorescence images with the corresponding scanning electron micrographs via a stochastic gold micro-pattern. Here, we provide detailed instructions for micro-pattern production and image processing, troubleshooting for critical intermediate steps, and examples of membrane ultra-structures aligned with the fluorescence signal of proteins enriched at such sites. Together, the presented method for correlative fluorescence – scanning electron microscopy is versatile, robust and easily integrated into existing workflows, permitting image alignment with accuracy comparable to existing approaches with negligible investment of time or capital. PMID:26647824

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

    PubMed

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

    2016-07-01

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

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

    PubMed

    Kilpatrick, Laura E; Hill, Stephen J

    2016-04-15

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

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

    PubMed Central

    Kilpatrick, Laura E.; Hill, Stephen J.

    2016-01-01

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

  11. Interaction of cinnamic acid derivatives with serum albumins: A fluorescence spectroscopic study

    NASA Astrophysics Data System (ADS)

    Singh, T. Sanjoy; Mitra, Sivaprasad

    2011-03-01

    Cinnamic acid (CA) derivatives are known to possess broad therapeutic applications including anti-tumor activity. The present study was designed to determine the underlying mechanism and thermodynamic parameters for the binding of two CA based intramolecular charge transfer (ICT) fluorescent probes, namely, 4-(dimethylamino) cinnamic acid (DMACA) and trans-ethyl p-(dimethylamino) cinnamate (EDAC), with albumins by fluorescence spectroscopy. Stern-Volmer analysis of the tryptophan fluorescence quenching data in presence of the added ligand reveals fluorescence quenching constant ( κq), Stern-Volmer constant ( KSV) and also the ligand-protein association constant ( Ka). The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated. The results show that the ligands bind into the sub-domain IIA of the proteins in 1:1 stoichiometry with an apparent binding constant value in the range of 10 4 dm 3 mol -1. In both the cases, the spontaneous ligand binding to the proteins occur through entropy driven mechanism, although the interaction of DMACA is relatively stronger in comparison with EDAC. The temperature dependence of the binding constant indicates the induced change in protein secondary structure.

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

    PubMed Central

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

    1997-01-01

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

  13. 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, +/-).

  14. Encapsulation of serotonin in β-cyclodextrin nano-cavities: Fluorescence spectroscopic and molecular modeling studies

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Sudip; Chakraborty, Sandipan; Sengupta, Pradeep K.

    2010-06-01

    Serotonin is a physiologically important biogenic amine, deficiency of which leads to mental disorders such as Alzheimer's disease, schizophrenia, infantile autism, and depression. Both β-cyclodextrin (β-CD) and its chemically substituted synthetic varieties (often possessing enhanced aqueous solubility and improved drug complexing abilities) are finding wide applications as drug delivery vehicles. Here we have studied the encapsulation of serotonin in β-CD and succinyl-2-hydroxypropyl β-cyclodextrin (SHP-β-CD) by exploiting the intrinsic serotonin fluorescence. Enhanced fluorescence emission intensity (which increases by ˜18% and 34% in β-CD and SHPβ-CD respectively) and anisotropy ( r) ( r = 0.075 and 0.1 in β-CD and SHPβ-CD respectively) are observed in presence of the cyclodextrins. From the fluorescence data host-guest interaction with 1:1 stoichiometry is evident, the association constants ( K) being 126.06 M -1 and 461.62 M -1 for β-CD and SHPβ-CD respectively. Additionally, molecular docking and semiempirical calculations have been carried out which provide, for the first time, detailed insights regarding the encapsulation process. In particular, it is evident that the indole ring is inserted within the β-CD cavity with the aliphatic amine side chain protruding towards the primary rim of the β-CD cavity. Docking calculations reveal that hydrogen bonding interactions are involved in the formation of the inclusion complex. Semiempirical calculations indicate that formation of the 1:1 inclusion complex is energetically favorable which is consistent with the fluorescence data.

  15. Identification of hematic cells by spectroscopic analysis of the intrinsic fluorescence

    NASA Astrophysics Data System (ADS)

    Monici, Monica; Agati, Giovanni; Fusi, Franco; Bernabei, Pietro A.; Caporale, Roberto; Ferrini, Pierluigi R.; Croce, Anna C.; Bottiroli, Giovanni F.; Cioncolini, Stefano; Innocenti, Alberto; Pratesi, Riccardo

    1994-12-01

    The determination of blood cell composition has been a valuable tool in diagnoses. In particular, both total and differential counts are considered the basic parameters that characterize the leukocyte population. Since 100 years ago, manual techniques were introduced that allow a morphological examination of blood smears. At present, the automated analysis has been proved to be particularly difficult to standardize. In fact, the identification and count of the five leukocyte populations are not completely solved problems in routine methods for hematological analysis. Optoelectronics could have a decisive role in the development of new techniques that can ensure characteristics of automation, reliability, accuracy and rapidity of execution. Fluorescence spectroscopy techniques could represent a valid approach. Recently, the evaluation of tissue and cell autofluorescence has been applied to the diagnosis of solid tissue neoplasies. In this work, we have considered the possibility to develop a reliable method of leukocyte analysis based on their intrinsic fluorescence emission properties. The study has been performed by applying both spectrofluorometric techniques to enriched suspensions of cells and microspectrofluorometric techniques to single leukocytes. The results obtained have shown the possibility to recognize some cell populations on the grounds of the intrinsic fluorescence characteristics.

  16. Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Zirak, P.; Penzkofer, A.; Schiereis, T.; Hegemann, P.; Jung, A.; Schlichting, I.

    2005-08-01

    The BLUF domain of the transcriptional anti-repressor protein AppA from the non-sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA 148 (consisting of amino-acid residues 1-148) and AppA 126 (amino-acid residues 1-126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark-adapted domains exist in two different active receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF r,f and BLUF r,sl) and a small non-interacting conformation (BLUF nc). The active receptor conformations are transformed to putative signalling states (BLUF s,f and BLUF s,sl) of low fluorescence efficiency and picosecond fluorescence lifetime by blue-light excitation (light-adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo-cycle scheme is presented including photo-induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation.

  17. Interaction of Sulfadiazine with Model Water Soluble Proteins: A Combined Fluorescence Spectroscopic and Molecular Modeling Approach.

    PubMed

    Islam, Mullah Muhaiminul; Moyon, N Shaemningwar; Gashnga, Pynsakhiat Miki; Mitra, Sivaprasad

    2014-03-01

    The binding behavior of antibacterial drug sulfadiazine (SDZ) with water soluble globular proteins like bovine as well as human serum albumin (BSA and HSA, respectively) and lysozyme (LYS) was monitored by fluorescence titration and molecular docking calculations. The experimental data reveal that the quenching of the intrinsic protein fluorescence in presence of SDZ is due to the strong interaction in the drug binding site of the respective proteins. The Stern-Volmer plot shows positive deviation at higher quencher concentration for all the proteins and was explained in terms of a sphere of action model. The calculated fluorophore-quencher distances vary within 4 ~ 11 Å in different cases. Fluorescence experiments at different temperature indicate thermodynamically favorable binding of SDZ with the proteins with apparently strong association constant (~10(4)-10(5) M(-1)) and negative free energy of interaction within the range of -26.0 ~ -36.8 kJ mol(-1). The experimental findings are in good agreement with the respective parameters obtained from best energy ranked molecular docking calculation results of SDZ with all the three proteins.

  18. Spectroscopic insights on imidazole substituted phthalocyanine photosensitizers: Fluorescence properties, triplet state and singlet oxygen generation

    NASA Astrophysics Data System (ADS)

    Zhang, Xian-Fu; Lin, Yong; Guo, Wenfeng; Zhu, Jingzhong

    2014-12-01

    Imidazole substituted metal phthalocyanine (Pc) complexes were synthesized. UV-vis absorption, steady state and time-resolved fluorescence, as well as laser flash photolysis were used to measure the photophysical and photosensitizing properties. All the imidazole-phthalocyanine conjugates show high ΦT (quantum yield of excited triplet formation), high ΦΔ (singlet oxygen formation yield, >0.50) and good fluorescence properties (quantum yield Φf > 0.20 and lifetime τf > 3.0 ns). Compared to the unsubstituted Pc, both α- and β-imidazole substitutions result in the remarkable decrease in Φf and τf, but the α-substitution is stronger. The imidazole substitution, on the other hand, causes the increase of ΦT, τT, and ΦΔ values. Magnesium phthalocyanine (MgPc) is more susceptible to the substitution than zinc phthalocyanine (ZnPc). The mechanism responsible for the result is suggested based on the involvement of intramolecular photoinduced electron transfer. The high ΦΔ and appropriate fluorescence properties make the Pcs good candidate for PDT photosensitizers.

  19. High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers☆

    PubMed Central

    Schellenberger, Pascale; Kaufmann, Rainer; Siebert, C. Alistair; Hagen, Christoph; Wodrich, Harald; Grünewald, Kay

    2014-01-01

    Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell. PMID:24262358

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

    PubMed

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

    2013-02-01

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

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

    PubMed

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

    2010-05-24

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

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

    PubMed Central

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

    2010-01-01

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

  3. Spectroscopic and fluorescence studies on Mn(II), Co(II), Ni(II) and Cu(II) complexes with NO donor fluorescence dyes

    NASA Astrophysics Data System (ADS)

    Refat, Moamen S.; El-Metwaly, Nashwa M.

    2011-10-01

    The reactions of the two common dyes [2TMPACT and 4PENI] with Mn(II), Co(II), Ni(II) and Cu(II) ions were done. All the isolated complexes have been characterized by physicochemical and spectroscopic techniques. The IR data reflect the bidentate mode of 2TMPACT towards the mononuclear complex [Mn(II)] even its tetradentate in binuclear complexes [Co(II) and Cu(II)]. However, the bidentate mode is the only behavior of 4PENI ligand towards each metal ion in its mononuclear complexes. The UV-vis spectral analysis beside the magnetic moment measurements are proposed different geometries concerning each metal ions with the two ligands under investigation, as the Mn(II)-2TMPACT complex is an octahedral but Mn(II)-4PENI is a tetrahedral geometry. All the synthesized compounds are thermogravimetrically investigated. The proposed thermal decomposition was discussed for each compound with each step as well as, the kinetic parameters were calculated for all preferrible decomposition steps. The mass spectroscopy tool was used to emphasis on the suitable molecular formula proposed and the fragmentation patterns were displayed. The fluorescence properties of the synthesized ligands and their complexes were studied in DMSO at room temperature.

  4. Spectroscopic and fluorescence studies on Mn(II), Co(II), Ni(II) and Cu(II) complexes with NO donor fluorescence dyes.

    PubMed

    Refat, Moamen S; el-Metwaly, Nashwa M

    2011-10-15

    The reactions of the two common dyes [2TMPACT and 4PENI] with Mn(II), Co(II), Ni(II) and Cu(II) ions were done. All the isolated complexes have been characterized by physicochemical and spectroscopic techniques. The IR data reflect the bidentate mode of 2TMPACT towards the mononuclear complex [Mn(II)] even its tetradentate in binuclear complexes [Co(II) and Cu(II)]. However, the bidentate mode is the only behavior of 4PENI ligand towards each metal ion in its mononuclear complexes. The UV-vis spectral analysis beside the magnetic moment measurements are proposed different geometries concerning each metal ions with the two ligands under investigation, as the Mn(II)-2TMPACT complex is an octahedral but Mn(II)-4PENI is a tetrahedral geometry. All the synthesized compounds are thermogravimetrically investigated. The proposed thermal decomposition was discussed for each compound with each step as well as, the kinetic parameters were calculated for all preferrible decomposition steps. The mass spectroscopy tool was used to emphasis on the suitable molecular formula proposed and the fragmentation patterns were displayed. The fluorescence properties of the synthesized ligands and their complexes were studied in DMSO at room temperature.

  5. Evaluation of transformer insulating oil quality using NIR, fluorescence, and NMR spectroscopic data fusion.

    PubMed

    Godinho, Mariana S; Blanco, Marcos R; Gambarra Neto, Francisco F; Lião, Luciano M; Sena, Marcelo M; Tauler, Romà; de Oliveira, Anselmo E

    2014-11-01

    Power transformers are essential components in electrical energy distribution. One of their most important parts is the insulation system, consisting of Kraft paper immersed in insulating oil. Interfacial tension and color are major parameters used for assessing oil quality and the system׳s degradation. This work proposes the use of near infrared (NIR), molecular fluorescence, and (1)H nuclear magnetic resonance (NMR) spectroscopy methods combined with chemometric multivariate calibration methods (Partial Least Squares - PLS) to predict interfacial tension and color in insulating mineral oil samples. Interfacial tension and color were also determined using tensiometry and colorimetry as standard reference methods, respectively. The best PLS model was obtained when NIR, fluorescence, and NMR data were combined (data fusion), demonstrating synergy among them. An optimal PLS model was calculated using the selected group of variables according to their importance on PLS projections (VIP). The root mean square errors of prediction (RMSEP) values of 2.9 mN m(-1) and 0.3 were estimated for interfacial tension and color, respectively. Mean relative standard deviations of 1.5% for interfacial tension and 6% for color were registered, meeting quality control requirements set by electrical energy companies. The methods proposed in this work are rapid and simple, showing great advantages over traditional approaches, which are slow and environmentally unfriendly due to chemical waste generation.

  6. Synthesis and spectroscopic characterization of fluorescent 4-aminoantipyrine analogues: Molecular docking and in vitro cytotoxicity studies

    NASA Astrophysics Data System (ADS)

    Premnath, D.; Mosae Selvakumar, P.; Ravichandiran, P.; Tamil Selvan, G.; Indiraleka, M.; Jannet Vennila, J.

    2016-01-01

    Two substituted aromatic carbonyl compounds (compounds 1 and 2) of 4-aminoantipyrine were synthesized by condensation of fluorine substituted benzoyl chlorides and 4-aminoantipyrine. The structures of synthesized derivatives were established on the basis of UV-Vis, IR, and Mass, 1H, 13C NMR and Fluorescence spectroscopy. Both compounds showed significant fluorescence emission and two broad emission bands were observed in the region at 340 nm and 450 nm on excitation at 280 nm. Theoretically to prove that the molecule has anticancer activity against cervical cancer cells, the compounds were analyzed for molecular docking interactions with HPV16-E7 target protein by Glide protocol. Furthermore, 4-aminoantipyrine derivatives were evaluated for their in vitro cytotoxic activity against human cervical cancer cells (SiHa) by MTT assay. Compound 1 showed two fold higher activity (IC50 = 0.912 μM) over compound 2, and its activity was similar to that of Pazopanib, suggesting that although the two compounds were chemically very similar the difference in substituent on the phenyl moiety caused changes in properties.

  7. Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

  8. Correlation coefficient mapping in fluorescence spectroscopy: tissue classification for cancer detection.

    PubMed

    Crowell, Ed; Wang, Gufeng; Cox, Jason; Platz, Charles P; Geng, Lei

    2005-03-01

    Correlation coefficient mapping has been applied to intrinsic fluorescence spectra of colonic tissue for the purpose of cancer diagnosis. Fluorescence emission spectra were collected of 57 colonic tissue sites in a range of 4 physiological conditions: normal (29), hyperplastic (2), adenomatous (5), and cancerous tissues (21). The sample-sample correlation was used to examine the ability of correlation coefficient mapping to determine tissue disease state. The correlation coefficient map indicates two main categories of samples. These categories were found to relate to disease states of the tissue. Sensitivity, selectivity, predictive value positive, and predictive value negative for differentiation between normal tissue and all other categories were all above 92%. This was found to be similar to, or higher than, tissue classification using existing methods of data reduction. Wavelength-wavelength correlation among the samples highlights areas of importance for tissue classification. The two-dimensional correlation map reveals absorption by NADH and hemoglobin in the samples as negative correlation, an effect not obvious from the one-dimensional fluorescence spectra alone. The integrity of tissue was examined in a time series of spectra of a single tissue sample taken after tissue resection. The wavelength-wavelength correlation coefficient map shows the areas of significance for each fluorophore and their relation to each other. NADH displays negative correlation to collagen and FAD, from the absorption of emission or fluorescence resonance energy transfer. The wavelength-wavelength correlation map for the decay set also clearly shows that there are only three fluorophores of importance in the samples, by the well-defined pattern of the map. The sample-sample correlation coefficient map reveals the changes over time and their impact on tissue classification. Correlation coefficient mapping proves to be an effective method for sample classification and cancer

  9. Vibrational nano-spectroscopic imaging correlating structure with intermolecular coupling and dynamics

    PubMed Central

    Pollard, Benjamin; Muller, Eric A.; Hinrichs, Karsten; Raschke, Markus B.

    2014-01-01

    Molecular self-assembly, the function of biomembranes and the performance of organic solar cells rely on nanoscale molecular interactions. Understanding and control of such materials have been impeded by difficulties in imaging their properties with the desired nanometre spatial resolution, attomolar sensitivity and intermolecular spectroscopic specificity. Here we implement vibrational scattering-scanning near-field optical microscopy with high spectral precision to investigate the structure–function relationship in nano-phase separated block copolymers. A vibrational resonance is used as a sensitive reporter of the local chemical environment and we image, with few nanometre spatial resolution and 0.2 cm−1 spectral precision, solvatochromic Stark shifts and line broadening correlated with molecular-scale morphologies. We discriminate local variations in electric fields between nano-domains with quantitative agreement with dielectric continuum models. This ability to directly resolve nanoscale morphology and associated intermolecular interactions can form a basis for the systematic control of functionality in multicomponent soft matter systems. PMID:24721995

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

    PubMed Central

    2015-01-01

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

  11. Detection of rheumatoid arthritis by evaluation of normalized variances of fluorescence time correlation functions

    NASA Astrophysics Data System (ADS)

    Dziekan, Thomas; Weissbach, Carmen; Voigt, Jan; Ebert, Bernd; MacDonald, Rainer; Bahner, Malte L.; Mahler, Marianne; Schirner, Michael; Berliner, Michael; Berliner, Birgitt; Osel, Jens; Osel, Ilka

    2011-07-01

    Fluorescence imaging using the dye indocyanine green as a contrast agent was investigated in a prospective clinical study for the detection of rheumatoid arthritis. Normalized variances of correlated time series of fluorescence intensities describing the bolus kinetics of the contrast agent in certain regions of interest were analyzed to differentiate healthy from inflamed finger joints. These values are determined using a robust, parameter-free algorithm. We found that the normalized variance of correlation functions improves the differentiation between healthy joints of volunteers and joints with rheumatoid arthritis of patients by about 10% compared to, e.g., ratios of areas under the curves of raw data.

  12. CFHTLenS and RCSLenS: testing photometric redshift distributions using angular cross-correlations with spectroscopic galaxy surveys

    NASA Astrophysics Data System (ADS)

    Choi, A.; Heymans, C.; Blake, C.; Hildebrandt, H.; Duncan, C. A. J.; Erben, T.; Nakajima, R.; Van Waerbeke, L.; Viola, M.

    2016-12-01

    We determine the accuracy of galaxy redshift distributions as estimated from photometric redshift probability distributions p(z). Our method utilizes measurements of the angular cross-correlation between photometric galaxies and an overlapping sample of galaxies with spectroscopic redshifts. We describe the redshift leakage from a galaxy photometric redshift bin j into a spectroscopic redshift bin i using the sum of the p(z) for the galaxies residing in bin j. We can then predict the angular cross-correlation between photometric and spectroscopic galaxies due to intrinsic galaxy clustering when i ≠ j as a function of the measured angular cross-correlation when i = j. We also account for enhanced clustering arising from lensing magnification using a halo model. The comparison of this prediction with the measured signal provides a consistency check on the validity of using the summed p(z) to determine galaxy redshift distributions in cosmological analyses, as advocated by the Canada-France-Hawaii Telescope Lensing Survey (CFHTLenS). We present an analysis of the photometric redshifts measured by CFHTLenS, which overlaps the Baryon Oscillation Spectroscopic Survey (BOSS). We also analyse the Red-sequence Cluster Lensing Survey, which overlaps both BOSS and the WiggleZ Dark Energy Survey. We find that the summed p(z) from both surveys are generally biased with respect to the true underlying distributions. If unaccounted for, this bias would lead to errors in cosmological parameter estimation from CFHTLenS by less than ˜4 per cent. For photometric redshift bins which spatially overlap in 3D with our spectroscopic sample, we determine redshift bias corrections which can be used in future cosmological analyses that rely on accurate galaxy redshift distributions.

  13. Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins

    PubMed Central

    Johnson, Errin; Seiradake, Elena; Jones, E. Yvonne; Davis, Ilan; Grünewald, Kay; Kaufmann, Rainer

    2015-01-01

    We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40–50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation. PMID:25823571

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

    PubMed Central

    Huang, Z; Thompson, N L

    1996-01-01

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

  15. Biodistribution, pharmacokinetic, and in-vivo fluorescence spectroscopic studies of photosensitizers

    NASA Astrophysics Data System (ADS)

    Moan, Johan; Peng, Qian; Iani, Vladimir; Ma, Li Wei; Horobin, Richard W.; Berg, Kristian; Kongshaug, Magne; Nesland, Jahn M.

    1996-01-01

    Some key data concerning the pharmacokinetics of PCT photosensitizers are reviewed. The following topics are discussed: The binding of photosensitizers to serum proteins, and the significance of LDL binding for tumor localization, the distribution of sensitizers among different tissue compartments and the significance of extracellular proteins and other stromal elements, such as macrophages, low tumor pH, leaky vasculature and poor lymphatic drainage for tumor selectivity of drugs, the retention and excretion of sensitizers, and intracellular pharmacokinetics. Furthermore, the usefulness of fluorescence measurements in the study of sensitizer pharmacokinetics is briefly discussed. A key observation is that 1O2 has a short radius of action. Since practically all PCT sensitizers act via the 1O2 pathway, only targets with significant sensitizer concentrations can be damaged. A given number of 1O2 entities generated in different organelles (mitochondria, lysosomes, plasma membrane, etc.) may lead to widely different effects with respect to cell inactivation. Similarly, sensitizers localizing in different compartments of tissues may have different photosensitizing efficiencies even under conditions of a similar 1O2 yield.

  16. Fluorescence Spectroscopic Studies on the Complexation of Antidiabetic Drugs with Glycosylated Serum Albumin

    NASA Astrophysics Data System (ADS)

    Seedher, N.; Kanojia, M.

    2013-11-01

    Glycosylation decreases the association constant values and hence the binding affinity of human serum albumin (HSA) for the antidiabetic drugs under study. The percentage of HAS-bound drug at physiological temperature was only about 21-38 % as compared to 46-74 % for non-glycosylated HSA. Thus the percentage of free drug available for an antihyperglycemic effect was about double (62-79 %) compared to the values for non-glycosylated HSA. Much higher free drug concentrations available for pharmacological effect can lead to the risk of hypoglycemia. Hydrophobic interactions were predominantly involved in the binding. In the binding of gliclazide, hydrogen bonding and electrostatic interactions were involved. Site specificity for glycosylated HSA was the same as that for non-glycosylated HSA; gliclazide and repaglinide bind only at site II whereas glimepiride and glipizide bind at both sites I and II. Glycosylation, however, caused conformational changes in albumin, and the binding region within site II was different for glycosylated and non-glycosylated albumin. Stern-Volmer analysis also indicated the conformational changes in albumin as a result of glycosylation and showed that the dynamic quenching mechanism was valid for fluorescence of both glycosylated and non-glycosylated HSA.

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

    PubMed

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

    2016-02-01

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

  18. Fluorescence correlation spectroscopy: Ultrasensitive detection in clear and turbid media

    NASA Astrophysics Data System (ADS)

    Tahari, Abdel Kader

    In this work, I describe the development of a simple, inexpensive, and powerful alternative technique to detect and analyze, without enrichment, extremely low concentrations of cells, bacteria, viruses, and protein aggregates in turbid fluids for clinical and biotechnological applications. The anticipated applications of this technique are many. They range from the determination of the somatic cell count in milk for the dairy industry, to the enumeration and characterization of microorganisms in environmental microbiology and the food industry, and to the fast and ultrasensitive detection of protein aggregates for the diagnosis of Alzheimer's and other neurodegenerative diseases in clinical medicine. A prototype instrument has been built and allowed the detection and quantification of particles down to a few per milliliter in short scanning times. It consists of a small microscope that has a horizontal geometry and a mechanical instrument that holds a cylindrical cuvette (1 cm in diameter) with two motors that provide a rotational and a slower vertical inversion motions. The illumination focus is centered about 200 mum from the wall of the cuvette inside the sample. The total volume that is explored is large (˜1ml/min for bright particles). The data is analyzed with a correlation filter program based on particle passage pattern recognition. I will also describe further work on improving the sensitivity of the technique, expanding it for multiple-species discrimination and enumeration, and testing the prototype device in actual clinical and biotechnological applications. The main clinical application of this project seeks to establish conditions and use this new technique to quantify and size-analyze oligomeric complexes of the Alzheimer's disease beta-peptide in cerebrospinal fluid and other body fluids as a molecular biomarker for persons at risk of Alzheimer's disease dementia. The technology could potentially be extended to the diagnosis and therapeutic

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

    PubMed

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

    2011-04-21

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

  20. BH2 revisited: New, extensive measurements of laser-induced fluorescence transitions and ab initio calculations of near-spectroscopic accuracy

    NASA Astrophysics Data System (ADS)

    Sunahori, Fumie X.; Gharaibeh, Mohammed; Clouthier, Dennis J.; Tarroni, Riccardo

    2015-05-01

    The spectroscopy of gas phase BH2 has not been explored experimentally since the pioneering study of Herzberg and Johns in 1967. In the present work, laser-induced fluorescence (LIF) spectra of the A ˜ 2 B 1 ( Π u ) - X ˜ 2A1 band system of 11BH2, 10BH2, 11BD2, and 10BD2 have been observed for the first time. The free radicals were "synthesized" by an electric discharge through a precursor mixture of 0.5% diborane (B2H6 or B2D6) in high pressure argon at the exit of a pulsed valve. A total of 67 LIF bands have been measured and rotationally analyzed, 62 of them previously unobserved. These include transitions to a wide variety of excited state bending levels, to several stretch-bend combination levels, and to three ground state levels which gain intensity through Renner-Teller coupling to nearby excited state levels. As an aid to vibronic assignment of the spectra, very high level hybrid ab initio potential energy surfaces were built starting from the coupled cluster singles and doubles with perturbative triples (CCSD(T))/aug-cc-pV5Z level of theory for this seven-electron system. In an effort to obtain the highest possible accuracy, the potentials were corrected for core correlation, extrapolation to the complete basis set limit, electron correlation beyond CCSD(T), and diagonal Born-Oppenheimer effects. The spin-rovibronic states of the various isotopologues of BH2 were calculated for energies up to 22 000 cm-1 above the X ˜ (000) level without any empirical adjustment of the potentials or fitting to experimental data. The agreement with the new LIF data is excellent, approaching near-spectroscopic accuracy (a few cm-1) and has allowed us to understand the complicated spin-rovibronic energy level structure even in the region of strong Renner-Teller resonances.

  1. Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of pearson's correlation coefficient.

    PubMed

    Barlow, Andrew L; Macleod, Alasdair; Noppen, Samuel; Sanderson, Jeremy; Guérin, Christopher J

    2010-12-01

    One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

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

    PubMed Central

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

    2007-01-01

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

  3. Determination of Equilibrium Constant and Relative Brightness in Fluorescence Correlation Spectroscopy by Considering Third-Order Correlations.

    PubMed

    Wu, Zhenqin; Bi, Huimin; Pan, Sichen; Meng, Lingyi; Zhao, Xin Sheng

    2016-11-17

    Fluorescence correlation spectroscopy (FCS) is a powerful tool to investigate molecular diffusion and relaxations, which may be utilized to study many problems such as molecular size and aggregation, chemical reaction, molecular transportation and motion, and various kinds of physical and chemical relaxations. This article focuses on a problem related to using the relaxation term to study a reaction. If two species with different fluorescence photon emission efficiencies are connected by a reaction, the kinetic and equilibrium properties will be manifested in the relaxation term of the FCS curve. However, the conventional FCS alone cannot simultaneously determine the equilibrium constant (K) and the relative fluorescence brightness (Q), both of which are indispensable in the extraction of thermodynamic and kinetic information from the experimental data. To circumvent the problem, an assumption of Q = 0 is often made for the weak fluorescent species, which may lead to numerous errors when the actual situation is not the case. We propose to combine the third-order FCS with the conventional second-order FCS to determine K and Q without invoking other resources. The strategy and formalism are verified by computer simulations and demonstrated in a classical example of the hairpin DNA-folding process.

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

    PubMed

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

    2013-03-21

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

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

    PubMed

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

    2011-08-11

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

  6. Acetylene-substituted two-photon absorbing molecules with rigid elongated pi-conjugation: synthesis, spectroscopic properties and two-photon fluorescence cell imaging applications.

    PubMed

    Liu, Bo; Zhang, Hai-Li; Liu, Jun; Huang, Zhen-Li; Zhao, Yuan-Di; Luo, Qing-Ming

    2007-09-01

    Two asymmetrical molecules with substituted acetylene as central rigid elongated conjugation are reported as potential chromophores for two-photon microscopic imaging. These molecules consist of a typical D-pi-A structure, have different donors (D), the same pi-conjugated center (pi) and the same acceptor (A). Structural characterization and spectroscopic properties, including single-photon (linear) absorption, quantum yields, single-photon fluorescence, and two-photon absorption spectra, were studied in solvents with different polarity. These acetylene-substituted molecules were found to have high two-photon absorption cross-sections (for example, 690 GM for molecule 1 in toluene), which were determined by a two-photon induced fluorescence method using a femtosecond Ti: sapphire laser as excitation source. Single- and two-photon cellular imaging experiments demonstrate that the substituted acetylene derivatives could be one kind of promising two-photon fluorescence probes for cellular imaging.

  7. Commercial counterboard for 10 ns software correlator for photon and fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Molteni, Matteo; Ferri, Fabio

    2016-11-01

    A 10 ns time resolution, multi-tau software correlator, capable of computing simultaneous autocorrelation (A-A, B-B) and cross (A-B) correlation functions at count rates up to ˜10 MHz, with no data loss, has been developed in LabVIEW and C++ by using the National Instrument timer/counterboard (NI PCIe-6612) and a fast Personal Computer (PC) (Intel Core i7-4790 Processor 3.60 GHz ). The correlator works by using two algorithms: for large lag times (τ ≳ 1 μs), a classical time-mode scheme, based on the measure of the number of pulses per time interval, is used; differently, for τ ≲ 1 μs a photon-mode (PM) scheme is adopted and the correlation function is retrieved from the sequence of the photon arrival times. Single auto- and cross-correlation functions can be processed online in full real time up to count rates of ˜1.8 MHz and ˜1.2 MHz, respectively. Two autocorrelation (A-A, B-B) and a cross correlation (A-B) functions can be simultaneously processed in full real time only up to count rates of ˜750 kHz. At higher count rates, the online processing takes place in a delayed modality, but with no data loss. When tested with simulated correlation data and latex spheres solutions, the overall performances of the correlator appear to be comparable with those of commercial hardware correlators, but with several nontrivial advantages related to its flexibility, low cost, and easy adaptability to future developments of PC and data acquisition technology.

  8. Commercial counterboard for 10 ns software correlator for photon and fluorescence correlation spectroscopy.

    PubMed

    Molteni, Matteo; Ferri, Fabio

    2016-11-01

    A 10 ns time resolution, multi-tau software correlator, capable of computing simultaneous autocorrelation (A-A, B-B) and cross (A-B) correlation functions at count rates up to ∼10 MHz, with no data loss, has been developed in LabVIEW and C++ by using the National Instrument timer/counterboard (NI PCIe-6612) and a fast Personal Computer (PC) (Intel Core i7-4790 Processor 3.60 GHz ). The correlator works by using two algorithms: for large lag times (τ ≳ 1 μs), a classical time-mode scheme, based on the measure of the number of pulses per time interval, is used; differently, for τ ≲ 1 μs a photon-mode (PM) scheme is adopted and the correlation function is retrieved from the sequence of the photon arrival times. Single auto- and cross-correlation functions can be processed online in full real time up to count rates of ∼1.8 MHz and ∼1.2 MHz, respectively. Two autocorrelation (A-A, B-B) and a cross correlation (A-B) functions can be simultaneously processed in full real time only up to count rates of ∼750 kHz. At higher count rates, the online processing takes place in a delayed modality, but with no data loss. When tested with simulated correlation data and latex spheres solutions, the overall performances of the correlator appear to be comparable with those of commercial hardware correlators, but with several nontrivial advantages related to its flexibility, low cost, and easy adaptability to future developments of PC and data acquisition technology.

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

    PubMed

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

    2005-12-01

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

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

    PubMed

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

    2017-01-23

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Correlation Between Scattering Properties of Silver Particle Arrays and Fluorescence Enhancement

    PubMed Central

    SZMACINSKI, HENRYK; LAKOWICZ, JOSEPH R.; CATCHMARK, JEFFREY M.; EID, KHALID; ANDERSON, JON P.; MIDDENDORF, LYLE

    2009-01-01

    We report on the nanofabrication of patterned silver particle arrays using electron-beam lithography and the evaluation of their optical properties using backscattering and fluorescence spectroscopy. The silver particles varied in size from 100 to 250 nm and were in the shape of circles, squares, and triangles. Three inter-particle separations, 40, 65, and 90 nm as measured from the side of one particle to the side of the next particle, were used. We observed distinctive patterns of backscattering and fluorescence intensity depending on the particle size, inter-particle spacing, and excitation/emission wavelength used. Our approach allows for a study of the correlation between the backscattering intensities and fluorescence enhancement of silver particle arrays, which can be used to optimize the arrays for multi-fluorophore configuration for advanced sensing designs. PMID:18935821

  14. CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography)

    PubMed Central

    Siegel, Nisan; Storrie, Brian; Bruce, Marc

    2016-01-01

    FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called “CINCH”. An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution. PMID:26839443

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

    NASA Astrophysics Data System (ADS)

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

    2001-09-01

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

  16. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    SciTech Connect

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-31

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

  17. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    DOE PAGES

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; ...

    2014-10-31

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

  18. Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

    PubMed

    Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

    2016-04-01

    A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

  19. Analysis of the Fluorescence Correlation Function of Quantum Rods with Different Lengths.

    PubMed

    Lee, Jaeran; Kim, Sok Won

    2015-11-01

    We built a polarization fluorescence correlation spectroscopy system to analyze the variation of the correlation function in rotational diffusion based on the length of rod-like fluorescent particles. Because the rotational diffusion of particles in liquid depends on the relative polarization states of the laser source and particle fluorescence, we compared the amplitudes of the rotational diffusion using the autocorrelation function in different polarization states. For experiments that depend on the length of the fluorescent particles, we prepared three kinds of quantum rod samples with a width of 6.5 ± 0.5 nm and lengths of 17 ± 3, 40 ± 3, and 46 ± 3 nm. Through the experiment, we obtained the hydrodynamic radii of each particle using the rotational diffusion coefficient: 10.7 ± 0.8, 13.4 ± 0.7, and 14.1 ± 0.4 nm with the length of the particles. All the obtained values for radii are 3 nm larger than the calculated equivalent radii of spheres with the same volume as the rod samples. Through a fraction analysis by polarization state, we confirmed that the ratio of rotational fraction for polarization increases with the aspect ratio of the actual particle.

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

    NASA Astrophysics Data System (ADS)

    Winkler, Roland G.

    2007-08-01

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

  1. A simple chlorophyll fluorescence parameter that correlates with the rate coefficient of photoinactivation of photosystem II.

    PubMed

    Hendrickson, Luke; Förster, Britta; Pogson, Barry J; Chow, Wah Soon

    2005-06-01

    A method of partitioning the energy in a mixed population of active and photoinactivated Photosystem II (PS II) complexes based on chlorophyll fluorescence measurements is presented. There are four energy fluxes, each with its quantum efficiency: a flux associated with photochemical electron flow in active PS II reaction centres (JPS II), thermal dissipation in photoinactivated, non-functional PS IIs (JNF), light-regulated thermal dissipation in active PS IIs (JNPQ) and a combined flux of fluorescence and constitutive, light-independent thermal dissipation (Jf,D). The four quantum efficiencies add up to 1.0, without the need to introduce an 'excess' term E, which in other studies has been claimed to be linearly correlated with the rate coefficient of photoinactivation of PS II (kpi). We examined the correlation of kpi with various fluxes, and found that the combined flux (JNPQ + Jf,D= Jpi) is as well correlated with kpi as is E. This combined flux arises from Fs/Fm ', the ratio of steady-state to maximum fluorescence during illumination, which represents the quantum efficiency of combined non-photochemical dissipation pathways in active PS IIs. Since Fs/Fm ' or its equivalent, Jpi, is a likely source of events leading to photoinactivation of PS II, we conclude that Fs/Fm ' is a simple predictor of kpi.

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

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  4. Speckle correlation resolution enhancement of wide-field fluorescence imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Yilmaz, Hasan

    2016-03-01

    Structured illumination enables high-resolution fluorescence imaging of nanostructures [1]. We demonstrate a new high-resolution fluorescence imaging method that uses a scattering layer with a high-index substrate as a solid immersion lens [2]. Random scattering of coherent light enables a speckle pattern with a very fine structure that illuminates the fluorescent nanospheres on the back surface of the high-index substrate. The speckle pattern is raster-scanned over the fluorescent nanospheres using a speckle correlation effect known as the optical memory effect. A series of standard-resolution fluorescence images per each speckle pattern displacement are recorded by an electron-multiplying CCD camera using a commercial microscope objective. We have developed a new phase-retrieval algorithm to reconstruct a high-resolution, wide-field image from several standard-resolution wide-field images. We have introduced phase information of Fourier components of standard-resolution images as a new constraint in our algorithm which discards ambiguities therefore ensures convergence to a unique solution. We demonstrate two-dimensional fluorescence images of a collection of nanospheres with a deconvolved Abbe resolution of 116 nm and a field of view of 10 µm × 10 µm. Our method is robust against optical aberrations and stage drifts, therefore excellent for imaging nanostructures under ambient conditions. [1] M. G. L. Gustafsson, J. Microsc. 198, 82-87 (2000). [2] H. Yilmaz, E. G. van Putten, J. Bertolotti, A. Lagendijk, W. L. Vos, and A. P. Mosk, Optica 2, 424-429 (2015).

  5. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  6. Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators.

    PubMed

    Visser, A J; Ykema, T; van Hoek, A; O'Kane, D J; Lee, J

    1985-03-12

    The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).

  7. Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy

    PubMed Central

    Miles, Benjamin T.; Greenwood, Alexander B.; Benito-Alifonso, David; Tanner, Hugh; Galan, M. Carmen; Verkade, Paul; Gersen, Henkjan

    2017-01-01

    Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle. This shows that the attached gold nanoparticle quenches the fluorescent signal by ~95%, or less likely that the complex has dissociated. In either scenario, the observed fluorescent signal in fact arises from a large population of untagged fluorophores; rendering these labels potentially ineffective and misleading to the field. PMID:28317888

  8. Correlation Between Structural, Spectroscopic, and Reactivity Properties Within a Series of Structurally Analogous Metastable Manganese(III)-Alkylperoxo Complexes

    PubMed Central

    Coggins, Michael K.; Martin-Diaconescu, Vlad; DeBeer, Serena; Kovacs, Julie A.

    2013-01-01

    Manganese–peroxos are proposed as key intermediates in a number of important biochemical and synthetic transformations. Our understanding of the structural, spectroscopic, and reactivity properties of these metastable species is limited, however, and correlations between these properties have yet to be established experimentally. Herein we report the crystallographic structures of a series of structurally related metastable Mn(III)–OOR compounds, and examine their spectroscopic and reactivity properties. The four reported Mn(III)–OOR compounds extend the number of known end-on Mn(III)–(η1-peroxos) to six. The ligand backbone is shown to alter the metal–ligand distances and modulate the electronic properties key to bonding and activation of the peroxo. The mechanism of thermal decay of these metastable species is examined via variable-temperature kinetics. Strong correlations between structural (O–O and Mn⋯Npy,quin distances), spectroscopic (E(πv*(O–O) → Mn CT band), νO–O), and kinetic (ΔH‡ and ΔS‡) parameters for these complexes provide compelling evidence for rate-limiting O–O bond cleavage. Products identified in the final reaction mixtures of Mn(III)–OOR decay are consistent with homolytic O–O bond scission. The N-heterocyclic amines and ligand backbone (Et vs Pr) are found to modulate structural and reactivity properties, and O–O bond activation is shown, both experimentally and theoretically, to track with metal ion Lewis acidity. The peroxo O–O bond is shown to gradually become more activated as the N-heterocyclic amines move closer to the metal ion causing a decrease in π-donation from the peroxo πv*(O–O) orbital. The reported work represents one of very few examples of experimentally verified relationships between structure and function. PMID:23432090

  9. A pilot validation of multi-echo based echo-planar correlated spectroscopic imaging in human calf muscles.

    PubMed

    Furuyama, Jon K; Nagarajan, Rajakumar; Roberts, Christian K; Lee, Cathy C; Hahn, Theodore J; Thomas, M Albert

    2014-10-01

    A current limitation of MR spectroscopic imaging of multiple skeletal muscles is prolonged scan duration. A significant reduction in the total scan duration using the echo-planar correlated spectroscopic imaging (EP-COSI) sequence was accomplished using two bipolar readout trains with different phase-encoded echoes for one of two spatial dimensions within a single repetition time (TR). The second bipolar readout was used for spatially encoding the outer k-space, whereas the first readout was used for the central k-space only. The performance of this novel sequence, called multi-echo based echo-planar correlated spectroscopic imaging (ME-EPCOSI), was demonstrated by localizing specific key features in calf muscles and bone marrow of 11 healthy volunteers and five subjects with type 2 diabetes (T2D). A 3 T MRI-MRS scanner equipped with a transmit-receive extremity coil was used. Localization of the ME-EPCOSI sequence was in good agreement with the earlier single-readout based EP-COSI sequence and the required scan time was reduced by a factor of two. In agreement with an earlier report using single-voxel based 2D MRS, significantly increased unsaturated pools of intramyocellular lipid (IMCL) and extramyocellular lipid (EMCL) and decreased IMCL and EMCL unsaturation indices (UIs) were observed in the soleus and tibialis anterior muscle regions of subjects with T2D compared with healthy controls. In addition, significantly decreased choline content was observed in the soleus of T2D subjects compared with healthy controls. Multi-voxel characterization of IMCL and EMCL ratios and UI in the calf muscle may be useful for the non-invasive assessment of altered lipid metabolism in the pathophysiology of T2D.

  10. Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

    PubMed Central

    Kohl, Tobias; Haustein, Elke; Schwille, Petra

    2005-01-01

    To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

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

    PubMed

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

    2015-09-22

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

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

    PubMed

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

    2008-09-15

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

  15. Experimental and theoretical DFT studies of structure, spectroscopic and fluorescence properties of a new imine oxime derivative

    NASA Astrophysics Data System (ADS)

    Kaya, Yunus; Yilmaz, Veysel T.; Arslan, Taner; Buyukgungor, Orhan

    2012-09-01

    A new imine oxime, (1E,2E)-phenyl-[(1-phenylethyl)imino]-ethanal oxime (I), is synthesized and characterized. The title compound crystallizes in the monoclinic space group P21/c with a = 12.3416(7), b = 9.5990(6), c = 11.9750(7), β = 92.417(4) and Z = 4. Crystallographic, vibrational (IR), and NMR (1H and 13C chemical shifts) data are compared with the results of density functional theory (DFT) method at the B3LYP/6-311++G(d,p) level. The structure of I is stabilized by intermolecular Osbnd H⋯N hydrogen bonds. The theoretical calculations show that the compound exhibits a number of isomers, and the molecular geometry of the most stable optimized isomer (s-trans-E,E) can well reproduce the X-ray structure. The calculated vibrational bands and NMR chemical shifts are consistent with the experimental results. The NBO/NPA atomic charges are performed to explore the possible coordination modes of the compound. The electronic (UV-vis) and photoluminescence spectra calculated using the TD-DFT method are correlated to the experimental spectra. The DMSO solutions of I are fluorescent at room temperature. The assignment and analysis of the frontier HOMO and LUMO orbitals indicates that both absorption and emission bands are originated mainly from the π-π* transitions.

  16. Infrared Spectroscopic Evidences of Strong Electronic Correlations in (Sr1−xLax)3Ir2O7

    PubMed Central

    Ahn, Gihyeon; Song, S. J.; Hogan, T.; Wilson, S. D.; Moon, S. J.

    2016-01-01

    We report on infrared spectroscopic studies of the electronic response of the (Sr1−xLax)3Ir2O7 system. Our experiments revealed hallmarks of strong electronic correlations in the evolution of the electronic response across the filling-controlled insulator-metal transition. We observed a collapse of the Jeff = 1/2 Mott gap accompanying the transfer of the spectral weight from the high-energy region to the gap region with electron doping. The intraband conductivity at the metallic side of the transition was found to consist of coherent Drude-like and incoherent responses. The sum rule and the extended Drude model analyses further indicated a large mass enhancement. Our results demonstrate a critical role of the electronic correlations in the charge dynamics of the (Sr1−xLax)3Ir2O7 system. PMID:27599573

  17. Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations.

    PubMed

    Zinchuk, Vadim; Wu, Yong; Grossenbacher-Zinchuk, Olga; Stefani, Enrico

    2011-09-15

    Interactions of proteins are examined by detecting their overlap using fluorescent markers. The observed overlap is then quantified to serve as a measure of spatial correlation. A major drawback of this approach is that it can produce false values because of the properties of the image background. To remedy this, we provide a protocol to reduce the contribution of image background and then apply a protein proximity index (PPI) and correlation coefficient to estimate colocalization. Background heterogeneity is reduced by the median filtering procedure, comprising two steps, to reduce random noise and background, respectively. Alternatively, background can be reduced by advanced thresholding. PPI provides separate values for each channel to characterize the contribution of each protein, whereas correlation coefficient determines the overall colocalization. The protocol is demonstrated using computer-simulated and real biological images. It minimizes human bias and can be universally applied to various cell types in which there is a need to understand protein-protein interactions. Background reductions require 3-5 min per image. Quantifications take <1 min. The entire procedure takes approximately 15-30 min.

  18. Towards correlative super-resolution fluorescence and electron cryo-microscopy.

    PubMed

    Wolff, Georg; Hagen, Christoph; Grünewald, Kay; Kaufmann, Rainer

    2016-09-01

    Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo-CLEM, the combination of fluorescence cryo-microscopy (cryo-FM) permitting for non-invasive specific multi-colour labelling, with electron cryo-microscopy (cryo-EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence-based information for guiding cryo-EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo-CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano-environment. However, a major obstacle of cryo-CLEM currently hindering many biological applications is the large resolution gap between cryo-FM (typically in the range of ∼400 nm) and cryo-EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super-resolution cryo-FM imaging and the correlation with cryo-EM. This opened the door towards super-resolution cryo-CLEM, and thus towards direct correlation of structural details from both imaging modalities.

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

    PubMed

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

    2010-05-03

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

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed

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

    2002-12-01

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

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

    PubMed

    Hu, Hongyan; Huang, Xiangyi; Ren, Jicun

    2016-05-01

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

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

    PubMed

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

    2017-02-01

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

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

    PubMed

    Hołyst, Robert; Poniewierski, Andrzej; Zhang, Xuzhu

    2017-02-08

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

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

    PubMed

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

    2010-12-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2006-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

  8. [Three-dimensional excitation emission matrix fluorescence spectroscopic characterization of dissolved organic matter in water of coal-mining area].

    PubMed

    Yang, Ce; Zhong, Ning-Ning; Shui, Yu-Lei; Wang, Fei-Yu; Chen, Dang-Yi

    2008-01-01

    Three-dimensional excitation emission matrix was applied to characterize the fluorescence properties of dissolved organic matter in various waters of Shilong coal-mining area. Fluorescence peak I (fulvic-like) and peak II (humic-like) were strong, while peak IV and peak V (protein-like) were weak or even undetected in some samples. Fluorescence peaks in various waters and different zones showed great difference in intensities and the fluorescence peaks in underground water tended to be much lower than those of surface waters. Furthermore, the fluorescence peaks of rivers and lakes were higher than those of mine drainage, and also the fluorescence peaks in coking zone and coal mining zone were higher than those in sewage-irrigated zone, or even much higher than those in farming zone. The reason may be that coal mining activities and coal industry can bring plenty of organic matter from coal to surroundings. Meanwhile, surface water would accept mine drainage, waste water of coal-washing and sewage from daily life easier than underground water, so surface water can be polluted seriously. Fluorescence peaks in waters from coal mining area are little influenced by pH of the water but can be influenced by the content of Ca2+ to water in some extent.

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

    SciTech Connect

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

    2013-12-21

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

  10. Characterization of two quinone radicals in the NADH:ubiquinone oxidoreductase from Escherichia coli by a combined fluorescence spectroscopic and electrochemical approach.

    PubMed

    Hielscher, Ruth; Yegres, Michelle; Voicescu, Mariana; Gnandt, Emmanuel; Friedrich, Thorsten; Hellwig, Petra

    2013-12-17

    The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. It was proposed that the electron transfer involves quinoid groups localized at the end of the electron transfer chain. To identify these groups, fluorescence excitation and emission spectra of Escherichia coli complex I and its fragments, namely, the NADH dehydrogenase fragment containing the flavin mononucleotide and six iron-sulfur (Fe-S) clusters, and the quinone reductase fragment containing three Fe-S clusters were measured. Signals sensitive to reduction by either NADH or dithionite were detected within the complex and the quinone reductase fragment and attributed to the redox transition of protonated ubiquinone radicals. A fluorescence spectroscopic electrochemical redox titration revealed midpoint potentials of -37 and- 235 mV (vs the standard hydrogen electrode) for the redox transitions of the quinone radicals in complex I at pH 6 with an absorption around 325 nm and a fluorescence emission at 460/475 nm. The role of these cofactor(s) for electron transfer is discussed.

  11. Combining spectroscopic and photometric surveys using angular cross-correlations - I. Algorithm and modelling

    NASA Astrophysics Data System (ADS)

    Eriksen, Martin; Gaztañaga, Enrique

    2015-09-01

    Weak lensing (WL) clustering is studied using 2D (angular) coordinates, while redshift space distortions (RSD) and baryon acoustic oscillations (BAO) use 3D coordinates, which requires a model-dependent conversion of angles and redshifts into comoving distances. This is the first paper of a series, which explore modelling multi-tracer galaxy clustering (of WL, BAO and RSD), using only angular (2D) cross-correlations in thin redshift bins. This involves evaluating many thousands cross-correlations, each a multidimensional integral, which is computationally demanding. We present a new algorithm that performs these calculations as matrix operations. Nearby narrow redshift bins are intrinsically correlated, which can be used to recover the full (radial) 3D information. We show that the Limber approximation does not work well for this task. In the exact calculation, both the clustering amplitude and the RSD effect increase when decreasing the redshift bin width. For narrow bins, the cross-correlation has a larger BAO peak than the auto-correlation because smaller scales are filtered out by the radial redshift separation. Moreover, the BAO peak shows a second (ghost) peak, shifted to smaller angles. We explore how WL, RSD and BAO contribute to the cross-correlations as a function of the redshift bin width and present a first exploration of non-linear effects and signal-to-noise ratio on these quantities. This illustrates that the new approach to clustering analysis provides new insights and is potentially viable in practice.

  12. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    PubMed

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues.

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

    PubMed

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

    2008-03-01

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

  14. Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).

    PubMed

    Rich, Ryan M; Mummert, Mark; Gryczynski, Zygmunt; Borejdo, Julian; Sørensen, Thomas Just; Laursen, Bo W; Foldes-Papp, Zeno; Gryczynski, Ignacy; Fudala, Rafal

    2013-05-01

    Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

  15. Elimination of autofluorescence in fluorescence Correlation spectroscopy by using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time correlated single photon counting (TCSPC)

    PubMed Central

    Rich, Ryan M.; Mummert, Mark; Gryczynski, Zygmunt; Borejdo, Julian; Sørensen, Thomas Just; Laursen, Bo W.; Foldes-Papp, Zeno; Gryczynski, Ignacy; Fudala, Rafal

    2013-01-01

    Fluorescence Correlation Spectroscopy (FCS) is a frequently applied technique that allows for precise and sensitive analyses of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time correlated single photon counting (TCSPC) methods. Here, we demonstrate the suppression of autofluorescence in FCS by using ADOTA labeled Hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time-gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time-gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the over-expression of which has been observed in many types of cancer. PMID:23564284

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

    PubMed

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

    2016-03-01

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

  17. Single-Particle Spectroscopic Study on Fluorescence Enhancement by Plasmon Coupled Gold Nanorod Dimers Assembled on DNA Origami.

    PubMed

    Zhang, Taishi; Gao, Nengyue; Li, Shuang; Lang, Matthew J; Xu, Qing-Hua

    2015-06-04

    Metal-enhanced fluorescence has attracted much attention due to its scientific importance and lots of potential applications. Plasmon coupled metal nanoparticles have been demonstrated to further improve the enhancement effects. Conventional studies of metal-enhanced fluorescence on the bulk systems are complicated by the ensemble average effects over many critical factors with large variations. Here, fluorescence enhancement of ATTO-655 by a plasmon coupled gold nanorod dimer fixed on a DNA origami nanobreadboard was studied on the single-particle level. A series of gold nanorod dimers with linear orientation and different gap distances ranging from 6.1 to 26.0 nm were investigated to explore the plasmon coupling effect on fluorescence enhancement. The results show that the dimer with the smallest gap (6.1 nm) gives the highest enhancement (470-fold), and the enhancement gradually decreases as the gap distance increases and eventually approaches that from a monomer (120-fold). This trend is consistent with the numerical calculation results. This study indicates that plasmon coupling in gold nanorod dimers offers further increased excitation efficiency to achieve large fluorescence enhancement.

  18. Fluorescence spectroscopic and calorimetry based approaches to characterize the mode of interaction of small molecules with DNA.

    PubMed

    Banerjee, Amrita; Singh, Jasdeep; Dasgupta, Dipak

    2013-07-01

    Ethidium bromide displacement assay by fluorescence is frequently used as a diagnostic tool to identify the intercalation ability of DNA binding small molecules. Here we have demonstrated that the method has pitfalls. We have employed fluorescence, absorbance and label free technique such as isothermal titration calorimetry to probe the limitations. Ethidium bromide, a non-specific intercalator, netropsin, a (A-T) specific minor groove binder, and sanguinarine, a (G-C) specific intercalator, have been used in our experiments to study the association of a ligand with DNA in presence of a competing ligand. Here we have shown that netropsin quenches the fluorescence intensity of an equilibrium mixture of ethidium bromide - calf thymus DNA via displacement of ethidium bromide. Isothermal titration calorimetry results question the accepted interpretation of the observed decrease in fluorescence of bound ethidium bromide in terms of competitive binding of two ligands to DNA. Furthermore, isothermal titration calorimetry experiments and absorbance measurements indicate that the fluorescence change might be due to formation of ternary complex and not displacement of one ligand by another.

  19. Synthesis, spectroscopic, fluorescence properties and biological evaluation of novel Pd(II) and Cd(II) complexes of NOON tetradentate Schiff bases.

    PubMed

    Ali, Omyma A M

    2014-01-01

    The solid complexes of Pd(II) and Cd(II) with N,N/bis(salicylaldehyde)4,5-dimethyl-1,2-phenylenediamine (H2L(1)), and N,N/bis(salicylaldehyde)4,5-dichloro-1,2-phenylenediamine (H2L(2)) have been synthesized and characterized by several techniques using elemental analysis (CHN), FT-IR, (1)H NMR, UV-Vis spectra and thermal analysis. Elemental analysis data proved 1:1 stoichiometry for the reported complexes while spectroscopic data indicated square planar and octahedral geometries for Pd(II) and Cd(II) complexes, respectively. The prepared ligands, Pd(II) and Cd(II) complexes exhibited intraligand (π-π(∗)) fluorescence and can potentially serve as photoactive materials. Thermal behavior of the complexes was studied and kinetic parameters were determined by Coats-Redfern method. Both the ligands and their complexes have been screened for antimicrobial activities.

  20. 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.

  1. Proton nuclear magnetic resonance and fluorescence spectroscopic studies of segmental mobility in aequorin and a green fluorescent protein from Aequorea forskalea

    SciTech Connect

    Nageswara Rao, B.D.; Kemple, M.D.; Prendergast, F.G.

    1980-10-01

    Aequorin is a protein of low molecular weight (20,000) isolated from the jellyfish Aequorea forskalea which emits blue light upon the binding of Ca/sup 2 +/ ions. This bioluminescence requires neither exogenous oxygen nor any other cofactors. The light emission occurs from an excited state of a chromophore (an imidazolopyrazinone) which is tightly and noncovalently bound to the protein. Apparently the binding of Ca/sup 2 +/ by the protein induces changes in the protein conformation which allow oxygen, already bound or otherwise held by the protein, to react with and therein oxidize the chromophore. The resulting discharged protein remains intact, with the Ca/sup 2 +/ and the chromophore still bound, but is incapable of further luminescence. The fluorescence spectrum of this discharged protein and the bioluminescence spectrum of the original charged aequorin are identical. A green fluorescent protein (GFP) of approx. 30,000 mol wt isolated from the same organism, functions in vivo as an acceptor of energy from aequorin and subsequently emits green light. We are applying proton nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy to examine structural details of, and fluctuations associated with the luminescent reaction of aequorin and the in vivo energy transfer from aequorin to the GFP.

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

    SciTech Connect

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

    1980-03-01

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

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

    PubMed

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

    2013-10-01

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

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

    PubMed

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

    2016-01-01

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

  5. Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector.

    PubMed

    Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

    2015-01-01

    Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

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

    NASA Astrophysics Data System (ADS)

    Swift, Kerry M.; Matayoshi, Edmund D.

    2008-02-01

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

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

    PubMed

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

    2011-07-01

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

  8. Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector

    PubMed Central

    Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

    2015-01-01

    Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD. PMID:26697410

  9. Guided fluorescence diagnosis of childhood caries: preliminary measures correlate with depth of carious decay

    NASA Astrophysics Data System (ADS)

    Timoshchuk, Mari-Alina; Zhang, Liang; Dickinson, Brian A.; Ridge, Jeremy S.; Kim, Amy S.; Baltuck, Camille T.; Nelson, Leonard Y.; Berg, Joel H.; Seibel, Eric J.

    2014-02-01

    The current rise in childhood caries worldwide has increased the demand for portable technologies that can quickly and accurately detect and diagnose early stage carious lesions. These lesions, if identified at an early stage, can be reversed with remineralization treatments, education, and improvements in home care. A multi-modal optical prototype for detecting and diagnosing occlusal caries demineralization in vivo has been developed and pilot tested. The device uses a 405-nm laser as a scanned illumination source to obtain high resolution and high surface contrast reflectance images, which allows the user to quickly image and screen for any signs of demineralized enamel. When a suspicious region is located, the device can be switched to perform dual laser fluorescence spectroscopy using 405-nm and 532-nm laser excitations. These spectra are used to compute an auto-fluorescence (AF) ratio of the suspicious region and the percent difference of AF ratios from a healthy region of the same tooth. The device was tested on 7 children's teeth in vivo with clinically diagnosed carious lesions. Lesion depth was then visually estimated from the video image using the 405-nm scanned light source, and within a month the maximum drill depth was assessed by a clinician. The researcher and clinicians were masked from previous measurements in a blinded study protocol. Preliminary results show that the ratiometric percent difference measurement of the AF spectrum of the tooth correlates with the severity of the demineralization as assessed by the clinician after drilling.

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

    PubMed

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

    2013-06-07

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

  11. Ultraviolet emission and excitation fluorescence spectroscopic characterization of DMBA-treated Swiss Albino mice skin carcinogenesis for measuring tissue transformation

    NASA Astrophysics Data System (ADS)

    Aruna, Prakasa R.; Hemamalini, Srinivasan; Ebenezar, Jeyasingh; Ganesan, Singaravelu

    2002-05-01

    The ultraviolet fluorescence emission spectra of skin tissues under different pathological conditions were measured at 280nm excitation. At this excitation wavelength, the normal skin showed a primary peak emission at 352nm and this primary peak emission from neoplastic skin shows a blue shift with respect to normal tissue. This blue shift increases as the stage of abnormality increases and it is maximum (19nm) for well-differentiated squamous cell carcinoma. This alteration is further confirmed from fluorescence excitation spectra of the tissues for 340nm emission. The study concludes that the change in the emission of tryptophan around 340nm may be due to partial unfolding of protein.

  12. A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy.

    PubMed

    Bacia, Kirsten; Schwille, Petra

    2003-01-01

    Fluorescence correlation spectroscopy (FCS) is becoming increasingly popular as a technique that aims at complementing live cell images with biophysical information. This article provides both a short overview over recent intracellular FCS applications and a practical guide for investigators, who are seeking to integrate FCS into live cell imaging to obtain information on particle mobility, local concentrations, and molecular interactions. A brief introduction to the principles of FCS is provided, particularly emphasizing practical aspects such as the choice of appropriate dyes and positioning of the measurement volume in the sample. Possibilities and limitations in extracting parameters from autocorrelation curves are discussed, and attention is drawn to potential artifacts, such as photobleaching and probe aggregation. The principle of dual-color cross-correlation is reviewed along with considerations for proper setup and adjustment. Practical implications of nonideal conditions including incomplete focus overlap and spectral cross-talk are considered. Recent examples of both auto- and cross-correlation applications demonstrate the potential of FCS for cell biology.

  13. Spectroscopic orbits of two short-period early-type binaries using two-dimensional cross-correlations

    NASA Astrophysics Data System (ADS)

    González, J. F.; Lapasset, E.

    2003-06-01

    We apply the two-dimensional cross-correlation technique TODCOR to derive spectroscopic orbits for the two B-type double-lined spectroscopic binaries HD 66066A and HD 315031, previously mentioned as blue straggler candidates of the open clusters NGC 2516 and NGC 6530, respectively. Reliable radial velocities for both components are measured even for orbital phases for which the separation between the spectral lines are about 0.5 times the quadratic sum of the full-width at half-maximum of the lines. Both binaries have circular orbits and the orbital periods are 1.67 and 1.38 days for HD 66066A and HD 315031, respectively. We calculate minimum masses with errors of 3-5% and obtain the projected radii from the line widths. We derive absolute stellar parameters which are consistent with the age and distance of the clusters. Both binary systems are formed by main-sequence stars and it is expected that they will experience mass-transfer between their components before the end of the core H-burning stage. HD 315031 is likely a triple system as suggested by the variation of the center-of-mass velocity. The observations presented here were obtained at the Complejo Astronómico El Leoncito (CASLEO), which is operated under agreement between the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET) and the National Universities of La Plata, Córdoba and San Juan.

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

    PubMed

    Arbour, Tyler J; Enderlein, Jörg

    2010-05-21

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  17. Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution.

    PubMed

    Löschberger, Anna; Franke, Christian; Krohne, Georg; van de Linde, Sebastian; Sauer, Markus

    2014-10-15

    Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

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

    PubMed

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

    2011-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2006-09-01

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

  20. Diffusion Tensor Analysis by Two-Dimensional Pair Correlation of Fluorescence Fluctuations in Cells.

    PubMed

    Di Rienzo, Carmine; Cardarelli, Francesco; Di Luca, Mariagrazia; Beltram, Fabio; Gratton, Enrico

    2016-08-23

    In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF yields an accurate description of the preferential diffusive routes. Furthermore, we combine this analysis with the image-derived mean-square displacement approach and gain information on the average nanoscopic molecular displacements in different directions. Through these quantities, we build a fluorescence-fluctuation-based diffusion tensor that contains information on speed and directionality of the local dynamical processes. Contrary to classical fluorescence correlation spectroscopy and related methods, this combined approach can distinguish between isotropic and anisotropic local diffusion. We argue that the measurement of this iMSD tensor will contribute to advance our understanding of the role played by the intracellular environment in the regulation of molecular diffusion at the nanoscale.

  1. Carotenoid-chlorophyll coupling and fluorescence quenching correlate with protein packing density in grana-thylakoids.

    PubMed

    Holleboom, Christoph-Peter; Yoo, Sunny; Liao, Pen-Nan; Compton, Ian; Haase, Winfried; Kirchhoff, Helmut; Walla, Peter Jomo

    2013-09-26

    The regulation of light-harvesting in photosynthesis under conditions of varying solar light irradiation is essential for the survival and fitness of plants and algae. It has been proposed that rearrangements of protein distribution in the stacked grana region of thylakoid membranes connected to changes in the electronic pigment-interaction play a key role for this regulation. In particular, carotenoid-chlorophyll interactions seem to be crucial for the down-regulation of photosynthetic light-harvesting. So far, it has been difficult to determine the influence of the dense protein packing found in native photosynthetic membrane on these interactions. We investigated the changes of the electronic couplings between carotenoids and chlorophylls and the quenching in grana thylakoids of varying protein packing density by two-photon spectroscopy, conventional chlorophyll fluorometry, low-temperature fluorescence spectroscopy, and electron micrographs of freeze-fracture membranes. We observed an increasing carotenoid-chlorophyll coupling and fluorescence quenching with increasing packing density. Simultaneously, the antennas size and excitonic connectivity of Photosystem II increased with increasing quenching and carotenoid-chlorophyll coupling whereas isolated, decoupled LHCII trimers decreased. Two distinct quenching data regimes could be identified that show up at different protein packing densities. In the regime corresponding to higher protein packing densities, quenching is strongly correlated to carotenoid-chlorophyll interactions whereas in the second regime, a weak correlation is apparent with low protein packing densities. Native membranes are in the strong-coupling data regime. Consequently, PSII and LHCII in grana membranes of plants are already quenched by protein crowding. We concluded that this ensures efficient electronic connection of all pigment-protein complexes for intermolecular energy transfer to the reaction centers and allows simultaneously

  2. Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells

    PubMed Central

    Hagen, Christoph; Guttmann, Peter; Klupp, Barbara; Werner, Stephan; Rehbein, Stefan; Mettenleiter, Thomas C.; Schneider, Gerd; Grünewald, Kay

    2012-01-01

    Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the ‘water-window’ wavelength region (2.34–4.37 nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach – the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only. PMID:22210307

  3. Revisiting oxo-centered carbonyl-triruthenium clusters: investigating CO photorelease and some spectroscopic and electrochemical correlations.

    PubMed

    Moreira, Mariete B; Da Silva, Camila F N; Pesci, Rafaela B P; Deflon, Victor M; Nikolaou, Sofia

    2016-10-25

    We synthesized and characterized a series of oxo-centered carbonyl-triruthenium complexes with the general formula [Ru3O(CH3COO)6(L)2(CO)], where L = 2,6-dimethylpyrazine (dmpz) (1), isonicotinamide (adpy) (2), 4-acetylpyridine (acpy) (3), 3-methylpyridine (3-pic) (4), 4-methylpyridine (4-pic) (5), 4-tert-butylpyridine (4-tbpy) (6), 4-(dimethyl)aminopyridine (dmap) (7), or 4-aminopyridine (ampy) (8); we also investigated the photoreactivity of these complexes. Single-crystal X-ray diffraction helped to elucidate the structures of 1·H2O, 7·C2H4Cl2, and 8. The unit cell of 8 is composed of four cluster units; the hydrogen bonds between the amino groups of the terminal ligand of a neighboring molecule and the oxygen atoms of CO or acetate bridging ligands hold these cluster units together. The spectroscopic (NMR, UV-visible, and IR) and the electrochemical properties (cyclic voltammetry) of these complexes correlated with the ancillary ligands in terms of their σ-donating and π-accepting characteristics. The molecular orbital and the electronic localized description of the [Ru3O]-CO unit helped to rationalize the correlations. The photoreactivity of compounds 1-8 was investigated by laser excitation at 377 nm. Given the CO photorelease quantum yields, σ-donor ligands and aqueous medium (more polar) stabilized the charge-transfer excited state that culminated in CO photosubstitution, leading to higher Φ values.

  4. Lightweight Raman spectroscope using time-correlated photon-counting detection.

    PubMed

    Meng, Zhaokai; Petrov, Georgi I; Cheng, Shuna; Jo, Javier A; Lehmann, Kevin K; Yakovlev, Vladislav V; Scully, Marlan O

    2015-10-06

    Raman spectroscopy is an important tool in understanding chemical components of various materials. However, the excessive weight and energy consumption of a conventional CCD-based Raman spectrometer forbids its applications under extreme conditions, including unmanned aircraft vehicles (UAVs) and Mars/Moon rovers. In this article, we present a highly sensitive, shot-noise-limited, and ruggedized Raman signal acquisition using a time-correlated photon-counting system. Compared with conventional Raman spectrometers, over 95% weight, 65% energy consumption, and 70% cost could be removed through this design. This technique allows space- and UAV-based Raman spectrometers to robustly perform hyperspectral Raman acquisitions without excessive energy consumption.

  5. Lightweight Raman spectroscope using time-correlated photon-counting detection

    PubMed Central

    Meng, Zhaokai; Petrov, Georgi I.; Cheng, Shuna; Jo, Javier A.; Lehmann, Kevin K.; Yakovlev, Vladislav V.; Scully, Marlan O.

    2015-01-01

    Raman spectroscopy is an important tool in understanding chemical components of various materials. However, the excessive weight and energy consumption of a conventional CCD-based Raman spectrometer forbids its applications under extreme conditions, including unmanned aircraft vehicles (UAVs) and Mars/Moon rovers. In this article, we present a highly sensitive, shot-noise–limited, and ruggedized Raman signal acquisition using a time-correlated photon-counting system. Compared with conventional Raman spectrometers, over 95% weight, 65% energy consumption, and 70% cost could be removed through this design. This technique allows space- and UAV-based Raman spectrometers to robustly perform hyperspectral Raman acquisitions without excessive energy consumption. PMID:26392538

  6. Ultra-narrow spectroscopic cells in atomic spectroscopy: reflection, transmission, fluorescence, and nonadiabatic transitions at the walls

    NASA Astrophysics Data System (ADS)

    Pazgalev, A.; Sarkisyan, D.; Cartaleva, S.; Przhibelskii, S.; Vartanyan, T.

    2014-11-01

    Ultra-narrow cells with the thicknesses in the range from several wavelengths to the small fractions of the wavelength brought a number of new opportunities for atomic spectroscopy. Depending on the cell thickness, spectral lines recorded in ultra-narrow cells are either Doppler-free or Doppler-broadened. With careful selection of the cell thickness hyperfine structure may be easily resolved without resorting on the multibeam nonlinear optical techniques. Moreover, frequent collisions with the walls leads to the important modifications of velocity selective optical pumping resonances. Finally, ultra-narrow cells provide with the unique opportunity to study collisions of the excited atoms with the solid surfaces. In this contribution several examples of the use of the ultra-narrow spectroscopic cells filled with the alkali atomic vapour is presented. First, we discuss general aspects of the transient polarisation that defines all peculiarities of an ultra-narrow cell as a spectroscopic tool. Second, we demonstrate the resolution of the magnetic sublevels in the transition from Zeeman to Paschen-Back regime in the Cs hyperfine structure. Third, new aspects of velocity selective optical pumping resonances in reflection and transmission of resonant radiation by the 6 wavelengths thick cell filled with Cs are discussed. Forth, the experimental evidences of the nonadiabatic transitions between excited states of Rb atoms in the course of collisions with the sapphire surface are presented.

  7. Tryptophan environment, secondary structure and thermal unfolding of the galactose-specific seed lectin from Dolichos lablab: fluorescence and circular dichroism spectroscopic studies.

    PubMed

    Sultan, Nabil Ali Mohammed; Rao, Rameshwaram Nagender; Nadimpalli, Siva Kumar; Swamy, Musti J

    2006-07-01

    Fluorescence and circular dichroism spectroscopic studies were carried out on the galactose-specific lectin from Dolichos lablab seeds (DLL-II). The microenvironment of the tryptophan residues in the lectin under native and denaturing conditions were investigated by quenching of the intrinsic fluorescence of the protein by a neutral quencher (acrylamide), an anionic quencher (iodide ion) and a cationic quencher (cesium ion). The results obtained indicate that the tryptophan residues of DLL-II are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains residing close to at least some of the tryptophan residues under the experimental conditions. Analysis of the far UV CD spectrum of DLL-II revealed that the secondary structure of the lectin consists of 57% alpha-helix, 21% beta-sheet, 7% beta-turns and 15% unordered structures. Carbohydrate binding did not significantly alter the secondary and tertiary structures of the lectin. Thermal unfolding of DLL-II, investigated by monitoring CD signals, showed a sharp transition around 75 degrees C both in the far UV region (205 nm) and the near UV region (289 nm), which shifted to ca. 77-78 degrees C in the presence of 0.1 M methyl-beta-D-galactopyranoside, indicating that ligand binding leads to a moderate stabilization of the lectin structure.

  8. Fluorescence spectroscopic and molecular docking studies of the binding interaction between the new anaplastic lymphoma kinase inhibitor crizotinib and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Abdelhameed, Ali S.; Alanazi, Amer M.; Bakheit, Ahmed H.; Darwish, Hany W.; Ghabbour, Hazem A.; Darwish, Ibrahim A.

    2017-01-01

    Binding of the recently introduced anti-cancer drug, crizotinib (CRB) with the bovine serum albumin (BSA) was comprehensively studied with the aid of fluorescence and UV-Vis spectroscopic as well as molecular docking techniques. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the CRB and BSA with binding constants of 104 L mol- 1. BSA conformational changes were investigated using three dimensional (3D) and synchronous fluorescence measurements. Moreover, the results of site marker competitive experiments and molecular docking, it could be deduced that CRB was inserted into the subdomain IIA (site I) of BSA yielding a more stabilized system. This was further confirmed with the molecular docking results which revealed that CRB is located in the active site residues Try149, Glu152, Ser191, Arg194, Arg198, Trp213, Arg217, Arg256, His287, Ala290, Glu291, Ser343, Asp450 within a radius of 6 Å. Combining the molecular docking studies and the computed thermodynamic parameters, it can be inferred that hydrophobic and electrostatic interactions are the major binding forces involved in formation of the CRB-BSA complex.

  9. Fluorescence spectroscopic and molecular docking studies of the binding interaction between the new anaplastic lymphoma kinase inhibitor crizotinib and bovine serum albumin.

    PubMed

    Abdelhameed, Ali S; Alanazi, Amer M; Bakheit, Ahmed H; Darwish, Hany W; Ghabbour, Hazem A; Darwish, Ibrahim A

    2017-01-15

    Binding of the recently introduced anti-cancer drug, crizotinib (CRB) with the bovine serum albumin (BSA) was comprehensively studied with the aid of fluorescence and UV-Vis spectroscopic as well as molecular docking techniques. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the CRB and BSA with binding constants of 10(4)Lmol(-1). BSA conformational changes were investigated using three dimensional (3D) and synchronous fluorescence measurements. Moreover, the results of site marker competitive experiments and molecular docking, it could be deduced that CRB was inserted into the subdomain IIA (site I) of BSA yielding a more stabilized system. This was further confirmed with the molecular docking results which revealed that CRB is located in the active site residues Try149, Glu152, Ser191, Arg194, Arg198, Trp213, Arg217, Arg256, His287, Ala290, Glu291, Ser343, Asp450 within a radius of 6Å. Combining the molecular docking studies and the computed thermodynamic parameters, it can be inferred that hydrophobic and electrostatic interactions are the major binding forces involved in formation of the CRB-BSA complex.

  10. An Exploratory Study of Spectroscopic Glutamatergic Correlates of Cortical Excitability in Depressed Adolescents

    PubMed Central

    Lewis, Charles P.; Port, John D.; Frye, Mark A.; Vande Voort, Jennifer L.; Ameis, Stephanie H.; Husain, Mustafa M.; Daskalakis, Zafiris J.; Croarkin, Paul E.

    2016-01-01

    Introduction: Transcranial magnetic stimulation (TMS) research has suggested dysfunction in cortical glutamatergic systems in adolescent depression, while proton magnetic resonance spectroscopy (1H-MRS) studies have demonstrated deficits in concentrations of glutamatergic metabolites in depressed individuals in several cortical regions, including the anterior cingulate cortex (ACC). However, few studies have combined TMS and MRS methods to examine relationships between glutamatergic neurochemistry and excitatory and inhibitory neural functions, and none have utilized TMS-MRS methodology in clinical populations or in youth. This exploratory study aimed to examine relationships between TMS measures of cortical excitability and inhibition and concentrations of glutamatergic metabolites as measured by 1H-MRS in depressed adolescents. Methods: Twenty-four adolescents (aged 11–18 years) with depressive symptoms underwent TMS testing, which included measures of the resting motor threshold (RMT), cortical silent period (CSP), short-interval intracortical inhibition (SICI), and intracortical facilitation (ICF). Fourteen participants from the same sample also completed 1H-MRS in a 3 T MRI scanner after TMS testing. Glutamate + glutamine (Glx) concentrations were measured in medial ACC and left primary motor cortex voxels with a TE-optimized PRESS sequence. Metabolite concentrations were corrected for cerebrospinal fluid (CSF) after tissue segmentation. Pearson product-moment and Spearman rank-order correlations were calculated to assess relationships between TMS measures and [Glx]. Results: In the left primary motor cortex voxel, [Glx] had a significant positive correlation with the RMT. In the medial ACC voxel, [Glx] had significant positive correlations with ICF at the 10-ms and 20-ms interstimulus intervals (ISIs). Conclusion: These preliminary data implicate glutamate in cortical excitatory processes measured by TMS. Limitations included small sample size, lack of

  11. Depolarized Photon Correlation Spectroscopic Study of the Glass-Forming Liquid Cumene at Very High Pressures

    NASA Astrophysics Data System (ADS)

    Lyon, Kevin; Ransom, Tim; Oliver, William

    2014-03-01

    In recent years full-spectrum analysis of light-scattering data has been utilized to explore the liquid-glass transition at variable temperatures and ambient pressure. We have developed methods for doing depolarized photon correlation spectroscopy (PCS) in the diamond anvil cell in order to probe directly the structural relaxation time of glass-forming liquids at very high pressures. Here we present results for liquid cumene at 25 C between 1 bar and pressures approaching the room-temperature glass transition at 2.1 GPa. Data along higher-temperature isotherms will also be presented. Methods for minimizing any undesired heterodyne component in the collected light as well as the use of the longitudinal modes of the Brillouin spectrum to aid in the acquisition and spatial filtering of the scattered light will be discussed. Intensity-intensity correlation data were found to be well represented by the KWW equation with a nearly constant stretching parameter of g = 0.66 for 25 C. Furthermore, the relaxation time as a function of pressure is described will using a modified VTF expression: (P)=0exp{DP/(P0-P)}, with values of 0 = 11.9 ps, D = 18.6, and P0 = 3.4 GPa at T = 25 °C. Thus, (P) has been obtained at 25 °C for Cumene over seven decades from about a microsecond to several seconds and is found to be in excellent agreement with previously determined values for the alpha relaxation at lower pressures obtained from Brillouin data [G. Li, et al., Phys. Rev. Lett. 74, 2280 (1995)]. Partially supported by NSF Grant Number: DMR 0552944.

  12. Ultraviolet-Visible (UV-Vis) and Fluorescence Spectroscopic Investigation of the Interactions of Ionic Liquids and Catalase.

    PubMed

    Dong, Xing; Fan, Yunchang; Yang, Peng; Kong, Jichuan; Li, Dandan; Miao, Juan; Hua, Shaofeng; Hu, Chaobing

    2016-11-01

    The inhibitory effects of nine ionic liquids (ILs) on the catalase activity were investigated using fluorescence, absorption ultraviolet-visible spectroscopy. The interactions of ILs and catalase on the molecular level were studied. The experimental results indicated that ILs could inhibit the catalase activity and their inhibitory abilities depended on their chemical structures. Fluorescence experiments showed that hydrogen bonding played an important role in the interaction process. The inhibitory abilities of ILs on catalase activity could be simply described by their hydrophobicity and hydrogen bonding abilities. Unexpected less inhibitory effects of trifluoromethanesulfonate (TfO(-)) might be ascribed to its larger size, which makes it difficult to go through the substrate channel of catalase to the active site.

  13. Correlations between arsenic in Maine groundwater and microbial populations as determined by fluorescence in situ hybridization.

    PubMed

    Weldon, Jennifer M; MacRae, Jean D

    2006-04-01

    Arsenic is known to cause serious health effects when consumed in drinking water. In the state of Maine, approximately half of the population relies on private groundwater wells for their drinking water. Of those wells, as many as 13% may contain arsenic levels above the current EPA maximum contaminant level of 10 microgl(-1). Microorganisms can potentially contribute to arsenic release into groundwater through several mechanisms. Some can reduce arsenate to arsenite, which is more toxic and may be more mobile. Sulfurospirillum species NP4, which was isolated from well water, respires arsenate and could act in this way. Microorganisms can also act indirectly by reducing bedrock surface coatings, such as iron oxyhydroxides, that adsorb arsenic in the groundwater environment. The genus Geobacter contains many species that are capable of iron reduction that could play a role in the indirect release of arsenic into groundwater. Water samples from Northport, ME and the Branch Lake region of Ellsworth, ME, which both have elevated groundwater arsenic levels, have been probed using fluorescence in situ hybridization (FISH), to determine the percentage of the population that is NP4 and the percentage that are Geobacter species. Geobacter abundance correlates well with the total arsenic concentration indicating that indirect mechanisms could be important in releasing arsenic. NP4 appears to be reducing arsenate since its prevalence correlates well with arsenite, the end product of arsenate respiration.

  14. Measurement of the hydrodynamic radius of quantum dots by fluorescence correlation spectroscopy excluding blinking.

    PubMed

    de Thomaz, A A; Almeida, D B; Pelegati, V B; Carvalho, H F; Cesar, C L

    2015-03-19

    One of the most important properties of quantum dots (QDs) is their size. Their size will determine optical properties and in a colloidal medium their range of interaction. The most common techniques used to measure QD size are transmission electron microscopy (TEM) and X-ray diffraction. However, these techniques demand the sample to be dried and under a vacuum. This way any hydrodynamic information is excluded and the preparation process may alter even the size of the QDs. Fluorescence correlation spectroscopy (FCS) is an optical technique with single molecule sensitivity capable of extracting the hydrodynamic radius (HR) of the QDs. The main drawback of FCS is the blinking phenomenon that alters the correlation function implicating in a QD apparent size smaller than it really is. In this work, we developed a method to exclude blinking of the FCS and measured the HR of colloidal QDs. We compared our results with TEM images, and the HR obtained by FCS is higher than the radius measured by TEM. We attribute this difference to the cap layer of the QD that cannot be seen in the TEM images.

  15. Fluorescence spectroscopic analysis of the structure and dynamics of Bacillus subtilis lipase A governing its activity profile under alkaline conditions.

    PubMed

    Kübler, Daniel; Ingenbosch, Kim N; Bergmann, Anna; Weidmann, Monika; Hoffmann-Jacobsen, Kerstin

    2015-12-01

    Because of their vast diversity of substrate specificity and reaction conditions, lipases are versatile materials for biocatalysis. Lipase A from Bacillus subtilis (BSLA) is the smallest lipase yet discovered. It has the typical α/β hydrolase fold but lacks a lid covering the substrate cleft. In this study, the pH-dependence of the activity, stability, structure, and dynamics of BSLA was investigated by fluorescence spectroscopy. By use of a fluorogenic substrate it was revealed that the optimum pH for BSLA activity is 8.5 whereas thermodynamic and kinetic stability are maximum at pH 10. The origin of this behavior was clarified by investigation of ANS (8-anilino-1-naphthalenesulfonic acid) binding and fluorescence quenching of the two single tryptophan mutants W31F and W42F. Variations in segmental dynamics were investigated by use of time-resolved fluorescence anisotropy. This analysis showed that the activity maximum is governed by high surface hydrophobicity and high segmental mobility of surface loops whereas the stability optimum is a result of low segmental mobility and surface hydrophobicity.

  16. Tuning the Spectroscopic Properties of Ratiometric Fluorescent Metal Indicators: Experimental and Computational Studies on Mag-fura-2 and Analogues.

    PubMed

    Zhang, Guangqian; Jacquemin, Denis; Buccella, Daniela

    2017-02-02

    In this joint theoretical and experimental work, we investigate the properties of Mag-fura-2 and seven structurally related fluorescent sensors designed for the ratiometric detection of Mg(2+) cations. The synthesis of three new compounds is described, and the absorption and emission spectra of all of the sensors in both their free and metal-bound forms are reported. A time-dependent density functional theory approach accounting for hydration effects using a hybrid implicit/explicit model is employed to calculate the absorption and fluorescence emission wavelengths, study the origins of the hypsochromic shift caused by metal binding for all of the sensors in this family, and investigate the auxochromic effects of various modifications of the "fura" core. The metal-free forms of the sensors are shown to undergo a strong intramolecular charge transfer upon light absorption, which is largely suppressed by metal complexation, resulting in predominantly locally excited states upon excitation of the metal complexes. Our computational protocol might aid in the design of new generations of fluorescent sensors with low-energy excitation and enhanced properties for ratiometric imaging of metal cations in biological samples.

  17. New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

    PubMed

    Schorb, Martin; Gaechter, Leander; Avinoam, Ori; Sieckmann, Frank; Clarke, Mairi; Bebeacua, Cecilia; Bykov, Yury S; Sonnen, Andreas F-P; Lihl, Reinhard; Briggs, John A G

    2017-02-01

    Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.

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

    PubMed

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

    2016-09-21

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

  19. Picosecond wide-field time-correlated single photon counting fluorescence microscopy with a delay line anode detector

    NASA Astrophysics Data System (ADS)

    Hirvonen, Liisa M.; Becker, Wolfgang; Milnes, James; Conneely, Thomas; Smietana, Stefan; Le Marois, Alix; Jagutzki, Ottmar; Suhling, Klaus

    2016-08-01

    We perform wide-field time-correlated single photon counting-based fluorescence lifetime imaging (FLIM) with a crossed delay line anode image intensifier, where the pulse propagation time yields the photon position. This microchannel plate-based detector was read out with conventional fast timing electronics and mounted on a fluorescence microscope with total internal reflection (TIR) illumination. The picosecond time resolution of this detection system combines low illumination intensity of microwatts with wide-field data collection. This is ideal for fluorescence lifetime imaging of cell membranes using TIR. We show that fluorescence lifetime images of living HeLa cells stained with membrane dye di-4-ANEPPDHQ exhibit a reduced lifetime near the coverslip in TIR compared to epifluorescence FLIM.

  20. Interplay of electron correlations and localization in disordered β-tantalum films: Evidence from dc transport and spectroscopic ellipsometry study

    SciTech Connect

    Kovaleva, N. N.; Chvostova, D.; Dejneka, A.; Bagdinov, A. V.; Petrova, M. G.; Demikhov, E. I.; Pudonin, F. A.

    2015-02-02

    We report the dc transport (5 K ≲ T ≲ 380 K) and spectroscopic ellipsometry (0.8 eV ≤ hν ≤ 8.5 eV, T ≃ 300 K) study of β-Ta films prepared by rf sputtering deposition as a function of their thickness in the range 2.5 nm ≲ d ≲ 200 nm. The dc transport of the β-Ta films with a thickness d ≳ 25 nm is characterized by negative temperature coefficient of resistivity (TCR) caused by localization effects peculiar of highly disordered metals. Their dielectric function spectra display non-metallic-like behavior due to the presence of the pronounced band at 2 eV. We found that with increasing TCR absolute value, specifying elevated degree disorder, the optical spectral weight (SW) of free charge carriers decreases. The associated SW is recovered in the range of Mott-Hubbard transitions, indicating the mechanism of localization enhancement by electronic correlations in disordered metals.

  1. Interplay of electron correlations and localization in disordered β-tantalum films: Evidence from dc transport and spectroscopic ellipsometry study

    NASA Astrophysics Data System (ADS)

    Kovaleva, N. N.; Chvostova, D.; Bagdinov, A. V.; Petrova, M. G.; Demikhov, E. I.; Pudonin, F. A.; Dejneka, A.

    2015-02-01

    We report the dc transport (5 K ≲ T ≲ 380 K) and spectroscopic ellipsometry (0.8 eV ≤ hν ≤ 8.5 eV, T ≃ 300 K) study of β-Ta films prepared by rf sputtering deposition as a function of their thickness in the range 2.5 nm ≲ d ≲ 200 nm. The dc transport of the β-Ta films with a thickness d ≳ 25 nm is characterized by negative temperature coefficient of resistivity (TCR) caused by localization effects peculiar of highly disordered metals. Their dielectric function spectra display non-metallic-like behavior due to the presence of the pronounced band at 2 eV. We found that with increasing TCR absolute value, specifying elevated degree disorder, the optical spectral weight (SW) of free charge carriers decreases. The associated SW is recovered in the range of Mott-Hubbard transitions, indicating the mechanism of localization enhancement by electronic correlations in disordered metals.

  2. The clustering of galaxies in the completed SDSS-III Baryon Oscillation Spectroscopic Survey: combining correlated Gaussian posterior distributions

    NASA Astrophysics Data System (ADS)

    Sánchez, Ariel G.; Grieb, Jan Niklas; Salazar-Albornoz, Salvador; Alam, Shadab; Beutler, Florian; Ross, Ashley J.; Brownstein, Joel R.; Chuang, Chia-Hsun; Cuesta, Antonio J.; Eisenstein, Daniel J.; Kitaura, Francisco-Shu; Percival, Will J.; Prada, Francisco; Rodríguez-Torres, Sergio; Seo, Hee-Jong; Tinker, Jeremy; Tojeiro, Rita; Vargas-Magaña, Mariana; Vazquez, Jose A.; Zhao, Gong-Bo

    2017-01-01

    The cosmological information contained in anisotropic galaxy clustering measurements can often be compressed into a small number of parameters whose posterior distribution is well described by a Gaussian. We present a general methodology to combine these estimates into a single set of consensus constraints that encode the total information of the individual measurements, taking into account the full covariance between the different methods. We illustrate this technique by applying it to combine the results obtained from different clustering analyses, including measurements of the signature of baryon acoustic oscillations and redshift-space distortions, based on a set of mock catalogues of the final SDSS-III Baryon Oscillation Spectroscopic Survey (BOSS). Our results show that the region of the parameter space allowed by the consensus constraints is smaller than that of the individual methods, highlighting the importance of performing multiple analyses on galaxy surveys even when the measurements are highly correlated. This paper is part of a set that analyses the final galaxy clustering data set from BOSS. The methodology presented here is used in Alam et al. to produce the final cosmological constraints from BOSS.

  3. The clustering of galaxies in the completed SDSS-III Baryon Oscillation Spectroscopic Survey: combining correlated Gaussian posterior distributions

    SciTech Connect

    Sánchez, Ariel G.; Grieb, Jan Niklas; Salazar-Albornoz, Salvador; Alam, Shadab; Beutler, Florian; Ross, Ashley J.; Brownstein, Joel R.; Chuang, Chia-Hsun; Cuesta, Antonio J.; Eisenstein, Daniel J.; Kitaura, Francisco-Shu; Percival, Will J.; Prada, Francisco; Rodríguez-Torres, Sergio; Seo, Hee-Jong; Tinker, Jeremy; Tojeiro, Rita; Vargas-Magaña, Mariana; Vazquez, Jose A.; Zhao, Gong-Bo

    2016-09-30

    The cosmological information contained in anisotropic galaxy clustering measurements can often be compressed into a small number of parameters whose posterior distribution is well described by a Gaussian. Here, we present a general methodology to combine these estimates into a single set of consensus constraints that encode the total information of the individual measurements, taking into account the full covariance between the different methods. We also illustrate this technique by applying it to combine the results obtained from different clustering analyses, including measurements of the signature of baryon acoustic oscillations and redshift-space distortions, based on a set of mock catalogues of the final SDSS-III Baryon Oscillation Spectroscopic Survey (BOSS). Our results show that the region of the parameter space allowed by the consensus constraints is smaller than that of the individual methods, highlighting the importance of performing multiple analyses on galaxy surveys even when the measurements are highly correlated. Our paper is part of a set that analyses the final galaxy clustering data set from BOSS. The methodology presented here is used in Alam et al. to produce the final cosmological constraints from BOSS.

  4. The clustering of galaxies in the completed SDSS-III Baryon Oscillation Spectroscopic Survey: combining correlated Gaussian posterior distributions

    DOE PAGES

    Sánchez, Ariel G.; Grieb, Jan Niklas; Salazar-Albornoz, Salvador; ...

    2016-09-30

    The cosmological information contained in anisotropic galaxy clustering measurements can often be compressed into a small number of parameters whose posterior distribution is well described by a Gaussian. Here, we present a general methodology to combine these estimates into a single set of consensus constraints that encode the total information of the individual measurements, taking into account the full covariance between the different methods. We also illustrate this technique by applying it to combine the results obtained from different clustering analyses, including measurements of the signature of baryon acoustic oscillations and redshift-space distortions, based on a set of mock cataloguesmore » of the final SDSS-III Baryon Oscillation Spectroscopic Survey (BOSS). Our results show that the region of the parameter space allowed by the consensus constraints is smaller than that of the individual methods, highlighting the importance of performing multiple analyses on galaxy surveys even when the measurements are highly correlated. Our paper is part of a set that analyses the final galaxy clustering data set from BOSS. The methodology presented here is used in Alam et al. to produce the final cosmological constraints from BOSS.« less

  5. Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

    PubMed Central

    Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

    2017-01-01

    In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample. PMID:28252673

  6. Correlative atomic force and confocal fluorescence microscopy: single molecule imaging and force induced spectral shifts (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Basché, Thomas; Hinze, Gerald; Stöttinger, Sven

    2016-09-01

    A grand challenge in nanoscience is to correlate structure or morphology of individual nano-sized objects with their photo-physical properties. An early example have been measurements of the emission spectra and polarization of single semiconductor quantum dots as well as their crystallographic structure by a combination of confocal fluorescence microscopy and transmission electron microscopy.[1] Recently, the simultaneous use of confocal fluorescence and atomic force microscopy (AFM) has allowed for correlating the morphology/conformation of individual nanoparticle oligomers or molecules with their photo-physics.[2, 3] In particular, we have employed the tip of an AFM cantilever to apply compressive stress to single molecules adsorbed on a surface and follow the effect of the impact on the electronic states of the molecule by fluorescence spectroscopy.[3] Quantum mechanical calculations corroborate that the spectral changes induced by the localized force can be associated to transitions among the different possible conformers of the adsorbed molecule.

  7. Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

    NASA Astrophysics Data System (ADS)

    Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

    2017-03-01

    In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

  8. Co-treatment of fruit and vegetable waste in sludge digesters: Chemical and spectroscopic investigation by fluorescence and Fourier transform infrared spectroscopy.

    PubMed

    Provenzano, Maria Rosaria; Cavallo, Ornella; Malerba, Anna Daniela; Di Maria, Francesco; Cucina, Mirko; Massaccesi, Luisa; Gigliotti, Giovanni

    2016-04-01

    In a previous work co-digestion of food waste and sewage sludge was performed in a pilot apparatus reproducing operating conditions of an existing full scale digester and processing waste mixed sludge (WMS) and fruit and vegetable waste (FVW) at different organic loading rates. An analysis of the relationship among bio-methane generation, process stability and digestate phytotoxicity was conducted. In this paper we considered humification parameters and spectroscopic analysis. Humification parameters indicated a higher not humified fraction (NH) and a lower degree of humification (DH) of FVW with respect to WMS (NH=19.22 and 5.10%; DH=36.65 and 61.94% for FVW and WMS, respectively) associated with their different chemical compositions and with the stabilization process previously undergone by sludge. FVW additions seemed to be favourable from an agronomical point of view since a lower percentage of organic carbon was lost. Fourier transform infrared spectra suggested consumption of aliphatics associated with rising in bio-methane generation followed by accumulation of aliphatics and carboxylic acids when the biogas production dropped. The trend of peaks ratios can be used as an indicator of the process efficiency. Fluorescence intensity of peak B associated with tryptophan-like substances and peak D associated with humic-like substances observed on tridimensional Excitation Emission Matrix maps increased up to sample corresponding to the highest rate of biogas production. Overall spectroscopic results provided evidence of different chemical pathways of anaerobic digestion associated with increasing amount of FVW which led to different levels of biogas production.

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

    PubMed

    Didier, Pascal; Godet, Julien; Mély, Yves

    2009-05-01

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

  10. Absorption and fluorescence spectroscopic characterisation of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry)

    NASA Astrophysics Data System (ADS)

    Shirdel, J.; Zirak, P.; Penzkofer, A.; Breitkreuz, H.; Wolf, E.

    2008-09-01

    The absorption and fluorescence behaviour of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry) in a pH 8 aqueous buffer solution is studied. The flavin adenine dinucleotide (FAD) cofactor of dCry is identified to be present in its oxidized form (FAD ox), and the 5,10-methenyltetrahydrofolate (MTHF) cofactor is found to be hydrolyzed and oxidized to 10-formyldihydrofolate (10-FDHF). The absorption and the fluorescence behaviour of dCry is investigated in the dark-adapted (receptor) state, the light-adapted (signalling) state, and under long-time violet light exposure. Photo-excitation of FAD ox in dCry causes a reductive electron transfer to the formation of anionic FAD semiquinone (FAD rad - ), and photo-excitation of the generated FAD rad - causes an oxidative electron transfer to the back formation of FAD ox. In light adapted dCry a photo-induced equilibrium between FAD ox and FAD rad - exists. The photo-cycle dynamics of signalling state formation and recovery is discussed. Quantum yields of photo-induced signalling state formation of about 0.2 and of photo-induced back-conversion of about 0.2 are determined. A recovery of FAD rad - to FAD ox in the dark with a time constant of 1.6 min at room temperature is found.

  11. Using fluorescence correlation spectroscopy to study diffusion in the presence of a hierarchy of membrane domains

    NASA Astrophysics Data System (ADS)

    Kalay, Ziya

    2014-03-01

    Fluorescence correlation spectroscopy (FCS) is a commonly used experimental technique to study molecular transport, especially in biological systems. FCS is particularly useful in two-dimensional systems such as the cell membrane, where molecules approximately move in a plane over several hundreds of nanometers, and the signal to noise ratio is high. Recent observations showed that proteins and lipids in the plasma membrane (the outermost membrane of a cell) can become temporarily confined in a hierarchy of membrane domains, induced by actin filaments and dynamic clusters formed by lipids and proteins (rafts). There has been considerable interest in measuring the characteristic size and lifetime of these domains via microscopy techniques, including FCS. Even though FCS is widely applicable, interpretation of the results is often indirect, as data has to be fit to model predictions in order to extract transport coefficients. In this talk, I will present our recent theoretical and computational findings on how FCS measurements would reflect diffusion in the simultaneous presence of cytoskeleton induced membrane compartments, and raft-like domains.

  12. Heat shock-induced interactions among nuclear HSFs detected by fluorescence cross-correlation spectroscopy

    SciTech Connect

    Pack, Chan-Gi; Ahn, Sang-Gun

    2015-07-31

    The cellular response to stress is primarily controlled in cells via transcriptional activation by heat shock factor 1 (HSF1). HSF1 is well-known to form homotrimers for activation upon heat shock and subsequently bind to target DNAs, such as heat-shock elements, by forming stress granules. A previous study demonstrated that nuclear HSF1 and HSF2 molecules in live cells interacted with target DNAs on the stress granules. However, the process underlying the binding interactions of HSF family in cells upon heat shock remains unclear. This study demonstrate for the first time that the interaction kinetics among nuclear HSF1, HSF2, and HSF4 upon heat shock can be detected directly in live cells using dual color fluorescence cross-correlation spectroscopy (FCCS). FCCS analyses indicated that the binding between HSFs was dramatically changed by heat shock. Interestingly, the recovery kinetics of interaction between HSF1 molecules after heat shock could be represented by changes in the relative interaction amplitude and mobility. - Highlights: • The binding interactions among nuclear HSFs were successfully detected. • The binding kinetics between HSF1s during recovery was quantified. • HSF2 and HSF4 strongly formed hetero-complex, even before heat shock. • Nuclear HSF2 and HSF4 bound to HSF1 only after heat shock.

  13. Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy.

    PubMed

    Clark, Natalie M; Hinde, Elizabeth; Winter, Cara M; Fisher, Adam P; Crosti, Giuseppe; Blilou, Ikram; Gratton, Enrico; Benfey, Philip N; Sozzani, Rosangela

    2016-06-11

    To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development.

  14. Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy

    PubMed Central

    Clark, Natalie M; Hinde, Elizabeth; Winter, Cara M; Fisher, Adam P; Crosti, Giuseppe; Blilou, Ikram; Gratton, Enrico; Benfey, Philip N; Sozzani, Rosangela

    2016-01-01

    To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development. DOI: http://dx.doi.org/10.7554/eLife.14770.001 PMID:27288545

  15. Exploring doxorubicin localization in eluting TiO2 nanotube arrays through fluorescence correlation spectroscopy analysis.

    PubMed

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

    2012-11-07

    Drug elution properties of TiO(2) nanotube arrays have been largely investigated by means of solely macroscopic observations. Controversial elution performances have been reported so far and a clear comprehension of these phenomena is still missing as a consequence of a lack of molecular investigation methods. Here we propose a way to discern drug elution properties of nanotubes through the evaluation of drug localization by Fluorescence Correlation Spectroscopy (FCS) analysis. We verified this method upon doxorubicin elution from differently loaded TiO(2) nanotubes. Diverse elution profiles were obtained from nanotubes filled by soaking and wet vacuum impregnation methods. Impregnated nanotubes controlled drug diffusion up to thirty days, while soaked samples completed elution in seven days. FCS analysis of doxorubicin motion in loaded nanotubes clarified that more than 90% of drugs dwell preferentially in inter-nanotube spaces in soaked samples due to decorrelation in a 2D fashion, while a 97% fraction of molecules showed 1D mobility ascribable to displacements along the nanotube vertical axis of wet vacuum impregnated nanotubes. The diverse drug localizations inferred from FCS measurements, together with distinct drug-surface interaction strengths resulting from diverse drug filling techniques, could explain the variability in elution kinetics.

  16. Evaluation of electrostatic binding of PAMAM dendrimers and charged phthalocyanines by fluorescence correlation spectroscopy.

    PubMed

    Garcia-Fernandez, Emilio; Paulo, Pedro M R; Costa, Sílvia M B

    2015-02-14

    We have assessed host-guest interactions between PAMAM dendrimers and charged phthalocyanine probes by Fluorescence Correlation Spectroscopy (FCS). Our results show strong binding in water at low ionic strength with an affinity that decreases from KB ∼ 10(9) to 10(8) M(-1) upon decreasing the phthalocyanine charge of z = -4, -2 and -1. The binding affinity also decreases significantly upon salt addition leading to KB values of ca. 10(5)-10(6) M(-1). The changes of binding affinity probed by varying the phthalocyanine charge, and by changing the ionic strength or pH conditions, allowed us to evaluate the electrostatic contribution (Kel) in dendrimer-phthalocyanine interactions. In particular, this approach afforded values of electrostatic potential for PAMAM dendrimers in water at low ionic strength and at dendrimer concentrations in the nanomolar range. The electrostatic potential of PAMAM generations 4 and 7 are around 50 mV in close agreement with theoretical estimates using the Poisson-Boltzmann cell model. Interestingly, the nonelectrostatic binding is significant and contributes even more than electrostatic binding to dendrimer-phthalocyanine interactions. The nonelectrostatic binding contributes to an affinity of KB above 10(5) M(-1), as measured under conditions of low dendrimer charge and high ionic strength, which makes these dendrimers promising hosts as drug carriers.

  17. Phospholipid Diffusion Coefficients of Cushioned Model Membranes determined via Z-Scan Fluorescence Correlation Spectroscopy

    PubMed Central

    Sterling, Sarah M.; Allgeyer, Edward S.; Fick, Jörg; Prudovsky, Igor; Mason, Michael D.; Neivandt, David J.

    2013-01-01

    Model cellular membranes enable the study of biological processes in a controlled environment and reduce the traditional challenges associated with live or fixed cell studies. However, model membrane systems based on the air/water or oil/solution interface do not allow for incorporation of transmembrane proteins, or for the study of protein transport mechanisms. Conversely, a phospholipid bilayer deposited via the Langmuir-Blodgett/Langmuir Schaefer method on a hydrogel layer is potentially an effective mimic of the cross-section of a biological membrane, and facilitates both protein incorporation and transport studies. Prior to application, however, such membranes must be fully characterized, particularly with respect to the phospholipid bilayer phase transition temperature. Here we present a detailed characterization of the phase transition temperature of the inner and outer leaflets of a chitosan supported model membrane system. Specifically, the lateral diffusion coefficient of each individual leaflet has been determined as a function of temperature. Measurements were performed utilizing z-scan fluorescence correlation spectroscopy (FCS), a technique that yields calibration-free diffusion information. Analysis via the method of Wawrezinieck and coworkers, revealed that phospholipid diffusion changes from raft-like to free diffusion as the temperature is increased; an insight into the dynamic behavior of hydrogel supported membranes not previously reported. PMID:23705855

  18. Effects of multiple scattering on fluorescence correlation spectroscopy measurements of particles moving within optically dense media

    NASA Astrophysics Data System (ADS)

    Zustiak, Silviya; Riley, Jason; Boukari, Hacène; Gandjbakhche, Amir; Nossal, Ralph

    2012-12-01

    Fluorescence correlation spectroscopy (FCS) is increasingly being used to assess the movement of particles diffusing in complex, optically dense surroundings, in which case measurement conditions may complicate data interpretation. It is considered how a single-photon FCS measurement can be affected if the sample properties result in scattering of the incident light. FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads are measured, which acted as scatterers. Data indicated that a scattering-linked increase in the illuminated volume, as much as two fold, resulted in minimal increase in diffusivity. To analyze the illuminated beam profile, Monte-Carlo simulations were employed, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, results indicate that multiple scattering does not result in FCS measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media.

  19. A Fluorescence Correlation Spectroscopy Study of the Cryoprotective Mechanism of Glucose on Hemocyanin

    NASA Astrophysics Data System (ADS)

    Hauger, Eric J.

    Cryopreservation is the method of preserving biomaterials by cooling and storing them at very low temperatures. In order to prevent the damaging effects of cooling, cryoprotectants are used to inhibit ice formation. Common cryoprotectants used today include ethylene glycol, propylene glycol, dimethyl sulfoxide, glycerol, and sugars. However, the mechanism responsible for the effectiveness of these cryoprotectants is poorly understood on the molecular level. The water replacement model predicts that water molecules around the surfaces of proteins are replaced with sugar molecules, forming a protective layer against the denaturing ice formation. Under this scheme, one would expect an increase in the hydrodynamic radius with increasing sugar concentration. In order to test this hypothesis, two-photon fluorescence correlation spectroscopy (FCS) was used to measure the hydrodynamic radius of hemocyanin (Hc), an oxygen-carrying protein found in arthropods, in glucose solutions up to 20wt%. FCS found that the hydrodynamic radius was invariant with increasing glucose concentration. Dynamic light scattering (DLS) results verified the hydrodynamic radius of hemocyanin in the absence of glucose. Although this invariant trend seems to indicate that the water replacement hypothesis is invalid the expected glucose layer around the Hc is smaller than the error in the hydrodynamic radius measurements for FCS. The expected change in the hydrodynamic radius with an additional layer of glucose is 1nm, however, the FCS standard error is +/-3.61nm. Therefore, the water replacement model cannot be confirmed nor refuted as a possible explanation for the cryoprotective effects of glucose on Hc.

  20. Mapping Liquid-liquid protein phase separation using ultra-fast-scanning fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Wei, Ming-Tzo; Elbaum-Garfinkle, Shana; Arnold, Craig B.; Priestley, Rodney D.; Brangwynne, Clifford P.

    Intrinsically disordered proteins (IDPs) are an understudied class of proteins that play important roles in a wide variety of biological processes in cells. We've previously shown that the C. elegans IDP LAF-1 phase separates into P granule-like droplets in vitro. However, the physics of the condensed phase remains poorly understood. Here, we use a novel technique, ultra-fast-scanning fluorescence correlation spectroscopy, to study the nano-scale rheological properties of LAF-1 droplets. Ultra-fast-scanning FCS uses a tunable acoustic gradient index of refraction (TAG) lens with an oil immersion objective to control axial movement of the focal point over a length of several micrometers at frequencies of 70kHz. Using ultra-fast-scanning FCS allows for the accurate determination of molecular concentrations and their diffusion coefficient, when the particle is passing through an excitation volume. Our work reveals an asymmetric LAF-1 phase diagram, and demonstrates that LAF-1 droplets are purely viscous phases which are highly tunable by salt concentration.

  1. Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy.

    PubMed

    Mori, Takayasu; Kikuchi, Eriko; Watanabe, Yuko; Fujii, Shinya; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Sohara, Eisei; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

    2013-11-01

    WNKs (with-no-lysine kinases) are the causative genes of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), and form a signal cascade with OSR1 (oxidative stress-responsive 1)/SPAK (STE20/SPS1-related proline/alanine-rich protein kinase) and Slc12a (solute carrier family 12) transporters. We have shown that this signal cascade regulates blood pressure by controlling vascular tone as well as renal NaCl excretion. Therefore agents that inhibit this signal cascade could be a new class of antihypertensive drugs. Since the binding of WNK to OSR1/SPAK kinases was postulated to be important for signal transduction, we sought to discover inhibitors of WNK/SPAK binding by screening chemical compounds that disrupt the binding. For this purpose, we developed a high-throughput screening method using fluorescent correlation spectroscopy. As a result of screening 17000 compounds, we discovered two novel compounds that reproducibly disrupted the binding of WNK to SPAK. Both compounds mediated dose-dependent inhibition of hypotonicity-induced activation of WNK, namely the phosphorylation of SPAK and its downstream transporters NKCC1 (Na/K/Cl cotransporter 1) and NCC (NaCl cotransporter) in cultured cell lines. The two compounds could be the promising seeds of new types of antihypertensive drugs, and the method that we developed could be applied as a general screening method to identify compounds that disrupt the binding of two molecules.

  2. Effect of ethanol-water mixture on the structure and dynamics of lysozyme: A fluorescence correlation spectroscopy study

    NASA Astrophysics Data System (ADS)

    Chattoraj, Shyamtanu; Mandal, Amit Kumar; Bhattacharyya, Kankan

    2014-03-01

    Effect of ethanol-water mixture on the hydrodynamic radius (rH) and conformational dynamics of lysozyme has been studied by circular dichroism, emission spectra, and fluorescence correlation spectroscopy. For this purpose, the protein lysozyme is covalently labeled near the active site with a fluorescent probe, alexa 488. The ethanol molecules are sequestered near the hydrophobic tryptophan residues as indicated by the blue shift of the emission maximum of tryptophan. It is observed that both size (rH) and time constant of conformational relaxation (τR) of lysozyme oscillate with increase in ethanol concentration. The rH of the protein fluctuates from 19 Å in the native state, to a minimum of 13 Å, and a maximum of 29 Å. It is proposed that the oscillating behavior arises from competition between mutual interaction among protein, ethanol, and water. The fluorescence intensity fluctuates because of quenching of the fluorescence of the probe (alexa) by the free amino group of certain residues (e.g., tryptophan). Rate of inter-conversion (folding dynamics) between the open (fluorescent) and closed (non-fluorescent) form has been determined and is found to exhibit similar oscillation with variation in ethanol content.

  3. Correlation of NADH fluorescence lifetime and oxidative phosphorylation metabolism in the osteogenic differentiation of human mesenchymal stem cell

    NASA Astrophysics Data System (ADS)

    Guo, Han-Wen; Yu, Jia-Sin; Hsu, Shu-Han; Wei, Yau-Huei; Lee, Oscar K.; Dong, Chen-Yuan; Wang, Hsing-Wen

    2015-01-01

    Reduced nicotinamide dinucleotide (NADH) fluorescence lifetime has been broadly used as a metabolic indicator for stem cell imaging. However, the direct relationship between NADH fluorescence lifetime and metabolic pathway and activity remains to be clarified. In this study, we measured the NADH fluorescence lifetime of human mesenchymal stem cells (hMSCs) as well as the metabolic indictors, such as adenosine triphosphate (ATP) level, oxygen consumption, and lactate release, up to 4 weeks under normal osteogenic differentiation and oxidative phosphorylation-attenuated/inhibited differentiation by oligomycin A (OA) treatment. NADH fluorescence lifetime was positively correlated with oxygen consumption and ATP level during energy transformation from glycolysis to oxidative phosphorylation. Under OA treatment, oxidative phosphorylation was attenuated/inhibited (i.e., oxygen consumption remained the same as controls or lower), cells showed attenuated differentiation under glycolysis, and NADH fluorescence lifetime change was not detected. Increased expression of the overall complex proteins was observed in addition to Complex I. We suggested special caution needs to be exercised while interpreting NADH fluorescence lifetime signal in terms of stem cell differentiation.

  4. Analytic expression of fluorescence ratio detection correlates with depth in multi-spectral sub-surface imaging

    PubMed Central

    Leblond, F; Ovanesyan, Z; Davis, S C; Valdés, P A; Kim, A; Hartov, A; Wilson, B C; Pogue, B W; Paulsen, K D; Roberts, D W

    2016-01-01

    Here we derived analytical solutions to diffuse light transport in biological tissue based on spectral deformation of diffused near-infrared measurements. These solutions provide a closed-form mathematical expression which predicts that the depth of a fluorescent molecule distribution is linearly related to the logarithm of the ratio of fluorescence at two different wavelengths. The slope and intercept values of the equation depend on the intrinsic values of absorption and reduced scattering of tissue. This linear behavior occurs if the following two conditions are satisfied: the depth is beyond a few millimeters, and the tissue is relatively homogenous. We present experimental measurements acquired with a broad-beam non-contact multi-spectral fluorescence imaging system using a hemoglobin-containing diffusive phantom. Preliminary results confirm that a significant correlation exists between the predicted depth of a distribution of protoporphyrin IX (PpIX) molecules and the measured ratio of fluorescence at two different wavelengths. These results suggest that depth assessment of fluorescence contrast can be achieved in fluorescence-guided surgery to allow improved intra-operative delineation of tumor margins. PMID:21971201

  5. Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

    PubMed

    Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

    2010-07-27

    Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

  6. Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

    PubMed Central

    Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

    2010-01-01

    Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

  7. Co(II), Ni(II) and Cu(II) complexes with coumarin-8-yl Schiff-bases: Spectroscopic, in vitro antimicrobial, DNA cleavage and fluorescence studies

    NASA Astrophysics Data System (ADS)

    Patil, Sangamesh A.; Unki, Shrishila N.; Kulkarni, Ajaykumar D.; Naik, Vinod H.; Badami, Prema S.

    2011-09-01

    A new series of Co(II), Ni(II) and Cu(II) complexes of the type ML·2H 2O of Schiff-bases derived from m-substituted thiosemicarbazides and 8-acetyl-7-hydroxy-4-methylcoumarin have been synthesized and characterized by spectroscopic studies. Schiff-bases exhibit thiol-thione tautomerism wherein sulphur plays an important role in the coordination. The coordination possibility of the Schiff-bases towards metal ions have been proposed in the light of elemental analyses, spectral (IR, UV-vis, FAB-mass, ESR and fluorescence), magnetic and thermal studies. The low molar conductance values in DMF indicate that, the metal complexes are non-electrolytes. The cyclic voltammetric studies suggested that, the Cu(II) and Ni(II) complexes are of single electron transfer quasi-reversible nature. The Schiff-bases and its metal complexes have been evaluated for their in vitro antibacterial ( Escherichia coli, Staphilococcus aureus, Bascillus subtilis and Salmonella typhi) and antifungal activities ( Candida albicans, Cladosporium and Aspergillus niger) by MIC method. The Schiff-base I and its metal complexes exhibited DNA cleavage activity on isolated DNA of A. niger.

  8. Co(II), Ni(II) and Cu(II) complexes with coumarin-8-yl Schiff-bases: spectroscopic, in vitro antimicrobial, DNA cleavage and fluorescence studies.

    PubMed

    Patil, Sangamesh A; Unki, Shrishila N; Kulkarni, Ajaykumar D; Naik, Vinod H; Badami, Prema S

    2011-09-01

    A new series of Co(II), Ni(II) and Cu(II) complexes of the type ML·2H2O of Schiff-bases derived from m-substituted thiosemicarbazides and 8-acetyl-7-hydroxy-4-methylcoumarin have been synthesized and characterized by spectroscopic studies. Schiff-bases exhibit thiol-thione tautomerism wherein sulphur plays an important role in the coordination. The coordination possibility of the Schiff-bases towards metal ions have been proposed in the light of elemental analyses, spectral (IR, UV-vis, FAB-mass, ESR and fluorescence), magnetic and thermal studies. The low molar conductance values in DMF indicate that, the metal complexes are non-electrolytes. The cyclic voltammetric studies suggested that, the Cu(II) and Ni(II) complexes are of single electron transfer quasi-reversible nature. The Schiff-bases and its metal complexes have been evaluated for their in vitro antibacterial (Escherichia coli, Staphilococcus aureus, Bascillus subtilis and Salmonella typhi) and antifungal activities (Candida albicans, Cladosporium and Aspergillus niger) by MIC method. The Schiff-base I and its metal complexes exhibited DNA cleavage activity on isolated DNA of A. niger.

  9. The correlation of the maximum intensity of fluorescence with pigment characteristics of leaves of Betula pendula

    NASA Astrophysics Data System (ADS)

    Zavoruev, V. V.; Zavorueva, E. N.

    2015-11-01

    Using fluorimeter Junior PAM (Heinz Walz GmbH, Germany) the fluorescence parameters of leaves of Betula pendula are investigated. A linear dependence of the maximum fluorescence (Fm) of leaves from the ratio of total chlorophylls concentration to concentration of carotenoids is obtained. Such dependence is found for samples collected during the period of vegetation and for simultaneous selection of colored leaves.

  10. Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach

    PubMed Central

    Lang, Kathrin; Rieder, Renate; Micura, Ronald

    2007-01-01

    Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures. PMID:17693433

  11. Changes in fluorescent dissolved organic matter upon interaction with anionic surfactant as revealed by EEM-PARAFAC and two dimensional correlation spectroscopy.

    PubMed

    Maqbool, Tahir; Hur, Jin

    2016-10-01

    Surfactants are present in significant amounts in both domestic and industrial wastewater, which may interact with dissolved organic matter (DOM). The present study investigated the interactions of sodium dodecyl sulfate (SDS) with three different DOM solutions, including bovine serum albumin (BSA), humic acid (HA), and the mixture of the two (BSA-HA), based on two advanced spectroscopic tools: excitation emission matrix (EEM) combined with parallel factor analysis (EEM-PARAFAC) and two dimensional correlation spectroscopy (2D-COS). The responses of two protein-like components to the addition of SDS differed depending the presence and the absence of HA. A decreasing and an increasing trend was observed for tryptophan-like (C1) and tyrosine-like (C2) components, respectively, in the BSA solution, while the BSA-HA mixture exhibited increasing fluorescence trends for both protein-like components. The conflicting results suggest that HA plays a secondary role in the protein-SDS interactions. No interaction between the SDS and humic-like component was found. 2D-COS combined with fluorescence spectra demonstrated that the protein-SDS interaction occurred on the order of C2 > C1 for the BSA solution but C1 > C2 for the BSA-HA mixture. Analyses of Scatchard plots confirmed the sequential order interpreted from 2D-COS, showing consistent trends in the binding constants. However, the presence of HA affected the protein-SDS interactions in different manners for C1 and C2, enhancing and reducing the binding constants, respectively. Circular dichroism spectra confirmed the occurrence of conformational changes in BSA with SDS. EEM-PARAFAC and 2D-COS successfully explained different interactions of surfactant with protein-like components in the presence of HA.

  12. Kinesin-1 inhibits the aggregation of amyloid-β peptide as detected by fluorescence cross-correlation spectroscopy.

    PubMed

    Zheng, Yanpeng; Tian, Shijun; Peng, Xianglei; Yang, Jingfa; Fu, Yuanhui; Jiao, Yueying; Zhao, Jiang; He, Jinsheng; Hong, Tao

    2016-04-01

    Although the exact etiology and pathogenesis of Alzheimer's disease (AD) are still unclear, amyloid-β (Aβ) generated by the proteolytic processing of amyloid-β precursor protein (APP) aggregate to form toxic amyloid species. Kinesin-1 is the first identified ATP-dependent axonal transport motor protein that has been proven to affect Aβ generation and deposition. In this paper, we applied dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) to investigate the direct interaction of Aβ with kinesin-1 at the single-molecule fluorescence level in vitro. The results showed that two kinds of enhanced green fluorescent protein (EGFP)-tagged kinesin light-chain subunits of kinesin-1(KLCs), KLC-E and E-KLC inhibited the aggregation of Aβ over a period of time, providing additional insight into the mechanism of axonal transport deficits in AD.

  13. Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment.

  14. Two-dimensional correlation spectroscopic analysis on the interaction between humic acids and TiO2 nanoparticles.

    PubMed

    Chen, Wei; Qian, Chen; Liu, Xiao-Yang; Yu, Han-Qing

    2014-10-07

    The elucidation of the interaction between TiO2 nanoparticles (NPs) and natural organic matter (NOM) can help one to better understand the fates, features, and environmental impacts of NPs. In this work, two-dimensional (2D) Fourier transformation infrared (FTIR) correlation spectroscopy (CoS) assisted by the fluorescence excitation-emission matrix (EEM) method is used to explore the interaction mechanism of humic acid (HA) with TiO2 NPs at a molecular level. The results show that the C═O bonds (carboxylate, amide, quinone, or ketone) and C-O bonds (phenol, aliphatic C-OH, and polysaccharide) of HA play important roles in their interaction with TiO2 NPs. The adsorption process of HA onto the surface of TiO2 NPs is different from the bonding process of the two species in solution. The forms of the relevant groups of HA and their consequent reaction with TiO2 NPs are affected to a great extent by the solution pH and the surface charge of NPs. The 2D-FTIR-CoS method is found to be able to construct a comprehensive picture about the NOM-TiO2 NPs interaction process. This 2D-FTIR-CoS approach might also be used to probe other complicated interaction processes in natural and engineered environments.

  15. Spatially Multiplexed Imaging: Fluorescence Correlation Spectroscopy for Efficient Measurement of Molecular Diffusion at Solid-Liquid Interfaces.

    PubMed

    Cooper, Justin T; Harris, Joel M

    2016-04-01

    Fluorescence correlation spectroscopy (FCS) has become an important technique for the characterization of molecular dynamics, especially at interfaces. Fluorescence correlation spectroscopy provides both temporal and spatial resolution for measuring fast processes at equilibrium through analysis of noise in fluorescence intensities from the statistical fluctuations in a small number of molecules. The small molecular populations produce very low-level fluorescence signals, where time-averaging the fluorescence autocorrelation function is needed to generate reasonable signal-to-noise (S/N) ratios. Recently imaging cameras have been adapted to FCS measurements of molecular dynamics at interfaces (membranes and surfaces) through the use of electron-multiplying charge-coupled device (EM-CCD) detectors for acquisition of fluorescence from addressable areas on the detector. This approach provides a major advantage over traditional focused-spot FCS by allowing electronic control over the location and area of the acquired region on the sample surface. Imaging-FCS can also provide a spatial multiplexing advantage through its ability to measure intensity data from larger areas in parallel with no loss of time resolution. In this work, this multiplexing advantage is exploited to determine molecular diffusion rates from the simultaneous measurement of multiple areas on a surface, the autocorrelation traces from which are averaged to improve the S/N ratio. As proof of concept, the diffusion of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) on a C18-modified interface was measured using this multiplexed method and compared to autocorrelation data acquired from a single spot. Due to the slow thermal recovery of the EM-CCD that inhibits fast time-averaging, spatial multiplexing in imaging-FCS provides an eightyfold time savings to reach the same S/N ratio as multiple (time-averaged) measurements from a single spot.

  16. Infrared and Fluorescence Spectroscopic Investigations of the Acyl Surface Modification of Hydrogel Beads for the Deposition of a Phospholipid Coating.

    PubMed

    Grossutti, Michael; Seenath, Ryan; Lipkowski, Jacek

    2015-10-27

    The scaffolded vesicle has been employed as an alternative means of developing natural model membranes and envisioned as a potential nutraceutical transporter. Furthering the research of the scaffolded vesicle system, a nucleophilic substitution reaction was implemented to form an ester linkage between palmitate and terminal hydroxyl groups of dextran in order to hydrophobically modify the hydrogel scaffold. An average tilt angle of 38° of the hydrophobic palmitate modifying layer on the surface of the hydrogel was determined from dichroic ratios obtained from infrared spectra collected in the attenuated total reflection (ATR) configuration. ATR-IR studies of the DMPC-coated acylated hydrogel demonstrated that the hydrocarbon chains of the DMPC coating was similar to those of the DMPC bilayers and that the underlying palmitate layer had a negligible effect on the average tilt angle (26°) of the DMPC coating. The permeability of this acylated hydrogel was investigated with fluorescence spectroscopy and the terbium/dipicolinic acid assay. The hydrophobic modification on the surface of the hydrogel bead allowed for an efficient deposition of a DMPC layer that served as an impermeable barrier to terbium efflux. About 72% of DMPC-coated acylated hydrogel beads showed ideal barrier properties. The remaining 28% were leaking, but the half-life of terbium efflux of the DMPC-coated acylated hydrogel was increasing, and the total amount of leaked terbium was decreasing with the incubation time. The half-life time and the retention were considered a marked improvement relative to past scaffolded vesicle preparations. The process of acylating hydrogel beads for efficient DMPC deposition has been identified as another viable method for controlling the permeability of the scaffolded vesicle.

  17. Spectroscopic constants of diatomic molecules computed correcting Hartree-Fock or general-valence-bond potential-energy curves with correlation-energy functionals

    NASA Astrophysics Data System (ADS)

    Pérez-Jordá, José M.; San-Fabián, Emilio; Moscardó, Federico

    1992-04-01

    The Kohn-Sham energy with exact exchange [using the exact Hartree-Fock (HF) exchange but an approximate correlation-energy functional] may be computed very accurately by adding the correlation obtained from the HF density to the total HF energy. Three density functionals are used: local spin density (LSD), LSD with self-interaction correction, and LSD with generalized gradient correction. This scheme has been extended (Lie-Clementi, Colle-Salvetti, and Moscardo-San-Fabian) to be used with general-valence-bond (GVB) energies and wave functions, so that the extra correlation included in the GVB energy is not counted again. The effect of all these approximate correlations on HF or GVB spectroscopic constants (Re,ωe, and De) is studied. Approximate relations showing how correlation affects them are derived, and may be summarized as follows: (1) the effect on Re and ωe depends only on the correlation derivative at Re, and (2) the effect on De depends mainly on the correlation difference between quasidissociated and equilibrium geometries. A consequence is that all the correlation corrections tested here give larger ωe and De and shorter Re than the uncorrected HF or GVB values. This trend is correct for De for both HF and GVB. For Re and ωe, it is correct in most cases for GVB, but it often fails for the HF cases. A comparison is made with Kohn-Sham calculations with both exchange and correlation approximated. As a final conclusion, it is found that, within the present scheme, a qualitatively correct HF or GVB potential-energy curve, together with a correlation-energy approximation with correct dissociation behavior, is crucial for obtaining good estimates of spectroscopic constants.

  18. 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.

  19. Reduced lifetimes are directly correlated with excitation irradiance in metal-enhanced fluorescence (MEF).

    PubMed

    Karolin, Jan O; Geddes, Chris D

    2012-11-01

    We describe a fundamental observation in Metal-Enhanced Fluorescence (MEF), which has become a leading technology in the life sciences today, namely, how the lifetime of fluorophores near-to metallic plasmon-supporting silver islands/nanoparticles, modulates as a function of excitation power irradiance. This finding is in stark contrast to that observed in classical far-field fluorescence spectroscopy, where excitation power does not influence fluorophore radiative decay/lifetime.

  20. 5-HT spatial distribution imaging with multiphoton excitation of 5-HT correlative visible fluorescence in live cells

    NASA Astrophysics Data System (ADS)

    Zhang, Zhihong; Zeng, Shaoqun; Liu, Yafeng; Zhou, Wei; Chen, Tongsheng; Luo, Qingming

    2002-04-01

    The autofluorescence of 5-Hydroxytryptamine (5-HT) loaded rat mucosal mast cells (RBL-2H3 cells) is imaged with multiphoton excitation laser scanning microscope (MPELSM). 5-HT correlative visible fluorescence (Fco-vis) excited with 740-nm multiphoton excitation is observed in live cells for the first time, and the generating mechanism of 5-HT Fco-vis is studied. The spatial distribution of 5-HT in live cells is imaged at high spatial resolution in our experiment, which provides a new way to study the correlation between 5-HT spatial distribution and content, and the cellular functional state in live tissue or cells.

  1. Investigating the Correlation between Miscibility and Physical Stability of Amorphous Solid Dispersions Using Fluorescence-Based Techniques.

    PubMed

    Tian, Bin; Tang, Xing; Taylor, Lynne S

    2016-11-07

    The purpose of this study was to investigate the feasibility of using a fluorescence-based technique to evaluate drug-polymer miscibility and to probe the correlation between miscibility and physical stability of amorphous solid dispersions (ASDs). Indomethacin-hydroxypropyl methylcellulose (IDM-HPMC), indomethacin-hydroxypropyl methylcellulose acetate succinate, and indomethacin-polyvinylpyrrolidone (IDM-PVP) were used as model systems. The miscibility of the IDM-polymer systems was evaluated by fluorescence spectroscopy, fluorescence imaging, differential scanning calorimetry (DSC), and infrared (IR) spectroscopy. The physical stability of IDM-polymer ASDs stored at 40 °C was evaluated using fluorescence imaging and X-ray diffraction (XRD). The experimentally determined miscibility limit of IDM with the polymers was 50-60%, 20-30%, and 70-80% drug loading for HPMC, HPMCAS, and PVP, respectively. The X-ray results showed that for IDM-HPMC ASDs, samples with a drug loading of less than 50% were maintained in amorphous form during the study period, while samples with drug loadings higher than 50% crystallized within 15 days. For IDM-HPMCAS ASDs, samples with drug loading less than 30% remained amorphous, while samples with drug loadings higher than 30% crystallized within 10 days. IDM-PVP ASDs were found to be resistant to crystallization for all compositions. Thus, a good correlation was observed between phase separation and reduced physical stability, suggesting that miscibility is indeed an important ASDs characteristic. In addition, fluorescence-based techniques show promise in the evaluation of drug-polymer miscibility.

  2. Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Michel, Patrik O; Gilbert, Leona; Lahtinen, Tomi; Marjomäki, Varpu; Hedman, Klaus; Vuento, Matti; Oker-Blom, Christian

    2004-01-01

    Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and past-immunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2 s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5 x 10(-8) cm2 s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a past-immunity serum sample gave an average diffusion coefficient of 3.5 x 10(-8) cm2 s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7 x 10(-7) to 1.6 x 10(-7) cm2 s(-1), which is much smaller than that observed with acute phase or past-immunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and past-immunity sera. FCS measurement of VLP-immune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.

  3. Lipid Diffusion in Supported Lipid Bilayers: A Comparison between Line-Scanning Fluorescence Correlation Spectroscopy and Single-Particle Tracking

    PubMed Central

    Rose, Markus; Hirmiz, Nehad; Moran-Mirabal, Jose M.; Fradin, Cécile

    2015-01-01

    Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 μm2 · s−1 and DSPT ~ 2 μm2 · s−1, respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D ≳ 1 μm2 · s−1 for FCS and D ≲ 5 μm2 · s−1 for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusion of loosely-bound proteins hovering above the membrane. SPT, on the other hand, is ideal to study the motions of membrane-inserted proteins, especially those presenting different conformations, but only allows studying lipid diffusion in relatively viscous membranes, such as supported lipid bilayers and cell membranes. PMID:26610279

  4. Artifact Free and Detection Profile Independent Higher Order Fluorescence Correlation Spectroscopy for Microsecond Resolved Kinetics. 2. Mixtures and Reactions.

    PubMed

    Abdollah-Nia, Farshad; Gelfand, Martin P; Van Orden, Alan K

    2017-02-09

    Fluorescence correlation spectroscopy (FCS) is a primary tool in the time-resolved analysis of non-reacting or reacting molecules in solution, based on fluorescence intensity fluctuations. However, conventional FCS alone is insufficient for complete determination of reaction or mixture parameters. In an accompanying article, a technique for computation of artifact-free higher-order correlations with microsecond time resolution was described. Here, we demonstrate applications of the technique to analyze systems of fast and slow reactions. As an example of slow- or non-reacting systems, the technique is applied to resolve two-component mixtures of labeled oligonucleotides. Next, the protonation reaction of fluorescein isothiocyanate (FITC) in phosphate buffer is analyzed as an example of fast reactions (relaxation time <1 μs ). By reference to an (apparent) non-reacting system, the simple factorized form of cumulant-based higher-order correlations is exploited to remove the dependence on the molecular detection function (MDF). Therefore, there is no need to model and characterize the experimental MDF, and the precision and the accuracy of the technique are enhanced. It is verified that higher-order correlation analysis enables complete and simultaneous determination of number and brightness parameters of mixing or reacting molecules, the reaction relaxation time, and forward and reverse reaction rates.

  5. Correlation of tryptophan fluorescence intensity decay parameters with sup 1 H NMR-determined rotamer conformations: (tryptophan sup 2 )oxytocin

    SciTech Connect

    Ross, J.B.A.; Schwartz, G.P.; Laws, W.R. ); Wyssbrod, H.R.; Porter, R.A. ); Michaels, C.A. )

    1992-02-18

    While the fluorescence decay kinetics of tyrosine model compounds can be explained in terms of heterogeneity derived from the three ground-state {chi}{sup 1} rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from {chi}{sup 2} rotations. In this paper, the time-resolved and steady-state fluorescence properties of (tryptophan{sup 2})oxytocin at pH 3 are presented and compared with {sup 1}H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the {sup 1}H NMR-determined {chi}{sup 1} rotamer populations of the indole side chain. {sup 15}N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the {chi}{sup 1} rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two {chi}{sup 2} populations.

  6. Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy.

    PubMed

    Papadopoulos, Dimitrios K; Krmpot, Aleksandar J; Nikolić, Stanko N; Krautz, Robert; Terenius, Lars; Tomancak, Pavel; Rigler, Rudolf; Gehring, Walter J; Vukojević, Vladana

    2015-11-01

    Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC). We show that the Hox transcription factor Sex combs reduced (Scr) forms dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. Our studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, we also provide mechanistic evidence using massively parallel FCS (mpFCS). We found that Scr dimers are predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. Our proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.

  7. Correlation Of Balkan Endemic Nephropathy With Fluorescent Organic Compounds In Shallow Groundwater

    NASA Astrophysics Data System (ADS)

    Goldberg, Marvin C.; Feder, Gerald L.; Radovanovic, Zoran

    1994-04-01

    Balkan endemic nephropathy (BEN) is a disease of intersitial nephropathy leading to end-stage renal failure. The disease occurs in persons living in villages on alluvial valleys of streams tributary to the Danube River in Rumania, Bulgaria and former Yugoslavia. The etiologic agent is not known, but a contaminant in shallow groundwater has become suspect. In this study, samples of drinking water from endemic and non-endemic village water supplies were analyzed by excitation/emission matrix (EEM) fluorescence spectroscopy. Spectra characteristic of groundwater from BEN households show elongated teardrop shapes in the fluorescence excitation/emission matrix. A sharp rise occurs in fluorescence emission between 380 and 400 nanometers (nm) and a trailing emission intensity from 400 to 550 nm. Spectra of groundwater samples from some BEN households have an additional excitation maxima at 300 nm, which further contributes to the emission intensity at 400 nm. Spectra of water samples from non-BEN households located in endemic villages show characteristics of BEN household waters, exhibiting the 250-nm excitation peak, even though the fluorophoric intensity is much less than that in samples from BEN household waters. Samples from non-endemic villages do not show the characteristic EEM spectra described as "teardrop shaped". The non-BEN households have lower concentrations of these fluorophores in the drinking water than the endemic households; hence, one of the factors in contracting the disease may be the concentration of these fluorescent materials in drinking water.

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

    PubMed

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

    2007-05-01

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

  9. In Situ Characterization of Protein Adsorption onto Nanoparticles by Fluorescence Correlation Spectroscopy.

    PubMed

    Shang, Li; Nienhaus, G Ulrich

    2017-02-21

    Nanotechnology holds great promise for applications in many fields including biology and medicine. Unfortunately, the processes occurring at the interface between nanomaterials and living systems are exceedingly complex and not yet well understood, which has significantly hampered the realization of many nanobiotechnology applications. Whenever nanoparticles (NPs) are incorporated by a living organism, a protein adsorption layer, also known as the "protein corona", forms on the NP surface. Accordingly, living organisms interact with protein-coated rather than bare NPs, and their biological responses depend on the nature of the protein corona. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP-protein interactions. In most studies, NPs are immersed in protein or biofluid (e.g., blood serum) solutions and then separated from the liquid for analysis. Because this approach may modify the composition and structure of the protein corona, our group has pioneered the use of fluorescence correlation spectroscopy (FCS) as an in situ technique, capable of examining NP-protein interactions while the NPs are suspended in biological fluids. FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. This Account aims at reviewing recent progress in the exploration of NP-protein interactions by using FCS. In vitro FCS studies of the adsorption of important serum proteins onto water-solubilized luminescent NPs always showed a stepwise increase of the NP radius upon protein binding in the form of a binding isotherm, regardless of the type of NP and its specific surface functionalization. This observation indicates formation of a protein monolayer on the NP. Structure-based calculations of protein surface potentials revealed that positively charged patches on the proteins interact electrostatically with

  10. ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy

    PubMed Central

    2016-01-01

    In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables ‘smart collection’ of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables ‘smart tracking’ of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes. PMID:28090593

  11. Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos.

    PubMed

    Pozzi, Paolo; Sironi, Laura; D'Alfonso, Laura; Bouzin, Margaux; Collini, Maddalena; Chirico, Giuseppe; Pallavicini, Piersandro; Cotelli, Franco; Foglia, Efrem A

    2014-06-01

    Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms-3 μm time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos' vessels.

  12. Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pozzi, Paolo; Sironi, Laura; D'Alfonso, Laura; Bouzin, Margaux; Collini, Maddalena; Chirico, Giuseppe; Pallavicini, Piersandro; Cotelli, Franco; Foglia, Efrem A.

    2014-06-01

    Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms-3 μm time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos' vessels.

  13. Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross correlation spectroscopy.

    PubMed

    Wu, Chia-Yan; Huang, Chuan-Keng; Chung, Chao-Yu; Huang, I-Ping; Hwu, Yeukuang; Yang, Chung-Shi; Lai, Yiu-Kay; Lo, Leu-Wei; Chiang, Su-Yu

    2011-05-21

    Dual color fluorescence cross correlation spectroscopy (FCCS) was used to investigate quantitatively the binding kinetics of tumor necrosis factor (TNFα) with TNFα antibody (anti-TNFα) following fluorescent labeling. Through the analysis of the auto correlation curves of fluorescence correlation spectroscopy (FCS), diffusion coefficients of 100.06 ± 4.9 μm(2) s(-1) and 48.96 ± 2.52 μm(2) s(-1) for Alexa488-TNFα and Atto647N-anti-TNFα were obtained. In addition, the calculated hydrodynamic diameters of the Alexa488-TNFα and Atto647N-anti-TNFα were approximately 4.89 ± 0.24 nm and 9.99 ± 0.52 nm, respectively, which agrees with the values of 5.20 ± 1.23 nm and 9.28 ± 0.86 nm for the native TNFα and the anti-TNFα as determined from dynamic light scattering measurements. For the binding kinetics, association (k(on)) and dissociation (k(off)) rate constants were (1.13 ± 0.08) × 10(4) M(-1) s(-1) and (1.53 ± 0.19) × 10(-3) s(-1) while the corresponding dissociation constant (K(d)) at 25 °C was (1.36 ± 0.10) × 10(-7) M. We believe this is the first report on the binding kinetics for TNFα-antibody recognition in the homogeneous phase. Using this technology, we have shown that controlled experiments can be performed to gain insight into molecular mechanisms involved in the immune response.

  14. Anomalous Diffusion in Polymer Solution as Probed by Fluorescence Correlation Spectroscopy and Its Universal Importance in Biological Systems

    NASA Astrophysics Data System (ADS)

    Ushida, Kiminori

    2008-02-01

    Experimental evidence of anomalous diffusion occurring in an inhomogeneous media (hyaluronan aquous solution) was obtained by use of fluorescence correlation spectroscopy (FCS) combined with other techniques (PFG-NMR and Photochemical reactions). The diffusion coefficient was obtained as a function of diffusion time or diffusion distance. Since this polymer solution can be regarded as a model system of extracellular matrices (ECMs), intercellular communication, which takes part in ECM, is greatly influenced by this anomalous diffusion mode. Therefore universal importance of anomalous diffusion in biological activity is identified in this series of independent experiments to measure diffusion coefficients.

  15. Adulteration screening of botanical materials by a sensitive and model-free approach using infrared spectroscopic imaging and two-dimensional correlation infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Chen, Jian-bo; Zhou, Qun; Sun, Su-qin

    2016-11-01

    Infrared (IR) spectroscopy is often used as a simple, fast, and green method for the adulteration screening of botanical materials for foods and herbs. However, the overlapping of absorption signals of various substances significantly decrease the sensitivity and specificity of IR spectroscopy in the detection of adulterated samples. In this research, a model-free approach is proposed for the sensitive and non-targeted screening of botanical materials adulterated by adding other plant materials. First, the spectra of the entities in the test sample are collected by near-infrared spectroscopic imaging and clustered by unsupervised pattern recognition methods. The sample may be adulterated if there are two or more clusters of the entities. Next, the entities of different clusters are characterized by mid-infrared spectroscopy to interpret the chemical compositions to determine the clustering is caused whether by adulteration or other reasons. Second derivative spectroscopy and two-dimensional correlation spectroscopy are often needed to resolve the overlapped bands mathematically or experimentally to find the characteristic signals to identify the authentic and adulterant entities. The feasibility of this approach was proved by the simulated adulterated sample of saffron. In conclusion, botanical materials adulterated by adding other plant materials can be detected by a simple, fast, sensitive, and green screening approach using IR spectroscopic imaging, two-dimensional correlation spectroscopy, and necessary chemometrics techniques.

  16. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  17. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  18. Characterization of binding site heterogeneity for copper within dissolved organic matter fractions using two-dimensional correlation fluorescence spectroscopy.

    PubMed

    Hur, Jin; Lee, Bo-Mi

    2011-06-01

    The heterogeneity of copper binding characteristics for dissolved organic matter (DOM) fractions was investigated based on the fluorescence quenching of the synchronous fluorescence spectra upon the addition of copper and two-dimensional correlation spectroscopy (2D-COS). Hydrophobic acid (HoA) and hydrophilic (Hi) fractions of two different DOM (algal and leaf litter DOM) were used for this study. For both DOM, fluorescence quenching occurred at a wider range of wavelengths for the HoA fractions compared to the Hi fractions. The combined information of the synchronous and asynchronous maps derived from 2D-COS provided a clear picture of the heterogeneous distribution of the copper binding sites within each DOM fraction, which was not readily recognized by a simple comparison of the changes in the synchronous fluorescence spectra upon the addition of copper. For the algal DOM, higher stability constants were exhibited for the HoA versus the Hi fractions. The logarithms of the stability constants ranged from 4.8 to 6.1 and from 4.5 to 5.0 for the HoA and the Hi fractions of the algal DOM, respectively, depending on the associated wavelength and the fitted models. In contrast, no distinctive difference in the binding characteristics was found between the two fractions of the leaf litter DOM. This suggests that influences of the structural and chemical properties of DOM on copper binding may differ for DOM from different sources. The relative difference of the calculated stability constants within the DOM fractions were consistent with the sequential orders interpreted from the asynchronous 2D-COS. It is expected that 2D-COS will be widely applied to other DOM studies requiring detailed information on the heterogeneous nature and subsequent effects under a range of environmental conditions.

  19. Fluorescence correlation spectroscopy reveals highly efficient cytosolic delivery of certain penta-arg proteins and stapled peptides.

    PubMed

    LaRochelle, Jonathan R; Cobb, Garrett B; Steinauer, Angela; Rhoades, Elizabeth; Schepartz, Alanna

    2015-02-25

    We used fluorescence correlation spectroscopy (FCS) to accurately and precisely determine the relative efficiencies with which three families of "cell-penetrating peptides" traffic to the cytosol of mammalian cells. We find that certain molecules containing a "penta-arg" motif reach the cytosol, intact, with efficiencies greater than 50%. This value is at least 10-fold higher than that observed for the widely studied cationic sequence derived from HIV Tat or polyarginine Arg8, and equals that of hydrocarbon-stapled peptides that are active in cells and animals. Moreover, we show that the efficiency with which stapled peptides reach the cytosol, as determined by FCS, correlates directly with their efficacy in cell-based assays. We expect that these findings and the associated technology will aid the design of peptides, proteins, and peptide mimetics that predictably and efficiently reach the interior of mammalian cells.

  20. Lack of Correlation Between the Spatial Distribution of A2E and Lipofuscin Fluorescence in the Human Retinal Pigment Epithelium

    PubMed Central

    Ablonczy, Zsolt; Higbee, Daniel; Anderson, David M.; Dahrouj, Mohammad; Grey, Angus C.; Gutierrez, Danielle; Koutalos, Yiannis; Schey, Kevin L.; Hanneken, Anne; Crouch, Rosalie K.

    2013-01-01

    Purpose. The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE. Methods. Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on flat-mounted RPE tissue sections and retinal cross-sections. Results. Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased toward the central region. High-resolution MALDI-IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels. Conclusions. This report to our knowledge is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging. PMID:23847313

  1. Fluorescence correlation spectroscopy: an efficient tool for measuring size, size-distribution and polydispersity of microemulsion droplets in solution.

    PubMed

    Pal, Nibedita; Dev Verma, Sachin; Singh, Moirangthem Kiran; Sen, Sobhan

    2011-10-15

    Fluorescence correlation spectroscopy (FCS) is an ideal tool for measuring molecular diffusion and size under extremely dilute conditions. However, the power of FCS has not been utilized to its best to measure diffusion and size parameters of complex chemical systems. Here, we apply FCS to measure the size, and, most importantly, the size distribution and polydispersity of a supramolecular nanostructure (i.e., microemulsion droplets, MEDs) in dilute solution. It is shown how the refractive index mismatch of a solution can be corrected in FCS to obtain accurate size parameters of particles, bypassing the optical matching problem of light scattering techniques that are used often for particle-size measurements. We studied the MEDs of 13 different W(0) values from 2 to 50 prepared in a ternary mixture of water, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and isooctane, with sulforhodamine-B as a fluorescent marker. We find that, near the optical matching point of MEDs, the dynamic light scattering (DLS) measurements underestimate the droplet sizes while FCS estimates the accurate ones. A Gaussian distribution model (GDM) and a maximum-entropy-based FCS data fitting model (MEMFCS) are used to analyze the fluorescence correlation curves that unfold Gaussian-type size distributions of MEDs in solution. We find the droplet size varies linearly with W(0) up to ~20, but beyond this W(0) value, the size variation deviates from this linearity. To explain nonlinear variation of droplet size for W(0) values beyond ~20, we invoke a model (the coated-droplet model) that incorporates the size polydispersity of the droplets.

  2. [Effect of temperature on the aggregation behavior of collagen solution by two-dimensional synchronous fluorescence correlation spectroscopy].

    PubMed

    Wu, Wan-ye; Wu, Kun; Li, Guo-ying

    2015-02-01

    The synchronous fluorescence spectroscopy and two dimensional correlation analysis method were applied to study the aggregation behavior of acid-soluble collagen solutions (0.2, 0.4 and 1.6 mg x mL(-1)) during the heating process of 10-70 degrees C. It was found that the fluorescence excited at 292 and 282 nm (delta lamda=9 nm) belongs to the tyrosine (Tyr) residues which participate in forming hydrogen bonds or not, respectively. The two dimensional correlation analysis with the temperature varying showed that with the temperature increased (10-30 degrees C) hydrogen bonds among collagen molecular with Tyr residues formed in the 0.2 mg x mL(-1) collagen solution, while the higher aggregations of collagen molecular and hydrophobic micro-domains appeared in the 0.4 and 1.6 mg x mL(-1) collagen solutions. With approaching the denatured temperature of collagen (36-38 degrees C), the hydrophobic micro-domain and aggregates seemed to be broken in the 0.4 and 1.6 mg x mL(-1) collagen solutions, however the hydrogen bonds in the 0.2 mg x mL(-1) were stable. Above the denaturation temperature of collagen, the triple-helix structure of collagen molecular in solution of each concentration tended to be loose. In the heating process of 45-70 degrees C, this trend was more obvious.

  3. Photon Correlation versus Interference of Single-Atom Fluorescence in a Half-Cavity

    SciTech Connect

    Dubin, Francois; Rotter, Daniel; Mukherjee, Manas; Russo, Carlos; Eschner, Juergen; Blatt, Rainer

    2007-05-04

    Photon correlations are investigated for a single laser-excited ion trapped in front of a mirror. Varying the relative distance between the ion and the mirror, photon correlation statistics can be tuned smoothly from an antibunching minimum to a bunchinglike maximum. Our analysis concerns the non-Markovian regime of the ion-mirror interaction and reveals the field establishment in a half-cavity interferometer.

  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. Correlated analysis of chemical variations with spectroscopic features of the K-Na jarosite solid solutions relevant to Mars

    NASA Astrophysics Data System (ADS)

    Ling, Zongcheng; Cao, Fengke; Ni, Yuheng; Wu, Zhongchen; Zhang, Jiang; Li, Bo

    2016-06-01

    Detailed chemical, structural and spectroscopic properties of jarosite solid solution minerals are key information for their potential discoveries by future remote sensing and in-situ detections on Mars. We successfully synthesized seven homogeneous K-Na jarosite solid solutions under hydrothermal conditions at 140 °C, whose phase identifications and chemical compositions are confirmed by X-ray diffraction (XRD) and inductively coupled plasma mass spectrometry (ICP-MS). The chemical ratios of K/(K+Na) in jarosite solid solutions lead to systematic shifts of their characteristic Raman peaks ν1 (SO4)2- (from 1006 to 1011.3 cm-1), ν3 (SO4)2- (from 1100.6 to 1111.2 cm-1), ν2 (SO4)2- (from 434.2 to 444.8 cm-1) with the increase of Na content. While the OH stretching mode decreases with even larger peak position variations (e.g., ∼3410 cm-1 peak shifts from 3410.5 to 3385.7 cm-1) as the K-Na jarosite solid solutions are enriched in Na content. Raman spectroscopic measurements of the seven K-Na jarosite solid solutions enabled us to build a calibration that uses Raman peak positions to estimate K-Na variation in jarosite, which is the key step for their possible applications in the future Raman applications on Mars' missions (e.g., ExoMars and Mars 2020 missions). The band assignments and compositional related variations of their XRD, near-infrared (NIR) and mid-infrared (MIR) spectra also provide informative clues for identifying the jarosite minerals and inferring their composition during martian in-situ and remote sensing measurements.

  6. Correlations between nuclear and fluorescent Imaging of mammary tumors in mice

    NASA Astrophysics Data System (ADS)

    Carroll, Robin; Stone, John; Blue, Eric; Bradley, Eric; Qian, Jianguo; Saha, Margaret; Welsh, Robert

    2008-10-01

    Progress with new imaging technologies permits the study of biological processes both in vivo and noninvasively. Two systems, a position-sensitive gamma camera and a cooled-CCD camera have been applied in this work. A C3H strain of mouse carrying the Mouse Mammary Tumor Virus (MMTV) was imaged using 800 nm Q-tracker fluorescent dots conjugated to a peptide targeting integrin αυβ C a mammary marker for angiogenesis. We subsequently imaged with the gamma camera to detect low levels of ^125I distribution, and hence, the activity of a trans-membrane protein called the sodium iodide symporter (NIS) responsible for iodine transport. Preliminary results indicate that the biodistribution of the tagged Q-tracker dots and ^125I co-localize very early in seemingly normal mammary glands of infected MMTV mice, while in larger palpable tumors the Q-dot signals are less apparent in comparison with the^125I signal.

  7. G-quadruplex hinders translocation of BLM helicase on DNA: a real-time fluorescence spectroscopic unwinding study and comparison with duplex substrates.

    PubMed

    Liu, Jia-quan; Chen, Chang-yue; Xue, Yong; Hao, Yu-hua; Tan, Zheng

    2010-08-04

    Sequences with the potential to form G-quadruplex structures are spread throughout genomic DNA. G-quadruplexes in promoter regions can play regulatory roles in gene expression. Expression of protein-encoding genes involves processing of DNA and RNA molecules at the level of transcription and translation, respectively. In order to examine how the G-quadruplex affects processing of nucleic acids, we established a real-time fluorescent assay and studied the unwinding of intramolecular G-quadruplex formed by the human telomere, ILPR and PSMA4 sequences by the BLM helicase. Through comparison with their corresponding duplex substrates, we found that the unwinding of intramolecular G-quadruplex structures was much less efficient than that of the duplexes. This result is in contrast to previous reports that multistranded intermolecular G-quadruplexes are far better substrates for the BLM and other RecQ family helicases. In addition, the unwinding efficiency varied significantly among the G-quadruplex structures, which correlated with the stability of the structures. These facts suggest that G-quadruplex has the capability to modulate the processing of DNA and RNA molecules in a stability-dependent manner and, as a consequence, may provide a mechanism to play regulatory roles in events such as gene expression.

  8. Structural studies on Si:H network before and after solid phase crystallization using spectroscopic ellipsometry: Correlation with Raman spectroscopy and transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Goswami, Romyani; Ray, Swati

    2013-10-01

    The structure of hydrogenated silicon films (Si:H) before and after solid phase crystallization (SPC) has been investigated by detailed study of spectroscopic ellipsometry (SE). The Si:H films have been deposited by radio frequency plasma enhanced chemical vapor deposition (RF PECVD) system varying deposition power density from 0.03 W/cm2 to 0.46 W/cm2, just below the onset of amorphous to nano-crystalline transition region. Solid phase crystallization of the Si:H network has been done by thermal annealing of the films in a vacuum furnace. Different bulk compositions of the as deposited Si:H network and annealed (polycrystalline) films have been calculated from the fitted parameters obtained from the simulation of the ellipsometry data by Bruggeman effective medium approximation (BEMA) method. More compact and void free structure in the bulk layer of the as deposited films has been observed at low power deposition region. Whereas void fraction in the bulk and surface roughness layer has increased with increase of deposition power density. For the annealed films higher crystallinity at the bulk layer with fewer voids has been observed at the low power region but in the surface roughness layer void fraction dominates in all the low and high power deposited films. The results obtained from the spectroscopic ellipsometry study have been correlated with Raman spectroscopy and transmission electron microscopy for both the as deposited and annealed films.

  9. Shedding light on the photostability of two intermolecular charge-transfer complexes between highly fluorescent bis-1,8-naphthalimide dyes and some π-acceptors: A spectroscopic study in solution and solid states

    NASA Astrophysics Data System (ADS)

    Refat, Moamen S.; Ismail, Lamia A.; Adam, Abdel Majid A.

    2015-01-01

    Given the great importance of the various uses of 1,8-naphthalimides in the trends of biology, medicine and industry, the current study focused on extending the scope of these dyes by introducing some of their charge-transfer (CT) complexes. For this purpose, two highly fluorescent bis-1,8-naphthalimide dyes and their complexes with some π-acceptors have been synthesized and characterized spectroscopically. The π-acceptors include picric acid (PA), chloranilic acid (CLA), tetracyanoquinodimethane (TCNQ) and dichlorodicyanobenzoquinone (DDQ). The molecular structure, spectroscopic and fluorescence properties as well as the binding modes were deduced from IR, UV-vis and 1H NMR spectral studies. The binding ratio of complexation was determined to be 1:1 according to the elemental analyses and photometric titrations. It has been found that the order of acceptance ability for the different acceptors is TCNQ > DDQ > CLA > PA. The photostability of 1,8-naphthalimide dye as a donor and its charge-transfer complex doped in polymethyl methacrylate/PMMA were exposed to UV-Vis radiation and the change in the absorption spectra was achieved at different times during irradiation period.

  10. Label-free fluorescence detection of aromatic compounds in chip electrophoresis applying two-photon excitation and time-correlated single-photon counting.

    PubMed

    Beyreiss, Reinhild; Geißler, David; Ohla, Stefan; Nagl, Stefan; Posch, Tjorben Nils; Belder, Detlev

    2013-09-03

    In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

  11. Fungal Biodegradative Oxidants in Lignocellulose: Fluorescence Mapping and Correlation With Gene Expression

    SciTech Connect

    Hammel, Kenneth E.; Ralph, John; Hunt, Christopher G.; Houtman, Carl J.

    2016-09-06

    This work focused on new methods for the detection of oxidation in natural substrates during the deconstruction of lignocellulose by microoganisms. Oxidation was the focus because all known biological systems that degrade lignin are oxidative. The detection methods involved the used of (a) micrometer-scale beads carrying a fluorescent dye that is sensitive to oxidation, (b) 13C-labeled synthetic lignins whose breakdown products can be assessed using mass spectrometry and nuclear magnetic resonance spectroscopy, and (c) a fluorometric stain that is highly sensitive to incipient oxidation during microbial attack. The results showed (a) that one white rot fungus, Phanerochaete chrysosporium, produces diffusible oxidants on wood, and that the onset of oxidation is coincident with the marked up-regulation of genes that encode ligninolytic peroxidases and auxiliary oxidative enzymes; (b) that a more selectively ligninolytic white rot fungus, Ceriporiopsis subvermispora, produces a highly diastereoselective oxidative system for attack on lignin; (c) that a brown rot fungus, Serpula lacrymans, uses extracellular hydroquinone metabolites to drive the production of lignocellulose-oxidizing free radicals; (d) that both white rot and brown rot fungi produce highly diffusible mild oxidants that modify lignocellulose at the earliest stage of substrate deconstruction; and (e) that lignin degradation in a tropical soil is not inhibited as much as expected during periods of flooding-induced hypoxia, which indicates that unknown mechanisms for attack on lignin remain to be discovered.

  12. Correlating structure with fluorescence emission in phase-separated conjugated-polymer blends.

    PubMed

    Chappell, John; Lidzey, David G; Jukes, Paul C; Higgins, Anthony M; Thompson, Richard L; O'Connor, Stephen; Grizzi, Ilaria; Fletcher, Robert; O'Brien, Jim; Geoghegan, Mark; Jones, Richard A L

    2003-09-01

    Blends of conjugated polymers are frequently used as the active semiconducting layer in light-emitting diodes and photovoltaic devices. Here we report the use of scanning near-field optical microscopy, scanning force microscopy and nuclear-reaction analysis to study the structure of a thin film of a phase-separated blend of two conjugated polymers prepared by spin-casting. We show that in addition to the well-known micrometre-scale phase-separated morphology of the blend, one of the polymers preferentially wets the surface and forms a 10-nm-thick, partially crystallized wetting layer. Using near-field microscopy we identify unexpected changes in the fluorescence emission from the blend that occurs in a 300-nm-wide band located at the interface between the different phase-separated domains. Our measurements provide an insight into the complex structure of phase-separated conjugated-polymer thin films. Characterizing and controlling the properties of the interfaces in such films will be critical in the further development of efficient optoelectronic devices.

  13. Artifact-Free and Detection-Profile-Independent Higher-Order Fluorescence Correlation Spectroscopy for Microsecond-Resolved Kinetics. 1. Multidetector and Sub-Binning Approach.

    PubMed

    Abdollah-Nia, Farshad; Gelfand, Martin P; Van Orden, Alan

    2017-03-10

    Fluorescence correlation spectroscopy (FCS) is a powerful tool in the time-resolved analysis of nonreacting or reacting molecules in solution, based on fluorescence intensity fluctuations. However, conventional (second-order) FCS alone is insufficient to measure all parameters needed to describe a reaction or mixture, including concentrations, fluorescence brightnesses, and forward and reverse rate constants. For this purpose, correlations of higher powers of fluorescence intensity fluctuations can be calculated to yield additional information from the single-photon data stream collected in an FCS experiment. To describe systems of diffusing and reacting molecules, considering cumulants of fluorescence intensity results in simple expressions in which the reaction and diffusion parts factorize. The computation of higher-order correlations in experiments is hindered by shot-noise and common detector artifacts, the effects of which become worse with increasing order. In this article, we introduce a technique to calculate artifact-free higher-order correlation functions with improved time resolution, and without any need for modeling and calibration of detector artifacts. The technique is formulated for general multidetector experiments and verified in both two-detector and single-detector configurations. Good signal-to-noise ratio is achieved down to 1 μs in correlation curves up to order (2, 2). This capability makes possible a variety of new measurements including multicomponent analysis and fast reaction kinetics, as demonstrated in a companion article (10.1021/acs.jpcb.7b00408).

  14. Correlative super-resolution fluorescence and metal replica transmission electron microscopy

    PubMed Central

    Sochacki, Kem A.; Shtengel, Gleb; van Engelenburg, Schuyler B.; Hess, Harald F.; Taraska, Justin W.

    2014-01-01

    Super-resolution localization microscopy is combined with a complementary imaging technique, transmission electron microscopy of metal replicas, to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. Robust correlation on the scale of 20 nm is validated by imaging endogenous clathrin (with 2D and 3D PALM/TEM) and the method is further used to find the previously unknown 3D position of epsin on clathrin coated structures. PMID:24464288

  15. Effects of humic substances on the bioconcentration of polycyclic aromatic hydrocarbons: Correlations with spectroscopic and chemical properties of humic substances

    USGS Publications Warehouse

    Haitzer, M.; Abbt-Braun, G.; Traunspurger, W.; Steinberg, C.E.W.

    1999-01-01

    The presence of dissolved humic substances (HS, fulvic and humic acids) generally reduces the uptake of hydrophobic organic compounds into aquatic organisms. The extent of this effect depends both on the concentration and on the origin of the HS. The aim of this study was to investigate the role of qualitative differences between HS from different origins. The effects of seven different HS on the bioconcentration of pyrene and benzo[a]pyrene (BaP) in the nematode Caenorhabditis elegans were related to the spectroscopic and chemical properties of the HS. The effect of each humic material on the bioconcentration of pyrene or BaP was quantified as a 'biologically determined' partition coefficient K(DOC). We observed significant linear relationships between K(DOC) and the atomic H/C ratio, the specific absorptivity at 254 nm, the content of aromatic carbons (as determined by 13C nuclear magnetic resonance spectroscopy, the copper-complexing capacity, the content of phenolic OH groups, and the molecular weight of the HS. There was no discernible relationship of K(DOC) with the atomic (N + O)/C ratio, an indicator of the polarity of HS. Taken together, our results show that the variability in the effects of HS from different origins could be related to variations in bulk properties of the HS. Parameters describing the aromaticity of the humic materials seemed to be most useful for estimating effects of HS on the bioconcentration of pyrene and BaP.

  16. Effects of humic substances on the bioconcentration of polycyclic aromatic hydrocarbons: Correlations with spectroscopic and chemical properties of humic substances

    SciTech Connect

    Haitzer, M.; Abbt-Braun, G.; Traunspurger, W.; Steinberg, C.E.W.

    1999-12-01

    The presence of dissolved humic substances (HS, fulvic and humic acids) generally reduces the uptake of hydrophobic organic compounds into aquatic organisms. The extent of this effect depends both on the concentration and on the origin of the HS. The aim of this study was to investigate the role of qualitative differences between HS from different origins. The effects of seven different HS on the bioconcentration of pyrene and benzo[a]pyrene (BaP) in the nematode Caenorhabditis elegans were related to the spectroscopic and chemical properties of the HS. The effect of each humic material on the bioconcentration of pyrene or BaP was quantified as a biologically determined partition coefficient K{sub DOC}. The authors observed significant linear relationships between K{sub DOC} and the atomic H/C ratio, the specific absorptivity at 254 nm, the content of aromatic carbons as determined by {sup 13}C nuclear magnetic resonance spectroscopy, the copper-complexing capacity, the content of phenolic OH groups, and the molecular weight of the HS. There was no discernible relationship of K{sub DOC} with the atomic (N + O)/C ratio, an indicator of the polarity of HS. Taken together, their results show that the variability in the effects of HS from different origins could be related to variations in bulk properties of the HS. Parameters describing the aromaticity of the humic materials seemed to be most useful for estimating effects of HS on the bioconcentration of pyrene and BaP.

  17. Correlated light and electron microscopy observations of the uterine epithelial cell actin cytoskeleton using fluorescently labeled resin-embedded sections.

    PubMed

    Moore, Chad L; Cheng, Delfine; Shami, Gerald J; Murphy, Christopher R

    2016-05-01

    In order to perform correlative light and electron microscopy (CLEM) more precisely, we have modified existing specimen preparation protocols allowing fluorescence retention within embedded and sectioned tissue, facilitating direct observation across length scales. We detail a protocol which provides a precise correlation accuracy using accessible techniques in biological specimen preparation. By combining a pre-embedding uranyl acetate staining step with the progressive lowering of temperature (PLT) technique, a methacrylate embedded tissue specimen is ultrathin sectioned and mounted onto a TEM finder grid for immediate viewing in the confocal and electron microscope. In this study, the protocol is applied to rat uterine epithelial cells in vivo during early pregnancy. Correlative overlay data was used to track changes in filamentous actin that occurs in these cells from fertilization (Day 1) to implantation on Day 6 as part of the plasma membrane transformation, a process essential in the development of uterine receptivity in the rat. CLEM confirmed that the actin cytoskeleton is disrupted as apical microvilli are progressively lost toward implantation, and revealed the thick and continuous terminal web is replaced by a thinner and irregular actin band, with individually distinguishable filaments connecting actin meshworks which correspond with remaining plasma membrane protrusions.

  18. Real-time autocorrelator for fluorescence correlation spectroscopy based on graphical-processor-unit architecture: method, implementation, and comparative studies

    NASA Astrophysics Data System (ADS)

    Laracuente, Nicholas; Grossman, Carl

    2013-03-01

    We developed an algorithm and software to calculate autocorrelation functions from real-time photon-counting data using the fast, parallel capabilities of graphical processor units (GPUs). Recent developments in hardware and software have allowed for general purpose computing with inexpensive GPU hardware. These devices are more suited for emulating hardware autocorrelators than traditional CPU-based software applications by emphasizing parallel throughput over sequential speed. Incoming data are binned in a standard multi-tau scheme with configurable points-per-bin size and are mapped into a GPU memory pattern to reduce time-expensive memory access. Applications include dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS) experiments. We ran the software on a 64-core graphics pci card in a 3.2 GHz Intel i5 CPU based computer running Linux. FCS measurements were made on Alexa-546 and Texas Red dyes in a standard buffer (PBS). Software correlations were compared to hardware correlator measurements on the same signals. Supported by HHMI and Swarthmore College

  19. Solar-induced chlorophyll fluorescence that correlates with canopy photosynthesis on diurnal and seasonal scales in a temperate deciduous forest

    NASA Astrophysics Data System (ADS)

    Yang, Xi; Tang, Jianwu; Mustard, John F.; Lee, Jung-Eun; Rossini, Micol; Joiner, Joanna; Munger, J. William; Kornfeld, Ari; Richardson, Andrew D.

    2015-04-01

    Previous studies have suggested that solar-induced chlorophyll fluorescence (SIF) is correlated with Gross Primary Production (GPP). However, it remains unclear to what extent this relationship is due to absorbed photosynthetically active radiation (APAR) and/or light use efficiency (LUE). Here we present the first time series of near-surface measurement of canopy-scale SIF at 760 nm in temperate deciduous forests. SIF correlated with GPP estimated with eddy covariance at diurnal and seasonal scales (r2 = 0.82 and 0.73, respectively), as well as with APAR diurnally and seasonally (r2 = 0.90 and 0.80, respectively). SIF/APAR is significantly positively correlated with LUE and is higher during cloudy days than sunny days. Weekly tower-based SIF agreed with SIF from the Global Ozone Monitoring Experiment-2 (r2 = 0.82). Our results provide ground-based evidence that SIF is directly related to both APAR and LUE and thus GPP, and confirm that satellite SIF can be used as a proxy for GPP.

  20. Fluorescence detection of esophageal neoplasia

    NASA Astrophysics Data System (ADS)

    Borisova, E.; Vladimirov, B.; Avramov, L.

    2008-06-01

    White-light endoscopy is well-established and wide used modality. However, despite the many technological advances that have been occurred, conventional endoscopy is suboptimal and usually detects advanced stage lesions. The limitations of standard endoscopy initiate development of spectroscopic techniques, additional to standard endoscopic equipment. One of the most sensitive approaches is fluorescence spectroscopy of gastrointestinal mucosa for neoplasia detection. In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus. The 5-ALA is administered per os six hours before measurements at dose 20 mg/kg weight. Excitation source has max of emission at 405 nm and light is delivered by the standard light guide of the endoscopic equipment. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to reabsorption of blood. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of 5-ALA/PpIX only in abnormal sites Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

  1. Dual-channel red/blue fluorescence dosimetry with broadband reflectance spectroscopic correction measures protoporphyrin IX production during photodynamic therapy of actinic keratosis

    NASA Astrophysics Data System (ADS)

    Kanick, Stephen Chad; Davis, Scott C.; Zhao, Yan; Hasan, Tayyaba; Maytin, Edward V.; Pogue, Brian W.; Chapman, M. Shane

    2014-07-01

    Dosimetry for aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy of actinic keratosis was examined with an optimized fluorescence dosimeter to measure PpIX during treatment. While insufficient PpIX generation may be an indicator of incomplete response, there exists no standardized method to quantitate PpIX production at depths in the skin during clinical treatments. In this study, a spectrometer-based point probe dosimeter system was used to sample PpIX fluorescence from superficial (blue wavelength excitation) and deeper (red wavelength excitation) tissue layers. Broadband white light spectroscopy (WLS) was used to monitor aspects of vascular physiology and inform a correction of fluorescence for the background optical properties. Measurements in tissue phantoms showed accurate recovery of blood volume fraction and reduced scattering coefficient from WLS, and a linear response of PpIX fluorescence versus concentration down to 1.95 and 250 nM for blue and red excitations, respectively. A pilot clinical study of 19 patients receiving 1-h ALA incubation before treatment showed high intrinsic variance in PpIX fluorescence with a standard deviation/mean ratio of >0.9. PpIX fluorescence was significantly higher in patients reporting higher pain levels on a visual analog scale. These pilot data suggest that patient-specific PpIX quantitation may predict outcome response.

  2. Dual-channel red/blue fluorescence dosimetry with broadband reflectance spectroscopic correction measures protoporphyrin IX production during photodynamic therapy of actinic keratosis

    PubMed Central

    Kanick, Stephen Chad; Davis, Scott C.; Zhao, Yan; Hasan, Tayyaba; Maytin, Edward V.; Pogue, Brian W.; Chapman, M. Shane

    2014-01-01

    Abstract. Dosimetry for aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy of actinic keratosis was examined with an optimized fluorescence dosimeter to measure PpIX during treatment. While insufficient PpIX generation may be an indicator of incomplete response, there exists no standardized method to quantitate PpIX production at depths in the skin during clinical treatments. In this study, a spectrometer-based point probe dosimeter system was used to sample PpIX fluorescence from superficial (blue wavelength excitation) and deeper (red wavelength excitation) tissue layers. Broadband white light spectroscopy (WLS) was used to monitor aspects of vascular physiology and inform a correction of fluorescence for the background optical properties. Measurements in tissue phantoms showed accurate recovery of blood volume fraction and reduced scattering coefficient from WLS, and a linear response of PpIX fluorescence versus concentration down to 1.95 and 250 nM for blue and red excitations, respectively. A pilot clinical study of 19 patients receiving 1-h ALA incubation before treatment showed high intrinsic variance in PpIX fluorescence with a standard deviation/mean ratio of >0.9. PpIX fluorescence was significantly higher in patients reporting higher pain levels on a visual analog scale. These pilot data suggest that patient-specific PpIX quantitation may predict outcome response. PMID:24996661

  3. Study of layer-by-layer films on thermoresponsive nanogels using temperature-controlled dual-focus fluorescence correlation spectroscopy.

    PubMed

    Wong, John E; Müller, Claus B; Díez-Pascual, Ana M; Richtering, Walter

    2009-12-10

    While a few studies have reported on the layer-by-layer (LbL) assembly of polyelectrolytes on soft and porous templates, none have really demonstrated direct proof that the layers are actually on the template. Thermoresponsive nanogels present challenges that render a quantitative proof of successful polyelectrolyte deposition extremely difficult. Additionally, the fate of the polyelectrolyte has never been investigated during the phase transition of the coated nanogel. Here, the auto- and cross-correlation functions of a labeled polyelectrolyte assembled via the LbL technique onto soft and porous thermoresponsive labeled nanogels using dual-focus fluorescence correlation spectroscopy (2f-FCS) are presented. Performing 2f-FCS as a function of temperature, hydrodynamic radii of nanogels coated with various numbers of layers are determined, which are found to be in excellent agreement with values obtained from dynamic light scattering. This study presents irrefutable quantitative evidence of successful LbL assembly on thermoresponsive nanogels and demonstrates that the layers are not stripped off during the phase transition of the nanogels. Forster Resonance Energy Transfer (FRET) detection also supports our findings.

  4. The clustering of galaxies in the completed SDSS-III Baryon Oscillation Spectroscopic Survey: observational systematics and baryon acoustic oscillations in the correlation function

    NASA Astrophysics Data System (ADS)

    Ross, Ashley J.; Beutler, Florian; Chuang, Chia-Hsun; Pellejero-Ibanez, Marcos; Seo, Hee-Jong; Vargas-Magaña, Mariana; Cuesta, Antonio J.; Percival, Will J.; Burden, Angela; Sánchez, Ariel G.; Grieb, Jan Niklas; Reid, Beth; Brownstein, Joel R.; Dawson, Kyle S.; Eisenstein, Daniel J.; Ho, Shirley; Kitaura, Francisco-Shu; Nichol, Robert C.; Olmstead, Matthew D.; Prada, Francisco; Rodríguez-Torres, Sergio A.; Saito, Shun; Salazar-Albornoz, Salvador; Schneider, Donald P.; Thomas, Daniel; Tinker, Jeremy; Tojeiro, Rita; Wang, Yuting; White, Martin; Zhao, Gong-bo

    2017-01-01

    We present baryon acoustic oscillation (BAO) scale measurements determined from the clustering of 1.2 million massive galaxies with redshifts 0.2 < z < 0.75 distributed over 9300 deg2, as quantified by their redshift-space correlation function. In order to facilitate these measurements, we define, describe, and motivate the selection function for galaxies in the final data release (DR12) of the SDSS III Baryon Oscillation Spectroscopic Survey (BOSS). This includes the observational footprint, masks for image quality and Galactic extinction, and weights to account for density relationships intrinsic to the imaging and spectroscopic portions of the survey. We simulate the observed systematic trends in mock galaxy samples and demonstrate that they impart no bias on BAO scale measurements and have a minor impact on the recovered statistical uncertainty. We measure transverse and radial BAO distance measurements in 0.2 < z < 0.5, 0.5 < z < 0.75, and (overlapping) 0.4 < z < 0.6 redshift bins. In each redshift bin, we obtain a precision that is 2.7 per cent or better on the radial distance and 1.6 per cent or better on the transverse distance. The combination of the redshift bins represents 1.8 per cent precision on the radial distance and 1.1 per cent precision on the transverse distance. This paper is part of a set that analyses the final galaxy clustering data set from BOSS. The measurements and likelihoods presented here are combined with others in Alam et al. to produce the final cosmological constraints from BOSS.

  5. Validation of diffuse correlation spectroscopic measurement of cerebral blood flow using phase-encoded velocity mapping magnetic resonance imaging

    PubMed Central

    Hance, Dalton; Pawlowski, Thomas; Lynch, Jennifer; Wilson, Felice B.; Mesquita, Rickson C.; Durduran, Turgut; Diaz, Laura K.; Putt, Mary E.; Licht, Daniel J.; Fogel, Mark A.; Yodh, Arjun G.

    2012-01-01

    Abstract. Diffuse correlation spectroscopy (DCS) is a novel optical technique that appears to be an excellent tool for assessing cerebral blood flow in a continuous and non-invasive manner at the bedside. We present new clinical validation of the DCS methodology by demonstrating strong agreement between DCS indices of relative cerebral blood flow and indices based on phase-encoded velocity mapping magnetic resonance imaging (VENC MRI) of relative blood flow in the jugular veins and superior vena cava. Data were acquired from 46 children with single ventricle cardiac lesions during a hypercapnia intervention. Significant increases in cerebral blood flow, measured both by DCS and by VENC MRI, as well as significant increases in oxyhemoglobin concentration, and total hemoglobin concentration, were observed during hypercapnia. Comparison of blood flow changes measured by VENC MRI in the jugular veins and by DCS revealed a strong linear relationship, R=0.88, p<0.001, slope=0.91±0.07. Similar correlations were observed between DCS and VENC MRI in the superior vena cava, R=0.77, slope=0.99±0.12, p<0.001. The relationship between VENC MRI in the aorta and DCS, a negative control, was weakly correlated, R=0.46, slope=1.77±0.45, p<0.001. PMID:22502579

  6. Spectroscopic evidence for negative electronic compressibility in a quasi-three-dimensional spin–orbit correlated metal

    SciTech Connect

    He, Junfeng; Hogan, T.; Mion, Thomas R.; Hafiz, H.; He, Y.; Denlinger, J. D.; Mo, S-K.; Dhital, C.; Chen, X.; Lin, Qisen; Zhang, Y.; Hashimoto, M.; Pan, H.; Lu, D. H.; Arita, M.; Shimada, K.; Markiewicz, R. S.; Wang, Z.; Kempa, K.; Naughton, M. J.; Bansil, A.; Wilson, S. D.; He, Rui-Hua

    2015-04-27

    Negative compressibility is a sign of thermodynamic instability of open1, 2, 3 or non-equilibrium4, 5 systems. In quantum materials consisting of multiple mutually coupled subsystems, the compressibility of one subsystem can be negative if it is countered by positive compressibility of the others. Manifestations of this effect have so far been limited to low-dimensional dilute electron systems6, 7, 8, 9, 10, 11. Here, we present evidence from angle-resolved photoemission spectroscopy (ARPES) for negative electronic compressibility (NEC) in the quasi-three-dimensional (3D) spin–orbit correlated metal (Sr1-xLax)3Ir2O7. Increased electron filling accompanies an anomalous decrease of the chemical potential, as indicated by the overall movement of the deep valence bands. Such anomaly, suggestive of NEC, is shown to be primarily driven by the lowering in energy of the conduction band as the correlated bandgap reduces. Our finding points to a distinct pathway towards an uncharted territory of NEC featuring bulk correlated metals with unique potential for applications in low-power nanoelectronics and novel metamaterials.

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  8. Correlative Energy-Dispersive X-Ray Spectroscopic Tomography and Atom Probe Tomography of the Phase Separation in an Alnico 8 Alloy

    SciTech Connect

    Guo, Wei; Sneed, Brian T.; Zhou, Lin; Tang, Wei; Kramer, Matthew J.; Cullen, David A.; Poplawsky, Jonathan D.

    2016-12-21

    Alnico alloys have long been used as strong permanent magnets because of their ferromagnetism and high coercivity. Understanding their structural details allows for better prediction of the resulting magnetic properties. However, quantitative three-dimensional characterization of the phase separation in these alloys is still challenged by the spatial quantification of nanoscale phases. Herein, we apply a dual tomography approach, where correlative scanning transmission electron microscopy (STEM) energy-dispersive X-ray spectroscopic (EDS) tomography and atom probe tomography (APT) are used to investigate the initial phase separation process of an alnico 8 alloy upon non-magnetic annealing. STEM-EDS tomography provides information on the morphology and volume fractions of Fe–Co-rich and Νi–Al-rich phases after spinodal decomposition in addition to quantitative information of the composition of a nanoscale volume. Subsequent analysis of a portion of the same specimen by APT offers quantitative chemical information of each phase at the sub-nanometer scale. Furthermore, APT reveals small, 2–4 nm Fe-rich α1 phases that are nucleated in the Ni-rich α2 matrix. From this information, we show that phase separation of the alnico 8 alloy consists of both spinodal decomposition and nucleation and growth processes. Lastly, we discuss the complementary benefits and challenges associated with correlative STEM-EDS and APT.

  9. Correlative Energy-Dispersive X-Ray Spectroscopic Tomography and Atom Probe Tomography of the Phase Separation in an Alnico 8 Alloy

    DOE PAGES

    Guo, Wei; Sneed, Brian T.; Zhou, Lin; ...

    2016-12-21

    Alnico alloys have long been used as strong permanent magnets because of their ferromagnetism and high coercivity. Understanding their structural details allows for better prediction of the resulting magnetic properties. However, quantitative three-dimensional characterization of the phase separation in these alloys is still challenged by the spatial quantification of nanoscale phases. Herein, we apply a dual tomography approach, where correlative scanning transmission electron microscopy (STEM) energy-dispersive X-ray spectroscopic (EDS) tomography and atom probe tomography (APT) are used to investigate the initial phase separation process of an alnico 8 alloy upon non-magnetic annealing. STEM-EDS tomography provides information on the morphology andmore » volume fractions of Fe–Co-rich and Νi–Al-rich phases after spinodal decomposition in addition to quantitative information of the composition of a nanoscale volume. Subsequent analysis of a portion of the same specimen by APT offers quantitative chemical information of each phase at the sub-nanometer scale. Furthermore, APT reveals small, 2–4 nm Fe-rich α1 phases that are nucleated in the Ni-rich α2 matrix. From this information, we show that phase separation of the alnico 8 alloy consists of both spinodal decomposition and nucleation and growth processes. Lastly, we discuss the complementary benefits and challenges associated with correlative STEM-EDS and APT.« less

  10. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

    SciTech Connect

    Leder, Verena; Lummer, Martina; Tegeler, Kathrin; Humpert, Fabian; Lewinski, Martin; Schüttpelz, Mark; Staiger, Dorothee

    2014-10-10

    Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R{sup 49} abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to binding. - Abstract: Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K{sub d} value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R{sup 49} that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2009-11-26

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

  13. Rational design, synthesis, and spectroscopic and photophysical properties of a visible-light-excitable, ratiometric, fluorescent near-neutral pH indicator based on BODIPY.

    PubMed

    Boens, Noël; Qin, Wenwu; Baruah, Mukulesh; De Borggraeve, Wim M; Filarowski, Aleksander; Smisdom, Nick; Ameloot, Marcel; Crovetto, Luis; Talavera, Eva M; Alvarez-Pez, Jose M

    2011-09-19

    A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the pH range 5-7 has been designed and synthesized by conjugatively linking the BODIPY fluorophore at the 3-position to the pH-sensitive ligand imidazole through an ethenyl bridge. The probe is available as cell membrane permeable methyl ester 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) and corresponding water-soluble sodium carboxylate, sodium 8-(4-carboxylatophenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (II). The fluorescence quantum yield Φ(f) of ester I is very high (0.8-1.0) in the organic solvents tested. The fluorescence lifetime (ca. 4 ns) of I in organic solvents with varying polarity/polarizability (from cyclohexane to acetonitrile) is independent of the solvent with a fluorescence rate constant k(f) of 2.4×10(8) s(-1). Probe I is readily loaded in the cytosol of live cells, where its high fluorescence intensity remains nearly constant over an extended time period. Water-soluble indicator II exhibits two acid-base equilibria in aqueous solution, characterized by pK(a) values of 6.0 and 12.6. The Φ(f) value of II in aqueous solution is high: 0.6 for the cationic and anionic forms of the imidazole ligand, and 0.8 for neutral imidazole. On protonation-deprotonation in the near-neutral pH range, UV/Vis absorption and fluorescence spectral shifts along with isosbestic and pseudo-isoemissive points are observed. This dual-excitation and dual-emission pH indicator emits intense green-yellow fluorescence at lower pH and intense orange fluorescence at higher pH. The influence of ionic strength and buffer concentration on the absorbance and steady-state fluorescence of II has also been investigated. The apparent pK(a) of the near-neutral acid-base equilibrium determined by spectrophotometric and fluorometric titration is nearly independent of

  14. Electron spectroscopic analysis of the SiO2/Si system and correlation with metal-oxide-semiconductor device characteristics

    NASA Astrophysics Data System (ADS)

    Iwata, Seiichi; Ishizaka, Akitoshi

    1996-05-01

    ESCA (electron spectroscopy for chemical analysis) measurement results on thin SiO2/Si samples are examined comprehensively, critically, and in detail to show that it is possible to correlate these results with MOS (metal-oxide-semiconductor) device characteristics such as flatband (threshold) voltage, oxide breakdown field, mobile-ion density, hole and electron trap density, and hot-carrier lifetime. Up to now, much effort has been made to detect SiOx phases at SiO2/Si interfaces since they are thought to have a significant effect on MOS device characteristics. However, correlating the SiOx phases with device characteristics is difficult and involves overcoming two problems. First, the chemical state is difficult to determine exactly due to x-ray irradiation effects. Second, the amount of defects and impurities which influence device characteristics is usually below the ESCA detection limit (1012-1013 cm-2) in device-quality SiO2/Si samples. Investigation of the first problem led to the conclusion that it is possible to correct for these effects from the x-ray intensity or oxide thickness dependence of the chemical shift. However, accurate (better than ±0.2 eV) chemical state determination is not easy. It is therefore necessary to approach this detection problem from a different viewpoint. Our first attempt involves measuring the ESCA thickness, which decreases when oxide defects like unoxidized Si or uneven thickness (or pinholes) are present, resulting in breakdown field degradation. Our second attempt started while we were studying how to interpret the measured chemical shift. The photoelectron peaks of the SiO2 and the Si can be observed to shift due to small amounts of charged defects and impurities, although they cannot be detected as peaks. This method is considered to be especially useful for characterizing ultrathin (a few nm thick) SiO2/Si samples which are difficult to characterize using conventional C-V (capacitance-voltage) measurements because of

  15. 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.

  16. A fluorescence spectroscopic study on the speciation of Cm(III) and Eu(III) in the presence of organic chelates in highly basic solutions

    SciTech Connect

    Wang, Zheming; Felmy, Andrew R.; Xia, Yuanxian; Mason, Marvin J.

    2003-07-15

    The speciation of Eu(III) and Cm(III) was investigated by time resolved laser fluorescence spectroscopy (TRLFS) over a range of base concentrations ranging from 0.01m NaOH to 7.5M NaOH and in the presence of several organic chelates including EDTA, HEDTA, NTA, and oxalate. The results of this work suggest that both Eu(III) and Cm(III) form strong mixed ligand complexes with organic chelates and the hydroxyl groups(s) in dilute NaOH solutions. However, in concentrated NaOH solutions, Eu(III)-/Cm(III)-containing colloidal nanoparticles are the primary cause for the measured Eu(III)/Cm(III) in the aqueous solutions. Therefore, the interpretation of these data solely in terms of the formation of amphoteric hydroxyl species (e.g. Eu(OH)4-) would appear to be inappropriate. The organic chelating ligands form strong complexes with surface Cm(III)/Eu(III) sites of the colloidal nanoparticles. For Cm(III), such surface complexes show largely red-shifted fluorescence spectra as compared with the aqueous complexes and unusually short fluorescence lifetimes. The decreased fluorescence lifetimes are likely due to the presence of transition metal ions, such as Fe3+, in the nanoparticle as well as reduced inter-nuclear distance between neighboring Cm(III) centers.

  17. Protein-surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS).

    PubMed

    Sonesson, Andreas W; Blom, Hans; Hassler, Kai; Elofsson, Ulla M; Callisen, Thomas H; Widengren, Jerker; Brismar, Hjalmar

    2008-01-15

    The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 microM C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

  18. Effects of surface functionalization on the adsorption of human serum albumin onto nanoparticles – a fluorescence correlation spectroscopy study

    PubMed Central

    Maffre, Pauline; Brandholt, Stefan; Nienhaus, Karin; Shang, Li; Parak, Wolfgang J

    2014-01-01

    Summary By using fluorescence correlation spectroscopy (FCS), we have studied the adsorption of human serum albumin (HSA) onto Fe–Pt nanoparticles (NPs, 6 nm radius), CdSe/ZnS quantum dots (QDs, 5 nm radius) and Au and Ag nanoclusters (1–4 nm radius), which are enshrouded by various water-solubilizing surface layers exposing different chemical functional groups (carboxyl, amino and both), thereby endowing the NPs with different surface charges. We have also measured the effects of modified surface functionalizations on the protein via succinylation and amination. A step-wise increase in hydrodynamic radius with protein concentration was always observed, revealing formation of protein monolayers coating the NPs, independent of their surface charge. The differences in the thickness of the protein corona were rationalized in terms of the different orientations in which HSA adsorbs onto the NPs. The midpoints of the binding transition, which quantifies the affinity of HSA toward the NP, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of specific Coulombic interactions between the proteins and the NP surfaces. PMID:25551031

  19. Direct observation of spatiotemporal dependence of anomalous diffusion in inhomogeneous fluid by sampling-volume-controlled fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Masuda, Akiko; Ushida, Kiminori; Okamoto, Takayuki

    2005-12-01

    The direct observation of a spatiotemporal behavior of anomalous diffusion in aqueous polymer [hyaluronan (HA)] solution was achieved by fluorescence correlation spectroscopy (FCS) using a modified instrument, enabling continuous change of the confocal volume of a microscope, namely, sampling-volume-controlled (SVC) FCS (SVC-FCS). Since HA chains form a mesh structure with a pore size of about 10-40nm , the observed diffusion coefficient (Dobs) is markedly dependent on the diffusion distance (L) . By SVC-FCS, the curve of the distance dependence of diffusion coefficient was directly obtained as a continuous profile in L=245-600nm showing evidence of anomalous diffusion. On plotting Dobs against either of the sampling time (τobs) or the diffusion distance (L) , Dobs turnover was observed near the anomalous diffusion area. The appearance of this turnover is attributed to the nonuniform mesh structure that can be observed only by a fast observation and that should be dynamically averaged by polymer motions with large τobs . This behavior is similar to that revealed in glass, colloidal systems, and gel solutions using dynamic light scattering, neutron scattering, and other techniques.

  20. Binding of human serum albumin to PEGylated liposomes: insights into binding numbers and dynamics by fluorescence correlation spectroscopy.

    PubMed

    Kristensen, Kasper; Urquhart, Andrew J; Thormann, Esben; Andresen, Thomas L

    2016-12-01

    Liposomes for medical applications are often administered by intravenous injection. Once in the bloodstream, the liposomes are covered with a "protein corona", which impacts the behavior and eventual fate of the liposomes. Currently, many aspects of the liposomal protein corona are not well understood. For example, there is generally a lack of knowledge about the liposome binding affinities and dynamics of common types of blood plasma proteins. Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that potentially can provide such knowledge. In this study, we have used FCS to investigate the binding of human serum albumin (HSA) to standard types of PEGylated fluid-phase liposomes (consisting of DOPC and DOPE-PEG2k) and PEGylated gel-phase liposomes (consisting of DSPC and DSPE-PEG2k) with various PEG chain surface densities. We detected no significant binding of HSA to the PEGylated fluid-phase liposomes. In contrast, we found that HSA bound tightly to the PEGylated gel-phase liposomes, although only a low number of HSA molecules could be accommodated per liposome. Overall, we believe that our data provides a useful benchmark for other researchers interested in studying the liposomal protein corona.

  1. A user's guide for characterizing plasma membrane subdomains in living cells by spot variation fluorescence correlation spectroscopy.

    PubMed

    Mailfert, S; Hamon, Y; Bertaux, N; He, H-T; Marguet, D

    2017-01-01

    Due to the intrinsic molecular Brownian agitation within plasma membrane and the vast diversity of membrane components, it is expected that the plasma membrane organization is highly heterogeneous with the formation of local complex multicomponent assemblies of lipids and proteins on different time scales. Still, deciphering this lateral organization on living cells and on the appropriate length and temporal scales has been challenging but is crucial to advance our knowledge on the biological function of the plasma membrane. Among the methodological developments based on biophotonics, the spot variation FCS (svFCS), a fluorescent correlation spectroscopy (FCS)-based method, has allowed the significant progress in the characterization of cell membrane lateral organization at the suboptical level, including to providing compelling evidence for the in vivo existence of lipid-dependent nanodomains. The aim of this chapter is to serve as a guide for setting and applying the svFCS methodology to study the plasma membrane of both adherent and nonadherent cell types.

  2. Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: Fluorescence correlation spectroscopy and femtosecond solvation dynamics

    NASA Astrophysics Data System (ADS)

    Chowdhury, Aritra; Mojumdar, Supratik Sen; Choudhury, Aparajita; Banerjee, Rajat; Das, Kali Pada; Sasmal, Dibyendu Kumar; Bhattacharyya, Kankan

    2012-04-01

    Structure and dynamics of acrylodan labeled αA-crystallin tetramer formed in the presence of a bile salt (sodium deoxycholate, NaDC) has been studied using fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion techniques. Using FCS it is shown that, the diffusion constant (Dt) of the αA-crystallin oligomer (mass ˜800 kDa) increases from ˜35 μm2 s-1 to ˜68 μm2 s-1. This corresponds to a decrease in hydrodynamic radius (rh) from ˜6.9 nm to ˜3.3 nm. This corresponds to about 10-fold decrease in molecular mass to ˜80 kDa and suggests formation of a tetramer (since mass of αA-crystallin monomer is ˜20 kDa). The steady state emission maximum and average solvation time (<τs>) of acrylodan labeled at cysteine 131 position of αA-crystallin is markedly affected on addition of NaDC, while the tryptophan (trp-9) becomes more exposed. This suggests that NaDC binds near the cys-131 and makes the terminal region of αA-crystallin exposed. This may explain the enhanced auto-phosphorylation activity of αA-crystallin near the terminus of the 173 amino acid protein (e.g., at the threonine 13, serine 45, or serine 169 and 172) and suggests that phosphorylation at ser-122 (close to cys-131) is relatively less important.

  3. Probing differential hydration of poly(vinylpyrrolidone) thin films using tracer mobility: an insight from fluorescence correlation spectroscopy.

    PubMed

    Bhattacharya, Sukanya; Dey, Arghya; Chowdhury, Arindam

    2014-05-15

    Dynamics of small probe molecules have been routinely used to unravel the intrinsic details of charged ion transport in polymer brushes and polyelectrolyte multilayer (PEM) thin films. However, corresponding morphological properties affected with absorption of moisture have been hardly dealt with despite numerous applications of isotropic thin films in material chemistry and medical purposes. We have explored the overall structural changes associated with plasticization of PVP thin films by probing dynamics of small reporter (rhodamine 6G, Rh6G) molecules using fluorescence correlation spectroscopy (FCS). It was observed that under lesser amounts of absorbed moisture, the rigidity of the film matrix was high enough to inhibit appreciable molecular mobility. Nonetheless, with gradual increase in the moisture level within the film, molecular movement became extremely facile, so much so that it almost attained close to a solution like state. Molecular mobility was found to be dependent on both the method of preparation and the thickness of the thin films. The diffusivities mostly followed anomalous subdiffusive behaviors, reminiscent of dynamics of tracers in crowded cellular environments. The mobility was found to be independent of any electrostatic interaction between probe and polymer thin film. Hence, the tracer dynamics was attributed most likely to the viscoelasticity of the thin film matrix.

  4. The oligomeric state and stability of the mannitol transporter, EnzymeIImtl, from Escherichia coli: A fluorescence correlation spectroscopy study

    PubMed Central

    Veldhuis, Gertjan; Hink, Mark; Krasnikov, Victor; van den Bogaart, Geert; Hoeboer, Jeroen; Visser, Antonie J.W.G.; Broos, Jaap; Poolman, Bert

    2006-01-01

    Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeIImtl, is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EIImtl functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EIImtl using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EIImtl is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect. PMID:16823033

  5. Room temperature synthesis of highly hemocompatible hydroxyapatite, study of their physical properties and spectroscopic correlation of particle size

    NASA Astrophysics Data System (ADS)

    Puvvada, Nagaprasad; Panigrahi, Pravas Kumar; Pathak, Amita

    2010-12-01

    Needle shaped nanoparticles of hydroxyapatite (HA) have been synthesized at room temperature using orthophosphoric acid as the source of (PO4)3- ions, while calcium chloride, the calcium source, is suitably complexed with citric acid/tartaric acid/acetic acid. The presence of ligands inhibits the growth along [001] and [100] directions of the crystal and thus, helps in formation of needle shaped nanoparticles. The chemical compositions of the samples have been established through AAS and FTIR spectroscopy, while the crystallinity has been assessed through XRD and by the spectral changes in the υ1 and υ3 frequencies of the phosphate group in the respective FTIR spectra. The particle sizes of the samples have been determined from line broadening studies and correlations have been established between the curve fitted percentage area of FTIR and full width half height (FWHH) of the XRD peaks. TEM studies revealed the particle to be needle-shaped with a length and diameter in the range of 20-65 nm and 4-11 nm respectively. Changes in the surface charge of the water dispersed HA samples have been determined at different pH and the isoelectric point for the samples have been found in the range of 3.1-3.4. Finally, the morphology, surface area and hemocompatibility characteristics of the HA samples, prepared by using different complexing agents, have been compared.Needle shaped nanoparticles of hydroxyapatite (HA) have been synthesized at room temperature using orthophosphoric acid as the source of (PO4)3- ions, while calcium chloride, the calcium source, is suitably complexed with citric acid/tartaric acid/acetic acid. The presence of ligands inhibits the growth along [001] and [100] directions of the crystal and thus, helps in formation of needle shaped nanoparticles. The chemical compositions of the samples have been established through AAS and FTIR spectroscopy, while the crystallinity has been assessed through XRD and by the spectral changes in the υ1 and υ3

  6. Two-dimensional correlation infrared spectroscopic study on the crystallization and gelation of poly(vinylidene fluoride) in cyclohexanone.

    PubMed

    Peng, Yun; Sun, Bingjie; Wu, Peiyi

    2008-03-01

    ) absorption is assigned to the CF2 group of PVDF, the 873 cm(-1) absorption involves the C-C group of PVDF, and the 796 cm(-1) band is attributed to the CH2 groups of PVDF. Thus, the CF2 functionalities change faster than the C-C and CH2 groups. However, the correlation cross-peaks between 762 cm(-1) and 873 cm(-1) and at 796 cm(-1) disappeared during the later state of the gelation process. At the same time, the bands of PVDF and solvent still varied, which suggests that it is a physical interaction process between PVDF chain and solvent.

  7. Airborne simultaneous spectroscopic detection of laser-induced water Raman backscatter and fluorescence from chlorophyll a and other naturally occurring pigments

    NASA Technical Reports Server (NTRS)

    Hoge, F. E.; Swift, R. N.

    1981-01-01

    The airborne laser-induced spectral emission bands obtained simultaneously from water Raman backscatter and the fluorescence of chlorophyll and other naturally occurring waterborne pigments are reported here for the first time. The importance of this type data lies not only in its single-shot multispectral character but also in the application of the Raman line for correction or calibration of the spatial variation of the laser penetration depth without the need for in situ water attenuation measurements. The entire laser-induced fluorescence and Raman scatter emissions resulting from each separate 532-nm 10-nsec laser pulse are collected and spectrally dispersed in a diffraction grating spectrometer having forty photomultiplier tube detectors. Results from field experiments conducted in the North Sea and the Chesapeake Bay/Potomac River are presented. Difficulties involving the multispectral resolution of the induced emissions are addressed, and feasible solutions are suggested together with new instrument configurations and future research directions.

  8. Pyrazolo[4,3-a]quinindoline as a new highly fluorescent heterocyclic system: Design, synthesis, spectroscopic characterization and DFT calculations

    NASA Astrophysics Data System (ADS)

    Alikhani, Elaheh; Pordel, Mehdi; Daghigh, Leila Rezaei

    2015-02-01

    After obtaining the desired precursors in several reactions, new N-alkyl-substituted heterocyclic system pyrazolo[4,3-a]quinindolines (pyrazolo[4,3-f]-indolo[2,3-b]quinolines) were synthesized by one-pot reaction of 1-alkyl-5-nitro-1H-indazole with 2-(1-alkyl-1H-3-indolyl)acetonitrile in MeOH/KOH solution via the nucleophilic substitution of hydrogen in excellent yields. Spectral (UV-Vis, FT-IR, NMR and fluorescence) and analytical data allowed the structures of the synthesized compounds to be established. The values of absorption and fluorescence maxima, extinction coefficients and fluorescence quantum yield of these new heterocyclic fluorophores were obtained and they show highlighting interesting photophysical properties. Density functional theory (DFT) calculations of one structure by using the B3LYP hybrid functional and the 6-311 + G(d,p) basis set were performed to provide the optimized geometry, relevant frontier orbitals and the prediction of 1H NMR chemical shifts. Calculated electronic absorption spectrum of one structure was also obtained by time-dependent density functional theory (TD-DFT) method. Solvatochromic properties of these dyes have been discussed and the results showed that the absorption and emission bands in polar solvents undergo a modest red shift.

  9. Correlation of electronic carotenoid-chlorophyll interactions and fluorescence quenching with the aggregation of native LHC II and chlorophyll deficient mutants

    NASA Astrophysics Data System (ADS)

    Liao, Pen-Nan; Bode, Stefan; Wilk, Laura; Hafi, Nour; Walla, Peter J.

    2010-07-01

    The aggregation dependent correlation between fluorescence quenching and the electronic carotenoid-chlorophyll interactions, ϕCouplingCar S-Chl, as measured by comparing chlorophyll fluorescence observed after two- and one-photon excitation, has been investigated using native LHC II samples as well as mutants lacking Chl 2 and Chl 13. For native LHC II the same linear correlation between ϕCouplingCar S-Chl and the fluorescence quenching was observed as previously reported for the pH and Zea-dependent quenching of LHC II [1]. In order to elucidate which carotenoid-chlorophyll pair might dominate this correlation we also investigated the mutants lacking Chl 2 and Chl 13. However, also with these mutants the same linear correlation as for native LHC II was observed. This provides indication that these two chlorophylls play only a minor role for the observed effects. Nevertheless, we also conclude that this does not exclude that their neighboured carotenoids, lutein 1 and neoxanthin, might interact electronically with other chlorophylls close by.

  10. Protoporphyrin IX fluorescence contrast in invasive glioblastomas is linearly correlated with Gd enhanced magnetic resonance image contrast but has higher diagnostic accuracy

    NASA Astrophysics Data System (ADS)

    Samkoe, Kimberley S.; Gibbs-Strauss, Summer L.; Yang, Harold H.; Khan Hekmatyar, S.; Jack Hoopes, P.; O'Hara, Julia A.; Kauppinen, Risto A.; Pogue, Brian W.

    2011-09-01

    The sensitivity and specificity of in vivo magnetic resonance (MR) imaging is compared with production of protoporphyrin IX (PpIX), determined ex vivo, in a diffusely infiltrating glioma. A human glioma transfected with green fluorescent protein, displaying diffuse, infiltrative growth, was implanted intracranially in athymic nude mice. Image contrast from corresponding regions of interest (ROIs) in in vivo MR and ex vivo fluorescence images was quantified. It was found that all tumor groups had statistically significant PpIX fluorescence contrast and that PpIX contrast demonstrated the best predictive power for tumor presence. Contrast from gadolinium enhanced T1-weighted (T1W+Gd) and absolute T2 images positively predicted the presence of a tumor, confirmed by the GFP positive (GFP+) and hematoxylin and eosin positive (H&E+) ROIs. However, only the absolute T2 images had predictive power from controls in ROIs that were GFP+ but H&E negative. Additionally, PpIX fluorescence and T1W+Gd image contrast were linearly correlated in both the GFP+ (r = 0.79, p<1×10-8) and H&E+ (r = 0.74, p<0.003) ROIs. The trace diffusion images did not have predictive power or significance from controls. This study indicates that gadolinium contrast enhanced MR images can predict the presence of diffuse tumors, but PpIX fluorescence is a better predictor regardless of tumor vascularity.

  11. BAT AGN Spectroscopic Survey - IV: Near-Infrared Coronal Lines, Hidden Broad Lines, and Correlation with Hard X-ray Emission

    NASA Astrophysics Data System (ADS)

    Lamperti, Isabella; Koss, Michael; Trakhtenbrot, Benny; Schawinski, Kevin; Ricci, Claudio; Oh, Kyuseok; Landt, Hermine; Riffel, Rogério; Rodríguez-Ardila, Alberto; Gehrels, Neil; Harrison, Fiona; Masetti, Nicola; Mushotzky, Richard; Treister, Ezequiel; Ueda, Yoshihiro; Veilleux, Sylvain

    2017-01-01

    We provide a comprehensive census of the near-Infrared (NIR, 0.8-2.4 μm) spectroscopic properties of 102 nearby (z < 0.075) active galactic nuclei (AGN), selected in the hard X-ray band (14-195 keV) from the Swift-Burst Alert Telescope (BAT) survey. With the launch of the James Webb Space Telescope this regime is of increasing importance for dusty and obscured AGN surveys. We measure black hole masses in 68% (69/102) of the sample using broad emission lines (34/102) and/or the velocity dispersion of the Ca II triplet or the CO band-heads (46/102). We find that emission line diagnostics in the NIR are ineffective at identifying bright, nearby AGN galaxies because ([Fe II] 1.257μm/Paβ and H2 2.12μm/Brγ) identify only 25% (25/102) as AGN with significant overlap with star forming galaxies and only 20% of Seyfert 2 have detected coronal lines (6/30). We measure the coronal line emission in Seyfert 2 to be weaker than in Seyfert 1 of the same bolometric luminosity suggesting obscuration by the nuclear torus. We find that the correlation between the hard X-ray and the [Fe II] coronal line luminosity is significantly better than with the [O III] λ5007 luminosity. Finally, we find 3/29 galaxies (10%) that are optically classified as Seyfert 2 show broad emission lines in the NIR. These AGN have the lowest levels of obscuration among the Seyfert 2s in our sample (log NH < 22.43 cm-2), and all show signs of galaxy-scale interactions or mergers suggesting that the optical broad emission lines are obscured by host galaxy dust.

  12. Fluorescence and CD spectroscopic analysis of the alpha-chymotrypsin stabilization by the ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide.

    PubMed

    De Diego, Teresa; Lozano, Pedro; Gmouh, Said; Vaultier, Michel; Iborra, José L

    2004-12-30

    The stability of alpha-chymotrypsin in the ionic liquid, 1-ethyl-3-methyl-imidizolium bis[(trifluoromethyl)sulfonyl]amide ([emim][NTf2]), was studied at 30 and 50 degrees C and compared with the stability in other liquid media, such as water, 3 M sorbitol, and 1-propanol. The kinetic analysis of the enzyme stability pointed to the clear denaturative effect of 1-propanol, while both 3M sorbitol and [emim][NTf2] displayed a strong stabilizing power. For the first time, it is shown that enzyme stabilization by ionic liquids seems to be related to the associated structural changes of the protein that can be observed by differential scanning calorimetry (DSC) and fluorescence and circular dichroism (CD). The [emim][NTf2] enhanced both the melting temperature and heat capacity of the enzyme compared to the other media assayed. The fluorescence spectra clearly showed the ability of [emim][NTf2] to compact the native structural conformation of alpha-chymotrypsin, preventing the usual thermal unfolding which occurs in other media. Changes in the secondary structure of this beta/beta protein, as quantified by the CD spectra, pointed to the great enhancement (up 40% with respect to that in water) of beta-strands in the presence of the ionic liquid, which reflects its stabilization power.

  13. Palus Somni - Anomalies in the correlation of Al/Si X-ray fluorescence intensity ratios and broad-spectrum visible albedos. [lunar surface mineralogy

    NASA Technical Reports Server (NTRS)

    Clark, P. E.; Andre, C. G.; Adler, I.; Weidner, J.; Podwysocki, M.

    1976-01-01

    The positive correlation between Al/Si X-ray fluorescence intensity ratios determined during the Apollo 15 lunar mission and a broad-spectrum visible albedo of the moon is quantitatively established. Linear regression analysis performed on 246 1 degree geographic cells of X-ray fluorescence intensity and visible albedo data points produced a statistically significant correlation coefficient of .78. Three distinct distributions of data were identified as (1) within one standard deviation of the regression line, (2) greater than one standard deviation below the line, and (3) greater than one standard deviation above the line. The latter two distributions of data were found to occupy distinct geographic areas in the Palus Somni region.

  14. Fate of biopolymers during rapeseed meal and wheat bran composting as studied by two-dimensional correlation spectroscopy in combination with multiple fluorescence labeling techniques.

    PubMed

    Wang, Li-Ping; Shen, Qi-Rong; Yu, Guang-Hui; Ran, Wei; Xu, Yang-Chun

    2012-02-01

    Detailed knowledge of the molecular events during composting is important in improving the efficiency of this process. By combining two-dimensional Fourier transform infrared (FTIR) correlation spectroscopy and multiple fluorescent labeling, it was possible to study the degradation of biopolymers during rapeseed meal and wheat bran composting. Two-dimensional FTIR correlation spectroscopy provided structural information and was used to deconvolute overlapping bands found in the compost FTIR spectra. The degradation of biopolymers in rapeseed meal and wheat bran composts followed the sequence: cellulose, heteropolysaccharides, and proteins. Fluorescent labeling suggested that cellulose formed an intact network-like structure and the other biopolymers were embedded in the core of this structure. The sequence of degradation of biopolymers during composting was related to their distribution patterns.

  15. Demonstration of phycobilisome mobility by the time- and space-correlated fluorescence imaging of a cyanobacterial cell.

    PubMed

    Yang, Shuzhen; Su, Zuqi; Li, Heng; Feng, Juanjuan; Xie, Jie; Xia, Andong; Gong, Yandao; Zhao, Jingquan

    2007-01-01

    The cell-wide mobility of PBSs was confirmed by synchronously monitoring the fluorescence recovery after photobleaching (FRAP) and the fluorescence loss in photobleaching (FLIP). On the other hand, a fluorescence recovery was still observed even if PBSs were immobile (PBSs fixed on the membranes by betaine and isolated PBSs fixed on the agar plate) or PBS mobility was unobservable (cell wholly bleached). Furthermore, it was proved that some artificial factors were involved not only in FRAP but also in FLIP, including renaturation of the reversibly denatured proteins, laser scanning-induced fluorescence loss and photo-damage to the cell. With consideration of the fast renaturation component in fluorescence recovery, the diffusion coefficient was estimated to be tenfold smaller than that without the component. Moreover, it was observed that the fluorescence intensity on the bleached area was always lower than that on the non-bleached area, even after 20 min, while it should be equal if PBSs were mobile freely. Based on the increasing proportion of the PBSs anti-washed to Triton X-100 (1%) with prolonged laser irradiation to the cells locked in light state 1 by PBQ, it was concluded that some PBSs became immobile due to photo-linking to PSII.

  16. Influence of Cd 2+, Hg 2+ and Pb 2+ on (+)-catechin binding to bovine serum albumin studied by fluorescence spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Peng, Mijun; Shi, Shuyun; Zhang, Yuping

    2012-01-01

    The effect of heavy metal ions, Cd 2+, Hg 2+ and Pb 2+ on (+)-catechin binding to bovine serum albumin (BSA) has been investigated by spectroscopic methods. The results indicated that the presence of heavy metal ions significantly affected the binding modes and binding affinities of (+)-catechin to BSA, and the effects depend on the types of heavy metal ion. One binding mode was found for (+)-catechin with and without Cd 2+, while two binding modes - a weaker one at low concentration and a stronger one at high concentration were found for (+)-catechin in the presence of Hg 2+ and Pb 2+. The presence of Cd 2+ decreased the binding affinities of (+)-catechin for BSA by 20.5%. The presence of Hg 2+ and Pb 2+ decreased the binding affinity of (+)-catechin for BSA by 8.9% and 26.7% in lower concentration, respectively, and increased the binding affinity of (+)-catechin for BSA by 5.2% and 9.2% in higher concentration, respectively. The changed binding affinity and binding distance of (+)-catechin for BSA in the presence of Cd 2+, Hg 2+ and Pb 2+ were mainly because of the conformational change of BSA induced by heavy metal ions. However, the quenching mechanism for (+)-catechin to BSA was based on static quenching combined with non-radiative energy transfer irrespective of the absence or presence of heavy metal ions.

  17. X-ray fluorescence mapping and micro-XANES spectroscopic characterization of exhaust particulates emitted from auto engines burning MMT-added gasoline.

    PubMed

    Mölders, N; Schilling, P J; Wong, J; Roos, J W; Smith, I L

    2001-08-01

    The elemental distribution and compositional homogeneity in auto exhaust particulates emitted from methylcyclopentadienyl manganese tricarbonyl-(MMT-)added gasoline engines have been investigated using a newly installed synchrotron X-ray microprobe. Two representative groups of exhaust particulate matter, as defined in a recent bulk X-ray absorption fine structure (XAFS) spectroscopic study at the Mn K-edge, were studied. The micro-X-ray absorption near-edge structure (XANES) spectra indicate a relatively homogeneous distribution of phases within a given particulate sample, down to a spatial extent of 40 microm (the resolution of microprobe). The micro-XANES also enabled analysis of several areas which displayed compositions different from the bulk sample, supporting the general theory describing manganese species formation in the exhaust. The ability to evaluate small regions also enabled direct verification of manganese sulfate from the S XANES despite the vast excess of sulfur present in other forms. The presence of a chloride compound, introduced through the sample dilution air and engine intake air, was also revealed. The study demonstrates the value of the combined X-ray microfluorescence with excitation by polychromatic radiation for elemental mapping and micro-XANES spectroscopy for chemical speciation in the study of dilute environmental materials containing low-Z constituents such as Cl, S, and P.

  18. Cobalt, nickel, copper and zinc complexes with 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde Schiff bases: antimicrobial, spectroscopic, thermal and fluorescence studies.

    PubMed

    Singh, Kiran; Kumar, Yogender; Puri, Parvesh; Kumar, Mahender; Sharma, Chetan

    2012-06-01

    Two new Schiff bases of 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde and 4-amino-5-mercapto-3-methyl/H-1,2,4-triazole [HL(1-2)] and their Cobalt, Nickel, Copper and Zinc complexes have been synthesized and characterized by elemental analyses, spectral (UV-vis, IR, (1)H NMR, Fluorescence) studies, thermal techniques and magnetic measurements. A square planar geometry for Cu(II) and octahedral geometry for Co(II), Ni(II) and Zn(II) complexes have been proposed. In order to evaluate the biological activity of Schiff bases and to assess the role of metal ion on biological activity, the pyrazole Schiff bases and their metal complexes have been studied in vitro antibacterial against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and antifungal against Aspergillus niger, and Aspergillus flavus. In most of the cases higher activity was exhibited upon coordination with metal ions.

  19. Super-resolution Stimulated Emission Depletion-Fluorescence Correlation Spectroscopy Reveals Nanoscale Membrane Reorganization Induced by Pore-Forming Proteins.

    PubMed

    Sarangi, Nirod Kumar; P, Ilanila I; Ayappa, K G; Visweswariah, Sandhya S; Basu, Jaydeep Kumar

    2016-09-20

    Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that

  20. Synchronous fluorescence spectroscopy combined with two-dimensional correlation and principle component analysis to characterize dissolved organic matter in an urban river.

    PubMed

    Yu, Huibin; Song, Yonghui; Pan, Hongwei; Peng, Jianfeng; Gao, Hongjie; Liu, Ruixia

    2016-10-01

    Synchronous fluorescence spectroscopy (SFS) combined with two-dimensional correlation and principle component analysis (PCA) can provide an excellent challenge to capture fluorescent components of dissolved organic matter (DOM) and reveal its spatial variations in an urban river. Water samples were collected from Baitapuhe River along human impact gradient, i.e., the rural, town, and urban regions. DOM in Baitapuhe River was composed of protein-like, microbial humic-like, fulvic-like, and humic-like fluorescent components. The protein-like was the dominant component, which consisted of tyrosine-like and tryptophan-like components. In the rural region, the variation of the microbial humic-like was higher than that of the protein-like according to the band changing order of 335 → 281 nm, and both components changed in the same direction. In the town region, the variation of the microbial humic-like was the highest followed by the protein-like and fulvic-like on the basis of the changing band order of 335 → 281 → 369 nm, and these components varied in the same trend too. In the urban region, the variation of the protein-like was the highest, followed by the microbial humic-like, fulvic-like, and humic-like based on the changing band order of 282 → 335 → 369 → 470 nm, and the protein-like variation was opposite to the other components. The SFS combined with PCA and two-dimensional correlation can be used as a powerful tool in investigating fluorescent components of DOM and revealing spatial variations of these fluorescent components.

  1. Spectroscopic and dynamical studies of highly energized small polyatomic molecules

    NASA Astrophysics Data System (ADS)

    Stimulated emission pumping (SEP) spectroscopy was used on acetylene and on formyl radical. An attempt was made for pattern recognition based on statistics; a method was invented that combined CNPI (complete nuclear permutation-inversion) group theory and SCC (spectral cross-correlation). But the direction away from statistical pattern recognition back to traditional spectroscopic pattern recognition was taken. Vibrational states and quantum numbers are discussed. For the formyl radical, the fluorescence excitation spectrum was recorded and a rotational analysis of the 0(sup 0)(sub 0) band performed.

  2. A dynamic model for ALA-PDT of skin: analysis of the correlation of fluorescence and singlet oxygen luminescence to spatial distribution of singlet oxygen

    NASA Astrophysics Data System (ADS)

    Liu, Baochang; Farrell, Thomas J.; Patterson, Michael S.

    2011-02-01

    Both photosensitizer fluorescence photobleaching and singlet oxygen luminescence (SOL) have been measured during ALA-PDT of skin in attempts to estimate PDT dose. However, the relationship of these detected signals to singlet oxygen (1O2) dose in a given volume and to its depth distribution are not well understood and difficult to verify experimentally because of the temporal and spatial variations of the essential parameters in PDT. A model for ALA-PDT of normal human skin was developed to simulate the dynamic progress of PDT. The model incorporates Monte Carlo simulations of excitation light fluence and both SOL and PpIX fluorescence signals, 1O2-mediated photobleaching mechanism, ground-state oxygen (3O2) diffusion and perfusion, a cumulative 1O2-dependent threshold vascular response and any initial distribution of PpIX. The simulated time-resolved evolution of the instantaneous PpIX fluorescence photobleaching and cumulative SOL signals are examined as functions of irradiance and related to both the time-resolved distribution of cumulative 1O2 production at various depths and the average dose in the dermis. The simulations used a green light source at 523 nm. The correlation of SOL signals with the average dose was found to be less irradiance-dependent than that of fluorescence photobleaching, which indicates the great potential of SOL as a clinical dosimetric tool in PDT.

  3. DNA cleavage, antimicrobial, spectroscopic and fluorescence studies of Co(II), Ni(II) and Cu(II) complexes with SNO donor coumarin Schiff bases

    NASA Astrophysics Data System (ADS)

    Patil, Sangamesh A.; Naik, Vinod H.; Kulkarni, Ajaykumar D.; Badami, Prema S.

    2010-01-01

    A series of Co(II), Ni(II) and Cu(II) complexes of the type ML 2 have been synthesized with Schiff bases derived from methylthiosemicarbazone and 5-formyl-6-hydroxy coumarin/8-formyl-7-Hydroxy-4-methylcoumarin. The complexes are insoluble in common organic solvents but soluble in DMF and DMSO. The measured molar conductance values in DMF indicate that, the complexes are non-electrolytes in nature. In view of analytical, spectral (IR, UV-vis, ESR, FAB-mass and fluorescence), magnetic and thermal studies, it has been concluded that, all the metal complexes possess octahedral geometry in which ligand is coordinated to metal ion through azomethine nitrogen, thione sulphur and phenolic oxygen atom via deprotonation. The redox behavior of the metal complexes was investigated by using cyclic voltammetry. The Schiff bases and their complexes have been screened for their antibacterial ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi) and antifungal activities ( Aspergillus niger, Aspergillus flavus and Cladosporium) by Minimum Inhibitory Concentration method. The DNA cleavage is studied by agarose gel electrophoresis method.

  4. A new rhodamine derived fluorescent sensor: Detection of Hg2+ at cellular level

    NASA Astrophysics Data System (ADS)

    Kuchlyan, Jagannath; Basak, Shyam; Dutta, Debabrata; Das, Amit Kumar; Mal, Dipakranjan; Sarkar, Nilmoni

    2017-04-01

    Mercury ion is of great threat to the human kind due to its high toxicity in live systems. Consequently, its detection at nanolevel is of current interest. The rhodamine derivative is one of the rarest examples of fluorescent chemosensors for Hg2+, wherein a phthalaldehydic acid moiety which shows antibacterial activity, is responsible for specific binding. By using fluorescence correlation spectroscopic (FCS) study of the probe, it has been possible to detect Hg2+ in solution at a level as low as ∼5 pM. This sensing, applicable to fluorescence lifetime imaging of live cells, provides a novel microscopic technique.

  5. Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells

    PubMed Central

    Hagen, Christoph; Werner, Stephan; Carregal-Romero, Susana; N. Malhas, Ashraf; G. Klupp, Barbara; Guttmann, Peter; Rehbein, Stefan; Henzler, Katja; C. Mettenleiter, Thomas; J. Vaux, David; J. Parak, Wolfgang; Schneider, Gerd; Grünewald, Kay

    2014-01-01

    Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness. PMID:24973653

  6. Note: a 4 ns hardware photon correlator based on a general-purpose field-programmable gate array development board implemented in a compact setup for fluorescence correlation spectroscopy.

    PubMed

    Kalinin, Stanislav; Kühnemuth, Ralf; Vardanyan, Hayk; Seidel, Claus A M

    2012-09-01

    We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

  7. Note: A 4 ns hardware photon correlator based on a general-purpose field-programmable gate array development board implemented in a compact setup for fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Kalinin, Stanislav; Kühnemuth, Ralf; Vardanyan, Hayk; Seidel, Claus A. M.

    2012-09-01

    We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

  8. Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components.

    PubMed Central

    Pyenta, P S; Holowka, D; Baird, B

    2001-01-01

    To investigate the structural basis for membrane interactions that occur between Lyn tyrosine kinase and IgE-Fc(epsilon)RI or other components of lipid rafts, we prepared a green fluorescent protein analog of Lyn (PM-EGFP) and used cross-correlation analysis to quantify co-redistributions of aggregates that occur after IgE-Fc(epsilon)RI is cross-linked on the cell surface. PM-EGFP, which contains minimally the palmitoylation and myristoylation sites on Lyn, was compared with another inner leaflet probe, EGFP-GG, which contains a prenylation site and a polybasic sequence similar to K-ras. Confocal fluorescence microscopy was used to examine co-redistributions of these inner leaflet components with IgE-Fc(epsilon)RI and outer leaflet raft components, ganglioside GD1b and glycosylphosphotidylinositol-linked Thy-1, under conditions where the latter were cross-linked externally to form large patches at the cell surface. The cross-correlation analysis was developed and characterized with simulations representing cell surface distributions, and parameters from the cross-correlation curves, rho(o) (peak height) and A (peak area), were shown to be reliable measures of the extent of co-redistributed aggregates and their size. Cross-correlation analysis was then applied to quantify co-redistributions of the fluorescently labeled inner and outer leaflet components on RBL-2H3 cells. As visually observed and parameterized in this manner, PM-EGFP was found to co-redistribute with lipid rafts significantly more than EGFP-GG or an endogenous prenylated protein, Cdc42. These quantitative results are consistent with previous analyses of Lyn co-redistributions and support the hypothesis that the functionally important interaction of Lyn with cross-linked IgE- Fc(epsilon)RI is due to their mutual co-association with lipid rafts. PMID:11325715

  9. In-cylinder temperature measurements via time-correlated single-photon counting of toluene laser-induced fluorescence through a fiber-based sensor.

    PubMed

    Friesen, Eugen; Gessenhardt, Christopher; Kaiser, Sebastian A; Dreier, Thomas; Schulz, Christof

    2012-12-15

    In a near-production internal combustion engine, the effective fluorescence lifetime of toluene was determined by time-correlated single-photon counting with a minimally invasive fiber-optic spark-plug sensor. The lifetime measurement provided continuous crank-angle-resolved measurements of gas temperature. Proof-of-concept experiments in a motored four-cylinder spark-ignition engine were evaluated with a time resolution of 500 μs, yielding temperature precision of 25 K (standard deviation) at top-dead center. In these experiments, 10% toluene was added to the nonfluorescent base fuel iso-octane. Fluorescence lifetimes were related to temperature via calibration measurements in a high temperature pressure vessel, with the data fitted to a functional dependence derived from a previously published phenomenological model.

  10. Fluorescence Correlation Spectroscopy Measurements of the Membrane Protein TetA in Escherichia coli Suggest Rapid Diffusion at Short Length Scales

    PubMed Central

    Chow, David; Guo, Lin; Gai, Feng; Goulian, Mark

    2012-01-01

    Structural inhomogeneities in biomembranes can lead to complex diffusive behavior of membrane proteins that depend on the length or time scales that are probed. This effect is well studied in eukaryotic cells, but has been explored only recently in bacteria. Here we used fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) to study diffusion of the membrane protein TetA-YFP in E. coli. We find that the diffusion constant determined from FRAP is comparable to other reports of inner membrane protein diffusion constants in E. coli. However, FCS, which probes diffusion on shorter length scales, gives a value that is almost two orders of magnitude higher and is comparable to lipid diffusion constants. These results suggest there is a population of TetA-YFP molecules in the membrane that move rapidly over short length scales (∼ 400 nm) but move significantly more slowly over the longer length scales probed by FRAP. PMID:23119068

  11. Ionic liquid-based zinc oxide nanofluid for vortex assisted liquid liquid microextraction of inorganic mercury in environmental waters prior to cold vapor atomic fluorescence spectroscopic detection.

    PubMed

    Amde, Meseret; Liu, Jing-Fu; Tan, Zhi-Qiang; Bekana, Deribachew

    2016-01-01

    Zinc oxide nanofluid (ZnO-NF) based vortex assisted liquid liquid microextraction (ZnO-NF VA-LLME) was developed and employed in extraction of inorganic mercury (Hg(2+)) in environmental water samples, followed by cold vapor atomic fluorescence spectrometry (CV-AFS). Unlike other dispersive liquid liquid microextraction techniques, ZnO-NF VA-LLME is free of volatile organic solvents and dispersive solvent consumption. Analytical signals were obtained without back-extraction from the ZnO-NF phase prior to CV-AFS determination. Some essential parameters of the ZnO-NF VA-LLME and cold vapor generation such as composition and volume of the nanofluid, vortexing time, pH of the sample solution, amount of the chelating agent, ionic strength and matrix interferences have been studied. Under optimal conditions, efficient extraction of 1ng/mL of Hg(2+) in 10mL of sample solution was achieved using 50μL of ZnO-NF. The enrichment factor before dilution, detection limits and limits of quantification of the method were about 190, 0.019 and 0.064ng/mL, respectively. The intra and inter days relative standard deviations (n=8) were found to be 4.6% and 7.8%, respectively, at 1ng/mL spiking level. The accuracy of the current method was also evaluated by the analysis of certified reference materials, and the measured Hg(2+) concentration of GBW08603 (9.6ng/mL) and GBW(E)080392 (8.9ng/mL) agreed well with their certified value (10ng/mL). The method was applied to the analysis of Hg(2+) in effluent, influent, lake and river water samples, with recoveries in the range of 79.8-92.8% and 83.6-106.1% at 1ng/mL and 5ng/mL spiking levels, respectively. Overall, ZnO-NF VA-LLME is fast, simple, cost-effective and environmentally friendly and it can be employed for efficient enrichment of the analyte from various water samples.

  12. Spectroscopic and photochemical properties of open-chain carotenoids.

    SciTech Connect

    Frank, H. A.; Josue, J. S.; Bautista, J. A.; van der Hoef, I.; Jansen, F. J.; Lugtenburg, J.; Wiederrecht, G.; Christensen, R. L.; Chemistry; Univ. of Connecticut; Leiden Univ.; Bowdoin College

    2002-02-28

    The spectroscopic properties of open-chain, all-trans-C{sub 30} carotenoids having seven, eight and nine {pi}-electron conjugated carbon-carbon double bonds were studied using steady-state absorption, fluorescence, fluorescence excitation and time-resolved absorption spectroscopy. These diapocarotenes were purified by high performance liquid chromatography (HPLC) prior to the spectroscopic experiments. The fluorescence data show a systematic crossover from dominant S{sub 1} {yields} S{sub 0} (2{sup 1}Ag{yields} 1{sup 1}Ag) emission to dominant S{sub 2} {yields} S{sub 0} (1{sup 1}Bu {yields} 1{sup 1}Ag) with increasing extent of conjugation. The low temperatures facilitated the determination of the spectral origins of the S{sub 1} {yields} S{sub 0} (2{sup 1}Ag {yields} 1{sup 1}Ag) emissions, which were assigned by Gaussian deconvolution of the experimental line shapes. The lifetimes of the S{sub 1} states of the molecules were measured by transient absorption spectroscopy and were found to decrease as the conjugated chain length increases. The energy gap law for radiationless transitions is used to correlate the S{sub 1} energies with the dynamics. These molecules provide a systematic series for understanding the structural features that control the photochemical properties of open-chain, diapocarotenoids. The implications of these results on the roles of carotenoids in photosynthetic organisms are discussed.

  13. Self-organising maps and correlation analysis as a tool to explore patterns in excitation-emission matrix data sets and to discriminate dissolved organic matter fluorescence components.

    PubMed

    Ejarque-Gonzalez, Elisabet; Butturini, Andrea

    2014-01-01

    Dissolved organic matter (DOM) is a complex mixture of organic compounds, ubiquitous in marine and freshwater systems. Fluorescence spectroscopy, by means of Excitation-Emission Matrices (EEM), has become an indispensable tool to study DOM sources, transport and fate in aquatic ecosystems. However the statistical treatment of large and heterogeneous EEM data sets still represents an important challenge for biogeochemists. Recently, Self-Organising Maps (SOM) has been proposed as a tool to explore patterns in large EEM data sets. SOM is a pattern recognition method which clusterizes and reduces the dimensionality of input EEMs without relying on any assumption about the data structure. In this paper, we show how SOM, coupled with a correlation analysis of the component planes, can be used both to explore patterns among samples, as well as to identify individual fluorescence components. We analysed a large and heterogeneous EEM data set, including samples from a river catchment collected under a range of hydrological conditions, along a 60-km downstream gradient, and under the influence of different degrees of anthropogenic impact. According to our results, chemical industry effluents appeared to have unique and distinctive spectral characteristics. On the other hand, river samples collected under flash flood conditions showed homogeneous EEM shapes. The correlation analysis of the component planes suggested the presence of four fluorescence components, consistent with DOM components previously described in the literature. A remarkable strength of this methodology was that outlier samples appeared naturally integrated in the analysis. We conclude that SOM coupled with a correlation analysis procedure is a promising tool for studying large and heterogeneous EEM data sets.

  14. Self-Organising Maps and Correlation Analysis as a Tool to Explore Patterns in Excitation-Emission Matrix Data Sets and to Discriminate Dissolved Organic Matter Fluorescence Components

    PubMed Central

    Ejarque-Gonzalez, Elisabet; Butturini, Andrea

    2014-01-01

    Dissolved organic matter (DOM) is a complex mixture of organic compounds, ubiquitous in marine and freshwater systems. Fluorescence spectroscopy, by means of Excitation-Emission Matrices (EEM), has become an indispensable tool to study DOM sources, transport and fate in aquatic ecosystems. However the statistical treatment of large and heterogeneous EEM data sets still represents an important challenge for biogeochemists. Recently, Self-Organising Maps (SOM) has been proposed as a tool to explore patterns in large EEM data sets. SOM is a pattern recognition method which clusterizes and reduces the dimensionality of input EEMs without relying on any assumption about the data structure. In this paper, we show how SOM, coupled with a correlation analysis of the component planes, can be used both to explore patterns among samples, as well as to identify individual fluorescence components. We analysed a large and heterogeneous EEM data set, including samples from a river catchment collected under a range of hydrological conditions, along a 60-km downstream gradient, and under the influence of different degrees of anthropogenic impact. According to our results, chemical industry effluents appeared to have unique and distinctive spectral characteristics. On the other hand, river samples collected under flash flood conditions showed homogeneous EEM shapes. The correlation analysis of the component planes suggested the presence of four fluorescence components, consistent with DOM components previously described in the literature. A remarkable strength of this methodology was that outlier samples appeared naturally integrated in the analysis. We conclude that SOM coupled with a correlation analysis procedure is a promising tool for studying large and heterogeneous EEM data sets. PMID:24906009

  15. In-vivo optical detection of brain tumor and tumor margin: a combined auto-fluorescence and diffuse reflectance spectroscopic study

    NASA Astrophysics Data System (ADS)

    Majumder, Shovan K.; Gebhart, Steven; Thompson, Reid; Weaver, Kyle D.; Johnson, Mahlon D.; Lin, Wei-Chiang; Mahadevan-Jansen, Anita

    2007-02-01

    Recently, optical spectroscopy has shown considerable promise to be used as a potential clinical tool for human brain tumor detection and therapeutic guidance. Our group showed for the first time the possibility of using combined autofluorescence and diffuse reflectance spectroscopy and established its applicability for human brain tumor demarcation in previous in vitro and in vivo studies. We report in this paper the results of a clinical study designed to further evaluate the efficacy of the approach for demarcation of brain tumors and tumor margins from normal brain tissues in intra-operative clinical setting. Using a portable system, optical spectra were collected from the brain of 110 patients undergoing craniotomy at the Vanderbilt University Medical Center. Spectral measurements were taken from multiple sites of tumor core, tumor margin and normal areas of brain tissues and the resulting spectra were correlated with the corresponding histopathologic diagnosis. Using histology as the gold standard, a probabilistic multi-class diagnostic algorithm was developed to simultaneously distinguish tumor core and tumor margin from normal brain tissue sites using independent training and validation sets of data. An unbiased estimate of the accuracy of the model indicates that combined autofluorescence and diffuse reflectance spectroscopy was able to distinguish tumor core and tumor margin from normal brain tissues with an average predictive accuracy of ~88%.

  16. Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.

    PubMed

    Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki; Aoki, Kazuhiro

    2014-09-01

    Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction.

  17. Application of Peak Intensity Analysis to Measurements of Protein Binding to Lipid Vesicles and Erythrocytes Using Fluorescence Correlation Spectroscopy: Dependence on Particle Size.

    PubMed

    Antonenko, Yuri N; Lapashina, Anna S; Kotova, Elena A; Ramonova, Alla A; Moisenovich, Mikhail M; Agapov, Igor I

    2017-02-01

    Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool for investigation of processes accompanied by changes in the mobility of molecules and complexes. In the present work, peak intensity analysis (PIA) in combination with the solution stirring using FCS setup was applied to explore the interaction between fluorescently labeled protein ligands and corresponding receptors located on membranes. In the system composed of biotinylated liposomes and fluorescently labeled streptavidin as a ligand, PIA allowed us to determine the optimum receptor concentration and demonstrate the essential dependence of the binding efficacy on the length of the linker between the biotin group and the polar head group of the lipid. The binding was dependent on the size of liposomes which was varied by lipid extrusion through filters of different pore diameters. The sensitivity of the method was higher with the liposomes of larger sizes. The PIA approach can be applied not only to liposomes but also to relatively large objects, e.g., erythrocytes or Sepharose beads derivatized with lactose as a receptor for the binding of viscumin and ricin.

  18. Spectroscopic detection

    DOEpatents

    Woskov, Paul P.; Hadidi, Kamal

    2003-01-01

    In embodiments, spectroscopic monitor monitors modulated light signals to detect low levels of contaminants and other compounds in the presence of background interference. The monitor uses a spectrometer that includes a transmissive modulator capable of causing different frequency ranges to move onto and off of the detector. The different ranges can include those with the desired signal and those selected to subtract background contributions from those with the desired signal. Embodiments of the system are particularly useful for monitoring metal concentrations in combustion effluent.

  19. Application of correlation constrained multivariate curve resolution alternating least-squares methods for determination of compounds of interest in biodiesel blends using NIR and UV-visible spectroscopic data.

    PubMed

    de Oliveira, Rodrigo Rocha; de Lima, Kássio Michell Gomes; Tauler, Romà; de Juan, Anna

    2014-07-01

    This study describes two applications of a variant of the multivariate curve resolution alternating least squares (MCR-ALS) method with a correlation constraint. The first application describes the use of MCR-ALS for the determination of biodiesel concentrations in biodiesel blends using near infrared (NIR) spectroscopic data. In the second application, the proposed method allowed the determination of the synthetic antioxidant N,N'-Di-sec-butyl-p-phenylenediamine (PDA) present in biodiesel mixtures from different vegetable sources using UV-visible spectroscopy. Well established multivariate regression algorithm, partial least squares (PLS), were calculated for comparison of the quantification performance in the models developed in both applications. The correlation constraint has been adapted to handle the presence of batch-to-batch matrix effects due to ageing effects, which might occur when different groups of samples were used to build a calibration model in the first application. Different data set configurations and diverse modes of application of the correlation constraint are explored and guidelines are given to cope with different type of analytical problems, such as the correction of matrix effects among biodiesel samples, where MCR-ALS outperformed PLS reducing the relative error of prediction RE (%) from 9.82% to 4.85% in the first application, or the determination of minor compound with overlapped weak spectroscopic signals, where MCR-ALS gave higher (RE (%)=3.16%) for prediction of PDA compared to PLS (RE (%)=1.99%), but with the advantage of recovering the related pure spectral profile of analytes and interferences. The obtained results show the potential of the MCR-ALS method with correlation constraint to be adapted to diverse data set configurations and analytical problems related to the determination of biodiesel mixtures and added compounds therein.

  20. Visualization of epidermal growth factor (EGF) receptor aggregation in plasma membranes by fluorescence resonance energy transfer. Correlation of receptor activation with aggregation.

    PubMed

    Carraway, K L; Koland, J G; Cerione, R A

    1989-05-25

    Fluorescence resonance energy transfer between epidermal growth factor (EGF) molecules, labeled with fluorescent reporter groups, was used as a monitor for EGF receptor-receptor interactions in plasma membranes isolated from human epidermoid A431 cells. Epidermal growth factor molecules labeled at the amino terminus with fluorescein isothiocyanate served as donor molecules in these energy transfer measurements, while EGF molecules labeled with eosin isothiocyanate at the amino terminus served as the energy acceptors. Both of these derivatives were shown to be active in binding to membrane receptors and in the activation of the endogenous receptor/tyrosine kinase activity. We found that membranes in the absence of added metal ion activators showed relatively little energy transfer (approximately 10% donor quenching) between the labeled growth factors. However, divalent metal ion activators of the EGF receptor/tyrosine kinase caused a significant increase in the extent of energy transfer between the labeled EGF molecules. Specifically, in the presence of 20 mM MgCl2, the extent of quenching of the donor fluorescence increased to 25% (from 10% in the absence of metal), while in the presence of 4 mM MnCl2, the extent of energy transfer was increased still further to 40-50%. The addition of an excess of EDTA resulted in the reversal of the observed energy transfer to basal levels. The increased energy transfer in the presence of these divalent cations correlated well with the ability of these metals to stimulate the EGF receptor/tyrosine kinase activity. However, the extent of receptor-receptor interactions measured by energy transfer was independent of receptor autophosphorylation. Overall, these results suggest that conditions under which the EGF receptor is primed to be active as a tyrosine kinase, within a lipid milieu, result in an increased aggregation of the receptor.

  1. Correlative Fluorescence and Scanning Electron Microscopy of Labelled Core Fucosylated Glycans Using Cryosections Mounted on Carbon-Patterned Glass Slides.

    PubMed

    Vancová, Marie; Nebesářová, Jana

    2015-01-01

    The aim of the study is co-localization of N-glycans with fucose attached to N-acetylglucosamine in α1,3 linkage, that belong to immunogenic carbohydrate epitopes in humans, and N-glycans with α1,6-core fucose typical for mammalian type of N-linked glycosylation. Both glycan epitopes were labelled in cryosections of salivary glands isolated from the tick Ixodes ricinus. Salivary glands secrete during feeding many bioactive molecules and influence both successful feeding and transmission of tick-borne pathogens. For accurate and reliable localization of labelled glycans in both fluorescence and scanning electron microscopes, we used carbon imprints of finder or indexed EM grids on glass slides. We discuss if the topographical images can provide information about labelled structures, the working setting of the field-emission scanning electron microscope and the influence of the detector selection (a below-the-lens Autrata improved YAG detector of back-scattered electrons; in-lens and conventional Everhart-Thornley detectors of secondary electrons) on the imaging of gold nanoparticles, quantum dots and osmium-stained membranes.

  2. Two-photon excitation fluorescence cross-correlation assay for ligand-receptor binding: cell membrane nanopatches containing the human micro-opioid receptor.

    PubMed

    Swift, Jody L; Burger, Melanie C; Massotte, Dominique; Dahms, Tanya E S; Cramb, David T

    2007-09-01

    Current ligand-receptor binding assays for G-protein coupled receptors cannot directly measure the system's dissociation constant, Kd, without purification of the receptor protein. Accurately measured Kd's are essential in the development of a molecular level understanding of ligand-receptor interactions critical in rational drug design. Here we report the introduction of two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to the direct analysis of ligand-receptor interactions of the human micro opioid receptor (hMOR) for both agonists and antagonists. We have developed the use of fluorescently distinct, dye-labeled hMOR-containing cell membrane nanopatches ( approximately 100-nm radius) and ligands, respectively, for this assay. We show that the output from TPE-FCCS data sets can be converted to the conventional Hill format, which provides Kd and the number of active receptors per nanopatch. When ligands are labeled with quantum dots, this assay can detect binding with ligand concentrations in the subnanomolar regime. Interestingly, conjugation to a bulky quantum dot did not adversely affect the binding propensity of the hMOR pentapeptide ligand, Leu-enkephalin.

  3. Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption

    NASA Astrophysics Data System (ADS)

    Hou, Jue; Wright, Heather J.; Chan, Nicole; Tran, Richard; Razorenova, Olga V.; Potma, Eric O.; Tromberg, Bruce J.

    2016-06-01

    Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells-the "optical redox ratio" (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.

  4. Investigation by two-photon fluorescence correlation spectroscopy of the interaction of the nucleocapsid protein of HIV-1 with hairpin loop DNA sequences

    NASA Astrophysics Data System (ADS)

    Mely, Yves; Azoulay, Joel; Beltz, Herve; Clamme, Jean-Pierre; Bernacchi, Serena; Ficheux, Damien; Roques, Bernard P.; Darlix, Jean-Luc

    2004-09-01

    The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two strand transfer reactions required during reverse transcription. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response element, TAR sequence, at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3" terminus of the early product of reverse transcription. To characterize NCp7-mediated nucleic acid destabilization, we investigated by steady-state and time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly-labelled cTAR sequence with NCp7. The conformational fluctuations observed in the absence of NCp7 were associated with the rapid opening and closing (fraying) of the double stranded terminal segment of cTAR. NCp7 destabilizes cTAR mainly through a large increase of the opening rate constant. Additionally, the various destabilizing structures (bulges, internal loop, mismatches) spread all over cTAR secondary structure were found to be critical for NCp7 chaperone activity. Taken together, our data enabled us to propose a molecular mechanism for the destabilizing activity of NCp7 on cTAR which is crucial for the formation of the cTAR-TAR complex during the first strand transfer reaction.

  5. A new color correlation method applied to XRF Al/Si ratios and other lunar remote sensing data. [X Ray Fluorescence

    NASA Technical Reports Server (NTRS)

    Clark, P. E.; Andre, C. G.; Adler, I.; Eliason, E.

    1978-01-01

    Orbital X-ray fluorescence Al/Si intensity ratios, corrected for variations in solar activity, are correlated with normal albedo, elevation measurements from laser altimetry data, and gamma ray data in the 2.75-8.60 MeV range. Each of these data sets is placed into a digital array consisting of 1/4 deg latitude by 1/4 deg longitude pixels. Information relative to the correlation of Al/Si ratios with each of the other data sets is presented in the following forms: (1) histograms are given for each data set to show the frequency distribution within the areas of common coverage; (2) density plots are produced from the plot of a two-dimensional array consisting of the Al/Si ratio vs the other parameter value for each pixel; and (3) color correlation maps are produced by placing the two-dimensional array into a 3 x 3 matrix consisting of nine equal subarrays containing an equal number of data points.

  6. Fluorescence correlation spectroscopy in thin films at reflecting substrates as a means to study nanoscale structure and dynamics at soft-matter interfaces

    NASA Astrophysics Data System (ADS)

    Täuber, Daniela; Radscheit, Kathrin; von Borczyskowski, Christian; Schulz, Michael; Osipov, Vladimir Al.

    2016-07-01

    Structure and dynamics at soft-matter interfaces play an important role in nature and technical applications. Optical single-molecule investigations are noninvasive and capable to reveal heterogeneities at the nanoscale. In this work we develop an autocorrelation function (ACF) approach to retrieve tracer diffusion parameters obtained from fluorescence correlation spectroscopy (FCS) experiments in thin liquid films at reflecting substrates. This approach then is used to investigate structure and dynamics in 100-nm-thick 8CB liquid crystal films on silicon wafers with five different oxide thicknesses. We find a different extension of the structural reorientation of 8CB at the solid-liquid interface for thin and for thick oxide. For the thin oxides, the perylenediimide tracer diffusion dynamics in general agrees with the hydrodynamic modeling using no-slip boundary conditions with only a small deviation close to the substrate, while a considerably stronger decrease of the interfacial tracer diffusion is found for the thick oxides.

  7. Fluorescence correlation spectroscopy at the oil-water interface: hard disk diffusion behavior in dilute beta-lactoglobulin layers precedes monolayer formation.

    PubMed

    Donsmark, Jesper; Rischel, Christian

    2007-06-05

    We have performed a thorough characterization of fluorescence correlations spectroscopy (FCS) applied to oil-water interfaces of viscous oil droplets in aqueous solution, including numerical wave-optical calculations of the detection geometry and regularized multicomponent analysis of sample data. It is shown how significant errors in the estimation of the surface concentration can be avoided when FCS is applied to an interface region. We present data on the adsorption dynamics of beta-lactoglobulin (BLG), a well-studied model system. It is found that electrostatic repulsion slows the adsorption process and reduces the initial saturation density far below the monolayer concentration. During the first stages of adsorption, the diffusion coefficients of adsorbed protein closely follow the 2D hard disk model of Lahtinen et al.1 in response to increased surface concentration, which suggests that protein-protein interactions are limited to long-range Coulombic interactions at this stage.

  8. Preparation of cesium targets for gamma-spectroscopic studies

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, S.; Basu, S. K.; Chanda, S.; Deb, P.; Eqbal, Md; Kundu, S.; Joseph, D.

    2000-11-01

    A procedure to prepare monoisotopic cesium compound targets for gamma-spectroscopic experiments is described. Using this procedure, uniform targets up to thicknesses of 0.6-1.2 mg/cm 2 were prepared and used for in-beam spectroscopic studies. The purity of the target was tested by energy dispersive X-ray fluorescence (EDXRF) measurements.

  9. Understanding Structure-Property Correlation in Monocationic and Dicationic Ionic Liquids through Combined Fluorescence and Pulsed-Field Gradient (PFG) and Relaxation NMR Experiments.

    PubMed

    Kumar Sahu, Prabhat; Ghosh, Arindam; Sarkar, Moloy

    2015-11-05

    Steady state, time-resolved fluorescence and NMR experiments are carried out to gain deeper insights into the structure-property correlation in structurally similar monocationic and dicationic room-temperature ionic liquids (RTILs). The excitation wavelength dependent fluorescence response of fluorophore in 1-methy-3-propyllimidazolium bis(trifluoromethylsulfonyl)amide [C3MIm][NTf2] is found to be different from that of 1,6-bis(3-methylimidazolium-1-yl)hexane bis(trifluoromethylsulfonyl)amide [C6(MIm)2][NTf2]2 and 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide [C6MIm][NTf2]. The outcomes of the present solvent dynamics study in [C3MIm][NTf2] when compared with those in [C6(MIm)2][NTf2]2 and in [C6MIm][NTf2] from our previous studies (Phys. Chem. Chem. Phys. 2014, 16, 12918-12928) indicate the involvement of dipolar rotation of imidazolium cation during solvation. To correlate the findings of solvation dynamics study with the dipolar rotation of the imidazolium ring, pulsed-field gradient (PFG)-NMR technique for translational diffusion coefficient measurement and (1)H as well as (19)F spin-lattice relaxation measurements are employed. NMR investigation reveals that an ultrafast component of solvation can be related to the dipolar rotation of imidazolium cation; hence, the role of dipolar rotation of cations in governing the dynamics of solvation in ILs cannot be ignored. Analysis of the rotational relaxation dynamics data by the Stokes-Einstein-Debye hydrodynamic theory unveils distinctive features of solute-solvent interaction in [C3MIm][NTf2] and [C6(MIm)2][NTf2]2.

  10. Correlation between Diarrhea Severity and Oocyst Count via Quantitative PCR or Fluorescence Microscopy in Experimental Cryptosporidiosis in Calves

    PubMed Central

    Operario, Darwin J.; Bristol, Lauren S.; Liotta, Janice; Nydam, Daryl V.; Houpt, Eric R.

    2015-01-01

    Cryptosporidium is an important diarrhea-associated pathogen, however the correlation between parasite burden and diarrhea severity remains unclear. We studied this relationship in 10 experimentally infected calves using immunofluorescence microscopy and real-time polymerase chain reaction (qPCR) (N = 124 fecal samples). The qPCR data were corrected for extraction/amplification efficiency and gene copy number to generate parasite counts. The qPCR and microscopic oocyst quantities exhibited significant correlation (R2 = 0.33, P < 0.05), however qPCR had increased sensitivity. Upon comparison with diarrhea severity scores (from 0 to 3), a PCR-based count of ≥ 2.6 × 105 parasites or an immunofluorescence microscopy count of ≥ 4.5 × 104 oocysts were discriminatory predictors of moderate-to-severe diarrhea (versus no-to-mild diarrhea), with accuracies and predictive values of 72–82%. In summary, a quantitative approach for Cryptosporidium can refine predictive power for diarrhea and appears useful for distinguishing clinical cryptosporidiosis versus subclinical infection. PMID:25371182

  11. Chemical correlation of some late Cenozoic tuffs of Northern and Central California by neutron activation analysis of glass and comparison with X-ray fluorescence analysis

    USGS Publications Warehouse

    Sarna-Wojcicki, Andrei M.; Bowman, Harry W.; Russell, Paul C.

    1979-01-01

    Glasses separated from several dacitic and rhyolitic late Cenozoic tuffs of northern and central California were analyzed by neutron activation for more than 43 elemental abundances. Eighteen elements--scandiurn, manganese, iron, zinc, rubidium, cesium, barium, lanthanum, cerium, samarium, europium, terbiurn, dysprosiurn, ytterbiurn, hafniurn, tantalurn, thorium and uranium--were selected as most suitable for purposes of chemical correlation on the basis of their natural variability in silicic tuffs and the precision obtainable in analysis. Stratigraphic relations between tuffs and replicate chemical analyses on individual tuffs make it possib1e to calibrate a quantitative parameter, the similarity coefficient, which indicates the degree of correlation for the tuffs studied. The highest similarity coefficient (0.99) was obtained for analyses of two tuffs (potassium-argon dated at about' 6.0 m.y.) exposed in the Merced(?) and Petaluma Formations of Sonoma County, which represent different paleoenvironments, shallow-water marine and fresh water or brackish marine, respectively. Corre1ation of these formations on the basis of criteria other than tephrochronoloqy would be difficult. Results of neutron activation analysis in general confirm earlier correlations made on the basis of analysis by X-ray fluorescence but also make it possible to resolve small compositional differences between chemically simi1ar tuffs in stratigraphic proximity. The Lawlor Tuff (potassium-argon dated at about 4.0 m.y.) is identified at two new localities: in a core sample obtained from a bore hole east of Suisun Bay, and from the Kettleman Hills of western San Joaquin Valley. This identification permits correlation of the uppermost part of the marine Etchegoin Formation in the San Joaquin Valley with the continental Livermore Gravels of Clark, the Tassajara Formation, and the upper part of the Sonoma Volcanics in the cel1tral Coast Ranges of California. A younger tuff near the top of the

  12. On the influence of crosslinker on template complexation in molecularly imprinted polymers: a computational study of prepolymerization mixture events with correlations to template-polymer recognition behavior and NMR spectroscopic studies.

    PubMed

    Shoravi, Siamak; Olsson, Gustaf D; Karlsson, Björn C G; Nicholls, Ian A

    2014-06-12

    Aspects of the molecular-level basis for the function of ethylene glycol dimethacrylate and trimethylolproprane trimethacrylate crosslinked methacrylic acid copolymers molecularly imprinted with (S)-propranolol have been studied using a series of all-component and all-atom molecular dynamics studies of the corresponding prepolymerization systems. The crosslinking agents were observed to contribute to template complexation, and the results were contrasted with previously reported template-recognition behavior of the corresponding polymers. Differences in the extent to which the two crosslinkers interacted with the functional monomer were identified, and correlations were made to polymer-ligand recognition behavior and the results of nuclear magnetic resonance spectroscopic studies studies. This study demonstrates the importance of considering the functional monomer-crosslinker interaction when designing molecularly imprinted polymers, and highlights the often neglected general contribution of crosslinker to determining the nature of molecularly imprinted polymer-template selectivity.

  13. On the Influence of Crosslinker on Template Complexation in Molecularly Imprinted Polymers: A Computational Study of Prepolymerization Mixture Events with Correlations to Template-Polymer Recognition Behavior and NMR Spectroscopic Studies

    PubMed Central

    Shoravi, Siamak; Olsson, Gustaf D.; Karlsson, Björn C. G.; Nicholls, Ian A.

    2014-01-01

    Aspects of the molecular-level basis for the function of ethylene glycol dimethacrylate and trimethylolproprane trimethacrylate crosslinked methacrylic acid copolymers molecularly imprinted with (S)-propranolol have been studied using a series of all-component and all-atom molecular dynamics studies of the corresponding prepolymerization systems. The crosslinking agents were observed to contribute to template complexation, and the results were contrasted with previously reported template-recognition behavior of the corresponding polymers. Differences in the extent to which the two crosslinkers interacted with the functional monomer were identified, and correlations were made to polymer-ligand recognition behavior and the results of nuclear magnetic resonance spectroscopic studies studies. This study demonstrates the importance of considering the functional monomer–crosslinker interaction when designing molecularly imprinted polymers, and highlights the often neglected general contribution of crosslinker to determining the nature of molecularly imprinted polymer-template selectivity. PMID:24927149

  14. Spectroscopic Studies of Fluorescence Effects in Bioactive 4-(5-Heptyl-1,3,4-Thiadiazol-2-yl)Benzene-1,3-Diol and 4-(5-Methyl-1,3,4-Thiadiazol-2-yl)Benzene-1,3-Diol Molecules Induced by pH Changes in Aqueous Solutions.

    PubMed

    Matwijczuk, Arkadiusz; Kluczyk, Dariusz; Górecki, Andrzej; Niewiadomy, Andrzej; Gagoś, Mariusz

    2017-03-01

    This paper presents the results of stationary fluorescence spectroscopy and time-resolved spectroscopy analyses of two 1,3,4-thiadiazole analogues, i.e. 4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C1) and 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C7) in an aqueous medium containing different concentrations of hydrogen ions. An interesting dual florescence effect was observed when both compounds were dissolved in aqueous solutions at pH below 7 for C1 and 7.5 for C7. In turn, for C1 and C7 dissolved in water at pH higher than the physiological value (mentioned above), single fluorescence was only noted. Based on previous results of investigations of the selected 1,3,4-thiadiazole compounds, it was noted that the presented effects were associated with both conformational changes in the analysed molecules and charge transfer (CT) effects, which were influenced by the aggregation factor. However, in the case of C1 and C7, the dual fluorescence effects were visible in a higher energetic region (different than that observed in the 1,3,4-thiadiazoles studied previously). Measurements of the fluorescence lifetimes in a medium characterised by different concentrations of hydrogen ions revealed clear lengthening of the excited-state lifetime in a pH range at which dual fluorescence effects can be observed. An important finding of the investigations presented in this article is the fact that the spectroscopic effects observed not only are interesting from the cognitive point of view but also can help in development of an appropriate theoretical model of molecular interactions responsible for the dual fluorescence effects in the analysed 1,3,4-thiadiazoles. Furthermore, the study will clarify a broad range of biological and pharmaceutical applications of these compounds, which are more frequently used in clinical therapies. Graphical Abstract Upper left corner - C7 molecule at high pH, right upper corner - fluorescence emission spectrum for C7 dissolved in H2

  15. Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis.

    PubMed

    Zhang, Xuzhu; Poniewierski, Andrzej; Jelińska, Aldona; Zagożdżon, Anna; Wisniewska, Agnieszka; Hou, Sen; Hołyst, Robert

    2016-10-04

    The equilibrium and rate constants of molecular complex formation are of great interest both in the field of chemistry and biology. Here, we use fluorescence correlation spectroscopy (FCS), supplemented by dynamic light scattering (DLS) and Taylor dispersion analysis (TDA), to study the complex formation in model systems of dye-micelle interactions. In our case, dyes rhodamine 110 and ATTO-488 interact with three differently charged surfactant micelles: octaethylene glycol monododecyl ether C12E8 (neutral), cetyltrimethylammonium chloride CTAC (positive) and sodium dodecyl sulfate SDS (negative). To determine the rate constants for the dye-micelle complex formation we fit the experimental data obtained by FCS with a new form of the autocorrelation function, derived in the accompanying paper. Our results show that the association rate constants for the model systems are roughly two orders of magnitude smaller than those in the case of the diffusion-controlled limit. Because the complex stability is determined by the dissociation rate constant, a two-step reaction mechanism, including the diffusion-controlled and reaction-controlled rates, is used to explain the dye-micelle interaction. In the limit of fast reaction, we apply FCS to determine the equilibrium constant from the effective diffusion coefficient of the fluorescent components. Depending on the value of the equilibrium constant, we distinguish three types of interaction in the studied systems: weak, intermediate and strong. The values of the equilibrium constant obtained from the FCS and TDA experiments are very close to each other, which supports the theoretical model used to interpret the FCS data.

  16. The application of k-shell x-ray fluorescence to determine bone lead burden and its correlation with hypertension among African Americans in Gadsden County, Florida

    NASA Astrophysics Data System (ADS)

    Jackson-Edwards, Patrice

    Photons from k shell x-ray fluorescence illuminates lead atoms by measuring the characteristic x-rays which indicate the abundance of 210Pb present in a sample. The measurement utilizes a 109Cd source and a low-energy germanium detector, which has emerged as the best available technique for estimating cumulative exposure to lead in adults and for predicting lead-associated risks for adult chronic disease outcomes such as hypertension. The main focus of this study, was to show the correlation between bone lead concentration at the tibia (mean +/- standard deviation of 7+/-1 ppm) and patella (mean +/- standard deviation of 6+/-1 ppm) bone sites and hypertension (mean +/- standard deviation of the systolic standing 143+/-18mmHg, systolic sitting 140+/-17mmHg, diastolic standing 88+/-14 mmHg, and diastolic sitting 81+/-9 mmHg), among the 67 Gadsden County subjects that participated in this study. This was accomplished using FAMU's setup for the detector. The gamma rays emitted by the 109Cd source are scattered by atomic electrons in the k-shell. Excited electrons in the k-shell then spontaneously fluoresce at 88 keV as a signature of lead in the bone. The 88 keV photons are then detected at an angle of 180 degrees with respect to the incident x-ray direction and are detected by the Canberra Germanium solid-state detector bathed in liquid nitrogen. Results show that in this population all lead biomarkers (tibia lead, patella lead, and blood lead) were not significant contributors to the occurrence of hypertension. In the final logistic regression analysis, age and gender were predictors for the occurrence of hypertension at the p<0.05 level in the overall population. This study will help contribute to the understanding of the body's management of lead toxicity and to KXRF techniques currently used in physics research.

  17. Spectroscopic properties of chlorophyll f.

    PubMed

    Li, Yaqiong; Cai, Zheng-Li; Chen, Min

    2013-09-26

    The absorption and fluorescence spectra of chlorophyll f (newly discovered in 2010) have been measured in acetone and methanol at different temperatures. The spectral analysis and assignment are compared with the spectra of chlorophyll a and d under the same experimental conditions. The spectroscopic properties of these chlorophylls have further been studied by the aid of density functional CAM-B3LYP and high-level symmetric adapted coupled-cluster configuration interaction calculations. The main Q and Soret bands and possible sidebands of chlorophylls have been determined. The photophysical properties of chlorophyll f are discussed.

  18. Multiparametric probing of microenvironment with solvatochromic fluorescent dyes.

    PubMed

    Klymchenko, Andrey S; Demchenko, Alexander P

    2008-01-01

    We describe new methodology for multiparametric probing of weak non-covalent interactions in the medium based on response of environment-sensitive fluorescent dyes. The commonly used approach is based on correlation of one spectroscopic parameter (e.g. wavelength shift) with environment polarity, which describes a superposition of universal and specific (such as hydrogen bonding) interactions. In our approach, by using several independent spectroscopic parameters of a dye, we monitor simultaneously each individual type of the interactions. For deriving these extra parameters the selected dye should exist in several excited and/or ground states. In the present work, we applied 4'-(diethylamino)-3-hydroxyflavone, which undergoes the excited-state intramolecular proton transfer (ESIPT) and thus exhibits an additional emission band belonging to an ESIPT product (tautomer) form of the dye. The spectroscopic characteristics of the excited normal and the tautomer states as well as of the ESIPT reaction of the dye are differently sensitive to the different types of interactions with microenvironment and therefore can be used for its multiparametric description. The new methodology allowed us to monitor simultaneously three fundamental physicochemical parameters of probe microenvironment: polarity, electronic polarizability and H-bond donor ability. The applications of this approach to binary solvent mixtures, reverse micelles, lipid bilayers and binding sites of proteins are presented and the limitations of this approach are discussed. We believe that the methodology of multiparametric probing will extend the capabilities of fluorescent probes as the tools in biomolecular and cellular research.

  19. Spectroscopic properties, energy transfer dynamics, and laser performance of thulium-holmium doped laser systems

    NASA Astrophysics Data System (ADS)

    Kalisky, Yehoshua Y.; Rotman, Stanley R.; Boulon, Georges; Pedrini, Christian; Brenier, Alain

    1994-07-01

    Spectroscopic studies using laser induced fluorescence and numerical modelling of energy transfer and back transfer mechanism are reported in Er:Tm:Ho:YLF, Cr:Tm:Ho:YAG and Cr:Tm:YAG laser crystals at various temperatures (10 K - 300 K). Direct energy transfer from Tm3+ excited states to Ho3+ 5I7 emitting level was observed and analyzed both in YAG and YLF. Further analysis of Cr3+ and Tm3+ time-dependent emission curves indicate a strong correlation of chromium- thulium pairs. Pulsed operation of holmium laser at high temperature will be presented.

  20. A prospective Phase II clinical trial of 5-aminolevulinic acid to assess the correlation of intraoperative fluorescence intensity and degree of histologic cellularity during resection of high-grade gliomas.

    PubMed

    Lau, Darryl; Hervey-Jumper, Shawn L; Chang, Susan; Molinaro, Annette M; McDermott, Michael W; Phillips, Joanna J; Berger, Mitchel S

    2016-05-01

    OBJECT There is evidence that 5-aminolevulinic acid (ALA) facilitates greater extent of resection and improves 6-month progression-free survival in patients with high-grade gliomas. But there remains a paucity of studies that have examined whether the intensity of ALA fluorescence correlates with tumor cellularity. Therefore, a Phase II clinical trial was undertaken to examine the correlation of intensity of ALA fluorescence with the degree of tumor cellularity. METHODS A single-center, prospective, single-arm, open-label Phase II clinical trial of ALA fluorescence-guided resection of high-grade gliomas (Grade III and IV) was held over a 43-month period (August 2010 to February 2014). ALA was administered at a dose of 20 mg/kg body weight. Intraoperative biopsies from resection cavities were collected. The biopsies were graded on a 4-point scale (0 to 3) based on ALA fluorescence intensity by the surgeon and independently based on tumor cellularity by a neuropathologist. The primary outcome of interest was the correlation of ALA fluorescence intensity to tumor cellularity. The secondary outcome of interest was ALA adverse events. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and Spearman correlation coefficients were calculated. RESULTS A total of 211 biopsies from 59 patients were included. Mean age was 53.3 years and 59.5% were male. The majority of biopsies were glioblastoma (GBM) (79.7%). Slightly more than half (52.5%) of all tumors were recurrent. ALA intensity of 3 correlated with presence of tumor 97.4% (PPV) of the time. However, absence of ALA fluorescence (intensity 0) correlated with the absence of tumor only 37.7% (NPV) of the time. For all tumor types, GBM, Grade III gliomas, and recurrent tumors, ALA intensity 3 correlated strongly with cellularity Grade 3; Spearman correlation coefficients (r) were 0.65, 0.66, 0.65, and 0.62, respectively. The specificity and PPV of ALA intensity 3 correlating

  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. Developing a targeting system for bacterial membranes: measuring receptor-phosphatidylglycerol interactions with (1)H NMR, ITC and fluorescence correlation spectroscopy.

    PubMed

    Alliband, Amanda; Wang, Zifan; Thacker, Christopher; English, Douglas S; Burns, Dennis H

    2015-01-14

    An ammonium picket porphyrin that targets bacterial membranes has been prepared and shown to bind to phosphatidylglycerol (PG), a bacterial lipid, when the lipid was in solution, contained within synthetic membrane vesicles, or when in Gram-negative and Gram-positive bacterial membranes. The multifunctional receptor was designed to interact with both the phosphate anion portion and neutral glycerol portion of the lipid headgroup. The receptor's affinity and selectivity for binding to surfactant vesicles or lipid vesicles that contain PG within their membranes was directly measured using fluorescence correlation spectroscopy (FCS). FCS demonstrated that the picket porphyrin's binding pocket was complementary for the lipid headgroup, since simple Coulombic interactions alone did not induce binding. (1)H NMR and isothermal titration calorimetry (ITC) were used to determine the receptor's binding stoichiometry, receptor-lipid complex structure, binding constant, and associated thermodynamic properties of complexation in solution. The lipid-receptor binding motif in solution was shown to mirror the binding motif of membrane-bound PG and receptor. Cell lysis assays with E. coli (Gram-negative) and Bacillus thuringiensis (Gram-positive) probed with UV/Visible spectrophotometry indicated that the receptor was able to penetrate either bacterial cell wall and to bind to the bacterial inner membrane.

  3. Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

    SciTech Connect

    Tiwari, Manisha; Mikuni, Shintaro; Muto, Hideki; Kinjo, Masataka

    2013-07-05

    Highlights: •We used two-laser-beam FCCS to determine the dissociation constant (K{sub d}) of IPT domain of p50/p65 heterodimer in living cell. •Interaction of p50 and p65 was analyzed in the cytoplasm and nucleus of single living cell. •Binding affinity of p50/p65 heterodimer is higher in cytoplasm than that of nucleus. -- Abstract: Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K{sub d}, for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K{sub d} values of mCherry{sub 2}- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.

  4. Exhaustive screening of the acid beta-glucosidase gene, by fluorescence-assisted mismatch analysis using universal primers: mutation profile and genotype/phenotype correlations in Gaucher disease.

    PubMed Central

    Germain, D P; Puech, J P; Caillaud, C; Kahn, A; Poenaru, L

    1998-01-01

    Gaucher disease (GD) is one of the most prevalent lysosomal storage disorders and one of the rare genetic diseases now accessible to therapy. Outside the Ashkenazi Jewish community, a high molecular diversity is observed, leaving approximately 30% of alleles undetected. Nevertheless, very few exhaustive methods have been developed for extensive gene screening of a large series of patients. Our approach for a complete search of mutations was the association of fluorescent chemical cleavage of mismatches with a universal strand-specific labeling system. The glucocerebrosidase (GBA) gene was scanned by use of a set of six amplicons, comprising 11 exons, all exon/intron boundaries, and the promoter region. By use of this screening strategy, the difficulties due to the existence of a highly homologous pseudogene were easily overcome, and both GD mutant alleles were identified in all 25 patients studied, thus attesting to a sensitivity that approaches 100%. A total of 18 different mutations and a new glucocerebrosidase haplotype were detected. The mutational spectrum included eight novel acid beta-glucosidase mutations: IVS2 G(+1)-->T, I119T, R170P, N188K, S237P, K303I, L324P, and A446P. These data further indicate the genetic heterogeneity of the lesions causing GD. Established genotype/phenotype correlations generally were confirmed, but notable disparities were disclosed in several cases, thus underlining the limitation in the prognostic value of genotyping. The observed influence of multifactorial control on this monogenic disease is discussed. PMID:9683600

  5. Characterizing the compositional variation of dissolved organic matter over hydrophobicity and polarity using fluorescence spectra combined with principal component analysis and two-dimensional correlation technique.

    PubMed

    Su, Ben-Sheng; Qu, Zhen; He, Xiao-Song; Song, Ying-Hao; Jia, Li-Min

    2016-05-01

    Dissolved organic matter (DOM) obtained from three leachates with different landfill ages was fractionated, and its compositional variation based on hydrophobicity and polarity was characterized by synchronous fluorescence spectra combined with principal component analysis (PCA) and two-dimensional correlation technique. The results showed that the bulk DOM and its fractions were comprised of tryosine-, tryptophan-, fulvic-, and humic-like substances. Tyrosine-like matter was dominant in the young leachate DOM and its fractions, while tryptophan-, fulvic-, and humic-like substances were the main components in the intermediate and old leachate DOMs and their fractions. Tryosine-, tryptophan-, fulvic-, and humic-like substances varied concurrently with the hydrophobicity and polarity. However, the change ratio of these substances was different for the three leachates. Tyrosine-like matter, humic-like materials, and fulvic-like substances were the most sensitive to the hydrophobicity and polarity in the young, intermediate, and old leachates, respectively. Such an integrated approach jointly enhances the characterization of the hydrophobicity- and polarity-dependent DOM fractions and provides a promising way to elucidate the environmental behaviors of different DOM species.

  6. Structural and spectroscopic characterization of methyl isocyanate, methyl cyanate, methyl fulminate, and acetonitrile N-oxide using highly correlated ab initio methods

    NASA Astrophysics Data System (ADS)

    Dalbouha, S.; Senent, M. L.; Komiha, N.; Domínguez-Gómez, R.

    2016-09-01

    Various astrophysical relevant molecules obeying the empirical formula C2H3NO are characterized using explicitly correlated coupled cluster methods (CCSD(T)-F12). Rotational and rovibrational parameters are provided for four isomers: methyl isocyanate (CH3NCO), methyl cyanate (CH3OCN), methyl fulminate (CH3ONC), and acetonitrile N-oxide (CH3CNO). A CH3CON transition state is inspected. A variational procedure is employed to explore the far infrared region because some species present non-rigidity. Second order perturbation theory is used for the determination of anharmonic frequencies, rovibrational constants, and to predict Fermi resonances. Three species, methyl cyanate, methyl fulminate, and CH3CON, show a unique methyl torsion hindered by energy barriers. In methyl isocyanate, the methyl group barrier is so low that the internal top can be considered a free rotor. On the other hand, acetonitrile N-oxide presents a linear skeleton, C3v symmetry, and free internal rotation. Its equilibrium geometry depends strongly on electron correlation. The remaining isomers present a bend skeleton. Divergences between theoretical rotational constants and previous parameters fitted from observed lines for methyl isocyanate are discussed on the basis of the relevant rovibrational interaction and the quasi-linearity of the molecular skeleton.

  7. Structural and spectroscopic characterization of methyl isocyanate, methyl cyanate, methyl fulminate, and acetonitrile N-oxide using highly correlated ab initio methods.

    PubMed

    Dalbouha, S; Senent, M L; Komiha, N; Domínguez-Gómez, R

    2016-09-28

    Various astrophysical relevant molecules obeying the empirical formula C2H3NO are characterized using explicitly correlated coupled cluster methods (CCSD(T)-F12). Rotational and rovibrational parameters are provided for four isomers: methyl isocyanate (CH3NCO), methyl cyanate (CH3OCN), methyl fulminate (CH3ONC), and acetonitrile N-oxide (CH3CNO). A CH3CON transition state is inspected. A variational procedure is employed to explore the far infrared region because some species present non-rigidity. Second order perturbation theory is used for the determination of anharmonic frequencies, rovibrational constants, and to predict Fermi resonances. Three species, methyl cyanate, methyl fulminate, and CH3CON, show a unique methyl torsion hindered by energy barriers. In methyl isocyanate, the methyl group barrier is so low that the internal top can be considered a free rotor. On the other hand, acetonitrile N-oxide presents a linear skeleton, C3v symmetry, and free internal rotation. Its equilibrium geometry depends strongly on electron correlation. The remaining isomers present a bend skeleton. Divergences between theoretical rotational constants and previous parameters fitted from observed lines for methyl isocyanate are discussed on the basis of the relevant rovibrational interaction and the quasi-linearity of the molecular skeleton.

  8. Fluorescent proteins: shine on, you crazy diamond.

    PubMed

    Dedecker, Peter; De Schryver, Frans C; Hofkens, Johan

    2013-02-20

    In this Perspective we discuss recent trends in the development and applications of fluorescent proteins. We start by providing a historical and structural perspective of their spectroscopic and structural aspects and describe how these properties have made fluorescent proteins essential as 'smart labels' for biosensing and advanced fluorescence imaging. We show that the strong link between the spectroscopic properties and protein structure and properties is a necessary element in these developments and that this dependence makes the proteins excellent model systems for a variety of fields. We pay particular attention to emerging or future research opportunities and unsolved questions.

  9. Fluorescence lifetime imaging of endogenous biomarker of oxidative stress

    PubMed Central

    Datta, Rupsa; Alfonso-García, Alba; Cinco, Rachel; Gratton, Enrico

    2015-01-01

    Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. Further, we performed spontaneous Raman spectral analysis at single points of the sample which provided molecular vibration information characteristics of lipid droplets. PMID:25993434

  10. Differences in fluorescence profiles from breast cancer tissues due to changes in relative tryptophan content via energy transfer: tryptophan content correlates with histologic grade and tumor size but not with lymph node metastases

    NASA Astrophysics Data System (ADS)

    Sordillo, Laura A.; Sordillo, Peter P.; Budansky, Yury; Pu, Yang; Alfano, Robert R.

    2014-12-01

    The correlation between histologic grade, an increasingly important measure of prognosis for patients with breast cancer, and tryptophan levels from tissues of 15 breast carcinoma patients was investigated. Changes in the relative content of key native organic biomolecule tryptophan were seen from the fluorescence spectra of cancerous and paired normal tissues with excitation wavelengths of 280 and 300 nm. Due to a large spectral overlap and matching excitation-emission spectra, fluorescence resonance energy transfer from tryptophan-donor to reduced nicotinamide adenine dinucleotides-acceptor was noted. We used the ratios of fluorescence intensities at their spectral emission peaks, or spectral fingerprint peaks, at 340, 440, and 460 nm. Higher ratios correlated strongly with high histologic grade, while lower-grade tumors had low ratios. Large tumor size also correlated with high ratios, while the number of lymph node metastases, a major factor in staging, was not correlated with tryptophan levels. High histologic grade correlates strongly with increased content of tryptophan in breast cancer tissues and suggests that measurement of tryptophan content may be useful as a part of the evaluation of these patients.

  11. Ground and excited state proton transfer of the bioactive plant flavonol robinetin in a protein environment: spectroscopic and molecular modeling studies.

    PubMed

    Pahari, Biswa Pathik; Chaudhuri, Sudip; Chakraborty, Sandipan; Sengupta, Pradeep K

    2015-02-12

    We performed spectroscopic and molecular modeling studies to explore the interaction of the bioactive plant flavonol robinetin (3,7,3',4',5'-OH flavone), with the carrier protein human serum albumin (HSA). Multiparametric fluorescence sensing, exploiting the intrinsic "two color" fluorescence of robinetin (comprising excited state intramolecular proton transfer (ESIPT) and charge transfer (CT) emissions) reveals that binding to HSA significantly affects the emission and excitation profiles, with strongly blue-shifted (∼29 nm) normal fluorescence and remarkable increase in the ESIPT fluorescence anisotropy (r) and lifetime (τ). Flavonol-induced HSA (tryptophan) fluorescence quenching data yield the dynamic quenching constant (KD) as 5.42 × 10(3) M(-1) and the association constant (Ks) as 5.59 × 10(4) M(-1). Time-resolved fluorescence anisotropy decay studies show dramatic (∼170 times) increase in the rotational correlation time (τ(rot)), reflecting greatly enhanced restrictions in motion of robinetin in the protein matrix. Furthermore, prominent induced circular dichroism (ICD) bands appear, indicating that the chiral environment of HSA strongly perturbs the electronic transitions of the intrinsically achiral robinetin molecule. Molecular docking calculations suggest that robinetin binds in subdomain IIA of HSA, where specific interactions with basic residues promote ground state proton abstraction and stabilize an anionic species, which is consistent with spectroscopic observations.

  12. Rapid identification of microorganisms by intrinsic fluorescence

    NASA Astrophysics Data System (ADS)

    Bhatta, Hemant; Goldys, Ewa M.; Learmonth, Robert

    2005-03-01

    Microbial contamination has serious consequences for the industries that use fermentation processes. Common contaminants such as faster growing lactic acid bacteria or wild yeast can rapidly outnumber inoculated culture yeast and produce undesirable end products. Our study focuses on a rapid method of identification of such contaminants based on autofluorescence spectroscopy of bacterial and yeast species. Lactic acid bacteria (Lac-tobacillus casei), and yeast (Saccharomyces cerevisiae) were cultured under controlled conditions and studied for variations in their autofluorescence. We observed spectral differences in the spectral range representative of tryptophan residues of proteins, with excitation at 290 nm and emission scanned in the 300 nm - 440 nm range. Excitation scans between 240 nm and 310 nm were also performed for the emission at 340 nm. Moreover, we observed clearly pronounced differences in the excitation and emission in the visible range, with 410 nm excitation. These results demonstrate that bacterial and yeast species can be differentiated using their intrinsic fluorescence both in UV and in the visible region. The comparative spectroscopic study of selected strains of Saccharomyces yeast showed clear differences between strains. Spectrally-resolved laser scanning microscopy was carried out to link the results obtained using ensembles of cells with spectral properties of individual cells. Strongly fluorescent subpopulation were observed for all yeast strains with excitation at 405 nm. The fluorescence spectra showed variations correlated with cell brightness. The presented results demonstrate that using autofluorescence, it is possible to differentiate between yeast and lactic acid bacteria and between different yeast species.

  13. Effects of essential oil treatments on the secondary protein structure of Vicia faba: a mid-infrared spectroscopic study supported by two-dimensional correlation analysis.

    PubMed

    Mecozzi, Mauro; Sturchio, Elena

    2015-02-25

    In this study we investigated the effects of essential oil treatments on the secondary protein structure of the Vicia faba roots, a bioindicator plant, in order to obtain information for the potential allelopathic uses of these oils as alternative to the use of pesticides in agriculture. We tested two mixtures of essential oils consisting of Tween 20-emulsions of tea tree oil (TTO) and Tween 20-emulsion of Clove and Rosemary (GARROM) essential oils respectively at three different oil concentrations each. The molecular modifications caused in Vicia faba by exposure to oil emulsions were investigated by FTIR spectroscopy in diffuse reflectance (DRIFT) mode. We considered the specific Amide I, Amide II and Amide VI bands by ordinary and second derivative spectroscopy and the results showed that both Tween 20-emulsion of GARROM and Tween 20-emulsion of TTO oils cause transitions among the secondary (α-helix, β-sheet and β-turn) structures with in addition the appearance of random coil structures in exposed samples. The Amide VI bands, placed between 500 and 600 cm(-1), confirmed the structural transitions observed for the Amide I bands. In addition we observed the presence of a protein oxidation effect for TTO treated samples, oxidation which resulted negligible instead for the GARROM oil samples. At last, FTIR spectra were also submitted to two-dimensional correlation analysis (2DCORR) and double two-dimensional correlation analysis (D2DCORR); the results confirmed the different effects caused by the two typologies of essential oils on the secondary protein structures of Vicia faba roots.

  14. Effects of essential oil treatments on the secondary protein structure of Vicia faba: A mid-infrared spectroscopic study supported by two-dimensional correlation analysis

    NASA Astrophysics Data System (ADS)

    Mecozzi, Mauro; Sturchio, Elena

    2015-02-01

    In this study we investigated the effects of essential oil treatments on the secondary protein structure of the Vicia faba roots, a bioindicator plant, in order to obtain information for the potential allelopathic uses of these oils as alternative to the use of pesticides in agriculture. We tested two mixtures of essential oils consisting of Tween 20-emulsions of tea tree oil (TTO) and Tween 20-emulsion of Clove and Rosemary (GARROM) essential oils respectively at three different oil concentrations each. The molecular modifications caused in Vicia faba by exposure to oil emulsions were investigated by FTIR spectroscopy in diffuse reflectance (DRIFT) mode. We considered the specific Amide I, Amide II and Amide VI bands by ordinary and second derivative spectroscopy and the results showed that both Tween 20-emulsion of GARROM and Tween 20-emulsion of TTO oils cause transitions among the secondary (α-helix, β-sheet and β-turn) structures with in addition the appearance of random coil structures in exposed samples. The Amide VI bands, placed between 500 and 600 cm-1, confirmed the structural transitions observed for the Amide I bands. In addition we observed the presence of a protein oxidation effect for TTO treated samples, oxidation which resulted negligible instead for the GARROM oil samples. At last, FTIR spectra were also submitted to two-dimensional correlation analysis (2DCORR) and double two-dimensional correlation analysis (D2DCORR); the results confirmed the different effects caused by the two typologies of essential oils on the secondary protein structures of Vicia faba roots.

  15. Fluorescent Biotin Analogues for Microstructure Patterning and Selective Protein Immobilization.

    PubMed

    Krishna, K Vijaya; Ghosh, Subhadip; Sharma, Bikramjit; Singh, Leeju; Mukherjee, Saptarshi; Verma, Sandeep

    2015-11-24

    Benzyl substitution on ureido nitrogens of biotin led to manifestation of aggregation-induced emission, which was studied by steady-state fluorescence, microscopy, and TD-DFT, providing a rationale into the observed photophysical behavior. Besides exhibiting solvatochromism, the biotin derivatives revealed emission peaks centered at ∼430 and 545 nm, which has been attributed to the π-π stacking interactions. Our TD-DFT results also correlate the spectroscopic data and quantify the nature of transitions involved. The isothermal titration calorimetry data substantiates that the binding of the biotin derivatives with avidin are pretty strong. These derivatives on lithographic patterning present a platform for site specific strept(avidin) immobilization, thus opening avenues for potential applications exploiting these interactions. The fluorescent biotin derivatives can thus find applications in cellular biology and imaging.

  16. Computer Modeling of the Structure and Spectra of Fluorescent Proteins

    PubMed Central

    Grigorenko, B.L.; Savitsky, A.P.

    2009-01-01

    Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers. PMID:22649601

  17. Correlation of polycrystalline Cu(In,Ga)Se{sub 2} device efficiency with homojunction depth and interfacial structure: X-ray photoemission and positron annihilation spectroscopic characterization

    SciTech Connect

    Nelson, A.J.; Sobol, P.E.; Gabor, A.M.; Contreras, M.A.; Asoka-Kumar, P.; Lynn, K.G.

    1994-06-01

    Angled-resolved high resolution photoemission measurements on valence band electronic structure and Cu 2p, In 3d, Ga 2p, and Se 3d core lines were used to evaluate surface and near-surface chemistry of CuInSe{sub 2} and Cu(In,Ga)Se{sub 2} device grade thin films. XPS compositional depth profiles were also acquired from the near-surface region, and bonding of the Cu, In, Ga, and Se was determined as a function of depth. A Cu-poor region was found, indicating CuIn{sub 5}Se{sub 8} or a CuIn{sub 3}Se{sub 5}-In{sub 2}Se{sub 3} mixture. Correlation between the depth of the Cu-poor region/bulk interface and device efficiency showed that the depth was 115 {angstrom} for a 16.4% CIGS device, 240 {angstrom} for a 15.0% CIGS, and 300 {angstrom} for 14.0% CIGS, with similar trends for CIS films. The surface region is n-type, the bulk is p-type, with a 0.5 eV valence band offset. Depth of homojunction may be the determining factor in device performance. Positron annihilation spectroscopy gave similarly illuminating results.

  18. Conformational Changes in Alamethicin Associated with Substitution of Its α-Methylalanines with Leucines: A FTIR Spectroscopic Analysis and Correlation with Channel Kinetics

    PubMed Central

    Haris, Parvez I.; Molle, Gérard; Duclohier, Hervé

    2004-01-01

    Alamethicin, a 20 residue-long peptaibol remains a favorite high voltage-dependent channel-forming peptide. However, the structural significance of its abundant noncoded residues (α-methylalanine or Aib) for its ion channel activity remains unknown, although a previous study showed that replacement of all Aib residues with leucines preserved the essential channel behavior except for much faster single-channel events. To correlate these functional properties with structural data, here we compare the secondary structures of an alamethicin derivative where all the eight Aibs were replaced by leucines and the native alamethicin. Fourier transform infrared (FTIR) spectra of these peptides were recorded in methanol and in aqueous phospholipid membranes. Results obtained show a significant conformational change in alamethicin upon substitution of its Aib residues with Leu. The amide I band occurs at a lower frequency for the Leu-derivative indicating that its α-helices are involved in stronger hydrogen-bonding. In addition, the structure of the Leu-derivative is quite sensitive to membrane fluidity changes. The amide I band shifts to higher frequencies when the lipids are in the fluid phase. This indicates either a decreased solvation due to a more complete peptide insertion or a peptide stretching to match the full thickness of the bilayer. These results contribute to explain the fast single-channel kinetics displayed by the Leu-derivative. PMID:14695266

  19. A deeper insight into an intriguing acetonitrile-water binary mixture: synergistic effect, dynamic Stokes shift, fluorescence correlation spectroscopy, and NMR studies.

    PubMed

    Koley, Somnath; Ghosh, Subhadip

    2016-11-30

    An insight study reveals the strong synergistic solvation behaviours from reporter dye molecules within the acetonitrile (ACN)-water (WT) binary mixture. Synergism of a binary mixture refers to some unique changes of the physical and thermodynamic properties of the solvent mixture, originating from the interactions among its cosolvents, which are absent within the pure cosolvents. Synergistic solvation of a binary mixture is likely to be fundamental for greater stabilization of an excited state solute dipole; at least to some extent greater as compared to one stabilized by any of its cosolvents alone. A dynamic Stokes shift due to the solvation of an excited dipole in the ACN-WT binary mixture is found to be highly relevant to the ground state physical properties of the solute molecule (polarity, hydrophilicity, acidity, etc.). Largely different solvation times in the ACN-WT mixture are observed from different dye molecules with widely varying polarities. However, earlier study shows that dye molecules, irrespective of their varying polarities, exhibit very similar solvation times within a pure solvent (J. Phys. Chem. B, 2014, 118, 7577-7785). On further study with fluorescence correlation spectroscopy (FCS) we observed that, unlike the translational diffusion coefficient (Dt) of a dye molecule within a pure solvent, which remains the same irrespective of the location of the dye molecule inside the solvent, a broad distribution among the Dt values of a dye molecule is obtained from different locations within the ACN-WT binary mixture. Lastly our (1)H NMR study in the ACN-WT binary mixture shows the existence of strong hydrogen bond interactions among the cosolvents in the ACN-WT mixture.

  20. Multiple Escherichia coli RecQ helicase monomers cooperate to unwind long DNA substrates: a fluorescence cross-correlation spectroscopy study.

    PubMed

    Li, Na; Henry, Etienne; Guiot, Elvire; Rigolet, Pascal; Brochon, Jean-Claude; Xi, Xu-Guang; Deprez, Eric

    2010-03-05

    The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3'-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 degrees C, whereas only modest or no cooperativity was observed at 25 degrees C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 degrees C but not at 25 degrees C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.

  1. Development of novel fluorescent probe 3-perylene diphenylphosphine for determination of lipid hydroperoxide with fluorescent image analysis

    SciTech Connect

    Chotimarkorn, Chatchawan; Nagasaka, Reiko; Ushio, Hideki . E-mail: hushio@s.kaiyodai.ac.jp; Ohshima, Toshiaki; Matsunaga, Shigeki

    2005-12-16

    A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, {sup 1}H NMR, and {sup 13}C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies. Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials.

  2. Synthesis and fluorescence properties of Tb(III) complex with a novel β-diketone ligand as well as spectroscopic studies on the interaction between Tb(III) complex and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Zhang, Zhenfeng; Tang, Ruiren

    2012-02-01

    A novel aromatic β-diketone ligand, 4-isopropyl-2,6-bisbenzoylactyl pyridine (L), and its corresponding Tb(III) complex Tb2(L)3·5H2O were synthesised in this paper. The ligand was characterized by FT-IR and 1H NMR. The complex was characterized with elemental analysis and FT-IR. The investigation of fluorescence property of the complex showed that the Tb(III) ion could be sensitized efficiently by the ligand. Furthermore, the interaction of Tb2(L)3·5H2O with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectra, UV-vis absorbance and synchronous fluorescence spectra. The fluorescence quenching mechanism of BSA by Tb2(L)3·5H2O was analyzed. The binding constants, binding site number and the corresponding thermodynamic parameters at different temperatures were calculated. The results indicated that the Van der Waals and hydrogen bond interactions were the predominant intermolecular forces in stabilizing the complex. Moreover, the effect of Tb2(L)3·5H2O on the conformation of BSA was analyzed according to synchronous fluorescence.

  3. Spectroscopic investigations on NO+(X1Σ+, a3Σ+, A1Π) ion using multi-reference configuration interaction method and correlation-consistent sextuple basis set augmented with diffuse functions

    NASA Astrophysics Data System (ADS)

    Zhang, Jin-Ping; Cheng, Xin-Lu; Zhang, Hong; Yang, Xiang-Dong

    2011-06-01

    Three low-lying electronic states (X1Σ+, a3Σ+, and A1Π) of NO+ ion are studied using the complete active space self-consistent-field (CASSCF) method followed by highly accurate valence internally contracted multi-reference configuration interaction (MRCI) approach in combination of the correlation-consistent sextuple basis set augmented with diffuse functions, aug-cc-pV6Z. The potential energy curves (PECs) of the NO+(X1Σ+, a3Σ+, A1Π) are calculated. Based on the PECs, the spectroscopic parameters Re, De, ωe, ωeχe, αe, Be, and D0 are reproduced, which are in excellent agreement with the available measurements. By numerically solving the radial Schrödinger equation of nuclear motion using the Numerov method, the first 20 vibrational levels, inertial rotation and centrifugal distortion constants of NO+(X1Σ+, a3Σ+, A1Π) ion are derived when the rotational quantum number J is equal to zero (J = 0) for the first time, which accord well with the available measurements. Finally, the analytical potential energy functions of these states are fitted, which are used to accurately derive the first 20 classical turning points when J = 0. These results are compared in detail with those of previous investigations reported in the literature.

  4. Spectroscopic-guided brain tumor resection

    NASA Astrophysics Data System (ADS)

    Lin, Wei-Chiang; Toms, Steven A.; Jansen, E. Duco; Mahadevan-Jansen, Anita

    2000-05-01

    A pilot in vivo study was conducted to investigate the feasibility of using optical spectroscopy for brain tumor margin detection. Fluorescence and diffuse reflectance spectra were acquired using a portable clinical spectroscopic system from normal brain tissues, tumors, and tumor margins in 21 brain tumor patients undergoing craniotomy. Results form this study show the potential of optical spectroscopy in detecting infiltrating tumor margins of primary brain tumors.

  5. Quantum chemical calculations and spectroscopic measurements of spectroscopic and thermodynamic properties of given uranyl complexes in aqueous solutions with possible environmental and industrial applications

    NASA Astrophysics Data System (ADS)

    Višňak, Jakub; Sobek, Lukáš

    2016-11-01

    A brief introduction into computational methodology and preliminary results for spectroscopic (excitation energies, vibrational frequencies in ground and excited electronic states) and thermodynamic (stability constants, standard enthalpies and entropies of complexation reactions) properties of some 1:1, 1:2 and 1:3 uranyl sulphato- and selenato- complexes in aqueos solutions will be given. The relativistic effects are included via Effective Core Potential (ECP), electron correlation via (TD)DFT/B3LYP (dispersion interaction corrected) and solvation is described via explicit inclusion of one hydration sphere beyond the coordinated water molecules. We acknowledge limits of this approximate description - more accurate calculations (ranging from semi-phenomenological two-component spin-orbit coupling up to four-component Dirac-Coulomb-Breit hamiltonian) and Molecular Dynamics simulations are in preparation. The computational results are compared with the experimental results from Time-resolved Laser-induced Fluorescence Spectroscopy (TRLFS) and UV-VIS spectroscopic studies (including our original experimental research on this topic). In case of the TRLFS and UV-VIS speciation studies, the problem of complex solution spectra decomposition into individual components is ill-conditioned and hints from theoretical chemistry could be very important. Qualitative agreement between our quantum chemical calculations of the spectroscopic properties and experimental data was achieved. Possible applications for geochemical modelling (e.g. safety studies of nuclear waste repositories, modelling of a future mining site) and analytical chemical studies (including natural samples) are discussed.

  6. Simultaneous spectroscopic measurements of the interior temperature and induced cargo release from pore-restricted mesoporous silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Dong, Juyao; Zink, Jeffrey I.

    2016-05-01

    Temperature changes initiated within nano structures are being increasingly used to externally activate responsive delivery vehicles. Yet, the precise measurement of the nano environment temperature increase and its correlation with the induced macroscopic cargo release are difficult to achieve. In this study, we focus on a photothermally activated drug delivery system based on mesoporous silica nanoparticles, and use an optical nanothermometer - NaYF4:Yb3+,Er3+ crystals - for a ratiometric temperature measurement. Using fluorescent dyes as the payload molecule, both the nanoparticle interior temperature change and the macroscopic cargo release amount are monitored simultaneously by fluorescent spectroscopy. We found that the cargo release lags the temperature increase by about 5 min, revealing the threshold temperature that the particles have to reach before a substantial release could happen. Using this spectroscopic method, we are able to directly compare and correlate a nano environment event with its stimulated macroscopic results.Temperature changes initiated within nano structures are being increasingly used to externally activate responsive delivery vehicles. Yet, the precise measurement of the nano environment temperature increase and its correlation with the induced macroscopic cargo release are difficult to achieve. In this study, we focus on a photothermally activated drug delivery system based on mesoporous silica nanoparticles, and use an optical nanothermometer - NaYF4:Yb3+,Er3+ crystals - for a ratiometric temperature measurement. Using fluorescent dyes as the payload molecule, both the nanoparticle interior temperature change and the macroscopic cargo release amount are monitored simultaneously by fluorescent spectroscopy. We found that the cargo release lags the temperature increase by about 5 min, revealing the threshold temperature that the particles have to reach before a substantial release could happen. Using this spectroscopic method, we are

  7. Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles.

    PubMed

    Richert, Ludovic; Humbert, Nicolas; Larquet, Eric; Girerd-Chambaz, Yves; Manin, Catherine; Ronzon, Frédéric; Mély, Yves

    2016-10-01

    Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.

  8. Spectroscopic characterization of Venus at the single molecule level.

    PubMed

    David, Charlotte C; Dedecker, Peter; De Cremer, Gert; Verstraeten, Natalie; Kint, Cyrielle; Michiels, Jan; Hofkens, Johan

    2012-02-01

    Venus is a recently developed, fast maturating, yellow fluorescent protein that has been used as a probe for in vivo applications. In the present work the photophysical characteristics of Venus were analyzed spectroscopically at the bulk and single molecule level. Through time-resolved single molecule measurements we found that single molecules of Venus display pronounced fluctuations in fluorescence emission, with clear fluorescence on- and off-times. These fluorescence intermittencies were found to occupy a broad range of time scales, ranging from milliseconds to several seconds. Such long off-times can complicate the analysis of single molecule counting experiments or single-molecule FRET experiments.

  9. Super-resolution spectroscopic microscopy via photon localization

    NASA Astrophysics Data System (ADS)

    Dong, Biqin; Almassalha, Luay; Urban, Ben E.; Nguyen, The-Quyen; Khuon, Satya; Chew, Teng-Leong; Backman, Vadim; Sun, Cheng; Zhang, Hao F.

    2016-07-01

    Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm--a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.

  10. Super-resolution spectroscopic microscopy via photon localization.

    PubMed

    Dong, Biqin; Almassalha, Luay; Urban, Ben E; Nguyen, The-Quyen; Khuon, Satya; Chew, Teng-Leong; Backman, Vadim; Sun, Cheng; Zhang, Hao F

    2016-07-25

    Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm-a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.

  11. Super-resolution spectroscopic microscopy via photon localization

    PubMed Central

    Dong, Biqin; Almassalha, Luay; Urban, Ben E.; Nguyen, The-Quyen; Khuon, Satya; Chew, Teng-Leong; Backman, Vadim; Sun, Cheng; Zhang, Hao F.

    2016-01-01

    Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm—a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra. PMID:27452975

  12. Spectroscopic study of 2-[2-(4-cyclaminophenyl)ethen-1-yl] benzothiazoles and their N-allylbenzothiazolium bromides. Solvent and substituent effects on their ultraviolet-visible and fluorescence spectra

    NASA Astrophysics Data System (ADS)

    Gáplovský, Anton; Donovalová, Jana; Magdolen, Peter; Toma, Štefan; Zahradník, Pavol

    2002-01-01

    UV-vis and fluorescence spectra of 2-[2-(4-cyclaminophenyl)ethen-1-yl] benzothiazoles 1 and their N-allylbenzothiazolium bromides 2 have been measured and interpreted. The substitution and solvent effects on electronic structure and spectra have been investigated. The benzothiazolium salts substituted with saturated cyclamines show strong push-pull character and can be used as potential NLO materials. Formation of aggregated structures was observed at higher concentrations of the benzothiazolium bromides.

  13. Selective excitation of tryptophan fluorescence decay in proteins using a subnanosecond 295 nm light-emitting diode and time-correlated single-photon counting

    NASA Astrophysics Data System (ADS)

    McGuinness, Colin D.; Sagoo, Kulwinder; McLoskey, David; Birch, David J. S.

    2005-06-01

    We demonstrate an AlGaN light-emitting diode (LED) giving pulses of ˜600ps full width half maximum, 0.35μW average power, 0.6mW peak power, and ˜12nm bandwidth at 295nm. This source is ideal for protein intrinsic tryptophan fluorescence decay research without the unwanted excitation of tyrosine and paves the way to lab-on-a-chip protein assays using fluorescence decay times. Fluorescence decay and anisotropy decay measurements of human serum albumin are reported and the usefulness of the 295nm LED demonstrated in comparisons with a nanosecond flashlamp and LEDs with nominal wavelength emission of 280nm.

  14. Integrated Fluorescence

    NASA Technical Reports Server (NTRS)

    Tuma, Margaret (Inventor); Gruhlke, Russell W. (Inventor)

    1998-01-01

    A detection method is integrated with a filtering method and an enhancement method to create a fluorescence sensor that can be miniaturized. The fluorescence sensor comprises a thin film geometry including a waveguide layer, a metal film layer and sensor layer. The thin film geometry of the fluorescence sensor allows the detection of fluorescent radiation over a narrow wavelength interval. This enables wavelength discrimination and eliminates the detection of unwanted light from unknown or spurious sources.

  15. Ultraviolet fluorescence monitor

    SciTech Connect

    Hargis, P.J. Jr.; Preppernau, B.L.; Aragon, B.P.

    1997-05-01

    A multispectral ultraviolet (UV) fluorescence imaging fluorometer and a pulsed molecular beam laser fluorometer were developed to detect volatile organic compounds of interest in environmental monitoring and drug interdiction applications. The UV fluorescence imaging fluorometer is a relatively simple instrument which uses multiple excitation wavelengths to measure the excitation/emission matrix for irradiated samples. Detection limits in the high part-per-million to low part-per-million range were measured for a number of volatile organic vapors in the atmosphere. Detection limits in the low part-per-million range were obtained using cryogenic cooling to pre-concentrate unknown samples before introducing them into the imaging fluorometer. A multivariate analysis algorithm was developed to analyze the excitation/emission matrix and used to determine the relative concentrations of species in computer synthesized mixtures containing up to five organic compounds. Analysis results demonstrated the utility of multispectral UV fluorescence in analytical measurements. A transportable UV fluorescence imaging fluorometer was used in two field tests. Field test results demonstrated that detection limits in the part-per-billion range were needed to reliably identify volatile organic compounds in realistic field test measurements. The molecular beam laser fluorometer, a more complex instrument with detection limits in the part-per-billion to part-per-trillion range, was therefore developed to satisfy detection sensitivity requirements for field test measurements. High-resolution spectroscopic measurements made with the molecular beam laser fluorometer demonstrated its utility in identifying volatile organic compounds in the atmosphere.

  16. Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

    PubMed Central

    Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

    2015-01-01

    Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving

  17. Optical Spectroscopic Monitoring of Parachute Yarn Aging

    SciTech Connect

    Tallant, D.R.; Garcia, M.J.; Simpson, R.L.; Behr, V.L.; Whinery, L.D.; Peng, L.W.

    1999-04-01

    Optical spectroscopic techniques were evaluated as nondestructive monitors of the aging of parachutes in nuclear weapons. We analyzed thermally aged samples of nylon and Kevlar webbing by photoluminescence spectroscopy and reflection spectroscopy. Infrared analysis was also performed to help understand the degradation mechanisms of the polymer materials in the webbing. The photoluminescence and reflection spectra were analyzed by chemometric data treatment techniques to see if aged-induced changes in the spectra correlated to changes in measured tensile strength. A correlation was found between the shapes of the photoluminescent bands and the measured tensile strengths. Photoluminescent spectra can be used to predict the tensile strengths of nylon and Kevlar webbing with sufficient accuracy to categorize the webbing sample as above rated tensile strength, marginal or below rated tensile strength. The instrumentation required to perform the optical spectroscopic measurement can be made rugged, compact and portable. Thus, optical spectroscopic techniques offer a means for nondestructive field monitoring of parachutes in the enduring stockpile/

  18. Spectroscopic characterization of dissolved organic matter isolates from sediments and the association with phenanthrene binding affinity.

    PubMed

    Hur, Jin; Lee, Bo-Mi; Shin, Kyung-Hoon

    2014-09-01

    In this study, selected spectroscopic characteristics of sediment organic matter (SOM) were compared and discussed with respect to their different isolation methods, the source discrimination capabilities, and the association with the extent of phenanthrene binding. A total of 16 sediments were collected from three categorized locations including a costal lake, industrial areas, and upper streams, each of which is likely influenced by the organic sources of algal production, industrial effluent, and terrestrial input, respectively. The spectroscopic properties related to aromatic structures and terrestrial humic acids were more pronounced for alkaline extractable organic matter (AEOM) isolates than for the SOM isolates based on water soluble extracts and pore water. The three categorized sampling locations were the most differentiated in the AEOM isolates, suggesting AEOM may be the most representative SOM isolates in terms of describing the chemical properties and the organic sources of SOM. Parallel factor analysis (PARAFAC) based on fluorescence excitation-emission matrix (EEM) showed that a combination of three fluorescent groups could represent all the fluorescence features of SOM. The three categorized sampling locations were well discriminated by the percent distributions of humic-like fluorescent groups of the AEOM isolates. The relative distribution of terrestrial humic-like fluorophores was well correlated with the extent of phenanthrene binding (r=0.571; p<0.05), suggesting that the presence of humic acids in SOM may contribute to the enhancement of binding with hydrophobic organic contaminants in sediments. Principal component analysis (PCA) further demonstrated that the extent of SOM's binding affinity might be affected by the degree of biogeochemical transformation in SOM.

  19. Interactions of Isophorone Derivatives with DNA: Spectroscopic Studies

    PubMed Central

    Deiana, Marco; Matczyszyn, Katarzyna; Massin, Julien; Olesiak-Banska, Joanna; Andraud, Chantal; Samoc, Marek

    2015-01-01

    Interactions of three new isophorone derivatives, Isoa Isob and Isoc with salmon testes DNA have been investigated using UV-Vis, fluorescence and circular dichroism spectroscopic methods. All the studied compounds interact with DNA through intercalative binding mode. The stoichiometry of the isophorone/DNA adducts was found to be 1:1. The fluorescence quenching data revealed a binding interaction with the base pairs of DNA. The CD data indicate that all the investigated isophorones induce DNA modifications. PMID:26069963

  20. Nine New Fluorescent Probes

    NASA Astrophysics Data System (ADS)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  1. Spectroscopic study of honey from Apis mellifera from different regions in Mexico.

    PubMed

    Frausto-Reyes, C; Casillas-Peñuelas, R; Quintanar-Stephano, J L; Macías-López, E; Bujdud-Pérez, J M; Medina-Ramírez, I

    2017-05-05

    The objective of this study was to analyze by Raman and UV-Vis-NIR Spectroscopic techniques, Mexican honey from Apis Mellífera, using representative samples with different botanic origins (unifloral and multifloral) and diverse climates. Using Raman spectroscopy together with principal components analysis, the results obtained represent the possibility to use them for determination of floral origin of honey, independently of the region of sampling. For this, the effect of heat up the honey was analyzed in relation that it was possible to greatly reduce the fluorescence background in Raman spectra, which allowed the visualization of fructose and glucose peaks. Using UV-Vis-NIR, spectroscopy, a characteristic spectrum profile of transmittance was obtained for each honey type. In addition, to have an objective characterization of color, a CIE Yxy and CIE L*a*b* colorimetric register was realized for each honey type. Applying the principal component analysis and their correlation with chromaticity coordinates allowed classifying the honey samples in one plot as: cutoff wavelength, maximum transmittance, tones and lightness. The results show that it is possible to obtain a spectroscopic record of honeys with specific characteristics by reducing the effects of fluorescence.

  2. Laser-induced fluorescence: quantitative analysis of atherosclerotic plaque chemical content in human aorta

    NASA Astrophysics Data System (ADS)

    Dai, Erbin; Wishart, David; Khoury, Samir; Kay, Cyril M.; Jugdutt, Bodh I.; Tulip, John; Lucas, Alexandra

    1996-05-01

    We have been studying laser-induced fluorescence as a technique for identification of selected changes in the chemical composition of atherosclerotic plaque. Formulae for quantification of chemical changes have been developed based upon analysis of fluorescence emission spectra using multiple regression analysis and the principal of least squares. The intima of human aortic necropsy specimens was injected with chemical compounds present in atherosclerotic plaque. Spectra recorded after injection of selected chemical components found in plaque (collagen I, III, IV, elastin and cholesterol) at varying concentrations (0.01 - 1.0 mg) were compared with saline injection. A single fiber system was used for both fluorescence excitation (XeCl excimer laser, 308 nm, 1.5 - 2.0 mJ/ pulse, 5 Hz) and fluorescence emission detection. Average spectra for each chemical have been developed and the wavelengths of peak emission intensity identified. Curve fitting analysis as well as multiple regression analysis were used to develop formulae for assessment of chemical content. Distinctive identifying average curves were established for each chemical. Excellent correlations were identified for collagen I, III, and IV, elastin, and cholesterol (R2 equals 0.92 6- 0.997). Conclusions: (1) Fluorescence spectra of human aortas were significantly altered by collagen I, collagen III, elastin and cholesterol. (2) Fluorescence spectroscopic analysis may allow quantitative assessment of atherosclerotic plaque chemical content in situ.

  3. Spectroscopic properties of alexandrite crystals

    NASA Astrophysics Data System (ADS)

    Powell, Richard C.; Xi, Lin; Gang, Xu; Quarles, Gregory J.; Walling, John C.

    1985-09-01

    Details of the optical-spectroscopic properties of alexandrite (BeAl2O4:Cr3+) crystals were studied by different laser-spectroscopy techniques. The temperature dependences of the fluorescence lifetimes and widths of the zero-phonon lines were found to be quite different for Cr3+ ions in the mirror and inversion crystal-field sites. The results indicate that direct phonon-absorption processes dominate both thermal line broadening and lifetime quenching for ions in the mirror sites while phonon-scattering processes dominate the line broadening of inversion-site ions and leave their lifetime independent of temperature. Tunable-dye-laser site-selection methods were used to obtain the excitation spectra of the Cr3+ ions in inversion sites at low temperature and to identify six types of exchange-coupled pairs of Cr3+ ions in the lattice. Time-resolved site-selection spectroscopy was used to monitor the energy transfer between Cr3+ ions in mirror and inversion sites at both low and high temperature. Finally, high-power, picosecond pulse excitation was used to produce two-photon absorption, and the resulting emission spectrum was found to exhibit a new fluorescence band in the 400-nm spectral region.

  4. Correlation of denitrification-accepted fraction of electrons with NAD(P)H fluorescence for Pseudomonas aeruginosa performing simultaneous denitrification and respiration at extremely low dissolved oxygen conditions.

    PubMed

    Chen, Fan; Xia, Qing; Ju, Lu-Kwang

    2004-01-01

    In cystic fibrosis airway infection, Pseudomonas aeruginosa forms a microaerobic biofilm and undergoes significant physiological changes. It is important to understand the bacterium's metabolism at microaerobic conditions. In this work, the culture properties and two indicators (the denitrification-accepted e- fraction and an NAD(P)H fluorescence fraction) for the culture's "fractional approach" to a fully anaerobic denitrifying state were examined in continuous cultures with practically zero DO but different aeration rates. With decreasing aeration, specific OUR decreased while specific NAR and NIR increased and kept Y(ATP/S) relatively constant. P. aeruginosa thus appeared to effectively compensate for energy generation at microaerobic conditions with denitrification. At the studied dilution rate of 0.06 h(-1), the maximum specific OUR was 2.8 mmol O2/g cells-h and the Monod constant for DO, in the presence of nitrate, was extremely low (<0.001 mg/L). The cell yield Y(X/S) increased significantly (from 0.24 to 0.34) with increasing aeration, attributed to a roughly opposite trend of Y(ATP/X) (ATP generation required for cell growth). As for the denitrification-accepted e- fraction and the fluorescence fraction, both decreased with increasing aeration as expected. The two fractions, however, were not directly proportional. The fluorescence fraction changed more rapidly than the e- fraction at very low aeration rates, whereas the opposite was true at higher aeration. The results demonstrated the feasibility of using online NAD(P)H fluorescence to monitor sensitive changes of cellular physiology and provided insights to the shift of e- -accepting mechanisms of P. aeruginosa under microaerobic conditions.

  5. Spectroscopic characterization of the Stentor photoreceptor.

    PubMed

    Walker, E B; Lee, T Y; Song, P S

    1979-09-20

    1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n-pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the pKa of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the pKa as the primary photoprocess, has been discussed.

  6. Spectroscopic chemical analysis methods and apparatus

    NASA Technical Reports Server (NTRS)

    Hug, William F. (Inventor); Reid, Ray D. (Inventor)

    2010-01-01

    Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. Chemical analysis instruments employed in some embodiments include capillary and gel plane electrophoresis, capillary electrochromatography, high performance liquid chromatography, flow cytometry, flow cells for liquids and aerosols, and surface detection instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted simultaneously with native fluorescence spectroscopy to provide high levels of sensitivity and specificity in the same instrument.

  7. Spectroscopic chemical analysis methods and apparatus

    NASA Technical Reports Server (NTRS)

    Hug, William F. (Inventor); Reid, Ray D. (Inventor)

    2009-01-01

    Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. Chemical analysis instruments employed in some embodiments include capillary and gel plane electrophoresis, capillary electrochromatography, high performance liquid chromatography, flow cytometry, flow cells for liquids and aerosols, and surface detection instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted simultaneously with native fluorescence spectroscopy to provide high levels of sensitivity and specificity in the same instrument.

  8. Spectroscopic Chemical Analysis Methods and Apparatus

    NASA Technical Reports Server (NTRS)

    Hug, William F. (Inventor); Reid, Ray D. (Inventor); Bhartia, Rohit (Inventor); Lane, Arthur L. (Inventor)

    2017-01-01

    Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted along with photoluminescence spectroscopy (i.e. fluorescence and/or phosphorescence spectroscopy) to provide high levels of sensitivity and specificity in the same instrument.

  9. Spectroscopic investigations of a novel tricyanofuran dye for nonlinear optics.

    PubMed

    Han, Likun; Jiang, Yadong; Li, Wei; Li, Yuanxun; Hao, Peng

    2008-11-01

    A novel tricyanofuran dye was synthesized and the dye-in-polymer films were fabricated by spin-coating process. The spectroscopic properties of the dye in the solutions and polymer films were investigated by the absorption spectra and fluorescence emission spectra. It is found that the absorption and fluorescence maxima are largely red-shifted along with the increase of the solvent polarity. And the low values of fluorescence quantum yield in higher polarity solvents suggest the presence of twisted intramolecular charge transfer states of the dye. Moreover, the second order polarizability value of the novel dye was estimated based on the quantum-mechanical two-level model.

  10. Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Pena, A.-M.; Strupler, M.; Boulesteix, T.; Schanne-Klein, M.-C.

    2005-08-01

    We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin.

  11. Alternative methods for estimating common descriptors for QSAR studies of dyes and fluorescent probes using molecular modeling software. 2. Correlations between log P and the hydrophilic/lipophilic index, and new methods for estimating degrees of amphiphilicity.

    PubMed

    Dapson, Richard W; Horobin, Richard W

    2013-11-01

    The log P descriptor, despite its usefulness, can be difficult to use, especially for researchers lacking skills in physical chemistry. Moreover this classic measure has been determined in numerous ways, which can result in inconsistant estimates of log P values, especially for relatively complex molecules such as fluorescent probes. Novel measures of hydrophilicity/lipophilicity (the Hydrophilic/Lipophilic Index, HLI) and amphiphilicity (hydrophilic/lipophilic indices for the head group and tail, HLIT and HLIHG, respectively) therefore have been devised. We compare these descriptors with measures based on log P, the standard method for quantitative structure activity relationships (QSAR) studies. HLI can be determined using widely available molecular modeling software, coupled with simple arithmetic calculations. It is based on partial atomic charges and is intended to be a stand-alone measure of hydrophilicity/lipophilicity. Given the wide application of log P, however, we investigated the correlation between HLI and log P using a test set of 56 fluorescent probes of widely different physicochemical character. Overall correlation was poor; however, correlation of HLI and log P for probes of narrowly specified charge types, i.e., non-ionic compounds, anions, conjugated cations, or zwitterions, was excellent. Values for probes with additional nonconjugated quaternary cations, however, were less well correlated. The newly devised HLI can be divided into domain-specific descriptors, HLIT and HLIHG in amphiphilic probes. Determinations of amphiphilicity, made independently by the authors using their respective methods, showed excellent agreement. Quantifying amphiphilicity from partial log P values of the head group (head group hydrophilicity; HGH) and tail (amphiphilicity index; AI) has proved useful for understanding fluorescent probe action. The same limitations of log P apply to HGH and AI, however. The novel descriptors, HLIT and HLIHG, offer analogous advantages

  12. 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.

  13. Laser induced fluorescence lifetime characterization of Bacillus endospore species using time correlated single photon counting analysis with the multi-exponential fit method

    NASA Astrophysics Data System (ADS)

    Smith, Clint; Edwards, Jarrod; Fisher, Andmorgan

    2010-04-01

    Rapid detection of biological material is critical for determining presence/absence of bacterial endospores within various investigative programs. Even more critical is that if select material tests positive for bacillus endospores then tests should provide data at the species level. Optical detection of microbial endospore formers such as Bacillus sp. can be heavy, cumbersome, and may only identify at the genus level. Data provided from this study will aid in characterization needed by future detection systems for further rapid breakdown analysis to gain insight into a more positive signature collection of Bacillus sp. Literature has shown that fluorescence spectroscopy of endospores could be statistically separated from other vegetative genera, but could not be separated among one another. Results of this study showed endospore species separation is possible using laser-induce fluorescence with lifetime decay analysis for Bacillus endospores. Lifetime decays of B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain were investigated. Using the Multi-Exponential fit method data showed three distinct lifetimes for each species within the following ranges, 0.2-1.3 ns; 2.5-7.0 ns; 7.5-15.0 ns, when laser induced at 307 nm. The four endospore species were individually separated using principle component analysis (95% CI).

  14. DNA-protected silver emitters: charge dependent switching of fluorescence.

    PubMed

    Berdakin, Matías; Taccone, Martin I; Pino, Gustavo A; Sánchez, Cristián G

    2017-02-22

    The relationship between the state of charge and spectroscopy of DNA-protected silver emitters is not yet well understood. This remains one of the major issues to unveil in order to fully disentangle the spectroscopic features of these novel systems. It is a well known fact that a fluorescence response arises upon chemical reduction of silver cations attached to DNA, leading to neutral (or partially oxidized) "bright" clusters. It is important to note that the absence of fluorescence in completely ionic complexes is universal in the sense that it does not depend on any experimental variable. This suggests that its origin may be founded on the nature of the interaction between DNA bases and silver cations. Nevertheless, to the best of our knowledge, no explanation exists for this charge dependent switching between dark completely ionic complexes and bright (neutral or partially oxidized) clusters. In this brief report we address this experimental fact on the basis of the electronic structure of the complex as a function of its charge and quantum dynamical simulations of the processes following photoexcitation. These data provide a dynamical picture of the correlation between charge and fluorescence.

  15. Rationalizing Inter- and Intracrystal Heterogeneities in Dealuminated Acid Mordenite Zeolites by Stimulated Raman Scattering Microscopy Correlated with Super-resolution Fluorescence Microscopy

    PubMed Central

    2014-01-01

    Dealuminated zeolites are widely used acid catalysts in research and the chemical industry. Bulk-level studies have revealed that the improved catalytic performance results from an enhanced molecular transport as well as from changes in the active sites. However, fully exploiting this information in rational catalyst design still requires insight in the intricate interplay between both. Here we introduce fluorescence and stimulated Raman scattering microscopy to quantify subcrystal reactivity as well as acid site distribution and to probe site accessibility in the set of individual mordenite zeolites. Dealumination effectively introduces significant heterogeneities between different particles and even within individual crystals. Besides enabling direct rationalization of the nanoscale catalytic performance, these observations reveal valuable information on the industrial dealumination process itself. PMID:25402756

  16. Reflectance and fluorescence characterization of maize species using field laboratory measurements and lidar remote sensing.

    PubMed

    Zhao, Guangyu; Duan, Zheng; Ming, Lian; Li, Yiyun; Chen, Ruipeng; Hu, Jiandong; Svanberg, Sune; Han, Yanlai

    2016-07-01

    Laser-induced fluorescence is an important technique to study photosynthesis and plants. Information on chlorophyll and other pigments can be obtained. We have been using a mobile laboratory in a Chinese experimental farm setting to study maize (Zea mays L.) leaves by reflectance and fluorescence measurements and correlated the spectroscopic signals to the amount of fertilizer supplied. Further, we studied five different species of maize using the remote monitoring of the fluorescence signatures obtained with the same mobile laboratory, but now in a laser radar remote-sensing configuration. The system separation from the target area was 50 m, and 355 nm pulsed excitation using the frequency-tripled output from an Nd:YAG laser was employed. Principal component analysis and linear discriminant analysis were combined to identify the different maize species using their fluorescence spectra. Likewise, the spectral signatures in reflectance and fluorescence frequently allowed us to separate different fertilizer levels applied to plants of the same species.

  17. Spectroscopic imaging in electron microscopy

    SciTech Connect

    Pennycook, Stephen J; Colliex, C.

    2012-01-01

    In the scanning transmission electron microscope, multiple signals can be simultaneously collected, including the transmitted and scattered electron signals (bright field and annular dark field or Z-contrast images), along with spectroscopic signals such as inelastically scattered electrons and emitted photons. In the last few years, the successful development of aberration correctors for the electron microscope has transformed the field of electron microscopy, opening up new possibilities for correlating structure to functionality. Aberration correction not only allows for enhanced structural resolution with incident probes into the sub-angstrom range, but can also provide greater probe currents to facilitate mapping of intrinsically weak spectroscopic signals at the nanoscale or even the atomic level. In this issue of MRS Bulletin, we illustrate the power of the new generation of electron microscopes with a combination of imaging and spectroscopy. We show the mapping of elemental distributions at atomic resolution and also the mapping of electronic and optical properties at unprecedented spatial resolution, with applications ranging from graphene to plasmonic nanostructures, and oxide interfaces to biology.

  18. Fluorescent refrigeration

    DOEpatents

    Epstein, Richard I.; Edwards, Bradley C.; Buchwald, Melvin I.; Gosnell, Timothy R.

    1995-01-01

    Fluorescent refrigeration is based on selective radiative pumping, using substantially monochromatic radiation, of quantum excitations which are then endothermically redistributed to higher energies. Ultimately, the populated energy levels radiatively deexcite emitting, on the average, more radiant energy than was initially absorbed. The material utilized to accomplish the cooling must have dimensions such that the exciting radiation is strongly absorbed, but the fluorescence may exit the material through a significantly smaller optical pathlength. Optical fibers and mirrored glasses and crystals provide this requirement.

  19. HER2 amplification in gastroesophageal adenocarcinoma: correlation of two antibodies using gastric cancer scoring criteria, H score, and digital image analysis with fluorescence in situ hybridization.

    PubMed

    Radu, Oana M; Foxwell, Tyler; Cieply, Kathleen; Navina, Sarah; Dacic, Sanja; Nason, Katie S; Davison, Jon M

    2012-04-01

    We assessed 103 resected gastroesophageal adenocarcinomas for HER2 amplification by fluorescence in situ hybridization (FISH) and 2 commercial immunohistochemical assays. Of 103, 30 (29%) were FISH-amplified. Both immunohistochemical assays had greater than 95% concordance with FISH. However, as a screening test for FISH amplification, the Ventana Medical Systems (Tucson, AZ) 4B5 antibody demonstrated superior sensitivity (87%) compared with the DAKO (Carpinteria, CA) A0485 (70%). Of the cases, 28 were immunohistochemically 3+ or immunohistochemically 2+/FISH-amplified with the 4B5 assay compared with only 22 cases with the A0485 assay, representing a large potential difference in patient eligibility for anti-HER2 therapy. Cases with low-level FISH amplification (HER2/CEP17, 2.2-4.0) express lower levels of HER2 protein compared with cases with high-level amplification (HER2/CEP17, ≥4.0), raising the possibility of a differential response to anti-HER2 therapy. The H score and digital image analysis may have a limited role in improving HER2 test performance.

  20. Fluorescence Correlation Spectroscopy and Photon Counting Histogram on membrane proteins: Functional dynamics of the GPI-anchored Urokinase Plasminogen Activator Receptor

    PubMed Central

    Malengo, Gabriele; Andolfo, Annapaola; Sidenius, Nicolai; Gratton, Enrico; Zamai, Moreno; Caiolfa, Valeria R

    2009-01-01

    The oligomerization of GPI-anchored proteins is thought to regulate their association with membrane microdomains, sub-cellular sorting and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in HEK293 cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining FCS and PCH analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, only for the active receptor the diffusion coefficient decreased in monomer-enriched fractions, suggesting that uPAR monomers might be preferentially engaged in multi-protein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus, and minimizing the overestimation of the molecular brightness. Joint to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady state conditions, at low expression levels, and in live, unperturbed cells. PMID:18601539

  1. In vitro anti-oxidant activity, fluorescence quenching study and structural features of carbohydrate polymers from Phyllanthus emblica.

    PubMed

    Chatterjee, Udipta Ranjan; Bandyopadhyay, Shruti S; Ghosh, Debjani; Ghosal, Pradyot K; Ray, Bimalendu

    2011-11-01

    The water-extracted carbohydrate polymers (WE) of Phyllanthus emblica are analyzed using chemical, chromatographic, and spectroscopic methods. Anion-exchange-chromatography of WE yielded four fractions (F1-F4) with different chemical compositions and all of them contain phenolics. The major fraction F4 possesses 50% polysaccharide and 26% phenol, and is a glycoconjugate. The antioxidant capacities of WE and F4 are comparable to standard anti-oxidants. Notably, activities of F1-F4 correlate with their phenol content. Evidence for the complexation of F4 with bovine serum albumin is presented by fluorescence quenching measurement. The results also indicate conformational change of protein at high carbohydrate polymer concentration.

  2. Visualization of soot inception in turbulent pressurized flames by simultaneous measurement of laser-induced fluorescence of polycyclic aromatic hydrocarbons and laser-induced incandescence, and correlation to OH distributions

    NASA Astrophysics Data System (ADS)

    Geigle, Klaus Peter; O'Loughlin, William; Hadef, Redjem; Meier, Wolfgang

    2015-03-01

    Distributions of polycyclic aromatic hydrocarbons (PAH) and their correlation with soot formation were studied in ethylene-air swirl flames stabilized in a gas turbine model combustor at increased pressure. The combustor can be operated with secondary air injection to study the influence of soot oxidation. We employed PAH laser-induced fluorescence using UV excitation simultaneously with IR-excited laser-induced incandescence to identify soot. PAH signatures typically appear discontinuous unlike OH, yet similar to soot but exhibit more uniform intensity and larger size. The correlation of both diagnostics allowed identification of a wide range of soot formation progress, including isolated soot or PAH, as well as PAH transitioning into soot. The occurrence of soot, PAH and OH and their spatial variations are strongly dependent on the properties of the flow field. In the bottom part of the inner recirculation zone and for the reference case, a rich flame with additional oxidation air, soot levels are relatively high, while PAH intensities in this region are minimal. This correlates well with high temperatures in this region published recently, which are unfavorable for soot formation as the precursors, PAH, decompose. Consequently, soot presence here is attributed to transport. In contrast to OH and soot distributions which change significantly upon addition of secondary air downstream of the primary combustion zone, PAH distributions for both cases look relatively similar. This is attributed to a downstream consumption of PAH by different processes. Without oxidation air, PAH completely transform into soot, while additional oxidation air leads to their oxidation.

  3. Spectroscopic investigations on the interaction of an anionic probe with nonionic micelles of Igepal surfactants in aqueous media

    NASA Astrophysics Data System (ADS)

    Moore, S. A.; Palepu, R. M.

    The behaviour of the anionic dye 8-anilino-1-napthalenesulfonic acid ammonium salt, or ANS, in aqueous solutions containing the Igepal series of polyoxyethylene nonionic surfactants was investigated using fluorescence spectroscopic technique. The interactions of the dye with the nonionic surfactants were examined in micellar media, to prevent dye aggregate formation and to ensure maximum dye and surfactant interaction. From the relative fluorescence enhancements, binding constants of the dye to the surfactant micelles and aggregation numbers of the micelles were determined. The aggregation numbers were also separately determined by static fluorescence quenching of pyrene by cetylpyridinium chloride in aqueous surfactant mixtures at a fixed concentration of surfactant, and compared with the value obtained from the present investigation of the interaction of the micelles with the ANS probe. The values of binding constants, micropolarity values sensed by pyrene and the Stern-Volmer constants for quenching of pyrene fluorescence by cetylpyridinium chloride were correlated with the number of ethylene oxide groups in the Igepal series.

  4. Spectroscopic characterization of dissolved organic matter derived from different biochars and their polycylic aromatic hydrocarbons (PAHs) binding affinity.

    PubMed

    Tang, Jianfeng; Li, Xinhu; Luo, Yan; Li, Gang; Khan, Sardar

    2016-06-01

    In recent years, biochar has received a great attention due to its high application in different sectors of environment. The feasibility of biochar applications is depended on its physical and chemical properties and biochar-derived dissolved organic matter (DOM) characteristics. This study was conducted to investigate the spectroscopic characteristics of biochar-derived DOM and its binding capacity of hydrophobic organic chemicals (HOCs). DOM solutions were isolated from five different biochars prepared through pyrolysis and analyzed for dissolved organic carbon (DOC) contents. The optical analysis with UV-visible absorption and excitation-emission matrix (EEM) fluorescence spectroscopes and DOC water distribution coefficient (KDOC) were calculated in the presence of PAHs and DOM. The DOC contents and the estimated aromaticity (SUVA254) were different for selected biochars. The DOM derived from soybean straw biochar (SBBC) showed the highest DOC contents followed by rice straw biochar (RSBC). The SBBC and RSBC peak position in the fluorescence excitation/emission matrix at longer wavelength corresponded to the peak position of other three biochars indicating that SBBC and RSBC had relatively higher degree of humification. This was well correlated with the observed KDOC values, suggesting that the KDOC value(')s dominant factor was the degree of biochar-derived DOM humification. The results of this study indicate that the optical analysis may provide valuable information regarding the characteristics of biochar-derived DOM and its application as environmental amendments for minimization of toxic organic compounds.

  5. Spectroscopic characterization of dissolved organic matter in coking wastewater during bio-treatment: full-scale plant study.

    PubMed

    Xu, Ronghua; Ou, Huase; Yu, Xubiao; He, Runsheng; Lin, Chong; Wei, Chaohai

    2015-01-01

    This paper taking a full-scale coking wastewater (CWW) treatment plant as a case study aimed to characterize removal behaviors of dissolved organic matter (DOM) by UV spectra and fluorescence excitation-emission matrix-parallel factor analysis (PARAFAC), and investigate the correlations between spectroscopic indices and water quality parameters. Efficient removal rates of chemical oxygen demand (COD), dissolved organic carbon (DOC) and total nitrogen (TN) after the bio-treatment were 91.3%, 87.3% and 69.1%, respectively. UV270 was proven to be a stable UV absorption peak of CWW that could reflect the mixture of phenols, heterocyclics, polynuclear aromatic hydrocarbons and their derivatives. Molecular weight and aromaticity were increased, and also the content of polar functional groups was greatly reduced after bio-treatment. Three fluorescent components were identified by PARAFAC: C1 (tyrosine-like), C2 (tryptophan-like) and C3 (humic-like). The removal rate of protein-like was higher than that of humic-like and C1 was identified as biodegradable substance. Correlation analysis showed UV270 had an excellent correlation with COD (r=0.921, n=60, P<0.01) and DOC (r=0.959, n=60, P<0.01) and significant correlation (r=0.875, n=60, P<0.01) was also found between C2 and TN. Therefore, spectroscopic characterization could provide novel insights into removal behaviors of DOM and potential to monitor water quality real-time during CWW bio-treatment.

  6. Detection of PLGA-based nanoparticles at a single-cell level by synchrotron radiation FTIR spectromicroscopy and correlation with X-ray fluorescence microscopy

    PubMed Central

    Pascolo, Lorella; Bortot, Barbara; Benseny-Cases, Nuria; Gianoncelli, Alessandra; Tosi, Giovanni; Ruozi, Barbara; Rizzardi, Clara; De Martino, Eleonora; Vandelli, Maria Angela; Severini, Giovanni Maria

    2014-01-01

    Poly-lactide-c