Photophysical Behaviors of Single Fluorophores Localized on Zinc Oxide Nanostructures

PubMed Central

Fu, Yi; Zhang, Jian; Lakowicz, Joseph R.

2012-01-01

Single-molecule fluorescence spectroscopy has now been widely used to investigate complex dynamic processes which would normally be obscured in an ensemble-averaged measurement. In this report we studied photophysical behaviors of single fluorophores in proximity to zinc oxide nanostructures by single-molecule fluorescence spectroscopy and time-correlated single-photon counting (TCSPC). Single fluorophores on ZnO surfaces showed enhanced fluorescence brightness to various extents compared with those on glass; the single-molecule time trajectories also illustrated pronounced fluctuations of emission intensities, with time periods distributed from milliseconds to seconds. We attribute fluorescence fluctuations to the interfacial electron transfer (ET) events. The fluorescence fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the interfacial electron transfer reaction processes. PMID:23109903

Characterization of brightness and stoichiometry of bright particles by flow-fluorescence fluctuation spectroscopy.

PubMed

Johnson, Jolene; Chen, Yan; Mueller, Joachim D

2010-11-03

Characterization of bright particles at low concentrations by fluorescence fluctuation spectroscopy (FFS) is challenging, because the event rate of particle detection is low and fluorescence background contributes significantly to the measured signal. It is straightforward to increase the event rate by flow, but the high background continues to be problematic for fluorescence correlation spectroscopy. Here, we characterize the use of photon-counting histogram analysis in the presence of flow. We demonstrate that a photon-counting histogram efficiently separates the particle signal from the background and faithfully determines the brightness and concentration of particles independent of flow speed, as long as undersampling is avoided. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used to determine stoichiometry of protein complexes in vivo and in vitro. We apply flow-FFS to determine the stoichiometry of the group specific antigen protein within viral-like particles of the human immunodeficiency virus type-1 from the brightness. Our results demonstrate that flow-FFS is a sensitive method for the characterization of complex macromolecular particles at low concentrations. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Velocity landscape correlation resolves multiple flowing protein populations from fluorescence image time series.

PubMed

Pandžić, Elvis; Abu-Arish, Asmahan; Whan, Renee M; Hanrahan, John W; Wiseman, Paul W

2018-02-16

Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy

PubMed Central

Youker, Robert T.; Teng, Haibing

2014-01-01

Abstract. Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques. PMID:25260867

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

PubMed Central

Macdonald, Patrick J.; Chen, Yan; Mueller, Joachim D.

2012-01-01

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction. PMID:22093611

Revealing time bunching effect in single-molecule enzyme conformational dynamics.

PubMed

Lu, H Peter

2011-04-21

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.

Eisosomes Are Dynamic Plasma Membrane Domains Showing Pil1-Lsp1 Heteroligomer Binding Equilibrium

PubMed Central

Olivera-Couto, Agustina; Salzman, Valentina; Mailhos, Milagros; Digman, Michelle A.; Gratton, Enrico; Aguilar, Pablo S.

2015-01-01

Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits. PMID:25863055

Characterization of cancer and normal tissue fluorescence through wavelet transform and singular value decomposition

NASA Astrophysics Data System (ADS)

Gharekhan, Anita H.; Biswal, Nrusingh C.; Gupta, Sharad; Pradhan, Asima; Sureshkumar, M. B.; Panigrahi, Prasanta K.

2008-02-01

The statistical and characteristic features of the polarized fluorescence spectra from cancer, normal and benign human breast tissues are studied through wavelet transform and singular value decomposition. The discrete wavelets enabled one to isolate high and low frequency spectral fluctuations, which revealed substantial randomization in the cancerous tissues, not present in the normal cases. In particular, the fluctuations fitted well with a Gaussian distribution for the cancerous tissues in the perpendicular component. One finds non-Gaussian behavior for normal and benign tissues' spectral variations. The study of the difference of intensities in parallel and perpendicular channels, which is free from the diffusive component, revealed weak fluorescence activity in the 630nm domain, for the cancerous tissues. This may be ascribable to porphyrin emission. The role of both scatterers and fluorophores in the observed minor intensity peak for the cancer case is experimentally confirmed through tissue-phantom experiments. Continuous Morlet wavelet also highlighted this domain for the cancerous tissue fluorescence spectra. Correlation in the spectral fluctuation is further studied in different tissue types through singular value decomposition. Apart from identifying different domains of spectral activity for diseased and non-diseased tissues, we found random matrix support for the spectral fluctuations. The small eigenvalues of the perpendicular polarized fluorescence spectra of cancerous tissues fitted remarkably well with random matrix prediction for Gaussian random variables, confirming our observations about spectral fluctuations in the wavelet domain.

Confocal fluorescence techniques in industrial application

NASA Astrophysics Data System (ADS)

Eggeling, Christian; Gall, Karsten; Palo, Kaupo; Kask, Peet; Brand, Leif

2003-06-01

The FCS+plus family of evaluation tools for confocal fluorescence spectroscopy, which was developed during recent years, offers a comprehensive view to a series of fluorescence properties. Originating in fluorescence correlation spectroscopy (FCS) and using similar experimental equipment, a system of signal processing methods such as fluorescence intensity distribution analysis (FIDA) was created to analyze in detail the fluctuation behavior of fluorescent particles within a small area of detection. Giving simultaneous access to molecular parameters like concentration, translational and rotational diffusion, molecular brightness, and multicolor coincidence, this portfolio was enhanced by more traditional techniques of fluorescence lifetime as well as time-resolved anisotropy determination. The cornerstones of the FCS+plus methodology will be shortly described. The inhibition of a phosphatase enzyme activity gives a comprehensive industrial application that demonstrates FCS+plus' versatility and its potential for pharmaceutical drug discovery.

Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

PubMed

Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

2014-07-16

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.

Scaling Behavior in Mitochondrial Redox Fluctuations

PubMed Central

Ramanujan, V. Krishnan; Biener, Gabriel; Herman, Brian A.

2006-01-01

Scale-invariant long-range correlations have been reported in fluctuations of time-series signals originating from diverse processes such as heart beat dynamics, earthquakes, and stock market data. The common denominator of these apparently different processes is a highly nonlinear dynamics with competing forces and distinct feedback species. We report for the first time an experimental evidence for scaling behavior in NAD(P)H signal fluctuations in isolated mitochondria and intact cells isolated from the liver of a young (5-month-old) mouse. Time-series data were collected by two-photon imaging of mitochondrial NAD(P)H fluorescence and signal fluctuations were quantitatively analyzed for statistical correlations by detrended fluctuation analysis and spectral power analysis. Redox [NAD(P)H / NAD(P)+] fluctuations in isolated mitochondria and intact liver cells were found to display nonrandom, long-range correlations. These correlations are interpreted as arising due to the regulatory dynamics operative in Krebs' cycle enzyme network and electron transport chain in the mitochondria. This finding may provide a novel basis for understanding similar regulatory networks that govern the nonequilibrium properties of living cells. PMID:16565066

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. This journal is © The Royal Society of Chemistry and Owner Societies 2012

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

Real-Time Nanoscopy by Using Blinking Enhanced Quantum Dots

PubMed Central

Watanabe, Tomonobu M.; Fukui, Shingo; Jin, Takashi; Fujii, Fumihiko; Yanagida, Toshio

2010-01-01

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices. PMID:20923631

Correction of cell-induced optical aberrations in a fluorescence fluctuation microscope

PubMed Central

Leroux, Charles-Edouard; Grichine, Alexei; Wang, Irène; Delon, Antoine

2013-01-01

We describe the effect of optical aberrations on fluorescence fluctuations microscopy (FFM), when focusing through a single living cell. FFM measurements are performed in an aqueous fluorescent solution, and prove to be a highly sensitive tool to assess the optical aberrations introduced by the cell. We demonstrate an adaptive optics (AO) system to remove the aberration-related bias in the FFM measurements. Our data show that AO is not only useful when imaging deep in tissues, but also when performing FFM measurements through a single cellular layer. PMID:23939061

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

NASA Astrophysics Data System (ADS)

Weger, Lukas; Hoffmann-Jacobsen, Kerstin

2017-09-01

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

Predictability and environmental drivers of chlorophyll fluctuations vary across different time scales and regions of the North Sea

NASA Astrophysics Data System (ADS)

Blauw, Anouk N.; Benincà, Elisa; Laane, Remi W. P. M.; Greenwood, Naomi; Huisman, Jef

2018-02-01

Phytoplankton concentrations display strong temporal variability at different time scales. Recent advances in automated moorings enable detailed investigation of this variability. In this study, we analyzed phytoplankton fluctuations at four automated mooring stations in the North Sea, which measured phytoplankton abundance (chlorophyll) and several environmental variables at a temporal resolution of 12-30 min for two to nine years. The stations differed in tidal range, water depth and freshwater influence. This allowed comparison of the predictability and environmental drivers of phytoplankton variability across different time scales and geographical regions. We analyzed the time series using wavelet analysis, cross correlations and generalized additive models to quantify the response of chlorophyll fluorescence to various environmental variables (tidal and meteorological variables, salinity, suspended particulate matter, nitrate and sea surface temperature). Hour-to-hour and day-to-day fluctuations in chlorophyll fluorescence were substantial, and mainly driven by sinking and vertical mixing of phytoplankton cells, horizontal transport of different water masses, and non-photochemical quenching of the fluorescence signal. At the macro-tidal stations, these short-term phytoplankton fluctuations were strongly driven by the tides. Along the Dutch coast, variation in salinity associated with the freshwater influence of the river Rhine played an important role, while in the central North Sea variation in weather conditions was a major determinant of phytoplankton variability. At time scales of weeks to months, solar irradiance, nutrient conditions and thermal stratification were the dominant drivers of changes in chlorophyll concentrations. These results show that the dominant drivers of phytoplankton fluctuations differ across marine environments and time scales. Moreover, our findings show that phytoplankton variability on hourly to daily time scales should not be dismissed as environmental noise, but is related to vertical and horizontal particle transport driven by winds and tides. Quantification of these transport processes contributes to an improved predictability of marine phytoplankton concentrations.

Consequences experimentales des effets des fluctuations du vide sur la fluorescence parametrique et la generation du second harmonique en milieu confine

NASA Astrophysics Data System (ADS)

Robichaud, Luc

Les fluctuations du vide, qui consistent en l'apparition momentanee de particules, ce qui est permit par le principe d'incertitude de Heisenberg, joue un role primordial dans les processus photoniques, en particulier les processus non-lineaires. Par la manipulation de ces fluctuations du vide a l'aide de confinement optique, on retrouve deux phenomenes particuliers : l'intensification de la fluorescence parametrique (Walker, 2008) et l'inhibition de la generation du second harmonique (Collette, 2013). Dans ce travail, on presente les resultats dans le cas classique ; c'est-a-dire sans fluctuations du vide et confinement. Par la suite, on presente les effets des fluctuations du vide et du confinement, ce qui mene aux deux effets mentionnes. Dans le cas de la fluorescence parametrique, le bruit quantique sur le champ interne et externe est calcule, le role du desaccord de phase dans le modele est expose et une generalisation tridimensionnelle est etudiee afin de generaliser la conception du modele d'un cas unidimensionnel a un cas tridimensionnel planaire. Dans le cas de la generation du second harmonique, les difficultes d'un modele purement tridimensionnel sont exposees et ensuite le cas limite planaire est etudie.

Two-photon excitation of nitric oxide fluorescence as a temperature indicator in unsteady gas-dynamic processes

NASA Technical Reports Server (NTRS)

Mckenzie, R. L.; Gross, K. P.

1980-01-01

A laser induced fluorescence technique, suitable for measuring fluctuating temperatures in cold turbulent flows containing very low concentrations of nitric oxide is described. Temperatures below 300 K may be resolved with signal to noise ratios greater than 50 to 1 using high peak power, tunable dye lasers. The method relies on the two photon excitation of selected ro-vibronic transitions. The analysis includes the effects of fluorescence quenching and shows the technique to be effective at all densities below ambient. Signal to noise ratio estimates are based on a preliminary measurement of the two photon absorptivity for a selected rotational transition in the NO gamma (0,0) band.

An intramolecular charge transfer process based fluorescent probe for monitoring subtle pH fluctuation in living cells.

PubMed

Sun, Mingtai; Du, Libo; Yu, Huan; Zhang, Kui; Liu, Yang; Wang, Suhua

2017-01-01

It is crucial to monitor intracellular pH values and their fluctuation since the organelles of cells have different pH distribution. Herein we construct a new small molecule fluorescent probe HBT-O for monitoring the subtle pH values within the scope of neutral to acid in living cells. The probe exhibited good water solubility, a marked turquoise to olivine emission color change in response to pH, and tremendous fluorescence hypochromatic shift of ∼50nm (1718cm -1 ) as well as the increased fluorescence intensity when the pH value changed from neutral to acid. Thus, the probe HBT-O can distinguish the subtle changes in the range of normal pH values from neutral to acid with significant fluorescence changes. These properties can be attributed to the intramolecular charge transfer (ICT) process of the probe upon protonation in buffer solutions at varied pH values. Moreover, the probe was reversible and nearly non-toxic for living cells. Then the probe was successfully used to detect pH fluctuation in living cells by exhibiting different fluorescence colors and intensity. These findings demonstrate that the probe will find useful applications in biology and biomedical research. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescence Fluctuation Approaches to the Study of Adhesion and Signaling

PubMed Central

Bachir, Alexia I.; Kubow, Kristopher E.; Horwitz, Alan R.

2013-01-01

Cell–matrix adhesions are large, multimolecular complexes through which cells sense and respond to their environment. They also mediate migration by serving as traction points and signaling centers and allow the cell to modify the surroucnding tissue. Due to their fundamental role in cell behavior, adhesions are germane to nearly all major human health pathologies. However, adhesions are extremely complex and dynamic structures that include over 100 known interacting proteins and operate over multiple space (nm–µm) and time (ms–min) regimes. Fluorescence fluctuation techniques are well suited for studying adhesions. These methods are sensitive over a large spatiotemporal range and provide a wealth of information including molecular transport dynamics, interactions, and stoichiometry from a single time series. Earlier chapters in this volume have provided the theoretical background, instrumentation, and analysis algorithms for these techniques. In this chapter, we discuss their implementation in living cells to study adhesions in migrating cells. Although each technique and application has its own unique instrumentation and analysis requirements, we provide general guidelines for sample preparation, selection of imaging instrumentation, and optimization of data acquisition and analysis parameters. Finally, we review several recent studies that implement these techniques in the study of adhesions. PMID:23280111

Note: Design of a full photon-timing recorder down to 1-ns resolution for fluorescence fluctuation measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimura, Goro, E-mail: gnishi@imd.es.hokudai.ac.jp

2015-10-15

A photon timing recorder was realized in a field programmable gate array to capture all timing data of photons on multiple channels with down to a 1-ns resolution and to transfer all data to a host computer in real-time through universal serial bus with more than 10 M events/s transfer rate. The main concept is that photon time series can be regarded as a serial communication data stream. This recorder was successfully applied for simultaneous measurements of fluorescence fluctuation and lifetime of near-infrared dyes in solution. This design is not only limited to the fluorescence fluctuation measurement but also applicablemore » to any kind of photon counting experiments in a nanosecond time range because of the simple and easily modifiable design.« less

Peculiarities of the statistics of spectrally selected fluorescence radiation in laser-pumped dye-doped random media

NASA Astrophysics Data System (ADS)

Yuvchenko, S. A.; Ushakova, E. V.; Pavlova, M. V.; Alonova, M. V.; Zimnyakov, D. A.

2018-04-01

We consider the practical realization of a new optical probe method of the random media which is defined as the reference-free path length interferometry with the intensity moments analysis. A peculiarity in the statistics of the spectrally selected fluorescence radiation in laser-pumped dye-doped random medium is discussed. Previously established correlations between the second- and the third-order moments of the intensity fluctuations in the random interference patterns, the coherence function of the probe radiation, and the path difference probability density for the interfering partial waves in the medium are confirmed. The correlations were verified using the statistical analysis of the spectrally selected fluorescence radiation emitted by a laser-pumped dye-doped random medium. Water solution of Rhodamine 6G was applied as the doping fluorescent agent for the ensembles of the densely packed silica grains, which were pumped by the 532 nm radiation of a solid state laser. The spectrum of the mean path length for a random medium was reconstructed.

Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

PubMed Central

McQuilken, Molly; La Riviere, Patrick J.; Occhipinti, Patricia; Verma, Amitabh; Oldenbourg, Rudolf; Gladfelter, Amy S.; Tani, Tomomi

2016-01-01

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells. PMID:27679846

Dual-Modal Colorimetric/Fluorescence Molecular Probe for Ratiometric Sensing of pH and Its Application.

PubMed

Wu, Luling; Li, Xiaolin; Huang, Chusen; Jia, Nengqin

2016-08-16

As traditional pH meters cannot work well for minute regions (such as subcellular organelles) and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. Here, we report a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) for ratiometric sensing of pH. Compared with previously reported pH probes, CPH bearing the benzyl group at the nitrogen position of the indolium group and the phenol, which is used as the acceptor for proton, could respond to pH changes immediately through both the ratiometric fluorescence signal readout and naked-eye colorimetric observation. The sensing process was highly stable and reversible. Most importantly, the suitable pKa value (6.44) allows CPH to presumably accumulate in lysosomes and become a lysosome-target fluorescent probe. By using CPH, the intralysosomal pH fluctuation stimulated by antimalaria drug chloroquine was successfully tracked in live cells through the ratiometric fluorescence images. Additionally, CPH could be immobilized on test papers, which exhibited a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis. This proof-of-concept study presents the potential application of CPH as a molecular tool for monitoring intralysosomal pH fluctuation in live cells, as well as paves the way for developing the economic, reusable, and fast-response optical pH meters for colorimetric sensing acid/base vapor with direct naked-eye observation.

Conformational fluctuations in a green fluorescent protein-like Akane family protein: a high-pressure fluorescence study at 0.1-700 MPa

NASA Astrophysics Data System (ADS)

Maeno, Akihiro; Kato, Yuko; Jimbo, Mitsuru; Amada, Kei; Mita, Hajime; Akasaka, Kazuyuki

2017-04-01

We have investigated conformational fluctuations in a new green fluorescent protein(GFP)-like protein rb-Akane found in a red-brown-colored octocoral, Scleronephthya gracillima (Kuekenthal)), with high pressure fluorescence spectroscopy at 0.1-700 MPa. Besides the green fluorescence at 510 nm, two red fluorescence peaks are observed at 590 and 629 nm, the relative intensity of which varies reversibly with pressure. The phenomenon is interpreted as representing the cis-trans isomerization of the chromophore accompanied by the conformational transition between two sub-states of the red fluorescence form of rb-Akane. The two sub-states are separated only marginally in free energy (ΔG0 = 1.9 ± 0.4 kJ mol-1), but significantly in partial molar volume (ΔV0 = -19.8 ± 1.4 ml mol-1) at 0.1 MPa (pH 7.5, 25°C). Above 500 MPa, the fluorescence at λmax 629 nm undergoes another reversible change with pressure, showing the onset of unfolding.

Chlorophyll fluorescence analysis revealed essential roles of FtsH 11 protease in regulation of the adaptive responses of photosynthetic systems to high temperature

USDA-ARS?s Scientific Manuscript database

Background: Photosynthetic systems are known to be sensitive to high temperature stress. To maintain a relatively “normal” level of photosynthetic activities, plants employ a variety of adaptive mechanisms in response to environmental temperature fluctuations. Previously, we reported that the chloro...

Frontiers in Fluctuation Spectroscopy: Measuring protein dynamics and protein spatio-temporal connectivity

NASA Astrophysics Data System (ADS)

Digman, Michelle

Fluorescence fluctuation spectroscopy has evolved from single point detection of molecular diffusion to a family of microscopy imaging correlation tools (i.e. ICS, RICS, STICS, and kICS) useful in deriving spatial-temporal dynamics of proteins in living cells The advantage of the imaging techniques is the simultaneous measurement of all points in an image with a frame rate that is increasingly becoming faster with better sensitivity cameras and new microscopy modalities such as the sheet illumination technique. A new frontier in this area is now emerging towards a high level of mapping diffusion rates and protein dynamics in the 2 and 3 dimensions. In this talk, I will discuss the evolution of fluctuation analysis from the single point source to mapping diffusion in whole cells and the technology behind this technique. In particular, new methods of analysis exploit correlation of molecular fluctuations originating from measurement of fluctuation correlations at distant points (pair correlation analysis) and methods that exploit spatial averaging of fluctuations in small regions (iMSD). For example the pair correlation fluctuation (pCF) analyses done between adjacent pixels in all possible radial directions provide a window into anisotropic molecular diffusion. Similar to the connectivity atlas of neuronal connections from the MRI diffusion tensor imaging these new tools will be used to map the connectome of protein diffusion in living cells. For biological reaction-diffusion systems, live single cell spatial-temporal analysis of protein dynamics provides a mean to observe stochastic biochemical signaling in the context of the intracellular environment which may lead to better understanding of cancer cell invasion, stem cell differentiation and other fundamental biological processes. National Institutes of Health Grant P41-RRO3155.

Enhanced labeling of microalgae cellular lipids by application of an electric field generated by alternating current.

PubMed

Su, Li-Chien; Hsu, Yi-Hsiang; Wang, Hsiang-Yu

2012-05-01

An alternating current was used to generate an electric field to enhance the fluorescent labeling of microalgae cellular lipids with Nile red and LipidTOX. The decay of the fluorescence intensity of Chlorella vulgaris cells in 0 V/cm was more than 50% after 10 min, and the intensity variation was as high as 7% in 20s. At 2000 V/cm, the decay rate decreased to 1.22% per minute and the intensity fluctuation was less than 1% for LipidTOX-labeled cells. For Spirulina sp. cells at 0 V/cm, the fluorescence intensity increased by 10% after 10 min, whereas at 2000 V/cm, labeling was more rapid and fluorescence intensity doubled. These results show that applying an electric field can improve the quality of fluorescence detection by alleviating decay and fluctuation or by enhancing signal intensity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Robust demarcation of basal cell carcinoma by dependent component analysis-based segmentation of multi-spectral fluorescence images.

PubMed

Kopriva, Ivica; Persin, Antun; Puizina-Ivić, Neira; Mirić, Lina

2010-07-02

This study was designed to demonstrate robust performance of the novel dependent component analysis (DCA)-based approach to demarcation of the basal cell carcinoma (BCC) through unsupervised decomposition of the red-green-blue (RGB) fluorescent image of the BCC. Robustness to intensity fluctuation is due to the scale invariance property of DCA algorithms, which exploit spectral and spatial diversities between the BCC and the surrounding tissue. Used filtering-based DCA approach represents an extension of the independent component analysis (ICA) and is necessary in order to account for statistical dependence that is induced by spectral similarity between the BCC and surrounding tissue. This generates weak edges what represents a challenge for other segmentation methods as well. By comparative performance analysis with state-of-the-art image segmentation methods such as active contours (level set), K-means clustering, non-negative matrix factorization, ICA and ratio imaging we experimentally demonstrate good performance of DCA-based BCC demarcation in two demanding scenarios where intensity of the fluorescent image has been varied almost two orders of magnitude. Copyright 2010 Elsevier B.V. All rights reserved.

Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting.

PubMed

Lamb, D C; Müller, B K; Bräuchle, C

2005-10-01

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.

Molecular imprinting ratiometric fluorescence sensor for highly selective and sensitive detection of phycocyanin.

PubMed

Wang, Xiaoyan; Yu, Jialuo; Kang, Qi; Shen, Dazhong; Li, Jinhua; Chen, Lingxin

2016-03-15

A facile strategy was developed to prepare molecular imprinting ratiometric fluorescence sensor for highly selective and sensitive detection of phycocyanin (PC) based on fluorescence resonance energy transfer (FRET), via a sol-gel polymerization process using nitrobenzoxadiazole (NBD) as fluorescent signal source. The ratio of two fluorescence peak emission intensities of NBD and PC was utilized to determine the concentration of PC, which could effectively reduce the background interference and fluctuation of diverse conditions. As a result, this sensor obtained high sensitivity with a low detection limit of 0.14 nM within 6 min, and excellent recognition specificity for PC over its analogues with a high imprinting factor of 9.1. Furthermore, the sensor attained high recoveries in the range of 93.8-110.2% at three spiking levels of PC, with precisions below 4.7% in seawater and lake water samples. The developed sensor strategy demonstrated simplicity, reliability, rapidity, high selectivity and high sensitivity, proving to be a feasible way to develop high efficient fluorescence sensors and thus potentially applicable for ultratrace analysis of complicated matrices. Copyright © 2015 Elsevier B.V. All rights reserved.

Feasibility of measuring temperature and density fluctuations in air using laser-induced O2 fluorescence

NASA Technical Reports Server (NTRS)

Massey, G. A.; Lemon, C. J.

1984-01-01

A tunable line-narrowed ArF laser can selectively excite several rotation al lines of the Schumann-Runge band system of O2 in air. The resulting ultraviolet fluorescence can be monitored at 90 deg to the laser beam axis, permitting space and time resolved observation of density and temperature fluctuations in turbulence. Experiments and calculations show that + or - 1 K, + or - 1 percent density, 1 cu mm spatial, and 1 microsecond temporal resolution can be achieved simultaneously under some conditions.

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. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Photorespiration plays an important role in the regulation of photosynthetic electron flow under fluctuating light in tobacco plants grown under full sunlight.

PubMed

Huang, Wei; Hu, Hong; Zhang, Shi-Bao

2015-01-01

Plants usually experience dynamic fluctuations of light intensities under natural conditions. However, the responses of mesophyll conductance, CO2 assimilation, and photorespiration to light fluctuation are not well understood. To address this question, we measured photosynthetic parameters of gas exchange and chlorophyll fluorescence in tobacco leaves at 2-min intervals while irradiance levels alternated between 100 and 1200 μmol photons m(-2) s(-1). Compared with leaves exposed to a constant light of 1200 μmol photons m(-2) s(-1), both stomatal and mesophyll conductances were significantly restricted in leaves treated with fluctuating light condition. Meanwhile, CO2 assimilation rate and electron flow devoted to RuBP carboxylation at 1200 μmol photons m(-2) s(-1) under fluctuating light were limited by the low chloroplast CO2 concentration. Analysis based on the C3 photosynthesis model indicated that, at 1200 μmol photons m(-2) s(-1) under fluctuating light, the CO2 assimilation rate was limited by RuBP carboxylation. Electron flow devoted to RuBP oxygenation at 1200 μmol photons m(-2) s(-1) under fluctuating light remained at nearly the maximum level throughout the experimental period. We conclude that fluctuating light restricts CO2 assimilation by decreasing both stomatal and mesophyll conductances. Under such conditions, photorespiration plays an important role in the regulation of photosynthetic electron flow.

Analysis of root surface properties by fluorescence/Raman intensity ratio.

PubMed

Nakamura, Shino; Ando, Masahiro; Hamaguchi, Hiro-O; Yamamoto, Matsuo

2017-11-01

The aim of this study is to evaluate the existence of residual calculus on root surfaces by determining the fluorescence/Raman intensity ratio. Thirty-two extracted human teeth, partially covered with calculus on the root surface, were evaluated by using a portable Raman spectrophotometer, and a 785-nm, 100-mW laser was applied for fluorescence/Raman excitation. The collected spectra were normalized to the hydroxyapatite Raman band intensity at 960 cm -1 . Raman spectra were recorded from the same point after changing the focal distance of the laser and the target radiating angle. In seven teeth, the condition of calculus, cementum, and dentin were evaluated. In 25 teeth, we determined the fluorescence/Raman intensity ratio following three strokes of debridement. Raman spectra collected from the dentin, cementum, and calculus were different. After normalization, spectra values were constant. The fluorescence/Raman intensity ratio of calculus region showed significant differences compared to the cementum and dentin (p < 0.05). The fluorescence/Raman intensity ratio decreased with calculus debridement. For this analysis, the delta value was defined as the difference between the values before and after three strokes, with the final 2 delta values close to zero, indicating a gradual asymptotic curve and the change in intensity ratio approximating that of individual constants. Fluorescence/Raman intensity ratio was effectively used to cancel the angle- and distance-dependent fluctuations of fluorescence collection efficiency during measurement. Changes in the fluorescence/Raman intensity ratio near zero suggested that cementum or dentin was exposed, and calculus removed.

Application of fluorescence spectroscopy and imaging in the detection of a photosensitizer in photodynamic therapy

NASA Astrophysics Data System (ADS)

Zang, Lixin; Zhao, Huimin; Zhang, Zhiguo; Cao, Wenwu

2017-02-01

Photodynamic therapy (PDT) is currently an advanced optical technology in medical applications. However, the application of PDT is limited by the detection of photosensitizers. This work focuses on the application of fluorescence spectroscopy and imaging in the detection of an effective photosenzitizer, hematoporphyrin monomethyl ether (HMME). Optical properties of HMME were measured and analyzed based on its absorption and fluorescence spectra. The production mechanism of its fluorescence emission was analyzed. The detection device for HMME based on fluorescence spectroscopy was designed. Ratiometric method was applied to eliminate the influence of intensity change of excitation sources, fluctuates of excitation sources and photo detectors, and background emissions. The detection limit of this device is 6 μg/L, and it was successfully applied to the diagnosis of the metabolism of HMME in the esophageal cancer cells. To overcome the limitation of the point measurement using fluorescence spectroscopy, a two-dimensional (2D) fluorescence imaging system was established. The algorithm of the 2D fluorescence imaging system is deduced according to the fluorescence ratiometric method using bandpass filters. The method of multiple pixel point addition (MPPA) was used to eliminate fluctuates of signals. Using the method of MPPA, SNR was improved by about 30 times. The detection limit of this imaging system is 1.9 μg/L. Our systems can be used in the detection of porphyrins to improve the PDT effect.

Intrinsic motions in the N-terminal domain of an ionotropic glutamate receptor detected by fluorescence correlation spectroscopy.

PubMed

Jensen, Mette H; Sukumaran, Madhav; Johnson, Christopher M; Greger, Ingo H; Neuweiler, Hannes

2011-11-18

Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function. Copyright © 2011 Elsevier Ltd. All rights reserved.

Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons.

PubMed

Weston, Kenneth D; Dyck, Martina; Tinnefeld, Philip; Müller, Christian; Herten, Dirk P; Sauer, Markus

2002-10-15

A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

PubMed Central

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W.; Knoch, Tobias A.; Waldeck, Waldemar; Langowski, Jörg

2003-01-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of ∼13 s. PMID:12719264

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

PubMed

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W; Knoch, Tobias A; Waldeck, Waldemar; Langowski, Jörg

2003-05-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Ratiometric detection of pH fluctuation in mitochondria with a new fluorescein/cyanine hybrid sensor.

PubMed

Chen, Yuncong; Zhu, Chengcheng; Cen, Jiajie; Bai, Yang; He, Weijiang; Guo, Zijian

2015-05-01

The homeostasis of mitochondrial pH (pH m ) is crucial in cell physiology. Developing small-molecular fluorescent sensors for the ratiometric detection of pH m fluctuation is highly demanded yet challenging. A ratiometric pH sensor, Mito-pH , was constructed by integrating a pH-sensitive FITC fluorophore with a pH-insensitive hemicyanine group. The hemicyanine group also acts as the mitochondria targeting group due to its lipophilic cationic nature. Besides its ability to target mitochondria, this sensor provides two ratiometric pH sensing modes, the dual excitation/dual emission mode (D ex /D em ) and dual excitation (D ex ) mode, and its linear and reversible ratiometric response range from pH 6.15 to 8.38 makes this sensor suitable for the practical tracking of pH m fluctuation in live cells. With this sensor, stimulated pH m fluctuation has been successfully tracked in a ratiometric manner via both fluorescence imaging and flow cytometry.

Single-Pixel Optical Fluctuation Analysis of Calcium Channel Function in Active Zones of Motor Nerve Terminals

PubMed Central

Luo, Fujun; Dittrich, Markus; Stiles, Joel R.; Meriney, Stephen D.

2011-01-01

We used high-resolution fluorescence imaging and single-pixel optical fluctuation analysis to estimate the opening probability of individual voltage-gated calcium (Ca2+) channels during an action potential and the number of such Ca2+ channels within active zones of frog neuromuscular junctions. Analysis revealed ~36 Ca2+ channels within each active zone, similar to the number of docked synaptic vesicles but far less than the total number of transmembrane particles reported based on freeze-fracture analysis (~200–250). The probability that each channel opened during an action potential was only ~0.2. These results suggest why each active zone averages only one quantal release event during every other action potential, despite a substantial number of docked vesicles. With sparse Ca2+ channels and low opening probability, triggering of fusion for each vesicle is primarily controlled by Ca2+ influx through individual Ca2+ channels. In contrast, the entire synapse is highly reliable because it contains hundreds of active zones. PMID:21813687

Implantable biosensors: analysis of fluorescent light propagation through skin

NASA Astrophysics Data System (ADS)

O'Neal, D. P.; McShane, Michael J.; Pishko, Michael V.; Cote, Gerard L.

2001-06-01

Progress towards a painless and hygienic glucose monitoring procedure for diabetics continues as the growth of diabetes mellitus reaches epidemic proportions in the American population. Utilizing an implantable fluorescence based glucose assay, the minimally invasive approach presented here has previously shown promise towards this goal in terms of glucose specificity and quantification for in vitro environments. However, in realistic physiological circumstances the depth of the implant can vary and optical properties of skin can change due to normal physiological conditions. Additionally, naturally occurring auto-fluorescence can obscure the sensor signal. An important concern under these conditions is that variations of fluorescent intensity due to these or other causes might be mistaken for glucose concentration fluctuations. New data shows that fluorescence-based glucose assays can be probed and interpreted in terms of glucose concentrations through pig skin at depths of up to 700 mm when immobilized in a bio-compatible polymer. When a combination of two fluorophores are employed as demonstrated here, reasonable changes in skin thickness and the confounding effects of the variations inherent in skin can be overcome for this glucose sensing application.

Watching Single Enzymes and Fluorescent Proteins in Action in Solution Using a Microfluidic Trap

NASA Astrophysics Data System (ADS)

Goldsmith, Randall

2012-02-01

Observation of dynamics of single biomolecules over a prolonged time without altering the biomolecule via immobilization is achieved with a specialized microfluidic device. This device, the Anti-Brownian ELectrokinetic (ABEL) Trap, uses real-time electrokinetic feedback to cancel Brownian motion of single objects in solution. First, we use the ABEL Trap to study Allophycocyanin (APC), a photosynthetic antenna-protein and popular fluorescent probe. A complex relationship between fluorescence intensity and lifetime is observed, suggesting light-induced conformational changes and radiative and non-radiative rate fluctuations. Second, we apply the ABEL Trap to single molecules of the multi-copper enzyme blue Nitrite Reductase where a fluorescent label reports on the oxidation state of the Type I Copper. Redox cycling is observed and kinetic analysis allows extraction of the microscopic rate constants in the kinetic scheme. Evidence of a substrate-induced shift of the intramolecular electron transfer rate is seen. Taken together, these observations provide windows of unprecedented detail into the dynamics of solution-phase biomolecules.

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

USDA-ARS?s Scientific Manuscript database

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

On the origin of shape fluctuations of the cell nucleus.

PubMed

Chu, Fang-Yi; Haley, Shannon C; Zidovska, Alexandra

2017-09-26

The nuclear envelope (NE) presents a physical boundary between the cytoplasm and the nucleoplasm, sandwiched in between two highly active systems inside the cell: cytoskeleton and chromatin. NE defines the shape and size of the cell nucleus, which increases during the cell cycle, accommodating for chromosome decondensation followed by genome duplication. In this work, we study nuclear shape fluctuations at short time scales of seconds in human cells. Using spinning disk confocal microscopy, we observe fast fluctuations of the NE, visualized by fluorescently labeled lamin A, and of the chromatin globule surface (CGS) underneath the NE, visualized by fluorescently labeled histone H2B. Our findings reveal that fluctuation amplitudes of both CGS and NE monotonously decrease during the cell cycle, serving as a reliable cell cycle stage indicator. Remarkably, we find that, while CGS and NE typically fluctuate in phase, they do exhibit localized regions of out-of-phase motion, which lead to separation of NE and CGS. To explore the mechanism behind these shape fluctuations, we use biochemical perturbations. We find the shape fluctuations of CGS and NE to be both thermally and actively driven, the latter caused by forces from chromatin and cytoskeleton. Such undulations might affect gene regulation as well as contribute to the anomalously high rates of nuclear transport by, e.g., stirring of molecules next to NE, or increasing flux of molecules through the nuclear pores.

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui, Yi; Hu, Dehong; Markillie, Lye Meng

Quantitative gene expression analysis in intact single cells can be achieved using single molecule- based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple FISH sub-probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific binding of sub-probes and tissue autofluorescence, limiting its accuracy. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on blinking frequencies of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses blinking frequency patterns, emitted frommore » a transcript bound to multiple sub-probes, which are distinct from blinking patterns emitted from partial or nonspecifically bound sub-probes and autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2, and their ratio (Nkx2-2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct blinking frequency patterns, laying the foundation for reliable single-cell transcriptomics.« less

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.

Measurement of temperature and density fluctuations in turbulence using an ultraviolet laser

NASA Technical Reports Server (NTRS)

Massey, G. A.

1984-01-01

Noninvasive measurement of density and temperature fluctuations in turbulent air flow was examined. The approach used fluorescence of oxygen molecules which are selectively excited by a tunable vacuum ultraviolet laser beam. The strength of the fluorescence signal and its dependence on laser wavelength vary with the density and temperature of the air in the laser beam. Because fluorescence can be detected at 90 degrees from the beam propagation direction, spatial resolution in three dimensions, rather than path-integrated measurements can be achieved. With spatial resolutions of the order of a millimeter and at supersonic air velocities it is necessary to perform each measurement in a time of the order of a microsecond; this is possible by by using laser pulses of ten nanosecond duration. In this method atmospheric O2 is excited by the emission of a tunable ArF excimer laser, and the fluorescence, which spans the 210 to 420 range, is detected by an ultraviolet phototube.

Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

NASA Astrophysics Data System (ADS)

Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

2018-03-01

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

Photorespiration plays an important role in the regulation of photosynthetic electron flow under fluctuating light in tobacco plants grown under full sunlight

PubMed Central

Huang, Wei; Hu, Hong; Zhang, Shi-Bao

2015-01-01

Plants usually experience dynamic fluctuations of light intensities under natural conditions. However, the responses of mesophyll conductance, CO2 assimilation, and photorespiration to light fluctuation are not well understood. To address this question, we measured photosynthetic parameters of gas exchange and chlorophyll fluorescence in tobacco leaves at 2-min intervals while irradiance levels alternated between 100 and 1200 μmol photons m−2 s−1. Compared with leaves exposed to a constant light of 1200 μmol photons m−2 s−1, both stomatal and mesophyll conductances were significantly restricted in leaves treated with fluctuating light condition. Meanwhile, CO2 assimilation rate and electron flow devoted to RuBP carboxylation at 1200 μmol photons m−2 s−1 under fluctuating light were limited by the low chloroplast CO2 concentration. Analysis based on the C3 photosynthesis model indicated that, at 1200 μmol photons m−2 s−1 under fluctuating light, the CO2 assimilation rate was limited by RuBP carboxylation. Electron flow devoted to RuBP oxygenation at 1200 μmol photons m−2 s−1 under fluctuating light remained at nearly the maximum level throughout the experimental period. We conclude that fluctuating light restricts CO2 assimilation by decreasing both stomatal and mesophyll conductances. Under such conditions, photorespiration plays an important role in the regulation of photosynthetic electron flow. PMID:26322062

Influence of non-line of sight luminescent emitters in visible light communication systems

NASA Astrophysics Data System (ADS)

Ghorai, Anaranya; Walvekar, Pratik; Nayak, Shreyas; Narayan, K. S.

2018-01-01

We introduce and demonstrate concepts which utilize the non-line of sight fraction of light incident on a detector assembly in a visible-light communication (VLC) system. In addition to ambient light, realistic enclosures where VLC is implemented consist of a sizable fraction of scattered and reflected light. We present results of VLC systems with detectors responding to contributions from the light source scattered off a surface embedded with fluorescent and phosphorescent emitters besides the direct line of sight signal. Contribution from the emitters takes a form of discernible fluctuations in the detector signal. The implication of our results from noise analysis of these fluctuations indicates the possibility of utilizing smart coatings to further tailor VLC capabilities.

An investigation of nonsimultaneous laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Fletcher, D. G.

1993-01-01

An alternative to simultaneous, two-line laser-induced fluorescence for thermodynamic property measurement is presented. This spectroscopic approach is similar to multiple-overheat hot-wire anemometry and is based on laser excitation of different fluorescence transitions for separate, sequential wind tunnel runs. Both fluctuating and mean thermodynamic property measurements seem to be achievable with this method without exciting the transitions during the same laser pulse.

Experimental nonlinear dynamical studies in cesium magneto-optical trap using time-series analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anwar, M., E-mail: mamalik2000@gmail.com; Islam, R.; Faisal, M.

2015-03-30

A magneto-optical trap of neutral atoms is essentially a dissipative quantum system. The fast thermal atoms continuously dissipate their energy to the environment via spontaneous emissions during the cooling. The atoms are, therefore, strongly coupled with the vacuum reservoir and the laser field. The vacuum fluctuations as well as the field fluctuations are imparted to the atoms as random photon recoils. Consequently, the external and internal dynamics of atoms becomes stochastic. In this paper, we have investigated the stochastic dynamics of the atoms in a magneto-optical trap during the loading process. The time series analysis of the fluorescence signal showsmore » that the dynamics of the atoms evolves, like all dissipative systems, from deterministic to the chaotic regime. The subsequent disappearance and revival of chaos was attributed to chaos synchronization between spatially different atoms in the magneto-optical trap.« less

A new approach to interpretation of heterogeneity of fluorescence decay in complex biological systems

NASA Astrophysics Data System (ADS)

Wlodarczyk, Jakub; Kierdaszuk, Borys

2005-08-01

Decays of tyrosine fluorescence in protein-ligand complexes are described by a model of continuous distribution of fluorescence lifetimes. Resulted analytical power-like decay function provides good fits to highly complex fluorescence kinetics. Moreover, this is a manifestation of so-called Tsallis q-exponential function, which is suitable for description of the systems with long-range interactions, memory effect, as well as with fluctuations of the characteristic lifetime of fluorescence. The proposed decay functions were applied to analysis of fluorescence decays of tyrosine in a protein, i.e. the enzyme purine nucleoside phosphorylase from E. coli (the product of the deoD gene), free in aqueous solution and in a complex with formycin A (an inhibitor) and orthophosphate (a co-substrate). The power-like function provides new information about enzyme-ligand complex formation based on the physically justified heterogeneity parameter directly related to the lifetime distribution. A measure of the heterogeneity parameter in the enzyme systems is provided by a variance of fluorescence lifetime distribution. The possible number of deactivation channels and excited state mean lifetime can be easily derived without a priori knowledge of the complexity of studied system. Moreover, proposed model is simpler then traditional multi-exponential one, and better describes heterogeneous nature of studied systems.

Metabolic Imaging in Multiple Time Scales

PubMed Central

Ramanujan, V Krishnan

2013-01-01

We report here a novel combination of time-resolved imaging methods for probing mitochondrial metabolism multiple time scales at the level of single cells. By exploiting a mitochondrial membrane potential reporter fluorescence we demonstrate the single cell metabolic dynamics in time scales ranging from milliseconds to seconds to minutes in response to glucose metabolism and mitochondrial perturbations in real time. Our results show that in comparison with normal human mammary epithelial cells, the breast cancer cells display significant alterations in metabolic responses at all measured time scales by single cell kinetics, fluorescence recovery after photobleaching and by scaling analysis of time-series data obtained from mitochondrial fluorescence fluctuations. Furthermore scaling analysis of time-series data in living cells with distinct mitochondrial dysfunction also revealed significant metabolic differences thereby suggesting the broader applicability (e.g. in mitochondrial myopathies and other metabolic disorders) of the proposed strategies beyond the scope of cancer metabolism. We discuss the scope of these findings in the context of developing portable, real-time metabolic measurement systems that can find applications in preclinical and clinical diagnostics. PMID:24013043

Single atoms in a MOT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meschede, Dieter; Ueberholz, Bernd; Gomer, Victor

1999-06-11

We are experimenting with individual neutral cesium atoms stored in a magneto-optical trap. The atoms are detected by their resonance fluorescence, and fluorescence fluctuations contain signatures of the atomic internal and external degrees of freedom. This noninvasive probe provides a rich source of information about atomic dynamics at all relevant time scales.

Rapid Analysis of Copper Ore in Pre-Smelter Head Flow Slurry by Portable X-ray Fluorescence.

PubMed

Burnett, Brandon J; Lawrence, Neil J; Abourahma, Jehad N; Walker, Edward B

2016-05-01

Copper laden ore is often concentrated using flotation. Before the head flow slurry can be smelted, it is important to know the concentration of copper and contaminants. The concentration of copper and other elements fluctuate significantly in the head flow, often requiring modification of the concentrations in the slurry prior to smelting. A rapid, real-time analytical method is needed to support on-site optimization of the smelter feedstock. A portable, handheld X-ray fluorescence spectrometer was utilized to determine the copper concentration in a head flow suspension at the slurry origin. The method requires only seconds and is reliable for copper concentrations of 2.0-25%, typically encountered in such slurries. © The Author(s) 2016.

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.

Amplified effect of Brownian motion in bacterial near-surface swimming

PubMed Central

Li, Guanglai; Tam, Lick-Kong; Tang, Jay X.

2008-01-01

Brownian motion influences bacterial swimming by randomizing displacement and direction. Here, we report that the influence of Brownian motion is amplified when it is coupled to hydrodynamic interaction. We examine swimming trajectories of the singly flagellated bacterium Caulobacter crescentus near a glass surface with total internal reflection fluorescence microscopy and observe large fluctuations over time in the distance of the cell from the solid surface caused by Brownian motion. The observation is compared with computer simulation based on analysis of relevant physical factors, including electrostatics, van der Waals force, hydrodynamics, and Brownian motion. The simulation reproduces the experimental findings and reveals contribution from fluctuations of the cell orientation beyond the resolution of present observation. Coupled with hydrodynamic interaction between the bacterium and the boundary surface, the fluctuations in distance and orientation subsequently lead to variation of the swimming speed and local radius of curvature of swimming trajectory. These results shed light on the fundamental roles of Brownian motion in microbial motility, nutrient uptake, and adhesion. PMID:19015518

Refractometric sensitivity and thermal stabilization of fluorescent core microcapillary sensors: theory and experiment.

PubMed

Lane, S; Marsiglio, F; Zhi, Y; Meldrum, A

2015-02-20

Fluorescent-core microcapillaries (FCMs) present a robust basis for the application of optical whispering gallery modes toward refractometric sensing. An important question concerns whether these devices can be rendered insensitive to local temperature fluctuations, which may otherwise limit their refractometric detection limits, mainly as a result of thermorefractive effects. Here, we first use a standard cylindrical cavity formalism to develop the refractometric and thermally limited detection limits for the FCM structure. We then measure the thermal response of a real device with different analytes in the channel and compare the result to the theory. Good stability against temperature fluctuations was obtained for an ethanol solvent, with a near-zero observed thermal shift for the transverse magnetic modes. Similarly good results could in principle be obtained for any other solvent (e.g., water), if the thickness of the fluorescent layer can be sufficiently well controlled.

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

PubMed

Chizhik, Anna M; Stein, Simon; Dekaliuk, Mariia O; Battle, Christopher; Li, Weixing; Huss, Anja; Platen, Mitja; Schaap, Iwan A T; Gregor, Ingo; Demchenko, Alexander P; Schmidt, Christoph F; Enderlein, Jörg; Chizhik, Alexey I

2016-01-13

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

Fluorescence Determination of Tryptophan Side-Chain Accessibility and Dynamics in Triple-Helical Collagen-Like Peptides

PubMed Central

Simon-Lukasik, Kristine V.; Persikov, Anton V.; Brodsky, Barbara; Ramshaw, John A. M.; Laws, William R.; Alexander Ross, J. B.; Ludescher, Richard D.

2003-01-01

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)n. About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5–9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter (∼3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels. PMID:12524302

Simultaneous imaging of fuel vapor mass fraction and gas-phase temperature inside gasoline sprays using two-line excitation tracer planar laser-induced fluorescence.

PubMed

Zigan, Lars; Trost, Johannes; Leipertz, Alfred

2016-02-20

This paper reports for the first time, to the best of our knowledge, on the simultaneous imaging of the gas-phase temperature and fuel vapor mass fraction distribution in a direct-injection spark-ignition (DISI) spray under engine-relevant conditions using tracer planar laser-induced fluorescence (TPLIF). For measurements in the spray, the fluorescence tracer 3-pentanone is added to the nonfluorescent surrogate fuel iso-octane, which is excited quasi-simultaneously by two different excimer lasers for two-line excitation LIF. The gas-phase temperature of the mixture of fuel vapor and surrounding gas and the fuel vapor mass fraction can be calculated from the two LIF signals. The measurements are conducted in a high-temperature, high-pressure injection chamber. The fluorescence calibration of the tracer was executed in a flow cell and extended significantly compared to the existing database. A detailed error analysis for both calibration and measurement is provided. Simultaneous single-shot gas-phase temperature and fuel vapor mass fraction fields are processed for the assessment of cyclic spray fluctuations.

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

DOE PAGES

Cui, Yi; Hu, Dehong; Markillie, Lye Meng; ...

2017-10-04

Here, quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes.more » This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio ( Nkx2- 2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.« less

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui, Yi; Hu, Dehong; Markillie, Lye Meng

Here, quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes.more » This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio ( Nkx2- 2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.« less

Mitochondrial dynamics and optical conformation changes in DsRed as studied by Fourier imaging correlation spectroscopy

NASA Astrophysics Data System (ADS)

Senning, Eric Nicolas

Novel experiments that probe the dynamics of intracellular species, including the center-of-mass displacements and internal conformational transitions of biological macromolecules, have the potential to reveal the complex biochemical mechanisms operating within the cell. This work presents the implementation and development of Fourier imaging correlation spectroscopy (FICS), a phase-selective approach to fluorescence spectroscopy that measures the collective coordinate fluctuations of fluorescently labeled microscopic particles. In FICS experiments, a spatially modulated optical grating excites a fluorescently labeled sample. Phase-synchronous detection of the fluorescence, with respect to the phase of the exciting optical grating, can be used to monitor the fluctuations of partially averaged spatial coordinates. These data are then analyzed by two-point and four-point time correlation functions to provide a statistically meaningful understanding of the dynamics under observation. FICS represents a unique route to elevate signal levels, while acquiring detailed information about molecular coordinate trajectories. Mitochondria of mammalian cells are known to associate with cytoskeletal proteins, and their motions are affected by the stability of microtubules and microfilaments. Within the cell it is possible to fluorescently label the mitochondria and study its dynamic behavior with FICS. The dynamics of S. cerevisiae yeast mitochondria are characterized at four discrete length scales (ranging from 0.6--1.19 mum) and provide detailed information about the influence of specific cytoskeletal elements. Using the microtubule and microfilament destabilizing agents, Nocodazole and Latrunculin A, it is determined that microfilaments are required for normal yeast mitochondrial motion while microtubules have no effect. Experiments with specific actin mutants revealed that actin is responsible for enhanced mobility on length scales greater than 0.6 mum. The versatility of FICS expands when individual molecules are labeled with fluorescent chromophores. In recent experiments on the tetrameric fluorescent protein DsRed, polarization-modulated FICS (PM-FICS) is demonstrated to separate conformational dynamics from molecular translational dynamics. The optical switching pathways of DsRed, a tetrameric complex of fluorescent protein subunits, are examined. An analysis of PM-FICS coordinate trajectories, in terms of 2D spectra and joint probability distributions, provides detailed information about the transition pathways between distinct dipole-coupled DsRed conformations. This dissertation includes co-authored and previously published material.

Mapping Diffusion in a Living Cell via the Phasor Approach

PubMed Central

Ranjit, Suman; Lanzano, Luca; Gratton, Enrico

2014-01-01

Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created. PMID:25517145

Endogenous fluorescence emission of the ovary

NASA Astrophysics Data System (ADS)

Utzinger, Urs; Kirkpatrick, Nathaniel D.; Drezek, Rebekah A.; Brewer, Molly A.

2005-03-01

Epithelial ovarian cancer has the highest mortality rate among the gynecologic cancers. Early detection would significantly improve survival and quality of life of women at increased risk to develop ovarian cancer. We have constructed a device to investigate endogenous signals of the ovarian tissue surface in the UV C to visible range and describe our initial investigation of the use of optical spectroscopy to characterize the condition of the ovary. We have acquired data from more than 33 patients. A table top spectroscopy system was used to collect endogenous fluorescence with a fiberoptic probe that is compatible with endoscopic techniques. Samples were broken into five groups: Normal-Low Risk (for developing ovarian cancer) Normal-High Risk, Benign, and Cancer. Rigorous statistical analysis was applied to the data using variance tests for direct intensity versus diagnostic group comparisons and principal component analysis (PCA) to study the variance of the whole data set. We conclude that the diagnostically most useful excitation wavelengths are located in the UV. Furthermore, our results indicate that UV B and C are most useful. A safety analysis indicates that UV-C imaging can be conducted at exposure levels below safety thresholds. We found that fluorescence excited in the UV-C and UV-B range increases from benign to normal to cancerous tissues. This is in contrast to the emission created with UV-A excitation which decreased in the same order. We hypothesize that an increase of protein production and a decrease of fluorescence contributions of the extracellular matrix could explain this behavior. Variance analysis also identified fluctuation of fluorescence at 320/380 which is associated with collagen cross link residues. Small differences were observed between the group at high risk and normal risk for ovarian cancer. High risk samples deviated towards the cancer group and low risk samples towards benign group.

Enhancement of Single Molecule Fluorescence Signals by Colloidal Silver Nanoparticles in Studies of Protein Translation

PubMed Central

Bharill, Shashank; Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Mandecki, Wlodek; Gryczynski, Ignacy; Gryczynski, Zygmunt; Cooperman, Barry S.; Goldman, Yale E.

2011-01-01

Metal enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold respectively. Fluorescence intensity fluctuations above shot noise, at 0.1 – 5 Hz, were greater on silver particles. Overall signal to noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G. PMID:21158483

Enhancement of single-molecule fluorescence signals by colloidal silver nanoparticles in studies of protein translation.

PubMed

Bharill, Shashank; Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Mandecki, Wlodek; Gryczynski, Ignacy; Gryczynski, Zygmunt; Cooperman, Barry S; Goldman, Yale E

2011-01-25

Metal-enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold, respectively. Fluorescence intensity fluctuations above shot noise, at 0.1-5 Hz, were greater on silver particles. Overall signal-to-noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G.

Thermal and Denaturation Studies of the Time-Resolved Fluorescence Decay of Human Superoxide Dismutase

NASA Astrophysics Data System (ADS)

Silva, Norberto De Jesus

Previous studies have shown that time-resolved fluorescence decay of various single tryptophan proteins is best described by a distribution of fluorescence lifetimes rather than one or two lifetimes. The thermal dependence of the lifetime distributions is consistent with the hypothesis that proteins fluctuate between a hierarchy of many conformational substates. With this scenario as a theoretical framework, the correlations between protein dynamic and structure are investigated by studying the time-resolved fluorescence and anisotropy decay of the single tryptophan (Trp) residue of human superoxide dismutase (HSOD) over a wide range of temperatures and at different denaturant concentrations. First, it is demonstrated that the center of the lifetime distribution can characterize the average deactivation environment of the excited Trp-protein system. A qualitative model is introduced to explain the time-resolved fluorescence decay of HSOD in 80% glycerol over a wide range of temperatures. The dynamical model features isoenergetic conformational substates separated by a hierarchy of energy barriers. The HSOD system is also investigated as a function of denaturant concentration in aqueous solution. As a function of guanidine hydrochloride (GdHCl), the width of the fluorescence lifetime distribution of HSOD displays a maximum which is not coincident with the fully denatured form of HSOD at 6.5M GdHCl. Furthermore, the width for the fully denatured form of HSOD is greater than that of the native form. This is consistent with the scenario that more conformational substates are being created upon denaturation of HSOD. HSOD is a dimeric protein and it was observed that the width of the lifetime distribution of HSOD at intermediate GdHCl concentrations increased with decreasing protein concentration. In addition, the secondary structure of HSOD at intermediate GdHCl concentration does not change with protein concentration. These results suggest that HSOD display structural microheterogeneity which is consistent with the hypothesis of conformational substates. Further analysis show that, during denaturation, the monomeric form of HSOD is an intermediate which displays native-like secondary structure and fluctuating tertiary structure; i.e., the monomeric form of HSOD is a molten globule.

The bright future of single-molecule fluorescence imaging

PubMed Central

Juette, Manuel F.; Terry, Daniel S.; Wasserman, Michael R.; Zhou, Zhou; Altman, Roger B.; Zheng, Qinsi; Blanchard, Scott C.

2014-01-01

Single-molecule Förster resonance energy transfer (smFRET) is an essential and maturing tool to probe biomolecular interactions and conformational dynamics in vitro and, increasingly, in living cells. Multi-color smFRET enables the correlation of multiple such events and the precise dissection of their order and timing. However, the requirements for good spectral separation, high time resolution, and extended observation times place extraordinary demands on the fluorescent labels used in such experiments. Together with advanced experimental designs and data analysis, the development of long-lasting, non-fluctuating fluorophores is therefore proving key to progress in the field. Recently developed strategies for obtaining ultra-stable organic fluorophores spanning the visible spectrum are underway that will enable multi-color smFRET studies to deliver on their promise of previously unachievable biological insights. PMID:24956235

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.

Dynamic nuclear protein interactions investigated using fluorescence lifetime and fluorescence fluctuation spectroscopy

NASA Astrophysics Data System (ADS)

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

2012-03-01

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic interactions in pituitary specific gene regulation.

Advances in superresolution optical fluctuation imaging (SOFI)

PubMed Central

Dertinger, Thomas; Pallaoro, Alessia; Braun, Gary; Ly, Sonny; Laurence, Ted A.; Weiss, Shimon

2013-01-01

We review the concept of superresolution optical fluctuation imaging (SOFI), discuss its attributes and trade-offs (in comparison with other superresolution methods), and present superresolved images taken on samples stained with quantum dots, organic dyes, and plasmonic metal nanoparticles. We also discuss the prospects of SOFI for live cell superresolution imaging and for imaging with other (non-fluorescent) contrasts. PMID:23672771

Periodic and stochastic thermal modulation of protein folding kinetics.

PubMed

Platkov, Max; Gruebele, Martin

2014-07-21

Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

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

Fourier-interpolation superresolution optical fluctuation imaging (fSOFi) (Conference Presentation)

NASA Astrophysics Data System (ADS)

Enderlein, Joerg; Stein, Simon C.; Huss, Anja; Hähnel, Dirk; Gregor, Ingo

2016-02-01

Stochastic Optical Fluctuation Imaging (SOFI) is a superresolution fluorescence microscopy technique which allows to enhance the spatial resolution of an image by evaluating the temporal fluctuations of blinking fluorescent emitters. SOFI is not based on the identification and localization of single molecules such as in the widely used Photoactivation Localization Microsopy (PALM) or Stochastic Optical Reconstruction Microscopy (STORM), but computes a superresolved image via temporal cumulants from a recorded movie. A technical challenge hereby is that, when directly applying the SOFI algorithm to a movie of raw images, the pixel size of the final SOFI image is the same as that of the original images, which becomes problematic when the final SOFI resolution is much smaller than this value. In the past, sophisticated cross-correlation schemes have been used for tackling this problem. Here, we present an alternative, exact, straightforward, and simple solution using an interpolation scheme based on Fourier transforms. We exemplify the method on simulated and experimental data.

Study of Linear and Nonlinear Wave Excitation

NASA Astrophysics Data System (ADS)

Chu, Feng; Berumen, Jorge; Hood, Ryan; Mattingly, Sean; Skiff, Frederick

2013-10-01

We report an experimental study of externally excited low-frequency waves in a cylindrical, magnetized, singly-ionized Argon inductively-coupled gas discharge plasma that is weakly collisional. Wave excitation in the drift wave frequency range is accomplished by low-percentage amplitude modulation of the RF plasma source. Laser-induced fluorescence is adopted to study ion-density fluctuations in phase space. The laser is chopped to separate LIF from collisional fluorescence. A single negatively-biased Langmuir probe is used to detect ion-density fluctuations in the plasma. A ring array of Langmuir probes is also used to analyze the spatial and spectral structure of the excited waves. We apply coherent detection with respect to the wave frequency to obtain the ion distribution function associated with externally generated waves. Higher-order spectra are computed to evaluate the nonlinear coupling between fluctuations at various frequencies produced by the externally generated waves. Parametric decay of the waves is observed. This work is supported by U.S. DOE Grant No. DE-FG02-99ER54543.

Dancing with the Tides: Fluctuations of Coastal Phytoplankton Orchestrated by Different Oscillatory Modes of the Tidal Cycle

PubMed Central

Blauw, Anouk N.; Benincà, Elisa; Laane, Remi W. P. M.; Greenwood, Naomi; Huisman, Jef

2012-01-01

Population fluctuations are often driven by an interplay between intrinsic population processes and extrinsic environmental forcing. To investigate this interplay, we analyzed fluctuations in coastal phytoplankton concentration in relation to the tidal cycle. Time series of chlorophyll fluorescence, suspended particulate matter (SPM), salinity and temperature were obtained from an automated measuring platform in the southern North Sea, covering 9 years of data at a resolution of 12 to 30 minutes. Wavelet analysis showed that chlorophyll fluctuations were dominated by periodicities of 6 hours 12 min, 12 hours 25 min, 24 hours and 15 days, which correspond to the typical periodicities of tidal current speeds, the semidiurnal tidal cycle, the day-night cycle, and the spring-neap tidal cycle, respectively. During most of the year, chlorophyll and SPM fluctuated in phase with tidal current speed, indicative of alternating periods of sinking and vertical mixing of algal cells and SPM driven by the tidal cycle. Spring blooms slowly built up over several spring-neap tidal cycles, and subsequently expanded in late spring when a strong decline of the SPM concentration during neap tide enabled a temporary “escape” of the chlorophyll concentration from the tidal mixing regime. Our results demonstrate that the tidal cycle is a major determinant of phytoplankton fluctuations at several different time scales. These findings imply that high-resolution monitoring programs are essential to capture the natural variability of phytoplankton in coastal waters. PMID:23166639

Dancing with the tides: fluctuations of coastal phytoplankton orchestrated by different oscillatory modes of the tidal cycle.

PubMed

Blauw, Anouk N; Benincà, Elisa; Laane, Remi W P M; Greenwood, Naomi; Huisman, Jef

2012-01-01

Population fluctuations are often driven by an interplay between intrinsic population processes and extrinsic environmental forcing. To investigate this interplay, we analyzed fluctuations in coastal phytoplankton concentration in relation to the tidal cycle. Time series of chlorophyll fluorescence, suspended particulate matter (SPM), salinity and temperature were obtained from an automated measuring platform in the southern North Sea, covering 9 years of data at a resolution of 12 to 30 minutes. Wavelet analysis showed that chlorophyll fluctuations were dominated by periodicities of 6 hours 12 min, 12 hours 25 min, 24 hours and 15 days, which correspond to the typical periodicities of tidal current speeds, the semidiurnal tidal cycle, the day-night cycle, and the spring-neap tidal cycle, respectively. During most of the year, chlorophyll and SPM fluctuated in phase with tidal current speed, indicative of alternating periods of sinking and vertical mixing of algal cells and SPM driven by the tidal cycle. Spring blooms slowly built up over several spring-neap tidal cycles, and subsequently expanded in late spring when a strong decline of the SPM concentration during neap tide enabled a temporary "escape" of the chlorophyll concentration from the tidal mixing regime. Our results demonstrate that the tidal cycle is a major determinant of phytoplankton fluctuations at several different time scales. These findings imply that high-resolution monitoring programs are essential to capture the natural variability of phytoplankton in coastal waters.

Fluctuating volume-current formulation of electromagnetic fluctuations in inhomogeneous media: Incandescence and luminescence in arbitrary geometries

NASA Astrophysics Data System (ADS)

Polimeridis, Athanasios G.; Reid, M. T. H.; Jin, Weiliang; Johnson, Steven G.; White, Jacob K.; Rodriguez, Alejandro W.

2015-10-01

We describe a fluctuating volume-current formulation of electromagnetic fluctuations that extends our recent work on heat exchange and Casimir interactions between arbitrarily shaped homogeneous bodies [A. W. Rodriguez, M. T. H. Reid, and S. G. Johnson, Phys. Rev. B 88, 054305 (2013), 10.1103/PhysRevB.88.054305] to situations involving incandescence and luminescence problems, including thermal radiation, heat transfer, Casimir forces, spontaneous emission, fluorescence, and Raman scattering, in inhomogeneous media. Unlike previous scattering formulations based on field and/or surface unknowns, our work exploits powerful techniques from the volume-integral equation (VIE) method, in which electromagnetic scattering is described in terms of volumetric, current unknowns throughout the bodies. The resulting trace formulas (boxed equations) involve products of well-studied VIE matrices and describe power and momentum transfer between objects with spatially varying material properties and fluctuation characteristics. We demonstrate that thanks to the low-rank properties of the associated matrices, these formulas are susceptible to fast-trace computations based on iterative methods, making practical calculations tractable. We apply our techniques to study thermal radiation, heat transfer, and fluorescence in complicated geometries, checking our method against established techniques best suited for homogeneous bodies as well as applying it to obtain predictions of radiation from complex bodies with spatially varying permittivities and/or temperature profiles.

Biological oscillations: Fluorescence monitoring by confocal microscopy

NASA Astrophysics Data System (ADS)

Chattoraj, Shyamtanu; Bhattacharyya, Kankan

2016-09-01

Fluctuations play a vital role in biological systems. Single molecule spectroscopy has recently revealed many new kinds of fluctuations in biological molecules. In this account, we focus on structural fluctuations of an antigen-antibody complex, conformational dynamics of a DNA quadruplex, effects of taxol on dynamics of microtubules, intermittent red-ox oscillations at different organelles in a live cell (mitochondria, lipid droplets, endoplasmic reticulum and cell membrane) and stochastic resonance in gene silencing. We show that there are major differences in these dynamics between a cancer cell and the corresponding non-cancer cell.

Adaptive handling of Rayleigh and Raman scatter of fluorescence data based on evaluation of the degree of spectral overlap

NASA Astrophysics Data System (ADS)

Hu, Yingtian; Liu, Chao; Wang, Xiaoping; Zhao, Dongdong

2018-06-01

At present the general scatter handling methods are unsatisfactory when scatter and fluorescence seriously overlap in excitation emission matrix. In this study, an adaptive method for scatter handling of fluorescence data is proposed. Firstly, the Raman scatter was corrected by subtracting the baseline of deionized water which was collected in each experiment to adapt to the intensity fluctuations. Then, the degrees of spectral overlap between Rayleigh scatter and fluorescence were classified into three categories based on the distance between the spectral peaks. The corresponding algorithms, including setting to zero, fitting on single or both sides, were implemented after the evaluation of the degree of overlap for individual emission spectra. The proposed method minimized the number of fitting and interpolation processes, which reduced complexity, saved time, avoided overfitting, and most importantly assured the authenticity of data. Furthermore, the effectiveness of this procedure on the subsequent PARAFAC analysis was assessed and compared to Delaunay interpolation by conducting experiments with four typical organic chemicals and real water samples. Using this method, we conducted long-term monitoring of tap water and river water near a dyeing and printing plant. This method can be used for improving adaptability and accuracy in the scatter handling of fluorescence data.

Continuous Measurements of Canopy-level Solar-Induced Chlorophyll Fluorescence for Inferring Diurnal and Seasonal Dynamics of Photosynthesis in Crop Fields in the Midwestern USA

NASA Astrophysics Data System (ADS)

Miao, G.; Guan, K.; Yang, X.; Bernacchi, C.; DeLucia, E. H.; Cai, Y.; Masters, M. D.; Peng, B.

2016-12-01

Plants emitted photons of red and far-red light, called chlorophyll fluorescence, after sunlight absorption for photosynthesis. This solar-induced fluorescence (SIF) is generated simultaneously while plants actively photosynthesize. The link between photosynthesis and SIF resulting from the competition for the same excitation energy has long been investigated and applied for inferring the rate of photosynthesis. Recent development of continuous SIF observational technology is furthering the inferring potential as well as our understandings of fluctuations of SIF and photosynthesis with changes in environmental conditions. To better understand this photosynthesis-SIF link at multiple time scales and their relationships with environmental drivers, we deployed two newly developed tower-based SIF systems (FluoSpec) in a corn (Zea mays L., C4 plant) field and a soybean (Glycine max L., C3 plant) field at University of Illinois Energy Farm and conducted continuous near-surface SIF measurements at canopy scale from mid-growing season of 2016. Eddy covariance flux towers were installed in parallel at both sites for canopy-scale gas exchange measurements. Relationship between SIF and flux tower photosynthesis will be analyzed to derive the empirical models for photosynthesis retrieval from SIF signals. Preliminary results indicate that canopy SIF can reflect diurnal and seasonal dynamics of photosynthesis. Mechanistic analysis on SIF fluctuations and responses to environmental variations will be conducted as well for a closer look at mechanism of photosynthetic responses. Corn and soybean SIF and photosynthesis-SIF relationship will be compared to investigate the difference between C4 and C3 plants.

Remote excitation fluorescence correlation spectroscopy using silver nanowires

NASA Astrophysics Data System (ADS)

Su, Liang; Yuan, Haifeng; Lu, Gang; Hofkens, Johan; Roeffaers, Maarten; Uji-i, Hiroshi

2014-11-01

Fluorescence correlation spectroscopy (FCS), a powerful tool to resolve local properties, dynamical process of molecules, rotational and translational diffusion motions, relies on the fluctuations of florescence observables in the observation volume. In the case of rare transition events or small dynamical fluctuations, FCS requires few molecules or even single molecules in the observation volume at a time to minimize the background signals. Metal nanoparticle which possess unique localized surface plasmon resonance (LSPR) have been used to reduce the observation volume down to sub-diffraction limited scale while maintain at high analyst concentration up to tens of micromolar. Nevertheless, the applications of functionalized nanoparticles in living cell are limited due to the continuous diffusion after cell uptake, which makes it difficult to target the region of interests in the cell. In this work, we demonstrate the use of silver nanowires for remote excitation FCS on fluorescent molecules in solution. By using propagation surface plasmon polaritons (SPPs) which supported by the silver nanowire to excite the fluorescence, both illumination and observation volume can be reduced simultaneously. In such a way, less perturbation is induced to the target region, and this will broaden the application scope of silver nanowire as tip in single cell endoscopy.

Scales of variability of bio-optical properties as observed from near-surface drifters

NASA Technical Reports Server (NTRS)

Abbott, Mark R.; Brink, Kenneth H.; Booth, C. R.; Blasco, Dolors; Swenson, Mark S.; Davis, Curtiss O.; Codispoti, L. A.

1995-01-01

A drifter equipped with bio-optical sensors and an automated water sampler was deployed in the California Current as part of the coastal transition zone program to study the biological, chemical, and physical dynamics of the meandering filaments. During deployments in 1987 and 1988, measurements were made of fluorescence, downwelling irradiance, upwelling radiance, and beam attenuation using several bio-optical sensors. Samples were collected by an automated sampler for later analysis of nutrients and phytoplankton species compositon. Large-scale spatial and temporal changes in the bio-optical and biological properties of the region were driven by changes in phytoplankton species composition which, in turn, were associated with the meandering circulation. Variance spectra of the bio-optical paramenters revealed fluctuations on both diel and semidiurnal scales, perhaps associated with solar variations and internal tides, respectively. Offshore, inertial-scale fluctuations were apparent in the variance spectra of temperature, fluorescence, and beam attenuation. Although calibration samples can help remove some of these variations, these results suggest that the use of bio-optical data from unattended platforms such as moorings and drifters must be analyzed carefully. Characterization of the scaled of phytoplankton variability must account for the scales of variability in the algorithms used to convert bio-optical measurments into biological quantities.

Spontaneous Fluctuations can Guide Drug Design Strategies for Structurally Disordered Proteins.

PubMed

Maity, Barun Kumar; Vishvakarma, Vicky; Surendran, Dayana; Rawat, Anoop; Das, Anirban; Pramanik, Shreya; Arfin, Najmul; Maiti, Sudipta

2018-06-21

Structure-based 'rational' drug-design strategies fail for diseases associated with intrinsically disordered proteins (IDPs). However, structural disorder allows large amplitude spontaneous intramolecular dynamics in a protein. We demonstrate a method that exploits this dynamics to provide quantitative information about the degree of interaction of an IDP with other molecules. A candidate ligand molecule may not bind strongly, but even momentary interactions can be expected to perturb the fluctuations. We measure the amplitude and frequency of the equilibrium fluctuations of fluorescently labeled small oligomers of hIAPP (an IDP associated with Type II diabetes) in a physiological solution, using nanosecond fluorescence cross-correlation spectroscopy. We show that the inter-terminal distance fluctuates at a characteristic timescale of 134 ± 10 ns, and 6.4 ± 0.2 % of the population is in the 'closed' (quenched) state at equilibrium. These fluctuations are affected in a dose-dependent manner by a series of small molecules known to reduce the toxicity of various amyloid peptides. The degree of interaction shows the following order: resveratrol < epicatechin ~ quercetin < congo red < epigallocatechin-3-gallate. Such ordering can provide a direction for exploring the chemical space for finding stronger-binding ligands. We test the biological relevance of these measurements by measuring the effect of these molecules on the affinity of hIAPP for lipid vesicles and cell membranes. We find that the ability of a molecule to modulate intramolecular fluctuations correlates well with its ability to lower membrane affinity. We conclude that structural disorder may provide new avenues for rational drug design for IDPs.

Self-assembly of BODIPY based pH-sensitive near-infrared polymeric micelles for drug controlled delivery and fluorescence imaging applications

NASA Astrophysics Data System (ADS)

Liu, Xiaodong; Chen, Bizheng; Li, Xiaojun; Zhang, Lifen; Xu, Yujie; Liu, Zhuang; Cheng, Zhenping; Zhu, Xiulin

2015-10-01

Responsive block copolymer micelles emerging as promising imaging and drug delivery systems show high stability and on-demand drug release activities. Herein, we developed self-assembled pH-responsive NIR emission micelles entrapped with doxorubicin (DOX) within the cores by the electrostatic interactions for fluorescence imaging and chemotherapy applications. The block copolymer, poly(methacrylic acid)-block-poly[(poly(ethylene glycol) methyl ether methacrylate)-co-boron dipyrromethene derivatives] (PMAA-b-P(PEGMA-co-BODIPY)), was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the molecular weight distribution of this copolymer was narrow (Mw/Mn = 1.31). The NIR fluorescence enhancement induced by the phenol/phenolate interconversion equilibrium works as a switch in response to the intracellular pH fluctuations. DOX-loaded PMAA-b-P(PEGMA-co-BODIPY) micelles can detect the physiological pH fluctuations with a pKa near physiological conditions (~7.52), and showed pH-responsive collapse and an obvious acid promoted anticancer drug release behavior (over 58.8-62.8% in 10 h). Real-time imaging of intracellular pH variations was performed and a significant chemotherapy effect was demonstrated against HeLa cells.Responsive block copolymer micelles emerging as promising imaging and drug delivery systems show high stability and on-demand drug release activities. Herein, we developed self-assembled pH-responsive NIR emission micelles entrapped with doxorubicin (DOX) within the cores by the electrostatic interactions for fluorescence imaging and chemotherapy applications. The block copolymer, poly(methacrylic acid)-block-poly[(poly(ethylene glycol) methyl ether methacrylate)-co-boron dipyrromethene derivatives] (PMAA-b-P(PEGMA-co-BODIPY)), was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the molecular weight distribution of this copolymer was narrow (Mw/Mn = 1.31). The NIR fluorescence enhancement induced by the phenol/phenolate interconversion equilibrium works as a switch in response to the intracellular pH fluctuations. DOX-loaded PMAA-b-P(PEGMA-co-BODIPY) micelles can detect the physiological pH fluctuations with a pKa near physiological conditions (~7.52), and showed pH-responsive collapse and an obvious acid promoted anticancer drug release behavior (over 58.8-62.8% in 10 h). Real-time imaging of intracellular pH variations was performed and a significant chemotherapy effect was demonstrated against HeLa cells. Electronic supplementary information (ESI) available: GPC, UV/vis, fluorescence, and MTT data of the as-prepared polymers; 1H NMR, 13C NMR, HRMS and FT-IR of organic molecules and polymers. See DOI: 10.1039/c5nr04655f

Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.

PubMed

Hebert, Benedict; Costantino, Santiago; Wiseman, Paul W

2005-05-01

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

The Temporal Resolution of Laser Induced Fluorescence Photobleaching Anemometer

NASA Astrophysics Data System (ADS)

Zhao, Wei; Yang, Fang; Wang, Guiren

2014-11-01

Recently, in microfluidics, electrokinetic flows are widely used on micromixer designing. However, there is unfortunately no valid velocimeter today that can measure the random velocity fluctuation at high temporal and spatial resolution simultaneously in the complicated flow circumstance. We recently introduced laser induced fluorescence photobleaching anemometer (LIFPA), which has been successfully used in the measurement of velocity field in AC electrically driven microflow. Here, we theoretically study the temporal resolution (TR) of and experimentally verify, LIFPA can have simultaneously ultrahigh temporal (~4 μs) and spatial (~203 nm) resolution and can measure velocity fluctuation up to at least 2 kHz, whose corresponding wave number is about 6 × 106 1/m in an electrokinetically forced unsteady flow in microfluidics. The measurement of LIFPA is also compared with the widely used micro Particle Imaging Velocimetry (μPIV). We found, at the inlet, due to multiple uncertainties, the velocity fluctuations by μPIV exhibits apparently smaller values than that by LIFPA. But at downstreams, where velocity fluctuation is much lower than at the inlet and the uncertainties of complicated electric field on particles becomes smaller, LIFPA and μPIV indicate similar measurement. The work was supported by NSF under grant no. CAREER CBET-0954977 and MRI CBET-1040227, respectively.

A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

PubMed Central

Pauly, Matthew D; Procario, Megan C; Lauring, Adam S

2017-01-01

Influenza virus’ low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7 × 10–6 - 3.0 × 10–5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8 × 10–4 s/n/r for PR8 (H1N1) and 2.5 × 10–4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7–3.6. Our data suggest that each replicated genome will have an average of 2–3 mutations and highlight the importance of mutational load in influenza virus evolution. DOI: http://dx.doi.org/10.7554/eLife.26437.001 PMID:28598328

On the origin of non-exponential fluorescence decays in enzyme-ligand complex

NASA Astrophysics Data System (ADS)

Wlodarczyk, Jakub; Kierdaszuk, Borys

2004-05-01

Complex fluorescence decays have usually been analyzed with the aid of a multi-exponential model, but interpretation of the individual exponential terms has not been adequately characterized. In such cases the intensity decays were also analyzed in terms of the continuous lifetime distribution as a consequence of an interaction of fluorophore with environment, conformational heterogeneity or their dynamical nature. We show that non-exponential fluorescence decay of the enzyme-ligand complexes may results from time dependent energy transport. The latter, to our opinion, may be accounted for by electron transport from the protein tyrosines to their neighbor residues. We introduce the time-dependent hopping rate in the form v(t)~(a+bt)-1. This in turn leads to the luminescence decay function in the form I(t)=Ioexp(-t/τ1)(1+lt/γτ2)-γ. Such a decay function provides good fits to highly complex fluorescence decays. The power-like tail implies the time hierarchy in migration energy process due to the hierarchical energy-level structure. Moreover, such a power-like term is a manifestation of so called Tsallis nonextensive statistic and is suitable for description of the systems with long-range interactions, memory effect as well as with fluctuations of characteristic lifetime of fluorescence. The proposed decay function was applied in analysis of fluorescence decays of tyrosine protein, i.e. the enzyme purine nucleoside phosphorylase from E. coli in a complex with formycin A (an inhibitor) and orthophosphate (a co-substrate).

A single pH fluorescent probe for biosensing and imaging of extreme acidity and extreme alkalinity.

PubMed

Chao, Jian-Bin; Wang, Hui-Juan; Zhang, Yong-Bin; Li, Zhi-Qing; Liu, Yu-Hong; Huo, Fang-Jun; Yin, Cai-Xia; Shi, Ya-Wei; Wang, Juan-Juan

2017-07-04

A simple tailor-made pH fluorescent probe 2-benzothiazole (N-ethylcarbazole-3-yl) hydrazone (Probe) is facilely synthesized by the condensation reaction of 2-hydrazinobenzothiazole with N-ethylcarbazole-3-formaldehyde, which is a useful fluorescent probe for monitoring extremely acidic and alkaline pH, quantitatively. The pH titrations indicate that Probe displays a remarkable emission enhancement with a pK a of 2.73 and responds linearly to minor pH fluctuations within the extremely acidic range of 2.21-3.30. Interestingly, Probe also exhibits strong pH-dependent characteristics with pK a 11.28 and linear response to extreme-alkalinity range of 10.41-12.43. In addition, Probe shows a large Stokes shift of 84 nm under extremely acidic and alkaline conditions, high selectivity, excellent sensitivity, good water-solubility and fine stability, all of which are favorable for intracellular pH imaging. The probe is further successfully applied to image extremely acidic and alkaline pH values fluctuations in E. coli cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescence fluctuation analysis of BACE1-GFP fusion protein in cultured HEK293 cells

NASA Astrophysics Data System (ADS)

Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-10-01

Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

A microplate assay for measuring cell death in C2C12 cells.

PubMed

Lima, Tanes; Silveira, Leonardo

2018-03-22

The main goal of this study was to develop a straightforward and rapid microplate assay for measuring propidium iodide (PI) in C2C12 cells. The PI method proves to be an efficient quantitative assay for analyzing cell viability through PI fluorescence analysis. Importantly, the protocol takes less than 30 minutes, and the results are reproducible. C2C12 cells were exposed to an increasing concentration of palmitate for a period of 24 hours to induce cell death, and the PI fluorescence increased in a concentration-dependent manner. Evaluation of mitochondrial function and reactive oxygen species production validated the deleterious effects of palmitate treatment. Also, the microplate PI assay demonstrated high sensitivity as indicated by the detection of modest fluctuations in cell viability in response to catalase overexpression in palmitate-treated cells. The microplate PI assay, therefore, offers an accurate method to be used for in vitro studies.

Probing short-range protein Brownian motion in the cytoplasm of living cells.

PubMed

Di Rienzo, Carmine; Piazza, Vincenzo; Gratton, Enrico; Beltram, Fabio; Cardarelli, Francesco

2014-12-23

The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.

Conjugated polymers: Watching polymers dance

NASA Astrophysics Data System (ADS)

Rothberg, Lewis

2011-06-01

Single-molecule spectroscopy allows fluctuations of conjugated polymer conformation to be monitored during solvent vapour annealing. Dramatic changes in fluorescence behaviour are observed and interpreted in terms of transformations between extended and collapsed polymer geometries.

Watching Individual Enzymes at Work

NASA Astrophysics Data System (ADS)

Blank, Kerstin; Rocha, Susana; De Cremer, Gert; Roeffaers, Maarten B. J.; Uji-i, Hiroshi; Hofkens, Johan

Single-molecule fluorescence experiments are a powerful tool to analyze reaction mechanisms of enzymes. Because of their unique potential to detect heterogeneities in space and time, they have provided unprecedented insights into the nature and mechanisms of conformational changes related to the catalytic reaction. The most important finding from experiments with single enzymes is the generally observed phenomenon that the catalytic rate constants fluctuate over time (dynamic disorder). These fluctuations originate from conformational changes occurring on time scales, which are similar to or slower than that of the catalytic reaction. Here, we summarize experiments with enzymes that show dynamic disorder and introduce new experimental strategies showing how single-molecule fluorescence experiments can be applied to address other open questions in medical and industrial enzymology, such as enzyme inactivation processes, reactant transfer in cascade reactions, and the mechanisms of interfacial catalysis.

Model-free uncertainty estimation in stochastical optical fluctuation imaging (SOFI) leads to a doubled temporal resolution

PubMed Central

Vandenberg, Wim; Duwé, Sam; Leutenegger, Marcel; Moeyaert, Benjamien; Krajnik, Bartosz; Lasser, Theo; Dedecker, Peter

2016-01-01

Stochastic optical fluctuation imaging (SOFI) is a super-resolution fluorescence imaging technique that makes use of stochastic fluctuations in the emission of the fluorophores. During a SOFI measurement multiple fluorescence images are acquired from the sample, followed by the calculation of the spatiotemporal cumulants of the intensities observed at each position. Compared to other techniques, SOFI works well under conditions of low signal-to-noise, high background, or high emitter densities. However, it can be difficult to unambiguously determine the reliability of images produced by any superresolution imaging technique. In this work we present a strategy that enables the estimation of the variance or uncertainty associated with each pixel in the SOFI image. In addition to estimating the image quality or reliability, we show that this can be used to optimize the signal-to-noise ratio (SNR) of SOFI images by including multiple pixel combinations in the cumulant calculation. We present an algorithm to perform this optimization, which automatically takes all relevant instrumental, sample, and probe parameters into account. Depending on the optical magnification of the system, this strategy can be used to improve the SNR of a SOFI image by 40% to 90%. This gain in information is entirely free, in the sense that it does not require additional efforts or complications. Alternatively our approach can be applied to reduce the number of fluorescence images to meet a particular quality level by about 30% to 50%, strongly improving the temporal resolution of SOFI imaging. PMID:26977356

Robust Distant Entanglement Generation Using Coherent Multiphoton Scattering

NASA Astrophysics Data System (ADS)

Chan, Ching-Kit; Sham, L. J.

2013-02-01

We describe a protocol to entangle two qubits at a distance by using resonance fluorescence. The scheme makes use of the postselection of large and distinguishable fluorescence signals corresponding to entangled and unentangled qubit states and has the merits of both high success probability and high entanglement fidelity owing to the multiphoton nature. Our result shows that the entanglement generation is robust against photon fluctuations in the fluorescence signals for a wide range of driving fields. We also demonstrate that this new protocol has an average entanglement duration within the decoherence time of corresponding qubit systems, based on current experimental photon efficiency.

Robust distant entanglement generation using coherent multiphoton scattering.

PubMed

Chan, Ching-Kit; Sham, L J

2013-02-15

We describe a protocol to entangle two qubits at a distance by using resonance fluorescence. The scheme makes use of the postselection of large and distinguishable fluorescence signals corresponding to entangled and unentangled qubit states and has the merits of both high success probability and high entanglement fidelity owing to the multiphoton nature. Our result shows that the entanglement generation is robust against photon fluctuations in the fluorescence signals for a wide range of driving fields. We also demonstrate that this new protocol has an average entanglement duration within the decoherence time of corresponding qubit systems, based on current experimental photon efficiency.

Time-resolved fluorescence microscopy to study biologically related applications using sol-gel derived and cellular media

NASA Astrophysics Data System (ADS)

Toury, Marion; Chandler, Lin; Allison, Archie; Campbell, David; McLoskey, David; Holmes-Smith, A. Sheila; Hungerford, Graham

2011-03-01

Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA Scientific DeltaDiode and DynaMyc).

A similarity measure for partially folded proteins: application to unfolded and native-like conformational fluctuations

NASA Astrophysics Data System (ADS)

Larios, Edgar; Yang, Wei Y.; Schulten, K.; Gruebele, M.

2004-12-01

Computing the root-mean-square deviation (RMSD) of a partially folded protein structure from the folded state requires the two structures to be translationally and rotationally aligned. We examine the constraint matrix L that preserves orthogonality of the rotation matrix during minimization of the RMSD. L is proportional to the sensitivity of the RMSD to the rotational alignment matrix. Its trace yields an isotropic reaction coordinate, while its off-diagonal matrix elements are related to the moment of inertia derivative tensor that encodes anisotropic information about the structure. We use L to compare λ-repressor fragment 6-85 (λ 6-85) to several partially folded structures obtained from molecular dynamics simulation (MD), and find that L as a reaction coordinate indeed encodes some information about protein topology. We also apply C α RMSD, L and tryptophan sidechain mobility as criteria for native state structural fluctuations of several λ 6-85 mutants. The mutants' denaturation curves and fluorescence quenching are measured experimentally for comparison. The results are in accord with a recent proposal that structural fluctuations near the chromophore can induce increased native state fluorescence or hyperfluorescence during unfolding of proteins.

Theoretical Studies Relating to the Interaction of Radiation with Matter

DTIC Science & Technology

1989-09-15

and Heisenberg picture calculations of resonance fluorescence in the presence of a strong field. Again, the Schrodinger calculation is quite...discussed in Sec. VIII. numerous effects have been discussed assuming fluctuat- ing radiation fields (resonant fluorescence , double reso- nance, multiphoton...tered at t12 =0. The peak has temporal width It 2 1 = rcl2 and, moreover, for fully correlated pulses (0) = 1) there is FIG. 6. Signal of order n = 1 as a

Time-Synchronized Continuous Wave Laser Induced Fluorescence Velocity Measurements of a 600 Watt Hall Thruster

DTIC Science & Technology

2015-07-01

channel and near- field plume region of a 600 W Hall thruster operating on xenon. Results show significant fluctuations in LIF signal intensity... LIF signal intensity (corre- lated with the density of the probed excited metastable state) in time during the discharge current cycle, with the peak...fluorescence ( LIF ).1 LIF provides the opportunity to investigate plasma sources non-intrusively with higher spatial resolution (typically < 1 mm) than

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

PubMed

Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O

2010-12-22

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

Conformational Switching in a Light-Harvesting Protein as Followed by Single-Molecule Spectroscopy

PubMed Central

Gall, Andrew; Ilioaia, Cristian; Krüger, Tjaart P.J.; Novoderezhkin, Vladimir I.; Robert, Bruno; van Grondelle, Rienk

2015-01-01

Among the ultimate goals of protein physics, the complete, experimental description of the energy paths leading to protein conformational changes remains a challenge. Single protein fluorescence spectroscopy constitutes an approach of choice for addressing protein dynamics, and, among naturally fluorescing proteins, light-harvesting (LH) proteins from purple bacteria constitute an ideal object for such a study. LHs bind bacteriochlorophyll a molecules, which confer on them a high intrinsic fluorescence yield. Moreover, the electronic properties of these pigment-proteins result from the strong excitonic coupling between their bound bacteriochlorophyll a molecules in combination with the large energetic disorder due to slow fluctuations in their structure. As a result, the position and probability of their fluorescence transition delicately depends on the precise realization of the disorder of the set of bound pigments, which is governed by the LH protein dynamics. Analysis of these parameters using time-resolved single-molecule fluorescence spectroscopy thus yields direct access to the protein dynamics. Applying this technique to the LH2 protein from Rhodovulum (Rdv.) sulfidophilum, the structure—and consequently the fluorescence properties—of which depends on pH, allowed us to follow a single protein, pH-induced, reversible, conformational transition. Hence, for the first time, to our knowledge, a protein transition can be visualized through changes in the electronic structure of the intrinsic cofactors, at a level of a single LH protein, which opens a new, to our knowledge, route for understanding the changes in energy landscape that underlie protein function and adaptation to the needs of living organisms. PMID:26039172

Maximizing the quantitative accuracy and reproducibility of Förster resonance energy transfer measurement for screening by high throughput widefield microscopy

PubMed Central

Schaufele, Fred

2013-01-01

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) provides insights into the proximities and orientations of FPs as surrogates of the biochemical interactions and structures of the factors to which the FPs are genetically fused. As powerful as FRET methods are, technical issues have impeded their broad adoption in the biologic sciences. One hurdle to accurate and reproducible FRET microscopy measurement stems from variable fluorescence backgrounds both within a field and between different fields. Those variations introduce errors into the precise quantification of fluorescence levels on which the quantitative accuracy of FRET measurement is highly dependent. This measurement error is particularly problematic for screening campaigns since minimal well-to-well variation is necessary to faithfully identify wells with altered values. High content screening depends also upon maximizing the numbers of cells imaged, which is best achieved by low magnification high throughput microscopy. But, low magnification introduces flat-field correction issues that degrade the accuracy of background correction to cause poor reproducibility in FRET measurement. For live cell imaging, fluorescence of cell culture media in the fluorescence collection channels for the FPs commonly used for FRET analysis is a high source of background error. These signal-to-noise problems are compounded by the desire to express proteins at biologically meaningful levels that may only be marginally above the strong fluorescence background. Here, techniques are presented that correct for background fluctuations. Accurate calculation of FRET is realized even from images in which a non-flat background is 10-fold higher than the signal. PMID:23927839

Self-assembly of BODIPY based pH-sensitive near-infrared polymeric micelles for drug controlled delivery and fluorescence imaging applications.

PubMed

Liu, Xiaodong; Chen, Bizheng; Li, Xiaojun; Zhang, Lifen; Xu, Yujie; Liu, Zhuang; Cheng, Zhenping; Zhu, Xiulin

2015-10-21

Responsive block copolymer micelles emerging as promising imaging and drug delivery systems show high stability and on-demand drug release activities. Herein, we developed self-assembled pH-responsive NIR emission micelles entrapped with doxorubicin (DOX) within the cores by the electrostatic interactions for fluorescence imaging and chemotherapy applications. The block copolymer, poly(methacrylic acid)-block-poly[(poly(ethylene glycol) methyl ether methacrylate)-co-boron dipyrromethene derivatives] (PMAA-b-P(PEGMA-co-BODIPY), was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the molecular weight distribution of this copolymer was narrow (Mw/Mn = 1.31). The NIR fluorescence enhancement induced by the phenol/phenolate interconversion equilibrium works as a switch in response to the intracellular pH fluctuations. DOX-loaded PMAA-b-P(PEGMA-co-BODIPY) micelles can detect the physiological pH fluctuations with a pKa near physiological conditions (∼7.52), and showed pH-responsive collapse and an obvious acid promoted anticancer drug release behavior (over 58.8-62.8% in 10 h). Real-time imaging of intracellular pH variations was performed and a significant chemotherapy effect was demonstrated against HeLa cells.

Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations

PubMed Central

Serag, Maged F.; Abadi, Maram; Habuchi, Satoshi

2014-01-01

Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields. PMID:25283876

Photoinduced changes in photosystem II pigments

NASA Astrophysics Data System (ADS)

Andreeva, Atanaska S.; Busheva, Mira C.; Stoitchkova, Katerina V.; Tzonova, Iren K.

2010-11-01

The photosynthetic apparatus in higher plants performs two seemingly opposing tasks: efficient harvest of sunlight, but also rapid and harmless dissipation of excess light energy as heat to avoid deleterious photodamage. In order to study this process in pigment-protein supercomplexes of photosystem II (PSII), 77 K fluorescence and room temperature resonance Raman (RR) spectroscopy were applied to investigate the changes in structure and spectral properties of the pigments in spinach PSII membranes. The high-light treatment results in a strong quenching of the fluorescence (being largest when the excitation is absorbed by carotenoids) and a red-shift of the main maximum. Decomposition of the fluorescence spectra into four bands revealed intensive quenching of F685 and F695 bands, possible bleaching of chlorophyll a, enhanced extent of light harvesting complexes (LHCII) aggregation and increased energy transfer to aggregated LHCII. The analysis of RR spectra revealed the predominant contribution of ß-carotene (ß-Car) upon 457.8 and 488 nm excitations and lutein (Lut) at 514.5 nm. During prolonged exposure to strong light no significant bleaching of ß-Car and weak photobleaching of Lut is observed. The results will contribute to the efforts to produce more efficient and robust solar cells when exposed to fluctuations in light intensity.

Motion estimation of subcellular structures from fluorescence microscopy images.

PubMed

Vallmitjana, A; Civera-Tregon, A; Hoenicka, J; Palau, F; Benitez, R

2017-07-01

We present an automatic image processing framework to study moving intracellular structures from live cell fluorescence microscopy. The system includes the identification of static and dynamic structures from time-lapse images using data clustering as well as the identification of the trajectory of moving objects with a probabilistic tracking algorithm. The method has been successfully applied to study mitochondrial movement in neurons. The approach provides excellent performance under different experimental conditions and is robust to common sources of noise including experimental, molecular and biological fluctuations.

Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

PubMed Central

Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek W.; Mai, Sabine

2017-01-01

Abstract Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions. PMID:28082388

Femtosecond pump/supercontinuum-probe spectroscopy: optimized setup and signal analysis for single-shot spectral referencing.

PubMed

Dobryakov, A L; Kovalenko, S A; Weigel, A; Pérez-Lustres, J L; Lange, J; Müller, A; Ernsting, N P

2010-11-01

A setup for pump/supercontinuum-probe spectroscopy is described which (i) is optimized to cancel fluctuations of the probe light by single-shot referencing, and (ii) extends the probe range into the near-uv (1000-270 nm). Reflective optics allow 50 μm spot size in the sample and upon entry into two separate spectrographs. The correlation γ(same) between sample and reference readings of probe light level at every pixel exceeds 0.99, compared to γ(consec)<0.92 reported for consecutive referencing. Statistical analysis provides the confidence interval of the induced optical density, ΔOD. For demonstration we first examine a dye (Hoechst 33258) bound in the minor groove of double-stranded DNA. A weak 1.1 ps spectral oscillation in the fluorescence region, assigned to DNA breathing, is shown to be significant. A second example concerns the weak vibrational structure around t=0 which reflects stimulated Raman processes. With 1% fluctuations of probe power, baseline noise for a transient absorption spectrum becomes 25 μOD rms in 1 s at 1 kHz, allowing to record resonance Raman spectra of flavine adenine dinucleotide in the S(0) and S(1) state.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation.

PubMed

Szczurek, Aleksander; Birk, Udo; Knecht, Hans; Dobrucki, Jurek; Mai, Sabine; Cremer, Christoph

2018-01-01

Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation

PubMed Central

Knecht, Hans; Dobrucki, Jurek; Mai, Sabine

2018-01-01

ABSTRACT Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine. PMID:29297245

Using Movies to Analyse Gene Circuit Dynamics in Single Cells

PubMed Central

Locke, James CW; Elowitz, Michael B

2010-01-01

Preface Many bacterial systems rely on dynamic genetic circuits to control critical processes. A major goal of systems biology is to understand these behaviours in terms of individual genes and their interactions. However, traditional techniques based on population averages wash out critical dynamics that are either unsynchronized between cells or driven by fluctuations, or ‘noise,’ in cellular components. Recently, the combination of time-lapse microscopy, quantitative image analysis, and fluorescent protein reporters has enabled direct observation of multiple cellular components over time in individual cells. In conjunction with mathematical modelling, these techniques are now providing powerful insights into genetic circuit behaviour in diverse microbial systems. PMID:19369953

Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murton, Jaclyn; Nagarajan, Aparna; Nguyen, Amelia Y.

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response tomore » nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. Lastly, we observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.« less

Probing short-range protein Brownian motion in the cytoplasm of living cells

PubMed Central

Di Rienzo, Carmine; Piazza, Vincenzo; Gratton, Enrico; Beltram, Fabio; Cardarelli, Francesco

2014-01-01

The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes. PMID:25532887

Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels

DOE PAGES

Murton, Jaclyn; Nagarajan, Aparna; Nguyen, Amelia Y.; ...

2017-07-21

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response tomore » nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. Lastly, we observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.« less

Mass action at the single-molecule level.

PubMed

Shon, Min Ju; Cohen, Adam E

2012-09-05

We developed a system to reversibly encapsulate small numbers of molecules in an array of nanofabricated "dimples". This system enables highly parallel, long-term, and attachment-free studies of molecular dynamics via single-molecule fluorescence. In studies of bimolecular reactions of small numbers of confined molecules, we see phenomena that, while expected from basic statistical mechanics, are not observed in bulk chemistry. Statistical fluctuations in the occupancy of sealed reaction chambers lead to steady-state fluctuations in reaction equilibria and rates. These phenomena are likely to be important whenever reactions happen in confined geometries.

The response of water quality variation in Poyang Lake (Jiangxi, People's Republic of China) to hydrological changes using historical data and DOM fluorescence.

PubMed

Yao, Xin; Wang, Shengrui; Ni, Zhaokui; Jiao, Lixin

2015-02-01

Poyang Lake is a unique wetland system that has evolved in response to natural seasonal fluctuations in water levels. To better characterize the response of water quality to hydrological variation, historical data were analyzed in combination with dissolved organic matter (DOM) fluorescence samplings conducted in situ. Historical data showed that long-term changes in water quality are mainly controlled by the sewage inputs to Poyang Lake. Monthly changes in water quality recorded during 2008 and 2012 suggest that water level may be the most important factor for water quality during a hydrological year. DOM fluorescence samples were identified as three humic-like components (C1, C2, and C3) and a protein-like component (C4). These obvious compositional changes in DOM fluorescence were considered to be related to the hydrodynamic differences controlled by water regimen. Principal component analysis (PCA) showed higher C1 and C2 signals during a normal season than the wet season, whereas C3 was lower, and C4 was higher in the dry season than in the wet or normal seasons. From the open lake to the Yangtze River mouth, increased C3 component carried by backflows of the Yangtze River to the lake resulted in these unique variations of PCA factor 2 scores during September. These obvious compositional changes in DOM fluorescence were considered to be related to the hydrodynamic differences controlled by water regimen. DOM fluorescence could be a proxy for capturing rapid changes in water quality and thereby provide an early warning signal for the quality of water supply.

Photoinduced electron transfer of oxazine 1/TiO2 nanoparticles at single molecule level by using confocal fluorescence microscopy.

PubMed

Chen, Yi-Ju; Tzeng, Hsin-Yu; Fan, Hsiu-Fang; Chen, Ming-Shiang; Huang, Jer-Shing; Lin, King-Chuen

2010-06-01

Kinetics of photoinduced electron transfer (ET) from oxazine 1 dye to TiO(2) nanoparticles (NPs) surface is studied at a single molecule level by using confocal fluorescence microscopy. Upon irradiation with a pulsed laser at 630 nm, the fluorescence lifetimes sampled among 100 different dye molecules are determined to yield an average lifetime of 2.9 +/- 0.3 ns, which is close to the value of 3.0 +/- 0.6 ns measured on the bare coverslip. The lifetime proximity suggests that most interfacial electron transfer (IFET) processes for the current system are inefficient, probably caused by physisorption between dye and the TiO(2) film. However, there might exist some molecules which are quenched before fluorescing and fail to be detected. With the aid of autocorrelation analysis under a three-level energy system, the IFET kinetics of single dye molecules in the conduction band of TiO(2) NPs is evaluated to be (1.0 +/- 0.1) x 10(4) s(-1) averaged over 100 single molecules and the back ET rate constant is 4.7 +/- 0.9 s(-1). When a thicker TiO(2) film is substituted, the resultant kinetic data do not make a significant difference. The trend of IFET efficacy agrees with the method of fluorescence lifetime measurements. The obtained forward ET rate constants are about ten times smaller than the photovoltage response measured in an assembled dye-sensitized solar cell. The discrepancy is discussed. The inhomogeneous and fluctuation characters for the IFET process are attributed to microenvironment variation for each single molecule. The obtained ET rates are much slower than the fluorescence relaxation. Such a small ET quantum yield is yet feasibly detectable at a single molecule level.

Fluorescence Fluctuations and Equivalence Classes of Ca 2+ Imaging Experiments

PubMed Central

Piegari, Estefanía; Lopez, Lucía; Perez Ipiña, Emiliano; Ponce Dawson, Silvina

2014-01-01

release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP3Rs) plays a relevant role in numerous physiological processes. IP3R-mediated signals involve -induced -release (CICR) whereby release through one open IP3R induces the opening of other channels. IP3Rs are apparently organized in clusters. The signals can remain localized (i.e., puffs) if CICR is limited to one cluster or become waves that propagate between clusters. puffs are the building blocks of waves. Thus, there is great interest in determining puff properties, especially in view of the current controversy on the spatial distribution of activatable IP3Rs. puffs have been observed in intact cells with optical techniques proving that they are intrinsically stochastic. Obtaining a correct picture of their dynamics then entails being able to detect the whole range of puff sizes. puffs are observed using visible single-wavelength dyes, slow exogenous buffers (e.g., EGTA) to disrupt inter-cluster CICR and UV-photolyzable caged IP3. Single-wavelength dyes increase their fluorescence upon calcium binding producing images that are strongly dependent on their kinetic, transport and photophysical properties. Determining the artifacts that the imaging setting introduces is particularly relevant when trying to analyze the smallest signals. In this paper we introduce a method to estimate the expected signal-to-noise ratio of imaging experiments that use single-wavelength dyes. The method is based on the Number and Brightness technique. It involves the performance of a series of experiments and their subsequent analysis in terms of a fluorescence fluctuation model with which the model parameters are quantified. Using the model, the expected signal-to-noise ratio is then computed. Equivalence classes between different experimental conditions that produce images with similar signal-to-noise ratios can then be established. The method may also be used to estimate the smallest signals that can reliably be observed with each setting. PMID:24776736

Optical spectroscopy of the bladder washout fluid to optimize fluorescence cystoscopy with Hexvix®.

PubMed

Martoccia, Carla; Zellweger, Matthieu; Lovisa, Blaise; Jichlinski, Patrice; van den Bergh, Hubert; Wagnières, Georges

2014-09-01

Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate thebladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degradesfluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studiedtheir fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C).A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320∕420 nm, FWHM =50∕100 nm) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455∕525 nm, FWHM = 80∕50 nm). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine’s main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370–430 nm to 395–415 nm would reduce the BWF background by a factor 2.

TLM-Quant: an open-source pipeline for visualization and quantification of gene expression heterogeneity in growing microbial cells.

PubMed

Piersma, Sjouke; Denham, Emma L; Drulhe, Samuel; Tonk, Rudi H J; Schwikowski, Benno; van Dijl, Jan Maarten

2013-01-01

Gene expression heterogeneity is a key driver for microbial adaptation to fluctuating environmental conditions, cell differentiation and the evolution of species. This phenomenon has therefore enormous implications, not only for life in general, but also for biotechnological applications where unwanted subpopulations of non-producing cells can emerge in large-scale fermentations. Only time-lapse fluorescence microscopy allows real-time measurements of gene expression heterogeneity. A major limitation in the analysis of time-lapse microscopy data is the lack of fast, cost-effective, open, simple and adaptable protocols. Here we describe TLM-Quant, a semi-automatic pipeline for the analysis of time-lapse fluorescence microscopy data that enables the user to visualize and quantify gene expression heterogeneity. Importantly, our pipeline builds on the open-source packages ImageJ and R. To validate TLM-Quant, we selected three possible scenarios, namely homogeneous expression, highly 'noisy' heterogeneous expression, and bistable heterogeneous expression in the Gram-positive bacterium Bacillus subtilis. This bacterium is both a paradigm for systems-level studies on gene expression and a highly appreciated biotechnological 'cell factory'. We conclude that the temporal resolution of such analyses with TLM-Quant is only limited by the numbers of recorded images.

The application of the HyPer fluorescent sensor in the real-time detection of H2O2 in Babesia bovis merozoites in vitro.

PubMed

Asada, Masahito; Hakimi, Hassan; Kawazu, Shin-Ichiro

2018-05-15

In recent years, genetically encoded fluorescent probes have allowed a dramatic advancement in time-lapse imaging, enabling this imaging modality to be used to investigate intracellular events in several apicomplexan parasite species. In this study, we constructed a plasmid vector to stably express a genetically encoded H 2 O 2 sensor probe called HyPer in Babesia bovis. The HyPer-transfected parasite population was successfully developed and subjected to a time-lapse imaging analysis under in vitro culture conditions. HyPer was capable of sensing an increasing H 2 O 2 concentration in the parasite cells which was induced by the administration of paraquat as a superoxide donor. HyPer fluorescence co-staining with MitoTracker Red indicated the mitochondria as the major source of reactive oxygen species (ROS) in parasite cells. The fluctuating ROS dynamics in the parasite gliding toward, attaching to, and invading the target red blood cell was visualized and monitored in real time with the HyPer expressing parasite population. This is the first report to describe the application of the HyPer probe in an imaging analysis involving Babesia parasites. Hyper-expressing parasites can be widely utilized in studies to investigate the mechanisms of emergence and the reduction of oxidative stress, as well as the signal transduction in the parasite cells during host invasion and intercellular development. Copyright © 2018 Elsevier B.V. All rights reserved.

Enhanced emission of fluorophores on shrink-induced wrinkled composite structures.

PubMed

Sharma, Himanshu; Digman, Michelle A; Felsinger, Natasha; Gratton, Enrico; Khine, Michelle

2014-01-01

We introduce a manufacturable and scalable method for creating tunable wrinkled ferromagnetic-metallic structures to enhance fluorescence signals. Thin layers of nickel (Ni) and gold (Au) were deposited onto a pre-stressed thermoplastic (shrink wrap film) polymer. Heating briefly forced the metal films to buckle when the thermoplastic retracted, resulting in multi-scale composite 'wrinkles'. This is the first demonstration of leveraging the plasmons in such hybrid nanostructures by metal enhanced fluorescence (MEF) in the near-infrared wavelengths. We observed more than three orders of magnitude enhancement in the fluorescence signal of a single molecule of goat anti-mouse immunoglobulin G (IgG) antibody conjugated to fluorescein isothiocyanate, FITC, (FITC-IgG) by two-photon excitation with these structures. These large enhancements in the fluorescence signal at the nanoscale gaps between the composite wrinkles corresponded to shortened lifetimes due to localized surface plasmons. To characterize these structures, we combined fluctuation correlation spectroscopy (FCS), fluorescence lifetime imaging microscopy (FLIM), and two-photon microscopy to spatially and temporally map the hot spots with high resolution.

Optimization of fluorescent imaging in the operating room through pulsed acquisition and gating to ambient background cycling

PubMed Central

Sexton, Kristian J.; Zhao, Yan; Davis, Scott C.; Jiang, Shudong; Pogue, Brian W.

2017-01-01

The design of fluorescence imaging instruments for surgical guidance is rapidly evolving, and a key issue is to efficiently capture signals with high ambient room lighting. Here, we introduce a novel time-gated approach to fluorescence imaging synchronizing acquisition to the 120 Hz light of the room, with pulsed LED excitation and gated ICCD detection. It is shown that under bright ambient room light this technique allows for the detection of physiologically relevant nanomolar fluorophore concentrations, and in particular reduces the light fluctuations present from the room lights, making low concentration measurements more reliable. This is particularly relevant for the light bands near 700nm that are more dominated by ambient lights. PMID:28663895

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.

Model Lipid Membranes on a Tunable Polymer Cushion

NASA Astrophysics Data System (ADS)

Smith, Hillary L.; Jablin, Michael S.; Vidyasagar, Ajay; Saiz, Jessica; Watkins, Erik; Toomey, Ryan; Hurd, Alan J.; Majewski, Jaroslaw

2009-06-01

A hydrated, surface-tethered polymer network capable of fivefold change in thickness over a 25-37°C temperature range has been demonstrated via neutron reflectivity and fluorescence microscopy to be a novel support for single lipid bilayers in a liquid environment. As the polymer swells from 170 to 900 Å, it promotes both in- and out-of-plane fluctuations of the supported membrane. The cushioned bilayer proved to be very robust, remaining structurally intact for 16 days and many temperature cycles. The promotion of membrane fluctuations offers far-reaching applications for this system as a surrogate biomembrane.

Fluorescence correlation spectroscopy: novel variations of an established technique.

PubMed

Haustein, Elke; Schwille, Petra

2007-01-01

Fluorescence correlation spectroscopy (FCS) is one of the major biophysical techniques used for unraveling molecular interactions in vitro and in vivo. It allows minimally invasive study of dynamic processes in biological specimens with extremely high temporal and spatial resolution. By recording and correlating the fluorescence fluctuations of single labeled molecules through the exciting laser beam, FCS gives information on molecular mobility and photophysical and photochemical reactions. By using dual-color fluorescence cross-correlation, highly specific binding studies can be performed. These have been extended to four reaction partners accessible by multicolor applications. Alternative detection schemes shift accessible time frames to slower processes (e.g., scanning FCS) or higher concentrations (e.g., TIR-FCS). Despite its long tradition, FCS is by no means dated. Rather, it has proven to be a highly versatile technique that can easily be adapted to solve specific biological questions, and it continues to find exciting applications in biology and medicine.

Detection of ultra-low oxygen concentration based on the fluorescence blinking dynamics of single molecules

NASA Astrophysics Data System (ADS)

Wu, Ruixiang; Chen, Ruiyun; Zhou, Haitao; Qin, Yaqiang; Zhang, Guofeng; Qin, Chengbing; Gao, Yan; Gao, Yajun; Xiao, Liantuan; Jia, Suotang

2018-01-01

We present a sensitive method for detection of ultra-low oxygen concentrations based on the fluorescence blinking dynamics of single molecules. The relationship between the oxygen concentration and the fraction of time spent in the off-state, stemming from the population and depopulation of triplet states and radical cationic states, can be fitted with a two-site quenching model in the Stern-Volmer plot. The oxygen sensitivity is up to 43.42 kPa-1 in the oxygen partial pressure region as low as 0.01-0.25 kPa, which is seven times higher than that of the fluorescence intensity indicator. This method avoids the limitation of the sharp and non-ignorable fluctuations that occur during the measurement of fluorescence intensity, providing potential applications in the field of low oxygen-concentration monitoring in life science and industry.

Dynamic Fluctuations of Protein-Carbohydrate Interactions Promote Protein Aggregation

PubMed Central

Voynov, Vladimir; Chennamsetty, Naresh; Kayser, Veysel; Helk, Bernhard; Forrer, Kurt; Zhang, Heidi; Fritsch, Cornelius; Heine, Holger; Trout, Bernhardt L.

2009-01-01

Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation. PMID:20037630

Spatial fluctuations in expression of the heterocyst differentiation regulatory gene hetR in Anabaena filaments.

PubMed

Corrales-Guerrero, Laura; Tal, Asaf; Arbel-Goren, Rinat; Mariscal, Vicente; Flores, Enrique; Herrero, Antonia; Stavans, Joel

2015-04-01

Under nitrogen deprivation, filaments of the cyanobacterium Anabaena undergo a process of development, resulting in a one-dimensional pattern of nitrogen-fixing heterocysts separated by about ten photosynthetic vegetative cells. Many aspects of gene expression before nitrogen deprivation and during the developmental process remain to be elucidated. Furthermore, the coupling of gene expression fluctuations between cells along a multicellular filament is unknown. We studied the statistics of fluctuations of gene expression of HetR, a transcription factor essential for heterocyst differentiation, both under steady-state growth in nitrogen-rich conditions and at different times following nitrogen deprivation, using a chromosomally-encoded translational hetR-gfp fusion. Statistical analysis of fluorescence at the individual cell level in wild-type and mutant filaments demonstrates that expression fluctuations of hetR in nearby cells are coupled, with a characteristic spatial range of circa two to three cells, setting the scale for cellular interactions along a filament. Correlations between cells predominantly arise from intercellular molecular transfer and less from cell division. Fluctuations after nitrogen step-down can build up on those under nitrogen-replete conditions. We found that under nitrogen-rich conditions, basal, steady-state expression of the HetR inhibitor PatS, cell-cell communication influenced by the septal protein SepJ and positive HetR auto-regulation are essential determinants of fluctuations in hetR expression and its distribution along filaments. A comparison between the expression of hetR-gfp under nitrogen-rich and nitrogen-poor conditions highlights the differences between the two HetR inhibitors PatS and HetN, as well as the differences in specificity between the septal proteins SepJ and FraC/FraD. Activation, inhibition and cell-cell communication lie at the heart of developmental processes. Our results show that proteins involved in these basic ingredients combine together in the presence of inevitable stochasticity in gene expression, to control the coupled fluctuations of gene expression that give rise to a one-dimensional developmental pattern in this organism.

Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

PubMed Central

Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

2015-01-01

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

NASA Astrophysics Data System (ADS)

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-01

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy.

PubMed

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-09

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Measurements of density, temperature, and their fluctuations in turbulent supersonic flow using UV laser spectroscopy

NASA Technical Reports Server (NTRS)

Fletcher, Douglas G.; Mckenzie, R. L.

1992-01-01

Nonintrusive measurements of density, temperature, and their turbulent fluctuation levels were obtained in the boundary layer of an unseeded, Mach 2 wind tunnel flow. The spectroscopic technique that was used to make the measurements is based on the combination of laser-induced oxygen fluorescence and Raman scattering by oxygen and nitrogen from the same laser pulse. Results from this demonstration experiment are compared with previous measurements obtained in the same facility using conventional probes and an earlier spectroscopic technique. Densities and temperatures measured with the current technique agree with the previous surveys to within 3 percent and 2 percent, respectively. The fluctuation amplitudes for both variables agree with the measurements obtained using the earlier spectroscopic technique and show evidence of an unsteady, weak shock wave that perturbs the boundary layer.

FPGA based demodulation of laser induced fluorescence in plasmas

NASA Astrophysics Data System (ADS)

Mattingly, Sean W.; Skiff, Fred

2018-04-01

We present a field programmable gate array (FPGA)-based system that counts photons from laser-induced fluorescence (LIF) on a laboratory plasma. This is accomplished with FPGA-based up/down counters that demodulate the data, giving a background-subtracted LIF signal stream that is updated with a new point as each laser amplitude modulation cycle completes. We demonstrate using the FPGA to modulate a laser at 1 MHz and demodulate the resulting LIF data stream. This data stream is used to calculate an LIF-based measurement sampled at 1 MHz of a plasma ion fluctuation spectrum.

Oil-Water Flow Investigations using Planar-Laser Induced Fluorescence and Particle Velocimetry

NASA Astrophysics Data System (ADS)

Ibarra, Roberto; Matar, Omar K.; Markides, Christos N.

2017-11-01

The study of the complex behaviour of immiscible liquid-liquid flow in pipes requires the implementation of advanced measurement techniques in order to extract detailed in situ information. Laser-based diagnostic techniques allow the extraction of high-resolution space- and time resolve phase and velocity information, which aims to improve the fundamental understanding of these flows and to validate closure relations for advanced multiphase flow models. This work shows a novel simultaneous planar-laser induced fluorescence and particle velocimetry in stratified oil-water flows using two laser light sheets at two different wavelengths for fluids with different refractive indices at horizontal and upward pipe inclinations (<5°) in stratified flow conditions (i.e. separated layers). Complex flow structures are extracted from 2-D instantaneous velocity fields, which are strongly dependent on the pipe inclination at low velocities. The analysis of mean wall-normal velocity profiles and velocity fluctuations suggests the presence of single- and counter-rotating vortices in the azimuthal direction, especially in the oil layer, which can be attributed to the influence of the interfacial waves. Funding from BP, and the TMF Consortium is gratefully acknowledged.

Fluorescence Correlation Spectroscopy and Nonlinear Stochastic Reaction-Diffusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 closedmore » 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.« less

TLM-Quant: An Open-Source Pipeline for Visualization and Quantification of Gene Expression Heterogeneity in Growing Microbial Cells

PubMed Central

Piersma, Sjouke; Denham, Emma L.; Drulhe, Samuel; Tonk, Rudi H. J.; Schwikowski, Benno; van Dijl, Jan Maarten

2013-01-01

Gene expression heterogeneity is a key driver for microbial adaptation to fluctuating environmental conditions, cell differentiation and the evolution of species. This phenomenon has therefore enormous implications, not only for life in general, but also for biotechnological applications where unwanted subpopulations of non-producing cells can emerge in large-scale fermentations. Only time-lapse fluorescence microscopy allows real-time measurements of gene expression heterogeneity. A major limitation in the analysis of time-lapse microscopy data is the lack of fast, cost-effective, open, simple and adaptable protocols. Here we describe TLM-Quant, a semi-automatic pipeline for the analysis of time-lapse fluorescence microscopy data that enables the user to visualize and quantify gene expression heterogeneity. Importantly, our pipeline builds on the open-source packages ImageJ and R. To validate TLM-Quant, we selected three possible scenarios, namely homogeneous expression, highly ‘noisy’ heterogeneous expression, and bistable heterogeneous expression in the Gram-positive bacterium Bacillus subtilis. This bacterium is both a paradigm for systems-level studies on gene expression and a highly appreciated biotechnological ‘cell factory’. We conclude that the temporal resolution of such analyses with TLM-Quant is only limited by the numbers of recorded images. PMID:23874729

ESC-Track: A computer workflow for 4-D segmentation, tracking, lineage tracing and dynamic context analysis of ESCs.

PubMed

Fernández-de-Manúel, Laura; Díaz-Díaz, Covadonga; Jiménez-Carretero, Daniel; Torres, Miguel; Montoya, María C

2017-05-01

Embryonic stem cells (ESCs) can be established as permanent cell lines, and their potential to differentiate into adult tissues has led to widespread use for studying the mechanisms and dynamics of stem cell differentiation and exploring strategies for tissue repair. Imaging live ESCs during development is now feasible due to advances in optical imaging and engineering of genetically encoded fluorescent reporters; however, a major limitation is the low spatio-temporal resolution of long-term 3-D imaging required for generational and neighboring reconstructions. Here, we present the ESC-Track (ESC-T) workflow, which includes an automated cell and nuclear segmentation and tracking tool for 4-D (3-D + time) confocal image data sets as well as a manual editing tool for visual inspection and error correction. ESC-T automatically identifies cell divisions and membrane contacts for lineage tree and neighborhood reconstruction and computes quantitative features from individual cell entities, enabling analysis of fluorescence signal dynamics and tracking of cell morphology and motion. We use ESC-T to examine Myc intensity fluctuations in the context of mouse ESC (mESC) lineage and neighborhood relationships. ESC-T is a powerful tool for evaluation of the genealogical and microenvironmental cues that maintain ESC fitness.

Mitochondrial fluctuations as a measure of active biomechanical properties of mammalian cells

NASA Astrophysics Data System (ADS)

Xu, Wenlong; Alizadeh, Elaheh; Castle, Jordan; Prasad, Ashok

A single-cell assay of mechanical properties would give significant insights into cellular processes. Force spectrum microscopy is one such technique, which involves both active and passive particle tracking microrheology on the same cells. Since active microrheology requires expensive instruments, it is of great interest to develop simpler alternatives. Here we study an alternative using endogenous mitochondrial fluctuations, rather than fluorescent beads, in particle tracking microrheology. Mitochondria of the C3H-10T1/2 cell line are labeled and tracked using confocal microscopy, their mean square displacement (MSD) measured, and mechanical parameters calculated. Active fluctuations are distinguished from passive fluctuations by treatment with ATP synthesis inhibitors. We find that the MSD of mitochondria resembles that of particles in viscoelastic media. However, comparisons of MSD between controls and cells disrupted in the actin or microtubule network showed surprisingly small effects, while ATP-depleted cells showed significantly decreased MSD, and characteristics of thermally driven fluctuations. Both active and ATP-depleted parameters showed heterogeneity among cells and between cell lines. This method is potentially very useful due to its simplicity. We gratefully acknowledge support from NSF CAREER Grant PHY-1151454 awarded to Ashok Prasad.

[Three-dimensional Fluorescence Spectral Characteristics of Different Molecular Weight Fractionations of Dissolved Organic Matter in the Water-level Fluctuation Zones of Three Gorges Reservoir Areas].

PubMed

Chen, Xue-shuang; Jiang, Tao; Lu, Song; Wei, Shi-qiang; Wang, Ding-yong; Yan, Jin-long

2016-03-15

The study of the molecular weight (MW) fractions of dissolved organic matter (DOM) in aquatic environment is of interests because the size plays an important role in deciding the biogeochemical characteristics of DOM. Thus, using ultrafiltration ( UF) technique combined with three-dimensional fluorescence spectroscopy, DOM samples from four sampling sites in typical water-level fluctuation zones of Three Gorge Reservoir areas were selected to investigate the differences of properties and sources of different DOM MW fractions. The results showed that in these areas, the distribution of MW fractions was highly dispersive, but the approximately equal contributions from colloidal (Mr 1 x 10³-0.22 µm) and true dissolved fraction (Mr < 1 x 10³) to the total DOC concentration were found. Four fluorescence signals (humic-like A and C; protein-like B and T) were observed in all MW fractions including bulk DOM, which showed the same distribution trend: true dissolved > low MW (Mr 1 x 10³-10 x 10³) > medium MW (Mr 10 x 10³-30 x 10³) > high MW (Mr 30 x 10³-0.22 µm). Additionally, with decreasing MW fraction, fluorescence index (FI) and freshness index (BIX) increased suggesting enhanced signals of autochthonous inputs, whereas humification index ( HIX) decreased indicating lowe humification degree. It strongly suggested that terrestrial input mainly affected the composition and property of higher MW fractions of DOM, as compared to lower MW and true dissolved fractions that were controlled by autochthonous sources such as microbial and alga activities, instead of allochthonous sources. Meanwhile, the riparian different land-use types also affected obviously on the characteristics of DOM. Therefore, higher diversity of land-use types, and also higher complexity of ecosystem and landscapes, induced higher heterogeneity of fluorescence components in different MW fraction of DOM.

Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors.

PubMed

Lukeš, Tomáš; Pospíšil, Jakub; Fliegel, Karel; Lasser, Theo; Hagen, Guy M

2018-03-01

Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared with organic dyes, which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented, including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM datasets using a different method: super-resolution optical fluctuation imaging (SOFI). The 2 modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. This dataset has potential for extensive reuse. Complete raw data from SMLM experiments have typically not been published. The YFP data exhibit low signal-to-noise ratios, making data analysis a challenge. These datasets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

Composition of dissolved organic matter (DOM) from periodically submerged soils in the Three Gorges Reservoir areas as determined by elemental and optical analysis, infrared spectroscopy, pyrolysis-GC-MS and thermally assisted hydrolysis and methylation.

PubMed

Jiang, Tao; Kaal, Joeri; Liang, Jian; Zhang, Yaoling; Wei, Shiqiang; Wang, Dingyong; Green, Nelson W

2017-12-15

Soil-derived dissolved organic matter (DOM) has a major influence in biogeochemical processes related to contaminant dynamics and greenhouse gas emissions, due to its reactivity and its bridging role between the soil and aquatic systems. Within the Three Gorges Reservoir (TGR, China) area, an extensive water-fluctuation zone periodically submerges the surrounding soils. Here we report a characterization study of soil-derived DOM across the TGR areas, using elemental and optical analysis, infrared spectroscopy (FTIR), pyrolysis-GC-MS (Py-GC-MS) and thermally assisted hydrolysis and methylation (THM-GC-MS). The results showed that the soil DOM from the TGR area is a mixture of "allochthonous" (i.e., plant-derived/terrigenous) and "autochthonous" (i.e., microbial) origins. The terrigenous DOM is composed primarily of phenolic and aliphatic structures from lignin and aliphatic biopolymers (i.e. cutin, suberin), respectively. Multivariate statistics differentiated between two fractions of the microbial DOM, i.e. chitin-derived, perhaps from fungi and arthropods in soil, and protein-derived, partially sourced from algal or aquatic organisms. Molecular proxies of source and degradation state were in good agreement with optical parameters such as SUVA 254 , the fluorescence index (FI) and the humification index (HIX). The combined use of elemental analysis, fluorescence spectroscopy, and Py-GC-MS provides rigorous and detailed DOM characterization, whereas THM-GC-MS is useful for more precise but qualitative identification of the different phenolic (cinnamyl, p-hydroxyphenyl, guaiacyl, syringyl and tannin-derived) and aliphatic materials. With the multi-methodological approach used in this study, FTIR was the least informative, in part, because of the interference of inorganic matter in the soil DOM samples. The soil DOM from the TGR's water fluctuation zone exhibited considerable compositional diversity, mainly related to the balance between DOM source (microbial- or plant-derived), local vegetation and anthropogenic activities (e.g., agriculture). Finally, the relationship between DOM composition and its potential reactivity with substances of environmental concerns in the TGR area are also discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel acidic pH fluorescent probe based on a benzothiazole derivative

NASA Astrophysics Data System (ADS)

Ma, Qiujuan; Li, Xian; Feng, Suxiang; Liang, Beibei; Zhou, Tiqiang; Xu, Min; Ma, Zhuoyi

2017-04-01

A novel acidic pH fluorescent probe 1 based on a benzothiazole derivative has been designed, synthesized and developed. The linear response range covers the acidic pH range from 3.44 to 6.46, which is valuable for pH researches in acidic environment. The evaluated pKa value of the probe 1 is 4.23. The fluorescence enhancement of the studied probe 1 with an increase in hydrogen ions concentration is based on the hindering of enhanced photo-induced electron transfer (PET) process. Moreover, the pH sensor possesses a highly selective response to H+ in the presence of metal ions, anions and other bioactive small molecules which would be interfere with its fluorescent pH response. Furthermore, the probe 1 responds to acidic pH with short response time that was less than 1 min. The probe 1 has been successfully applied to confocal fluorescence imaging in live HeLa cells and can selectively stain lysosomes. All of such good properties prove it can be used to monitoring pH fluctuations in acidic environment with high sensitivity, pH dependence and short response time.

Enhanced emission of fluorophores on shrink-induced wrinkled composite structures

PubMed Central

Sharma, Himanshu; Digman, Michelle A.; Felsinger, Natasha; Gratton, Enrico

2014-01-01

We introduce a manufacturable and scalable method for creating tunable wrinkled ferromagnetic-metallic structures to enhance fluorescence signals. Thin layers of nickel (Ni) and gold (Au) were deposited onto a pre-stressed thermoplastic (shrink wrap film) polymer. Heating briefly forced the metal films to buckle when the thermoplastic retracted, resulting in multi-scale composite ‘wrinkles’. This is the first demonstration of leveraging the plasmons in such hybrid nanostructures by metal enhanced fluorescence (MEF) in the near-infrared wavelengths. We observed more than three orders of magnitude enhancement in the fluorescence signal of a single molecule of goat anti-mouse immunoglobulin G (IgG) antibody conjugated to fluorescein isothiocyanate, FITC, (FITC-IgG) by two-photon excitation with these structures. These large enhancements in the fluorescence signal at the nanoscale gaps between the composite wrinkles corresponded to shortened lifetimes due to localized surface plasmons. To characterize these structures, we combined fluctuation correlation spectroscopy (FCS), fluorescence lifetime imaging microscopy (FLIM), and two-photon microscopy to spatially and temporally map the hot spots with high resolution. PMID:25383253

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions.

PubMed

Zheng, Haocheng; Goldner, Lori S; Leuba, Sanford H

2007-03-01

Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

Getting back to nature: a reality check for experiments in controlled environments.

PubMed

Annunziata, Maria Grazia; Apelt, Federico; Carillo, Petronia; Krause, Ursula; Feil, Regina; Mengin, Virginie; Lauxmann, Martin A; Köhl, Karin; Nikoloski, Zoran; Stitt, Mark; Lunn, John E; Raines, Christine

2017-07-20

Irradiance from sunlight changes in a sinusoidal manner during the day, with irregular fluctuations due to clouds, and light-dark shifts at dawn and dusk are gradual. Experiments in controlled environments typically expose plants to constant irradiance during the day and abrupt light-dark transitions. To compare the effects on metabolism of sunlight versus artificial light regimes, Arabidopsis thaliana plants were grown in a naturally illuminated greenhouse around the vernal equinox, and in controlled environment chambers with a 12-h photoperiod and either constant or sinusoidal light profiles, using either white fluorescent tubes or light-emitting diodes (LEDs) tuned to a sunlight-like spectrum as the light source. Rosettes were sampled throughout a 24-h diurnal cycle for metabolite analysis. The diurnal metabolite profiles revealed that carbon and nitrogen metabolism differed significantly between sunlight and artificial light conditions. The variability of sunlight within and between days could be a factor underlying these differences. Pairwise comparisons of the artificial light sources (fluorescent versus LED) or the light profiles (constant versus sinusoidal) showed much smaller differences. The data indicate that energy-efficient LED lighting is an acceptable alternative to fluorescent lights, but results obtained from plants grown with either type of artificial lighting might not be representative of natural conditions. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Inhomogeneity in the excited-state torsional disorder of a conjugated macrocycle.

PubMed

Yang, Jaesung; Ham, Sujin; Kim, Tae-Woo; Park, Kyu Hyung; Nakao, Kazumi; Shimizu, Hideyuki; Iyoda, Masahiko; Kim, Dongho

2015-03-12

The photophysics of conjugated polymers has generally been explained based on the interactions between the component conjugated chromophores in a tangled chain. However, conjugated chromophores are entities with static and dynamic structural disorder, which directly affects the conjugated polymer photophysics. Here we demonstrate the impact of chain structure torsional disorder on the spectral characteristics for a macrocyclic oligothiophene 1, which is obscured in conventional linear conjugated chromophores by diverse structural disorders such as those in chromophore size and shape. We used simultaneous multiple fluorescence parameter measurement for a single molecule and quantum-mechanical calculations to show that within the fixed conjugation length across the entire ring an inhomogeneity from torsional disorder in the structure of 1 plays a crucial role in causing its energetic disorder, which affords the spectral broadening of ∼220 meV. The torsional disorder in 1 fluctuated on the time scale of hundreds of milliseconds, typically accompanied by spectral drifts on the order of ∼10 meV. The fluctuations could generate torsional defects and change the electronic structure of 1 associated with the ring symmetry. These findings disclose the fundamental nature of conjugated chromophore that is the most elementary spectroscopic unit in conjugated polymers and suggest the importance of engineering structural disorder to optimize polymer-based device photophysics. Additionally, we combined defocused wide-field fluorescence microscopy and linear dichroism obtained from the simultaneous measurements to show that 1 emits polarized light with a changing polarization direction based on the torsional disorder fluctuations.

Direct fluorescence characterisation of a picosecond seeded optical parametric amplifier

NASA Astrophysics Data System (ADS)

Stuart, N. H.; Bigourd, D.; Hill, R. W.; Robinson, T. S.; Mecseki, K.; Patankar, S.; New, G. H. C.; Smith, R. A.

2015-02-01

The temporal intensity contrast of high-power lasers based on optical parametric amplification (OPA) can be limited by parametric fluorescence from the non-linear gain stages. Here we present a spectroscopic method for direct measurement of unwanted parametric fluorescence widely applicable from unseeded to fully seeded and saturated OPA operation. Our technique employs simultaneous spectroscopy of fluorescence photons slightly outside the seed bandwidth and strongly attenuated light at the seed central wavelength. To demonstrate its applicability we have characterised the performance of a two-stage picosecond OPA pre-amplifier with 2.8×105 gain, delivering 335 μJ pulses at 1054 nm. We show that fluorescence from a strongly seeded OPA is reduced by ~500× from the undepleted to full pump depletion regimes. We also determine the vacuum fluctuation driven noise term seeding this OPA fluorescence to be 0.7±0.4 photons ps-1 nm-1 bandwidth. The resulting shot-to-shot statistics highlights a 1.5% probability of a five-fold and 0.3% probability of a ten-fold increase of fluorescence above the average value. Finally, we show that OPA fluorescence can be limited to a few-ps pedestal with 3×10-9 temporal intensity contrast 1.3 ps ahead of an intense laser pulse, a level highly attractive for large scale chirped-pulse OPA laser systems.

Fluorescent lighting with aluminum nitride phosphors

DOEpatents

Cherepy, Nerine J.; Payne, Stephen A.; Seeley, Zachary M.; Srivastava, Alok M.

2016-05-10

A fluorescent lamp includes a glass envelope; at least two electrodes connected to the glass envelope; mercury vapor and an inert gas within the glass envelope; and a phosphor within the glass envelope, wherein the phosphor blend includes aluminum nitride. The phosphor may be a wurtzite (hexagonal) crystalline structure Al.sub.(1-x)M.sub.xN phosphor, where M may be drawn from beryllium, magnesium, calcium, strontium, barium, zinc, scandium, yttrium, lanthanum, cerium, praseodymium, europium, gadolinium, terbium, ytterbium, bismuth, manganese, silicon, germanium, tin, boron, or gallium is synthesized to include dopants to control its luminescence under ultraviolet excitation. The disclosed Al.sub.(1-x)M.sub.xN:Mn phosphor provides bright orange-red emission, comparable in efficiency and spectrum to that of the standard orange-red phosphor used in fluorescent lighting, Y.sub.2O.sub.3:Eu. Furthermore, it offers excellent lumen maintenance in a fluorescent lamp, and does not utilize "critical rare earths," minimizing sensitivity to fluctuating market prices for the rare earth elements.

State-dependent fluorescence of neutral atoms in optical potentials

NASA Astrophysics Data System (ADS)

Martinez-Dorantes, M.; Alt, W.; Gallego, J.; Ghosh, S.; Ratschbacher, L.; Meschede, D.

2018-02-01

Recently we have demonstrated scalable, nondestructive, and high-fidelity detection of the internal state of 87Rb neutral atoms in optical dipole traps using state-dependent fluorescence imaging [M. Martinez-Dorantes, W. Alt, J. Gallego, S. Ghosh, L. Ratschbacher, Y. Völzke, and D. Meschede, Phys. Rev. Lett. 119, 180503 (2017), 10.1103/PhysRevLett.119.180503]. In this paper we provide experimental procedures and interpretations to overcome the detrimental effects of heating-induced trap losses and state leakage. We present models for the dynamics of optically trapped atoms during state-dependent fluorescence imaging and verify our results by comparing Monte Carlo simulations with experimental data. Our systematic study of dipole force fluctuations heating in optical traps during near-resonant illumination shows that off-resonant light is preferable for state detection in tightly confining optical potentials.

Effect of probe diffusion on the SOFI imaging accuracy.

PubMed

Vandenberg, Wim; Dedecker, Peter

2017-03-23

Live-cell super-resolution fluorescence imaging is becoming commonplace for exploring biological systems, though sample dynamics can affect the imaging quality. In this work we evaluate the effect of probe diffusion on super-resolution optical fluctuation imaging (SOFI), using a theoretical model and numerical simulations based on the imaging of live cells labelled with photochromic fluorescent proteins. We find that, over a range of physiological conditions, fluorophore diffusion results in a change in the amplitude of the SOFI signal. The magnitude of this change is approximately proportional to the on-time ratio of the fluorophores. However, for photochromic fluorescent proteins this effect is unlikely to present a significant distortion in practical experiments in biological systems. Due to this lack of distortions, probe diffusion strongly enhances the SOFI imaging by avoiding spatial undersampling caused by the limited labeling density.

In vivo sodium concentration continuously monitored with fluorescent sensors.

PubMed

Dubach, J Matthew; Lim, Edward; Zhang, Ning; Francis, Kevin P; Clark, Heather

2011-02-01

Sodium balance is vital to maintaining normal physiological function. Imbalances can occur in a variety of diseases, during certain surgical operations or during rigorous exercise. There is currently no method to continuously monitor sodium concentration in patients who may be susceptible to hyponatremia. Our approach was to design sodium specific fluorescent sensors capable of measuring physiological fluctuations in sodium concentration. The sensors are submicron plasticized polymer particles containing sodium recognition components that are coated with biocompatible poly(ethylene) glycol. Here, the sensors were brought up in saline and placed in the subcutaneous area of the skin of mice by simple injection. The fluorescence was monitored in real time using a whole animal imager to track changes in sodium concentrations. This technology could be used to monitor certain disease states or warn against dangerously low levels of sodium during exercise.

Resonance energy transfer process in nanogap-based dual-color random lasing

NASA Astrophysics Data System (ADS)

Shi, Xiaoyu; Tong, Junhua; Liu, Dahe; Wang, Zhaona

2017-04-01

The resonance energy transfer (RET) process between Rhodamine 6G and oxazine in the nanogap-based random systems is systematically studied by revealing the variations and fluctuations of RET coefficients with pump power density. Three working regions stable fluorescence, dynamic laser, and stable laser are thus demonstrated in the dual-color random systems. The stable RET coefficients in fluorescence and lasing regions are generally different and greatly dependent on the donor concentration and the donor-acceptor ratio. These results may provide a way to reveal the energy distribution regulars in the random system and to design the tunable multi-color coherent random lasers for colorful imaging.

Nonintrusive Temperature and Velocity Measurements in a Hypersonic Nozzle Flow

NASA Technical Reports Server (NTRS)

OByrne, S.; Danehy, P. M.; Houwing, A. F. P.

2002-01-01

Distributions of nitric oxide vibrational temperature, rotational temperature and velocity have been measured in the hypersonic freestream at the exit of a conical nozzle, using planar laser-induced fluorescence. Particular attention has been devoted to reducing the major sources of systematic error that can affect fluorescence tempera- ture measurements, including beam attenuation, transition saturation effects, laser mode fluctuations and transition choice. Visualization experiments have been performed to improve the uniformity of the nozzle flow. Comparisons of measured quantities with a simple one-dimensional computation are made, showing good agreement between measurements and theory given the uncertainty of the nozzle reservoir conditions and the vibrational relaxation rate.

Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

NASA Astrophysics Data System (ADS)

Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti

2016-08-01

Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.

Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes.

PubMed

Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti

2016-08-28

Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.

Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu

Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kineticsmore » resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.« less

Probing the non-equilibrium force fluctuation spectrum of actomyosin cortices in vivo

NASA Astrophysics Data System (ADS)

Tan, Tzer Han; Swartz, Zachary; Keren, Kinneret; Fakhri, Nikta

Mechanics of the cortex govern the shape of animal cells, and its dynamics underlie cell migration, cytokinesis and embryogenesis. The molecular players involved are largely known, yet it is unclear how their collective dynamics give rise to large scale behavior. This is mostly due to the lack of experimental tools to probe the spatially varying active mechanical properties of the cortex. Here, we introduce a novel technique based on fluorescent single walled carbon nanotubes to generate non-equilibrium force fluctuation spectrum of actomysion cortices in starfish oocytes. The quantitative measurements combined with a theoretical model reveal the role of stress organization in active mechanics and dynamics of the cortex.

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.

Plasma Turbulence Imaging via Beam Emission Spectroscopy in the Core of the DIII-D Tokamak

NASA Astrophysics Data System (ADS)

McKee, George R.; Fonck, Raymond J.; Gupta, Deepak K.; Schlossberg, David J.; Shafer, Morgan W.; Boivin, Réjean L.; Solomon, Wayne

Beam Emission Spectroscopy (BES), a high-sensitivity, good spatial resolution imaging diagnostic system, has been deployed and recently upgraded and expanded at the DIII-D tokamak to better understand density fluctuations arising from plasma turbulence. The currently deployed system images density fluctuations over an approximately 5 × 7 cm region at the plasma mid-plane (radially scannable over 0.2 < r/a ≤ 1) with a 5 × 6 (radial × poloidal) grid of rectangular detection channels, with one microsecond time resolution. BES observes collisionally-induced, Doppler-shifted Dα fluorescence (λ = 652-655 nm) of injected deuterium neutral beam atoms. The diagnostic wavenumber sensitivity is approximately k⊥ < 2.5 cm-1, allowing measurement of longwavelength (k⊥ρI < 1) density fluctuations. The recent upgrade includes expanded fiber optics bundles, customdesigned high-transmission, sharp-edge interference filters, ultra fast collection optics, and enlarged photodiode detectors that together provide nearly an order of magnitude increase in sensitivity relative to an earlier generation BES system. The high sensitivity allows visualization of turbulence at normalized density fluctuation amplitudes of ‾n/n < 1%, typical of fluctuation levels in the core region. The imaging array allows for sampling over 2-3 turbulent eddy scale lengths, which captures the essential dynamics of eddy evolution, interaction and shearing.

Analysis of dynamic multiplicity fluctuations at PHOBOS

NASA Astrophysics Data System (ADS)

Chai, Zhengwei; PHOBOS Collaboration; Back, B. B.; Baker, M. D.; Ballintijn, M.; Barton, D. S.; Betts, R. R.; Bickley, A. A.; Bindel, R.; Budzanowski, A.; Busza, W.; Carroll, A.; Chai, Z.; Decowski, M. P.; García, E.; George, N.; Gulbrandsen, K.; Gushue, S.; Halliwell, C.; Hamblen, J.; Heintzelman, G. A.; Henderson, C.; Hofman, D. J.; Hollis, R. S.; Holynski, R.; Holzman, B.; Iordanova, A.; Johnson, E.; Kane, J. L.; Katzy, J.; Khan, N.; Kucewicz, W.; Kulinich, P.; Kuo, C. M.; Lin, W. T.; Manly, S.; McLeod, D.; Mignerey, A. C.; Nouicer, R.; Olszewski, A.; Pak, R.; Park, I. C.; Pernegger, H.; Reed, C.; Remsberg, L. P.; Reuter, M.; Roland, C.; Roland, G.; Rosenberg, L.; Sagerer, J.; Sarin, P.; Sawicki, P.; Skulski, W.; Steinberg, P.; Stephans, G. S. F.; Sukhanov, A.; Tang, J. L.; Trzupek, A.; Vale, C.; van Nieuwenhuizen, G. J.; Verdier, R.; Wolfs, F. L. H.; Wosiek, B.; Wozniak, K.; Wuosmaa, A. H.; Wyslouch, B.

2005-01-01

This paper presents the analysis of the dynamic fluctuations in the inclusive charged particle multiplicity measured by PHOBOS for Au+Au collisions at surdsNN = 200GeV within the pseudo-rapidity range of -3 < η < 3. First the definition of the fluctuations observables used in this analysis is presented, together with the discussion of their physics meaning. Then the procedure for the extraction of dynamic fluctuations is described. Some preliminary results are included to illustrate the correlation features of the fluctuation observable. New dynamic fluctuations results will be available in a later publication.

Controllable synthesis of green and blue fluorescent carbon nanodots for pH and Cu(2+) sensing in living cells.

PubMed

Shi, Lihong; Li, Yanyan; Li, Xiaofeng; Zhao, Bo; Wen, Xiangping; Zhang, Guomei; Dong, Chuan; Shuang, Shaomin

2016-03-15

We report a controllable strategy for fabrication of green and blue fluorescent carbon nanodots (CDs), and demonstrate their applications for pH and Cu(2+) sensing in living cells. Green and blue fluorescent CDs have been synthesized by hydrothermal method and pyrolysis of leeks, respectively, providing an easy way for the production of CDs without the request of tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. Green fluorescent CDs (G-CDs) exhibit high tolerance to pH values and external cations. Blue fluorescent CDs (B-CDs) can be applied to pH and Cu(2+) sensing. The linear range of Cu(2+) detection is 0.01-10.00 μM and the detection limit is 0.05 μM. For pH detection, there is a good linearity in the pH range of 3.5-10.0. The linear and rapid response of B-CDs to Cu(2+) and pH is valuable for Cu(2+) and pH sensing in living cells. Confocal fluorescent imaging of human cervical carcinoma cells indicates that B-CDs could visualize Cu(2+) and pH fluctuations in living cells with negligible autofluorescence. Copyright © 2015 Elsevier B.V. All rights reserved.

Diurnal Variability in Chlorophyll-a, Carotenoids, CDOM and SO₄(2-) Intensity of Offshore Seawater Detected by an Underwater Fluorescence-Raman Spectral System.

PubMed

Chen, Jing; Ye, Wangquan; Guo, Jinjia; Luo, Zhao; Li, Ying

2016-07-13

A newly developed integrated fluorescence-Raman spectral system (λex = 532 nm) for detecting Chlorophyll-a (chl-a), Chromophoric Dissolved Organic Matter (CDOM), carotenoids and SO₄(2-) in situ was used to successfully investigate the diurnal variability of all above. Simultaneously using the integration of fluorescence spectroscopy and Raman spectroscopy techniques provided comprehensive marine information due to the complementarity between the different excitation mechanisms and different selection rules. The investigation took place in offshore seawater of the Yellow Sea (36°05'40'' N, 120°31'32'' E) in October 2014. To detect chl-a, CDOM, carotenoids and SO₄(2-), the fluorescence-Raman spectral system was deployed. It was found that troughs of chl-a and CDOM fluorescence signal intensity were observed during high tides, while the signal intensity showed high values with larger fluctuations during ebb-tide. Chl-a and carotenoids were influenced by solar radiation within a day cycle by different detection techniques, as well as displaying similar and synchronous tendency. CDOM fluorescence cause interference to the measurement of SO₄(2-). To avoid such interference, the backup Raman spectroscopy system with λex = 785 nm was employed to detect SO₄(2-) concentration on the following day. The results demonstrated that the fluorescence-Raman spectral system has great potential in detection of chl-a, carotenoids, CDOM and SO₄(2-) in the ocean.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

PubMed

Schneidereit, D; Vass, H; Reischl, B; Allen, R J; Friedrich, O

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

Anomalous subdiffusion in fluorescence photobleaching recovery: a Monte Carlo study.

PubMed Central

Saxton, M J

2001-01-01

Anomalous subdiffusion is hindered diffusion in which the mean-square displacement of a diffusing particle is proportional to some power of time less than one. Anomalous subdiffusion has been observed for a variety of lipids and proteins in the plasma membranes of a variety of cells. Fluorescence photobleaching recovery experiments with anomalous subdiffusion are simulated to see how to analyze the data. It is useful to fit the recovery curve with both the usual recovery equation and the anomalous one, and to judge the goodness of fit on log-log plots. The simulations show that the simplest approximate treatment of anomalous subdiffusion usually gives good results. Three models of anomalous subdiffusion are considered: obstruction, fractional Brownian motion, and the continuous-time random walk. The models differ significantly in their behavior at short times and in their noise level. For obstructed diffusion the approach to the percolation threshold is marked by a large increase in noise, a broadening of the distribution of diffusion coefficients and anomalous subdiffusion exponents, and the expected abrupt decrease in the mobile fraction. The extreme fluctuations in the recovery curves at and near the percolation threshold result from extreme fluctuations in the geometry of the percolation cluster. PMID:11566793

Simultaneous velocity and concentration measurements in the near field of a turbulent low-pressure jet by digital particle image velocimetry-planar laser-induced fluorescence

NASA Astrophysics Data System (ADS)

Borg, A.; Bolinder, J.; Fuchs, L.

The main purpose of this work is to develop a method for simultaneous measurement of velocity and passive scalar concentration by means of digital particle image velocimetry and planar laser-induced fluorescence. Details of the implementation of the method are given, and the technique is applied to measurements of concentration and velocity in the centre-plane of a liquid jet with a Reynolds number of 6,000. The measurements are compared with large eddy simulations. Mean velocities and concentrations, fluctuating velocities and concentrations, and correlation between fluctuating velocities and concentrations are analysed for the first six diameters downstream of the jet exit. The general agreement between measured and simulated results was found to be good, in particular for mean quantities. Mean profiles are also found to be in good agreement with other experimental work on jets reported in the literature. The ``whole-plane'' measurement method was found to be very useful for detailed comparisons of turbulent statistics with simulated data. The inadequacy of models for turbulent mass transport based on the standard gradient diffusion concept is demonstrated through the experimental data.

A reversible fluorescent probe for real-time live-cell imaging and quantification of endogenous hydropolysulfides.

PubMed

Umezawa, Keitaro; Kamiya, Mako; Urano, Yasuteru

2018-05-23

The chemical biology of reactive sulfur species, including hydropolysulfides, has been a subject undergoing intense study in recent years, but further understanding of their 'intact' function in living cells has been limited due to a lack of appropriate analytical tools. In order to overcome this limitation, we developed a new type of fluorescent probe which reversibly and selectively reacts to hydropolysulfides. The probe enables live-cell visualization and quantification of endogenous hydropolysulfides without interference from intrinsic thiol species such as glutathione. Additionally, real-time reversible monitoring of oxidative-stress-induced fluctuation of intrinsic hydropolysulfides has been achieved with a temporal resolution in the order of seconds, a result which has not yet been realized using conventional methods. These results reveal the probe's versatility as a new fluorescence imaging tool to understand the function of intracellular hydropolysulfides. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Near-Infrared Fluorescent Nanoprobes for Revealing the Role of Dopamine in Drug Addiction.

PubMed

Feng, Peijian; Chen, Yulei; Zhang, Lei; Qian, Cheng-Gen; Xiao, Xuanzhong; Han, Xu; Shen, Qun-Dong

2018-02-07

Brain imaging techniques enable visualizing the activity of central nervous system without invasive neurosurgery. Dopamine is an important neurotransmitter. Its fluctuation in brain leads to a wide range of diseases and disorders, like drug addiction, depression, and Parkinson's disease. We designed near-infrared fluorescence dopamine-responsive nanoprobes (DRNs) for brain activity imaging during drug abuse and addiction process. On the basis of light-induced electron transfer between DRNs and dopamine and molecular wire effect of the DRNs, we can track the dynamical change of the neurotransmitter level in the physiological environment and the releasing of the neurotransmitter in living dopaminergic neurons in response to nicotine stimulation. The functional near-infrared fluorescence imaging can dynamically track the dopamine level in the mice midbrain under normal or drug-activated condition and evaluate the long-term effect of addictive substances to the brain. This strategy has the potential for studying neural activity under physiological condition.

Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy.

PubMed

Haustein, Elke; Schwille, Petra

2003-02-01

Fluorescence correlation spectroscopy (FCS) extracts information about molecular dynamics from the tiny fluctuations that can be observed in the emission of small ensembles of fluorescent molecules in thermodynamic equilibrium. Employing a confocal setup in conjunction with highly dilute samples, the average number of fluorescent particles simultaneously within the measurement volume (approximately 1 fl) is minimized. Among the multitude of chemical and physical parameters accessible by FCS are local concentrations, mobility coefficients, rate constants for association and dissociation processes, and even enzyme kinetics. As any reaction causing an alteration of the primary measurement parameters such as fluorescence brightness or mobility can be monitored, the application of this noninvasive method to unravel processes in living cells is straightforward. Due to the high spatial resolution of less than 0.5 microm, selective measurements in cellular compartments, e.g., to probe receptor-ligand interactions on cell membranes, are feasible. Moreover, the observation of local molecular dynamics provides access to environmental parameters such as local oxygen concentrations, pH, or viscosity. Thus, this versatile technique is of particular attractiveness for researchers striving for quantitative assessment of interactions and dynamics of small molecular quantities in biologically relevant systems.

Observing Quantum State Diffusion by Heterodyne Detection of Fluorescence

NASA Astrophysics Data System (ADS)

Campagne-Ibarcq, P.; Six, P.; Bretheau, L.; Sarlette, A.; Mirrahimi, M.; Rouchon, P.; Huard, B.

2016-01-01

A qubit can relax by fluorescence, which prompts the release of a photon into its electromagnetic environment. By counting the emitted photons, discrete quantum jumps of the qubit state can be observed. The succession of states occupied by the qubit in a single experiment, its quantum trajectory, depends in fact on the kind of detector. How are the quantum trajectories modified if one measures continuously the amplitude of the fluorescence field instead? Using a superconducting parametric amplifier, we perform heterodyne detection of the fluorescence of a superconducting qubit. For each realization of the measurement record, we can reconstruct a different quantum trajectory for the qubit. The observed evolution obeys quantum state diffusion, which is characteristic of quantum measurements subject to zero-point fluctuations. Independent projective measurements of the qubit at various times provide a quantitative verification of the reconstructed trajectories. By exploring the statistics of quantum trajectories, we demonstrate that the qubit states span a deterministic surface in the Bloch sphere at each time in the evolution. Additionally, we show that when monitoring fluorescence field quadratures, coherent superpositions are generated during the decay from excited to ground state. Counterintuitively, measuring light emitted during relaxation can give rise to trajectories with increased excitation probability.

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells.

PubMed

Wang, Sheng; Chen, Xuanze; Chang, Lei; Ding, Miao; Xue, Ruiying; Duan, Haifeng; Sun, Yujie

2018-06-05

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

The consistent difference in red fluorescence in fishes across a 15 m depth gradient is triggered by ambient brightness, not by ambient spectrum.

PubMed

Harant, Ulrike Katharina; Michiels, Nicolaas Karel; Anthes, Nils; Meadows, Melissa Grace

2016-02-17

Organisms adapt to fluctuations or gradients in their environment by means of genetic change or phenotypic plasticity. Consistent adaptation across small spatial scales measured in meters, however, has rarely been reported. We recently found significant variation in fluorescence brightness in six benthic marine fish species across a 15 m depth gradient. Here, we investigate whether this can be explained by phenotypic plasticity alone, using the triplefin Tripterygion delaisi as a model species. In two separate experiments, we measure change in red fluorescent brightness to spectral composition and ambient brightness, two central parameters of the visual environment that change rapidly with depth. Changing the ambient spectra simulating light at -5 or -20 m depth generated no detectable changes in mean fluorescence brightness after 4-6 weeks. In contrast, a reduction in ambient brightness generated a significant and reversible increase in mean fluorescence, most of this within the first week. Although individuals can quickly up- and down-regulate their fluorescence around this mean value using melanosome aggregation and dispersal, we demonstrate that this range around the mean remained unaffected by either treatment. We show that the positive association between fluorescence and depth observed in the field can be fully explained by ambient light brightness, with no detectable additional effect of spectral composition. We propose that this change is achieved by adjusting the ratio of melanophores and fluorescent iridophores in the iris.

Imaging and quantification of trans-membrane protein diffusion in living bacteria.

PubMed

Oswald, Felix; L M Bank, Ernst; Bollen, Yves J M; Peterman, Erwin J G

2014-07-07

The cytoplasmic membrane forms the barrier between any cell's interior and the outside world. It contains many proteins that enable essential processes such as the transmission of signals, the uptake of nutrients, and cell division. In the case of prokaryotes, which do not contain intracellular membranes, the cytoplasmic membrane also contains proteins for respiration and protein folding. Mutual interactions and specific localization of these proteins depend on two-dimensional diffusion driven by thermal fluctuations. The experimental investigation of membrane-protein diffusion in bacteria is challenging due to their small size, only a few times larger than the resolution of an optical microscope. Here, we review fluorescence microscopy-based methods to study diffusion of membrane proteins in living bacteria. The main focus is on data-analysis tools to extract diffusion coefficients from single-particle tracking data obtained by single-molecule fluorescence microscopy. We introduce a novel approach, IPODD (inverse projection of displacement distributions), to obtain diffusion coefficients from the usually obtained 2-D projected diffusion trajectories of the highly 3-D curved bacterial membrane. This method provides, in contrast to traditional mean-squared-displacement methods, correct diffusion coefficients and allows unravelling of heterogeneously diffusing populations.

First Results from the Telescope Array RAdar (TARA) Detector

NASA Astrophysics Data System (ADS)

Myers, Isaac

2014-03-01

The TARA cosmic ray detector has been in operation for about a year and a half. This bi-static radar detector was designed with the goal of detecting cosmic rays in coincidence with Telescope Array (TA). A new high power (25 kW, 5 MW effective radiated power) transmitter and antenna array and 250 MHz fPGA-based DAQ have been operational since August 2013. The eight-Yagi antenna array broadcasts a 54.1 MHz tone across the TA surface detector array toward our receiver station 50 km away at the Long Ridge fluorescence detector. Receiving antennas feed an intelligent DAQ that self-adjusts to the fluctuating radio background and which employs a bank of matched filters that search in real-time for chirp radar echoes. Millions of triggers have been collected in this mode. A second mode is a forced trigger scheme that uses the trigger status of the fluorescence telescope. Of those triggers collected in FD-triggered mode, about 800 correspond with well-reconstructed TA events. I will describe recent advancements in calibrating key components in the transmitter and receiver RF chains and the analysis of FD-triggered data. Work supported by W.M. Keck Foundation and NSF.

Single-molecule RNA observation in vivo reveals dynamics of co-transcriptional splicing

NASA Astrophysics Data System (ADS)

Ferguson, M. L.; Coulon, A.; de Turris, V.; Palangat, M.; Chow, C. C.; Singer, R. H.; Larson, D. R.

2013-03-01

The synthesis of pre-mRNA and the splicing of that pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the transcription elongation complex and the spliceosome). How this coordination occurs in vivo is unknown. Here we report the first experimental observation of transcription and splicing occurring at the same gene in living cells. By utilizing the PP7/MS2 fluorescent RNA reporter system, we can directly observe two distinct regions of the nascent RNA, allowing us to measure the rise and fall time of the intron and exon of a reporter gene stably integrated into a human cell line. The reporter gene consists of a beta globin gene where we have inserted a 24 RNA hairpin cassette into the intron/exon. Upon synthesis, the RNA hairpins are tightly bound by fluorescently-labeled PP7/MS2 bacteriophage coat proteins. After gene induction, a single locus of active transcription in the nucleus shows fluorescence intensity changes characteristic of the synthesis and excision of the intron/exon. Using fluctuation analysis, we determine the elongation rate to be 1.5 kb/min. From the temporal cross correlation function, we determine that splicing of this gene must be co-transcriptional with a splicing time of ~100 seconds before termination and a ~200 second pause at termination. We propose that dual-color RNA imaging may be extended to investigate other mechanisms of transcription, gene regulation, and RNA processing.

Probing the conformational dynamics of photosystem I in unconfined and confined spaces.

PubMed

Das, Gaurav; Chattoraj, Shyamtanu; Nandi, Somen; Mondal, Prasenjit; Saha, Abhijit; Bhattacharyya, Kankan; Ghosh, Surajit

2017-12-20

The fluorescence dynamics of Photosystem I (PSI) in bulk water and inside a confined environment like a liposome have been investigated using time resolved confocal microscopy. In bulk water, PSI exhibits a major emission peak at ∼680 nm, while in the liposome it exhibits a markedly blue shifted emission maximum at ∼485 nm. This is indicative of conformational changes due to entrapment and emergence of a stressed conformation of PSI inside the liposome. The observed time constants for the fluorescence lifetime of PSI inside the liposome are significantly high as opposed to PSI in bulk water. More interestingly, the fluorescence intensity of PSI in bulk water exhibits strong fluctuations with many high intensity jumps and these are anti-correlated with the fluorescence lifetime of PSI. In contrast, inside the liposome, no such anti-correlated behaviour is observed. We further demonstrated that PSI exhibits at least two conformational states in bulk water, whereas a single conformation is observed inside the liposome, indicating the conformational rigidity and locking of the PSI complex inside a liposome.

Nanog Fluctuations in Embryonic Stem Cells Highlight the Problem of Measurement in Cell Biology.

PubMed

Smith, Rosanna C G; Stumpf, Patrick S; Ridden, Sonya J; Sim, Aaron; Filippi, Sarah; Harrington, Heather A; MacArthur, Ben D

2017-06-20

A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, because temporal changes in intracellular protein abundances cannot be measured directly in live cells, fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systematically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog, we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight the problem of measurement in live cells. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fluctuations in Cerebral Hemodynamics

DTIC Science & Technology

2003-12-01

Determination of scaling properties Detrended Fluctuations Analysis (see (28) and references therein) is commonly used to determine scaling...pressure (averaged over a cardiac beat) of a healthy subject. First 1000 values of the time series are shown. (b) Detrended fluctuation analysis (DFA...1000 values of the time series are shown. (b) Detrended fluctuation analysis of the time series shown in (a). Fig . 3 Side-by-side boxplot for the

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa

PubMed Central

Vass, H.; Reischl, B.; Allen, R. J.; Friedrich, O.

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules Δdv¯ is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence. PMID:27764134

Diurnal Variability in Chlorophyll-a, Carotenoids, CDOM and SO42− Intensity of Offshore Seawater Detected by an Underwater Fluorescence-Raman Spectral System

PubMed Central

Chen, Jing; Ye, Wangquan; Guo, Jinjia; Luo, Zhao; Li, Ying

2016-01-01

A newly developed integrated fluorescence-Raman spectral system (λex = 532 nm) for detecting Chlorophyll-a (chl-a), Chromophoric Dissolved Organic Matter (CDOM), carotenoids and SO42− in situ was used to successfully investigate the diurnal variability of all above. Simultaneously using the integration of fluorescence spectroscopy and Raman spectroscopy techniques provided comprehensive marine information due to the complementarity between the different excitation mechanisms and different selection rules. The investigation took place in offshore seawater of the Yellow Sea (36°05′40′′ N, 120°31′32′′ E) in October 2014. To detect chl-a, CDOM, carotenoids and SO42−, the fluorescence-Raman spectral system was deployed. It was found that troughs of chl-a and CDOM fluorescence signal intensity were observed during high tides, while the signal intensity showed high values with larger fluctuations during ebb-tide. Chl-a and carotenoids were influenced by solar radiation within a day cycle by different detection techniques, as well as displaying similar and synchronous tendency. CDOM fluorescence cause interference to the measurement of SO42−. To avoid such interference, the backup Raman spectroscopy system with λex = 785 nm was employed to detect SO42− concentration on the following day. The results demonstrated that the fluorescence-Raman spectral system has great potential in detection of chl-a, carotenoids, CDOM and SO42− in the ocean. PMID:27420071

Analyzing near infrared scattering from human skin to monitor changes in hematocrit

NASA Astrophysics Data System (ADS)

Chaiken, Joseph; Deng, Bin; Goodisman, Jerry; Shaheen, George; Bussjager, R. J.

2012-01-01

The leading preventable cause of death, world-wide, civilian or military, for all people between the ages of 18-45 is undetected internal hemorrhage. Autonomic compensation mechanisms mask changes such as e.g. hematocrit fluctuations that could give early warning if only they could be monitored continuously with reasonable degrees of precision and relative accuracy. Probing tissue with near infrared radiation (NIR) simultaneously produces remitted fluorescence and Raman scattering (IE) plus Rayleigh/Mie light scattering (EE) that noninvasively give chemical and physical information about the materials and objects within. We model tissue as a three-phase system: plasma and red blood cell (RBC) phases that are mobile and a static tissue phase. In vivo, any volume of tissue naturally experiences spatial and temporal fluctuations of blood plasma and RBC content. Plasma and RBC fractions may be discriminated from each other on the basis of their physical, chemical and optical properties. Thus IE and EE from NIR probing yield information about these fractions. Assuming there is no void volume in viable tissue, or that void volume is constant, changes in plasma and RBC volume fractions may be calculated from simultaneous measurements of the two observables, EE and IE. In a previously published analysis we showed the underlying phenomenology but did not provide an algorithm for calculating volume fractions from experimental data. Here we present a simple analysis that allows continuous monitoring of fluid fraction and hematocrit (Hct) changes by measuring IE and EE, and apply it to some experimental in vivo measurements.

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

PubMed

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

2017-11-15

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

New solutions for standardization, monitoring and quality management of fluorescence-based imaging systems (Conference Presentation)

NASA Astrophysics Data System (ADS)

Royon, Arnaud; Papon, Gautier

2016-03-01

Fluorescence microscopes have become ubiquitous in life sciences laboratories, including those focused on pharmaceuticals, diagnosis, and forensics. For the past few years, the need for both performance guarantees and quantifiable results has driven development in this area. However, the lack of appropriate standards and reference materials makes it difficult or impossible to compare the results of two fluorescence microscopes, or to measure performance fluctuations of one microscope over time. Therefore, the operation of fluorescence microscopes is not monitored as often as their use warrants - an issue that is recognized by both systems manufacturers and national metrology institutes. We have developed a new process that enables the etching of long-term stable fluorescent patterns with sub-micrometer sizes in three dimensions inside glass. In this paper, we present, based on this new process, a fluorescent multi-dimensional ruler and a dedicated software that are suitable for monitoring and quality management of fluorescence-based imaging systems (wide-field, confocal, multiphoton, high content machines). In addition to fluorescence, the same patterns exhibit bright- and dark-field contrast, DIC, and phase contrast, which make them also relevant to monitor these types of microscopes. Non-exhaustively, this new solution enables the measurement of: The stage repositioning accuracy; The illumination and detection homogeneities; The field flatness; The detectors' characteristics; The lateral and axial spatial resolutions; The spectral response (spectrum, intensity and lifetime) of the system. Thanks to the stability of the patterns, microscope performance assessment can be carried out as well in a daily basis as in the long term.

Localizer: fast, accurate, open-source, and modular software package for superresolution microscopy

PubMed Central

Duwé, Sam; Neely, Robert K.; Zhang, Jin

2012-01-01

Abstract. We present Localizer, a freely available and open source software package that implements the computational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algorithms as they become available, while maintaining the same interface and performance. We provide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies. PMID:23208219

Advantages of statistical analysis of giant vesicle flickering for bending elasticity measurements.

PubMed

Méléard, P; Pott, T; Bouvrais, H; Ipsen, J H

2011-10-01

We show how to greatly improve precision when determining bending elasticity of giant unilamellar vesicles. Taking advantage of the well-known quasi-spherical model of liposome flickering, we analyze the full probability distributions of the configurational fluctuations instead of limiting the analysis to the second moment measurements only as usually done in previously published works. This leads to objective criteria to reject vesicles that do not behave according to the model. As a result, the confidence in the bending elasticity determination of individual vesicles that fit the model is improved and, consequently, the reproducibility of this measurement for a given membrane system. This approach uncovers new possibilities for bending elasticity studies like detection of minute influences by solutes in the buffer or into the membrane. In the same way, we are now able to detect the inhomogeneous behavior of giant vesicle systems such as the hazardous production of peroxide in bilayers containing fluorescent dyes. © EDP Sciences / Società Italiana di Fisica / Springer-Verlag 2011

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

PubMed Central

Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

2014-01-01

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

Microwave-assisted solid-phase synthesis of highly fluorescent carbon nanoparticles and its application in intracellular pH sensing.

PubMed

Yang, Shenghong; Chen, Xiao; Liu, Shuqin; Wang, Fuxin; Ouyang, Gangfeng

2018-08-15

Fluorescent carbon nanoparticles (FCNPs) have been deeply researched and widely applied in recent years due to their good optics performance, chemical stability and biocompatibility. Herein, a green and rapid microwave-assisted solid-phase synthesis (solvent-free) approach was proposed for the fabrication of highly FCNPs in a very short period of time, 4 min. The as-prepared FCNPs can emit a blue emission with quantum yield of up to 63.2% in water solution and show yellow fluorescence in the solid state. The FCNPs also exhibit special solvent effect that the fluorescence emission can be adjusted by controlling the solvent ratio of ethanol and water. Most importantly, the FCNPs possess a narrow-range pH response. The probe responds linearly and rapidly to minor pH fluctuations within the range of 3.47-5.10 and the correlation coefficient is above 0.99. The proposed FCNPs also exhibit high photostability and reusability. As expected, the cell imaging and intracellular pH monitoring was achieved successfully in living SMMC 7721 hepatoma cells by this probe. The FCNPs is promising as a convenient and general fluorescent pH sensor for bioimaging applications. Copyright © 2018. Published by Elsevier B.V.

Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke (Conference Presentation)

NASA Astrophysics Data System (ADS)

Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S.

2016-03-01

Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

Breathable NIPAAm Network with Controllable Hydration Supports Model Lipid Membrane

NASA Astrophysics Data System (ADS)

Jablin, Michael; Smith, Hillary; Zhernenkov, Mikhail; Vidyasagar, Ajay; Toomey, Ryan; Saiz, Jessica; Toperverg, Boris; Watkins, Erik; Kuhl, Tonya; Hurd, Alan; Majewski, Jaroslaw

2009-03-01

The interaction of a model lipid bilayer composed of DPPC with a surface-tethered poly(N-isopropylacrylamide) (NIPAAm) was explored with neutron reflectometry (NR). The Langmuir-Blodgett / Langmuir-Schaeffer method was used to deposit a lipid bilayer onto the polymer. NR measurements were used to probe the in- and out-of-plane structure of the system as a function of temperature. NR with fluorescence microscopy show that the polymer supports a lipid bilayer, and hydration of the support can be controlled. At low temp. the membrane develops out-of-plane undulations visible in off-specular scattering. Analysis of the off-specular reveals in-plane correlation of the bilayer fluctuations. The separation of the lipid bilayer from the solid support of a substrate constitutes a significant step towards a more realistic model of biological membranes.

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating

PubMed Central

Böcking, Till; Aguet, François; Harrison, Stephen C.; Kirchhausen, Tomas

2010-01-01

Heat shock cognate protein 70 (Hsc70) supports remodeling of protein complexes -- for example, disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by electron cryomicroscopy, in the vicinity of each occupied site and that accumulation of local strains destabilises the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes. PMID:21278753

Achieving 100% Efficient Postcolumn Hydride Generation for As Speciation Analysis by Atomic Fluorescence Spectrometry.

PubMed

Marschner, Karel; Musil, Stanislav; Dědina, Jiří

2016-04-05

An experimental setup consisting of a flow injection hydride generator coupled to an atomic fluorescence spectrometer was optimized in order to generate arsanes from tri- and pentavalent inorganic arsenic species (iAs(III), iAs(V)), monomethylarsonic acid (MAs(V)), and dimethylarsinic acid (DMAs(V)) with 100% efficiency with the use of only HCl and NaBH4 as the reagents. The optimal concentration of HCl was 2 mol L(-1); the optimal concentration of NaBH4 was 2.5% (m/v), and the volume of the reaction coil was 8.9 mL. To prevent excessive signal noise due to fluctuations of hydride supply to an atomizer, a new design of a gas-liquid separator was implemented. The optimized experimental setup was subsequently interfaced to HPLC and employed for speciation analysis of arsenic. Two chromatography columns were tested: (i) ion-pair chromatography and (ii) ion exchange chromatography. The latter offered much better results for human urine samples without a need for sample dilution. Due to the equal hydride generation efficiency (and thus the sensitivities) of all As species, a single species standardization by DMAs(V) standard was feasible. The limits of detection for iAs(III), iAs(V), MAs(V), and DMAs(V) were 40, 97, 57, and 55 pg mL(-1), respectively. Accuracy of the method was tested by the analysis of the standard reference material (human urine NIST 2669), and the method was also verified by the comparative analyses of human urine samples collected from five individuals with an independent reference method.

Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin.

PubMed

Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T; Rao, Madan; Mayor, Satyajit

2015-11-05

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24-37 °C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an "active actin-membrane composite" cell surface. © 2015 Saha et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Ultrafast Hydration Dynamics and Coupled Water-Protein Fluctuations in Apomyoglobin

NASA Astrophysics Data System (ADS)

Yang, Yi; Zhang, Luyuan; Wang, Lijuan; Zhong, Dongping

2009-06-01

Protein hydration dynamics are of fundamental importance to its structure and function. Here, we characterize the global solvation dynamics and anisotropy dynamics around the apomyoglobin surface in different conformational states (native and molten globule) by measuring the Stokes shift and anisotropy decay of tryptophan with femtosecond-resolved fluorescence upconversion. With site-directed mutagenesis, we designed sixteen mutants with one tryptophan in each, and placed the probe at a desirable position ranging from buried in the protein core to fully solvent-exposed on the protein surface. In all protein sites studied, two distinct solvation relaxations (1-8 ps and 20-200 ps) were observed, reflecting the initial collective water relaxation and subsequent hydrogen-bond network restructuring, respectively, and both are strongly correlated with protein's local structures and chemical properties. The hydration dynamics of the mutants in molten globule state are faster than those observed in native state, indicating that the protein becomes more flexible and less structured when its conformation is converted from fully-folded native state to partially-folded molten globule state. Complementary, fluorescence anisotropy dynamics of all mutants in native state show an increasing trend of wobbling times (40-260 ps) when the location of the probe is changed from a loop, to a lateral helix, and then, to the compact protein core. Such an increase in wobbling times is related to the local protein structural rigidity, which relates the interaction of water with side chains. The ultrafast hydration dynamics and related side-chain motion around the protein surface unravel the coupled water-protein fluctuations on the picosecond time scales and indicate that the local protein motions are slaved by hydrating water fluctuations.

Förster-type energy transfer as a probe for changes in local fluctuations of the protein matrix.

PubMed

Somogyi, B; Matkó, J; Papp, S; Hevessy, J; Welch, G R; Damjanovich, S

1984-07-17

Much evidence, on both theoretical and experimental sides, indicates the importance of local fluctuations (in energy levels, conformational substates, etc.) of the macromolecular matrix in the biological activity of proteins. We describe here a novel application of the Förster-type energy-transfer process capable of monitoring changes both in local fluctuations and in conformational states of macromolecules. A new energy-transfer parameter, f, is defined as an average transfer efficiency, [E], normalized by the actual average quantum efficiency of the donor fluorescence, [phi D]. A simple oscillator model (for a one donor-one acceptor system) is presented to show the sensitivity of this parameter to changes in amplitudes of local fluctuations. The different modes of averaging (static, dynamic, and intermediate cases) occurring for a given value of the average transfer rate, [kt], and the experimental requirements as well as limitations of the method are also discussed. The experimental tests were performed on the ribonuclease T1-pyridoxamine 5'-phosphate conjugate (a one donor-one acceptor system) by studying the change of the f parameter with temperature, an environmental parameter expectedly perturbing local fluctuations of proteins. The parameter f increased with increasing temperature as expected on the basis of the oscillator model, suggesting that it really reflects changes of fluctuation amplitudes (significant changes in the orientation factor, k2, as well as in the spectral properties of the fluorophores can be excluded by anisotropy measurements and spectral investigations). Possibilities of the general applicability of the method are also discussed.

Note: Demonstration of an external-cavity diode laser system immune to current and temperature fluctuations.

PubMed

Miao, Xinyu; Yin, Longfei; Zhuang, Wei; Luo, Bin; Dang, Anhong; Chen, Jingbiao; Guo, Hong

2011-08-01

We demonstrate an external-cavity laser system using an anti-reflection coated laser diode as gain medium with about 60 nm fluorescence spectrum, and a Rb Faraday anomalous dispersion optical filter (FADOF) as frequency-selecting element with a transmission bandwidth of 1.3 GHz. With 6.4% optical feedback, a single stable longitudinal mode is obtained with a linewidth of 69 kHz. The wavelength of this laser is operating within the center of the highest transmission peak of FADOF over a diode current range from 55 mA to 142 mA and a diode temperature range from 15 °C to 35 °C, thus it is immune to the fluctuations of current and temperature.

Electrogenerated Chemiluminescence and Fluorescence Lifetime Spatial Heterogeneity of Poly (2-methoxy-5-(2-ethylhexyloxy)-1, 4-phenylenevinylene) in Presence of [6,6]-phenyl-C61-butyric Acid Methyl Ester

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geng, Hongwei; Pan, Shanlin; Hu, Dehong

In this paper, we studied the luminescence property and fluorescence lifetime mapping of MEH-PPV/PCBM system by using electrogenerated chemiluminescence (ECL) and time-correlated single photo counting (TC-SPC) technologies. The ECL results showed that the oxidation peak of MEH-PPV near 0.7 V (vs. SCE) and ECL response of films shifted positively towards 1.2 V when in the presence of PCBM. At the same time, the oxidation peak current density of MEH-PPV increases while the ECL response decreased with the loading of PCBM in the composite films. The fluorescence lifetime images clearly show that the lifetime fluctuation is effected by different substrates andmore » MEH-PPV/PCBM ratios. Meanwhile, the lifetime of MEH-PPV decreases with the increasing of film thickness. The lifetimes of MEH-PPV films on TiO2 substrate are lower than them of films on cover slips.« less

Watching conformational- and photo-dynamics of single fluorescent proteins in solution.

PubMed

Goldsmith, Randall H; Moerner, W E

2010-03-01

Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the Anti-Brownian ELectrokinetic (ABEL) Trap, which allows extended investigation of solution-phase biomolecules - without immobilization -through real-time electrokinetic feedback. Here we apply the ABEL trap to study an important photosynthetic antenna protein, Allophycocyanin (APC). The technique allows the observation of single molecules of solution-phase APC for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation, and biomaterials for solar energy harvesting.

A solution-based single-molecule study of surface-bound PBIs: solvent-mediated environmental effects on molecular flexibility.

PubMed

Lee, Ji-Eun; Han, Ye Ri; Ham, Sujin; Jun, Chul-Ho; Kim, Dongho

2017-11-08

We have investigated the fundamental photophysical properties of surface-bound perylene bisimide (PBI) molecules in a solution-phase at the single-molecule level. By efficient immobilization of single PBIs on glass, we were able to simultaneously monitor fluorescence intensity trajectories, fluorescence lifetimes, and emission spectra of individual PBIs in organic and aqueous media using confocal microscopy. We showed that the fluorescence dynamics of single PBIs in the solution phase is highly dependent on their local and chemical environments. Furthermore, we visualized different spatial-fluctuations of surface-bound PBIs using defocused wide-field imaging. While PBIs show more steric flexibility in organic media, the flexible motion of PBI molecules in aqueous solution is relatively prohibited due to a cage effect by a hydrogen bonding network, which is previously unobserved. Our method opens up a new possibility to investigate the photophysical properties of multi-chromophoric systems in various solvents at the single-molecule level for developing optimal molecular devices such as water-proof devices.

Watching conformational- and photo-dynamics of single fluorescent proteins in solution

PubMed Central

Goldsmith, Randall H.

2010-01-01

Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the Anti-Brownian ELectrokinetic (ABEL) Trap, which allows extended investigation of solution-phase biomolecules - without immobilization -through real-time electrokinetic feedback. Here we apply the ABEL trap to study an important photosynthetic antenna protein, Allophycocyanin (APC). The technique allows the observation of single molecules of solution-phase APC for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation, and biomaterials for solar energy harvesting. PMID:20625479

Chip-based wide field-of-view nanoscopy

NASA Astrophysics Data System (ADS)

Diekmann, Robin; Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Huser, Thomas R.; Schüttpelz, Mark; Ahluwalia, Balpreet S.

2017-04-01

Present optical nanoscopy techniques use a complex microscope for imaging and a simple glass slide to hold the sample. Here, we demonstrate the inverse: the use of a complex, but mass-producible optical chip, which hosts the sample and provides a waveguide for the illumination source, and a standard low-cost microscope to acquire super-resolved images via two different approaches. Waveguides composed of a material with high refractive-index contrast provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging. As chip-based nanoscopy separates the illumination and detection light paths, total-internal-reflection fluorescence excitation is possible over a large field of view, with up to 0.5 mm × 0.5 mm being demonstrated. Using multicolour chip-based nanoscopy, we visualize fenestrations in liver sinusoidal endothelial cells.

Single-molecule fluorimetry and gating currents inspire an improved optical voltage indicator

PubMed Central

Treger, Jeremy S; Priest, Michael F; Bezanilla, Francisco

2015-01-01

Voltage-sensing domains (VSDs) underlie the movement of voltage-gated ion channels, as well as the voltage-sensitive fluorescent responses observed from a common class of genetically encoded voltage indicators (GEVIs). Despite the widespread use and potential utility of these GEVIs, the biophysical underpinnings of the relationship between VSD movement and fluorophore response remain unclear. We investigated the recently developed GEVI ArcLight, and its close variant Arclight', at both the single-molecule and macroscopic levels to better understand their characteristics and mechanisms of activity. These studies revealed a number of previously unobserved features of ArcLight's behavior, including millisecond-scale fluorescence fluctuations in single molecules as well as a previously unreported delay prior to macroscopic fluorescence onset. Finally, these mechanistic insights allowed us to improve the optical response of ArcLight to fast or repetitive pulses with the development of ArcLightning, a novel GEVI with improved kinetics. DOI: http://dx.doi.org/10.7554/eLife.10482.001 PMID:26599732

Watching conformational- and photodynamics of single fluorescent proteins in solution

NASA Astrophysics Data System (ADS)

Goldsmith, Randall H.; Moerner, W. E.

2010-03-01

Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the anti-Brownian electrokinetic trap, which allows extended investigation of solution-phase biomolecules-without immobilization-through real-time electrokinetic feedback. Here we apply the trap to study an important photosynthetic antenna protein, allophycocyanin. The technique allows the observation of single molecules of solution-phase allophycocyanin for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation and biomaterials for solar energy harvesting.

High-resolution single-molecule fluorescence imaging of zeolite aggregates within real-life fluid catalytic cracking particles.

PubMed

Ristanović, Zoran; Kerssens, Marleen M; Kubarev, Alexey V; Hendriks, Frank C; Dedecker, Peter; Hofkens, Johan; Roeffaers, Maarten B J; Weckhuysen, Bert M

2015-02-02

Fluid catalytic cracking (FCC) is a major process in oil refineries to produce gasoline and base chemicals from crude oil fractions. The spatial distribution and acidity of zeolite aggregates embedded within the 50-150 μm-sized FCC spheres heavily influence their catalytic performance. Single-molecule fluorescence-based imaging methods, namely nanometer accuracy by stochastic chemical reactions (NASCA) and super-resolution optical fluctuation imaging (SOFI) were used to study the catalytic activity of sub-micrometer zeolite ZSM-5 domains within real-life FCC catalyst particles. The formation of fluorescent product molecules taking place at Brønsted acid sites was monitored with single turnover sensitivity and high spatiotemporal resolution, providing detailed insight in dispersion and catalytic activity of zeolite ZSM-5 aggregates. The results point towards substantial differences in turnover frequencies between the zeolite aggregates, revealing significant intraparticle heterogeneities in Brønsted reactivity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging

PubMed Central

Annibale, Paolo; Gratton, Enrico

2015-01-01

Multi-cell biochemical assays and single cell fluorescence measurements revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Here by employing high-speed 3D fluorescence nanoimaging techniques we resolve and track at the single cell level multiple, distinct regions of mRNA synthesis within the model system of a large transgene array. We demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm. Using fluctuation spectroscopy and the phasor analysis approach we were able to extract the local PolII elongation rate at each site as a function of time. We measured a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell, demonstrating a correlation between local transcription kinetics and the movement of the transcription site. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability. PMID:25788248

Covalent dye attachment influences the dynamics and conformational properties of flexible peptides

PubMed Central

Crevenna, Alvaro H.; Bomblies, Rainer; Lamb, Don C.

2017-01-01

Fluorescence spectroscopy techniques like Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET). The attachment of fluorophores to the molecule of interest can potentially alter the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP). Using the intrinsically disordered S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties. We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the open to closed transition rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulations and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules, at least for inherently flexible short peptides. The presented protocol is not only useful for comparing PET-FCS experiments with simulation results but provides a strategy to minimize the influence on molecular properties when chosing labeling positions for fluorescence experiments. PMID:28542243

Spectral characterization of Dictyostelium autofluorescence.

PubMed

Engel, Ruchira; Van Haastert, Peter J M; Visser, Antonie J W G

2006-03-01

Dictyostelium discoideum is used extensively as a model organism for the study of chemotaxis. In recent years, an increasing number of studies of Dictyostelium chemotaxis have made use of fluorescence-based techniques. One of the major factors that can interfere with the application of these techniques in cells is the cellular autofluorescence. In this study, the spectral properties of Dictyostelium autofluorescence have been characterized using fluorescence microscopy. Whole cell autofluorescence spectra obtained using spectral imaging microscopy show that Dictyostelium autofluorescence covers a wavelength range from approximately 500 to 650 nm with a maximum at approximately 510 nm, and thus, potentially interferes with measurements of green fluorescent protein (GFP) fusion proteins with fluorescence microscopy techniques. Further characterization of the spatial distribution, intensity, and brightness of the autofluorescence was performed with fluorescence confocal microscopy and fluorescence fluctuation spectroscopy (FFS). The autofluorescence in both chemotaxing and nonchemotaxing cells is localized in discrete areas. The high intensity seen in cells incubated in the growth medium HG5 reduces by around 50% when incubated in buffer, and can be further reduced by around 85% by photobleaching cells for 5-7 s. The average intensity and spatial distribution of the autofluorescence do not change with long incubations in the buffer. The cellular autofluorescence has a seven times lower molecular brightness than eGFP. The influence of autofluorescence in FFS measurements can be minimized by incubating cells in buffer during the measurements, pre-bleaching, and making use of low excitation intensities. The results obtained in this study thus offer guidelines to the design of future fluorescence studies of Dictyostelium. Microsc. Res. Tech. 69:168-174, 2006. (c) 2006 Wiley-Liss, Inc.

Mass composition results from the Pierre Auger Observatory

NASA Astrophysics Data System (ADS)

Riggi, Simone; Pierre Auger Collaboration

2011-03-01

The present paper reports the recent composition results obtained by the Pierre Auger Observatory using both hybrid and surface detector data. The reconstruction of the shower longitudinal profile and depth of maximum with the fluorescence detector is described. The measured average depth of maximum and its fluctuations as function of the primary energy is presented. The sensitivity of rise time parameters measured with the ground stations and the obtained composition results are discussed.

Conformational fluctuation of Synaptotagmin-1 observed with single molecule fluorescence resonance energy transfer (smFRET)

NASA Astrophysics Data System (ADS)

Choi, Ucheor; Weninger, Keith

2008-10-01

Calcium dependent neurotransmitter release at the synapses involves a synaptic vesicle protein synaptotagmin-1, a calcium sensor, to regulate exocytosis. It has been known that Synaptotagmin-1 interacts with assembled SNARE complexes, but it is unclear how their molecular mechanisms are coupled. X-ray studies in the absence of calcium revealed a closed conformation of synaptotagmin-1 and with calcium bound to the C2 domains of synaptotagmin-3 found extensive interactions holding the domains open. Suggesting the two conformations can be the key to the two functions of synaptotagmin in regulating neurotransmission. Here we use single molecule fluorescence resonance energy transfer (smFRET) to study synaptotagmin interactions with SNARE complexes and the spontaneous conformational changes of synaptotagmin-1 when calcium is induced.

Long-range correlation in cosmic microwave background radiation.

PubMed

Movahed, M Sadegh; Ghasemi, F; Rahvar, Sohrab; Tabar, M Reza Rahimi

2011-08-01

We investigate the statistical anisotropy and gaussianity of temperature fluctuations of Cosmic Microwave Background (CMB) radiation data from the Wilkinson Microwave Anisotropy Probe survey, using the Multifractal Detrended Fluctuation Analysis, Rescaled Range, and Scaled Windowed Variance methods. Multifractal Detrended Fluctuation Analysis shows that CMB fluctuations has a long-range correlation function with a multifractal behavior. By comparing the shuffled and surrogate series of CMB data, we conclude that the multifractality nature of the temperature fluctuation of CMB radiation is mainly due to the long-range correlations, and the map is consistent with a gaussian distribution.

Observation of microscopic dynamics of phase transition in ferroelectric crystals using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Sedarous, Salah S.

1996-03-01

Despite the large quantity of data on the macroscopic changes in the physical properties of ferroelectric crystals during phase transition, there is a continued need for understanding their microscopic origin. Here we describe a novel method for examining the microscopic dynamics of the ferroelectric phase transition using time-resolved fluorescence spectroscopy. The fluorescence properties of organic chromophores embedded in the ferroelectric crystals triglycine sulfate and potassium dihydrogen phosphate are altered in response to the structural phase transitions. The lifetime and the fractional intensity decay show large changes around Tc and the order of the phase transition is readily recovered (first or second order). To explain the fluorescence lifetime data we present a novel theoretical model based on the concept of polaritons in these crystals. Deactivation of the excited state chromophore involves the participation of the vibrational modes of the chromophore. These modes are coupled to the polarization dispersion of the matrix and facilitate the coupling of the excited state to the collective modes in the crystal. The net result is the flow of energy from the excited state chromophore to the lattice phonon. The data indicate that changes in fluorescence lifetime can be used to examine directly the collective modes in these crystals. Our work provides important insight into the emergence of macroscopic phase transition behavior out of microscopic fluctuations.

Microelectrokinetic turbulence in microfluidics at low Reynolds number.

PubMed

Wang, Guiren; Yang, Fang; Zhao, Wei

2016-01-01

There is commonly no turbulence in microfluidics, and the flows are believed to be either laminar or chaotic, since Reynolds number (Re) in microflows is usually on the order of unity or lower. However, we recently demonstrated that it is possible to achieve turbulence with low Re (based on the measured flow velocity and the width of the channel entrance) when a pressure-driven flow is electrokinetically forced in a quasi T-microchannel. To be able to measure high frequency velocity fluctuations in microchannels, a velocimeter with submicrometer spatial resolution and microsecond temporal resolution, called a laser-induced fluorescence photobleaching anemometer, is developed. Here we characterize the microelectrokinetic turbulence and observe some typical and important features of high Re flows, such as Kolmogorov -5/3 spectrum of velocity fluctuation, which usually can be realized only at very high Re in macroturbulent flows.

Body sway, aim point fluctuation and performance in rifle shooters: inter- and intra-individual analysis.

PubMed

Ball, Kevin A; Best, Russell J; Wrigley, Tim V

2003-07-01

In this study, we examined the relationships between body sway, aim point fluctuation and performance in rifle shooting on an inter- and intra-individual basis. Six elite shooters performed 20 shots under competition conditions. For each shot, body sway parameters and four aim point fluctuation parameters were quantified for the time periods 5 s to shot, 3 s to shot and 1 s to shot. Three parameters were used to indicate performance. An AMTI LG6-4 force plate was used to measure body sway parameters, while a SCATT shooting analysis system was used to measure aim point fluctuation and shooting performance. Multiple regression analysis indicated that body sway was related to performance for four shooters. Also, body sway was related to aim point fluctuation for all shooters. These relationships were specific to the individual, with the strength of association, parameters of importance and time period of importance different for different shooters. Correlation analysis of significant regressions indicated that, as body sway increased, performance decreased and aim point fluctuation increased for most relationships. We conclude that body sway and aim point fluctuation are important in elite rifle shooting and performance errors are highly individual-specific at this standard. Individual analysis should be a priority when examining elite sports performance.

Influence analysis of fluctuation parameters on flow stability based on uncertainty method

NASA Astrophysics Data System (ADS)

Meng, Tao; Fan, Shangchun; Wang, Chi; Shi, Huichao

2018-05-01

The relationship between flow fluctuation and pressure in a flow facility is studied theoretically and experimentally in this paper, and a method for measuring the flow fluctuation is proposed. According to the synchronicity of pressure and flow fluctuation, the amplitude of the flow fluctuation is calculated using the pressure measured in the flow facility and measurement of the flow fluctuation in a wide range of frequency is realized. Based on the method proposed, uncertainty analysis is used to evaluate the influences of different parameters on the flow fluctuation by the help of a sample-based stochastic model established and the parameters that have great influence are found, which can be a reference for the optimization design and the stability improvement of the flow facility.

Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

PubMed Central

Pallavicini, Carla; Levi, Valeria; Wetzler, Diana E.; Angiolini, Juan F.; Benseñor, Lorena; Despósito, Marcelo A.; Bruno, Luciana

2014-01-01

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells. PMID:24940780

A tunable ratiometric pH sensor based on carbon nanodots for the quantitative measurement of the intracellular pH of whole cells.

PubMed

Shi, Wen; Li, Xiaohua; Ma, Huimin

2012-06-25

The whole picture: Carbon nanodots labeled with two fluorescent dyes have been developed as a tunable ratiometric pH sensor to measure intracellular pH. The nanosensor shows good biocompatibility and cellular dispersibility. Quantitative determinations on intact HeLa cells and pH fluctuations associated with oxidative stress were performed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detecting Crop Functional Response to a Heat Wave using Airborne Reflectance and Sun-induced Chlorophyll Fluorescence Measurements

NASA Astrophysics Data System (ADS)

Yang, P.; Van der Tol, C.; Rascher, U.; Damm, A.; Schickling, A.; Verhoef, W.

2016-12-01

This study presents an analysis of airborne measured reflectance (R) and solar-induced chlorophyll fluorescence (SIF) as indicators of high temperature stress in agricultural crops. We used atmospherically corrected R and retrievals of SIF in the O2-A band as obtained from HyPlant data over C3 crops (rapeseed, wheat and barley) and a C4 crop (corn) in Germany before (30th June) and during (2nd July) a heat wave in 2015. The availability of airborne data during this heat wave allowed us to detect fluorescence emission efficiency changes as an indicator of crop photosynthetic performance in response to temperature fluctuations. We found that SIF is affected relatively stronger by heat stress than R. This is according to expectation, because the R spectrum is determined by leaf properties and canopy structure, whereas top-of-canopy (TOC) SIF is also affected by the temperature dependent efficiencies of photochemical and non-photochemical quenching of fluorescence. With the model 'Soil Canopy Observation of Photosynthesis and Energy fluxes (SCOPE), we differentiated leaf optical parameters and canopy structure from the fluorescence quantum emission efficiency (FQE), i.e. the ratio of fluorescence production to light absorption of photosystems. The leaf optical and canopy structure parameters were retrieved from R by inversion of the radiative transfer module 'RTMo' of SCOPE. The retrieved parameters were further used to estimate the FQE from SIF measurements. It appeared that both the leaf water content CW and the FQE responded to the heat wave, but the responses were different for C3 and C4 crops. A slight reduction of CW occurred in C3 crops between the two days, but not in the C4 crop. The reduction of FQE was only significant in C3 crops, and ranged from 18% to 31% for various C3 species. These findings agree with the general knowledge that C4 plants are better adapted to high temperature than C3 plants, and comply with simulations from a biochemical model for C3 and C4 crops in SCOPE. It is concluded that the combination of hyperspectral R and SIF enables the differentiation of long-term and short term responses to heat stress.

Linearized spectrum correlation analysis for line emission measurements

NASA Astrophysics Data System (ADS)

Nishizawa, T.; Nornberg, M. D.; Den Hartog, D. J.; Sarff, J. S.

2017-08-01

A new spectral analysis method, Linearized Spectrum Correlation Analysis (LSCA), for charge exchange and passive ion Doppler spectroscopy is introduced to provide a means of measuring fast spectral line shape changes associated with ion-scale micro-instabilities. This analysis method is designed to resolve the fluctuations in the emission line shape from a stationary ion-scale wave. The method linearizes the fluctuations around a time-averaged line shape (e.g., Gaussian) and subdivides the spectral output channels into two sets to reduce contributions from uncorrelated fluctuations without averaging over the fast time dynamics. In principle, small fluctuations in the parameters used for a line shape model can be measured by evaluating the cross spectrum between different channel groupings to isolate a particular fluctuating quantity. High-frequency ion velocity measurements (100-200 kHz) were made by using this method. We also conducted simulations to compare LSCA with a moment analysis technique under a low photon count condition. Both experimental and synthetic measurements demonstrate the effectiveness of LSCA.

Non-Gaussian statistics and nanosecond dynamics of electrostatic fluctuations affecting optical transitions in proteins.

PubMed

Martin, Daniel R; Matyushov, Dmitry V

2012-08-30

We show that electrostatic fluctuations of the protein-water interface are globally non-Gaussian. The electrostatic component of the optical transition energy (energy gap) in a hydrated green fluorescent protein is studied here by classical molecular dynamics simulations. The distribution of the energy gap displays a high excess in the breadth of electrostatic fluctuations over the prediction of the Gaussian statistics. The energy gap dynamics include a nanosecond component. When simulations are repeated with frozen protein motions, the statistics shifts to the expectations of linear response and the slow dynamics disappear. We therefore suggest that both the non-Gaussian statistics and the nanosecond dynamics originate largely from global, low-frequency motions of the protein coupled to the interfacial water. The non-Gaussian statistics can be experimentally verified from the temperature dependence of the first two spectral moments measured at constant-volume conditions. Simulations at different temperatures are consistent with other indicators of the non-Gaussian statistics. In particular, the high-temperature part of the energy gap variance (second spectral moment) scales linearly with temperature and extrapolates to zero at a temperature characteristic of the protein glass transition. This result, violating the classical limit of the fluctuation-dissipation theorem, leads to a non-Boltzmann statistics of the energy gap and corresponding non-Arrhenius kinetics of radiationless electronic transitions, empirically described by the Vogel-Fulcher-Tammann law.

The source of electrostatic fluctuations in the solar-wind

NASA Technical Reports Server (NTRS)

Lemons, D. S.; Asbridge, J. R.; Bame, S. J.; Feldman, W. C.; Gary, S. P.; Gosling, J. T.

1979-01-01

Solar wind electron and ion distribution functions measured simultaneously with or close to times of intense electrostatic fluctuations are subjected to a linear Vlasov stability analysis. Although all distributions tested were found to be stable, the analysis suggests that the ion beam instability is the most likely source of the fluctuations.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies.

PubMed

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia ( pml ) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

PubMed

Chowdhury, Rajdeep; Amin, Md Asif; Bhattacharyya, Kankan

2015-08-27

Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

Fluorescence Intermittency and Nanodot Evolution in Graphene Oxide

NASA Astrophysics Data System (ADS)

Ruth, Anthony; Michitoshi, Hayashi; McDonald, Matthew; Si, Jixin; Morozov, Yuri; Zapol, Peter; Kuno, Masaru; Janko, Boldizsar

In recent experiments, micron-sized reduced graphene oxide (rGO) flakes were observed to exhibit strong photoluminescence intensity fluctuations, or blinking. Although blinking has been observed in a wide variety of nanoscale emitters, and striking universalities exist across these very different systems, rGO is the first quasi-two dimensional emitter that shows blinking. Despite the widespread presence of blinking at nanoscale, a microscopic mechanism behind this phenomenon remains elusive. Here we provide density functional theory results, analytical calculations, and Monte Carlo simulations to connect the fluorescence trajectories observed in the experiment to microscopic processes. Through Monte Carlo simulations of chemical processes occurring on the graphene oxide surface, we observe the formation and destruction of carbon nanodots. Finally, we use emission characteristics of carbon nanodots from Ab Initio methods to reconstruct the photoluminescence of the macroscopic flake. In particular, we are investigating whether fluorescence intermittency in reduced graphene oxide is an intrinsic optoelectronic property of the nanodot constituents or the result of reversible chemical processes capable of changing the size and number of graphene nanodots. This work was supported by a NASA Space Technology Research Fellowship.

Solution-based single molecule imaging of surface-immobilized conjugated polymers.

PubMed

Dalgarno, Paul A; Traina, Christopher A; Penedo, J Carlos; Bazan, Guillermo C; Samuel, Ifor D W

2013-05-15

The photophysical behavior of conjugated polymers used in modern optoelectronic devices is strongly influenced by their structural dynamics and conformational heterogeneity, both of which are dependent on solvent properties. Single molecule studies of these polymer systems embedded in a host matrix have proven to be very powerful to investigate the fundamental fluorescent properties. However, such studies lack the possibility of examining the relationship between conformational dynamics and photophysical response in solution, which is the phase from which films for devices are deposited. By developing a synthetic strategy to incorporate a biotin moiety as a surface attachment point at one end of a polyalkylthiophene, we immobilize it, enabling us to make the first single molecule fluorescence measurements of conjugated polymers for long periods of time in solution. We identify fluctuation patterns in the fluorescence signal that can be rationalized in terms of photobleaching and stochastic transitions to reversible dark states. Moreover, by using the advantages of solution-based imaging, we demonstrate that the addition of oxygen scavengers improves optical stability by significantly decreasing the photobleaching rates.

2D ratiometric fluorescent pH sensor for tracking of cells proliferation and metabolism.

PubMed

Ma, Jun; Ding, Changqin; Zhou, Jie; Tian, Yang

2015-08-15

Extracellular pH plays a vital role no matter in physiological or pathological studies. In this work, a hydrogel, CD@Nile-FITC@Gel (Gel sensor), entrapping the ratiometric fluorescent probe CD@Nile-FITC was developed. The Gel sensor was successfully used for real-time extracellular pH monitoring. In the case of CD@Nile-FITC, pH-sensitive fluorescent dye fluorescein isothiocyanate (FITC) was chosen as the response signal for H(+) and Nile blue chloride (Nile) as the reference signal. The developed fluorescent probe exhibited high selectivity for pH over other metal ions and amino acids. Meanwhile, the carbon-dots-based inorganic-organic probe demonstrated excellent photostability against long-term light illumination. In order to study the extracellular pH change in processes of cell proliferation and metabolism, CD@Nile-FITC probe was entrapped in sodium alginate gel and consequently formed CD@Nile-FITC@Gel. The MTT assay showed low cytotoxicity of the Gel and the pH titration indicated that it could monitor the pH fluctuations linearly and rapidly within the pH range of 6.0-9.0, which is valuable for physiological pH determination. As expected, the real-time bioimaging of the probe was successfully achieved. Copyright © 2015 Elsevier B.V. All rights reserved.

Spatial and temporal dynamics of receptor for advanced glycation endproducts, integrins, and actin cytoskeleton as probed with fluorescence-based imaging techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Syed, Aleem

Systematic spatial and temporal fluctuations are a fundamental part of any biological process. For example, lateral diffusion of membrane proteins is one of the key mechanisms in their cellular function. Lateral diffusion governs how membrane proteins interact with intracellular, transmembrane, and extracellular components to achieve their function. Herein, fluorescence-based techniques are used to elucidate the dynamics of receptor for advanced glycation end-products (RAGE) and integrin membrane proteins. RAGE is a transmembrane protein that is being used as a biomarker for various diseases. RAGE dependent signaling in numerous pathological conditions is well studied. However, RAGE lateral diffusion in the cell membranemore » is poorly understood. For this purpose, effect of cholesterol, cytoskeleton dynamics, and presence of ligand on RAGE lateral diffusion is investigated.« less

A fluctuation-induced plasma transport diagnostic based upon fast-Fourier transform spectral analysis

NASA Technical Reports Server (NTRS)

Powers, E. J.; Kim, Y. C.; Hong, J. Y.; Roth, J. R.; Krawczonek, W. M.

1978-01-01

A diagnostic, based on fast Fourier-transform spectral analysis techniques, that provides experimental insight into the relationship between the experimentally observable spectral characteristics of the fluctuations and the fluctuation-induced plasma transport is described. The model upon which the diagnostic technique is based and its experimental implementation is discussed. Some characteristic results obtained during the course of an experimental study of fluctuation-induced transport in the electric field dominated NASA Lewis bumpy torus plasma are presented.

Changes in Chromatin Compaction During the Cell Cycle Revealed by Micrometer-Scale Measurement of Molecular Flow in the Nucleus

PubMed Central

Hinde, Elizabeth; Cardarelli, Francesco; Digman, Michelle A.; Gratton, Enrico

2012-01-01

We present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca2+ intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes. PMID:22325293

Molecular cloning, characterization and expression analysis of coagulation factor VII gene in grass carp (Ctenopharyngodon idella).

PubMed

Liu, Qiaolin; Xu, Baohong; Xiao, Tiaoyi; Su, Jianming; Zhong, Lei

2013-08-01

Coagulation factor VII has been studied in several species but, to date, not in grass carp (Ctenopharyngodon idella), a commercially important freshwater fish found in China. In this study, the full-length cDNA of grass carp coagulation factor VII (GcCFVII) was cloned using a RACE-Ready cDNA Kit, grass carp were challenged with a hemorrhagic virus, and temporal expression profiles of GcCFVII in the thymus, gills, liver, spleen, and head kidney were examined at 0 h, 24 h, 48 h, 72 h, 96 h, and 138 h using fluorescence quantitative PCR. The results showed the 1480 bp GcCFVII to contain three conservative motifs: Gla, EGF-CA, and Tryp-SPc, similar to other species. Phylogenetic analysis showed the evolution of GcCFVII gene to be consistent with the evolution of the species. After viral challenge, GcCFVII expression in five tissues of grass carp showed different patterns of fluctuation. These results provide a solid basis for further investigation of GcCFVII and its relationship with grass carp hemorrhage. Copyright © 2013 Elsevier Ltd. All rights reserved.

Markov Chain Monte Carlo in the Analysis of Single-Molecule Experimental Data

NASA Astrophysics Data System (ADS)

Kou, S. C.; Xie, X. Sunney; Liu, Jun S.

2003-11-01

This article provides a Bayesian analysis of the single-molecule fluorescence lifetime experiment designed to probe the conformational dynamics of a single DNA hairpin molecule. The DNA hairpin's conformational change is initially modeled as a two-state Markov chain, which is not observable and has to be indirectly inferred. The Brownian diffusion of the single molecule, in addition to the hidden Markov structure, further complicates the matter. We show that the analytical form of the likelihood function can be obtained in the simplest case and a Metropolis-Hastings algorithm can be designed to sample from the posterior distribution of the parameters of interest and to compute desired estiamtes. To cope with the molecular diffusion process and the potentially oscillating energy barrier between the two states of the DNA hairpin, we introduce a data augmentation technique to handle both the Brownian diffusion and the hidden Ornstein-Uhlenbeck process associated with the fluctuating energy barrier, and design a more sophisticated Metropolis-type algorithm. Our method not only increases the estimating resolution by several folds but also proves to be successful for model discrimination.

Energetic Interrelationship between Spontaneous Low-Frequency Fluctuations in Regional Cerebral Blood Volume, Arterial Blood Pressure, Heart Rate, and Respiratory Rhythm

NASA Astrophysics Data System (ADS)

Katura, Takusige; Yagyu, Akihiko; Obata, Akiko; Yamazaki, Kyoko; Maki, Atsushi; Abe, Masanori; Tanaka, Naoki

2007-07-01

Strong spontaneous fluctuations around 0.1 and 0.3 Hz have been observed in blood-related brain-function measurements such as functional magnetic resonance imaging and optical topography (or functional near-infrared spectroscopy). These fluctuations seem to reflect the interaction between the cerebral circulation system and the systemic circulation system. We took an energetic viewpoint in our analysis of the interrelationships between fluctuations in cerebral blood volume (CBV), mean arterial blood pressure (MAP), heart rate (HR), and respiratory rhythm based on multivariate autoregressive modeling. This approach involves evaluating the contribution of each fluctuation or rhythm to specific ones by performing multivariate spectral analysis. The results we obtained show MAP and HR can account slightly for the fluctuation around 0.1 Hz in CBV, while the fluctuation around 0.3 Hz is derived mainly from the respiratory rhythm. During our presentation, we will report on the effects of posture on the interrelationship between the fluctuations and the respiratory rhythm.

Summing up the noise in gene networks

NASA Astrophysics Data System (ADS)

Paulsson, Johan

2004-01-01

Random fluctuations in genetic networks are inevitable as chemical reactions are probabilistic and many genes, RNAs and proteins are present in low numbers per cell. Such `noise' affects all life processes and has recently been measured using green fluorescent protein (GFP). Two studies show that negative feedback suppresses noise, and three others identify the sources of noise in gene expression. Here I critically analyse these studies and present a simple equation that unifies and extends both the mathematical and biological perspectives.

Multifractal detrended Cross Correlation Analysis of Foreign Exchange and SENSEX fluctuation in Indian perspective

NASA Astrophysics Data System (ADS)

Dutta, Srimonti; Ghosh, Dipak; Chatterjee, Sucharita

2016-12-01

The manuscript studies autocorrelation and cross correlation of SENSEX fluctuations and Forex Exchange Rate in respect to Indian scenario. Multifractal detrended fluctuation analysis (MFDFA) and multifractal detrended cross correlation analysis (MFDXA) were employed to study the correlation between the two series. It was observed that the two series are strongly cross correlated. The change of degree of cross correlation with time was studied and the results are interpreted qualitatively.

From force-fields to photons: MD simulations of dye-labeled nucleic acids and Monte Carlo modeling of FRET

NASA Astrophysics Data System (ADS)

Milas, Peker; Gamari, Ben; Parrot, Louis; Buckman, Richard; Goldner, Lori

2011-11-01

Fluorescence resonance energy transfer (FRET) is a powerful experimental technique for understanding the structural fluctuations and transformations of RNA, DNA and proteins. Molecular dynamics (MD) simulations provide a window into the nature of these fluctuations on a faster time scale inaccessible to experiment. We use Monte Carlo methods to model and compare FRET data from dye-labeled RNA with what might be predicted from the MD simulation. With a few notable exceptions, the contribution of fluorophore and linker dynamics to these FRET measurements has not been investigated. We include the dynamics of the ground state dyes and linkers along with an explicit water solvent in our study of a 16mer double-stranded RNA. Cyanine dyes are attached at either the 3' or 5' ends with a three carbon linker, providing a basis for contrasting the dynamics of similar but not identical molecular structures.

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

PubMed Central

Price, Allen C.; Pilkiewicz, Kevin R.; Graham, Thomas G.W.; Song, Dan; Eaves, Joel D.; Loparo, Joseph J.

2015-01-01

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length. PMID:25992731

A surface-bound molecule that undergoes optically biased Brownian rotation.

PubMed

Hutchison, James A; Uji-i, Hiroshi; Deres, Ania; Vosch, Tom; Rocha, Susana; Müller, Sibylle; Bastian, Andreas A; Enderlein, Jörg; Nourouzi, Hassan; Li, Chen; Herrmann, Andreas; Müllen, Klaus; De Schryver, Frans; Hofkens, Johan

2014-02-01

Developing molecular systems with functions analogous to those of macroscopic machine components, such as rotors, gyroscopes and valves, is a long-standing goal of nanotechnology. However, macroscopic analogies go only so far in predicting function in nanoscale environments, where friction dominates over inertia. In some instances, ratchet mechanisms have been used to bias the ever-present random, thermally driven (Brownian) motion and drive molecular diffusion in desired directions. Here, we visualize the motions of surface-bound molecular rotors using defocused fluorescence imaging, and observe the transition from hindered to free Brownian rotation by tuning medium viscosity. We show that the otherwise random rotations can be biased by the polarization of the excitation light field, even though the associated optical torque is insufficient to overcome thermal fluctuations. The biased rotation is attributed instead to a fluctuating-friction mechanism in which photoexcitation of the rotor strongly inhibits its diffusion rate.

Feedback control of persistent-current oscillation based on the atomic-clock technique

NASA Astrophysics Data System (ADS)

Yu, Deshui; Dumke, Rainer

2018-05-01

We propose a scheme of stabilizing the persistent-current Rabi oscillation based on the flux qubit-resonator-atom hybrid structure. The low-Q L C resonator weakly interacts with the flux qubit and maps the persistent-current Rabi oscillation of the flux qubit onto the intraresonator electric field. This oscillating electric field is further coupled to a Rydberg-Rydberg transition of the 87Rb atoms. The Rabi-frequency fluctuation of the flux qubit is deduced from measuring the atomic population via the fluorescence detection and stabilized by feedback controlling the external flux bias. Our numerical simulation indicates that the feedback-control method can efficiently suppress the background fluctuations in the flux qubit, especially in the low-frequency limit. This technique may be extensively applicable to different types of superconducting circuits, paving a way to long-term-coherence superconducting quantum information processing.

Chromosome and mitotic spindle dynamics in fission yeast kinesin-8 mutants

NASA Astrophysics Data System (ADS)

Crapo, Ammon M.; Gergley, Zachary R.; McIntosh, J. Richard; Betterton, M. D.

2014-03-01

Fission yeast proteins Klp5p and Klp6p are plus-end directed motors of the kinesin-8 family which promote microtubule (MT) depolymerization and also affect chromosome segregation, but the mechanism of these activities is not well understood. Using live-cell time-lapse fluorescence microscopy of fission yeast wild-type (WT) and klp5/6 mutant strains, we quantify and compare the dynamics of kinetochore motion and mitotic spindle length in 3D. In WT cells, the spindle, once formed, remains a consistent size and chromosomes are correctly organized and segregated. In kinesin-8 mutants, spindles undergo large length fluctuations of several microns. Kinetochore motions are also highly fluctuating, with kinetochores frequently moving away from the spindle rather than toward it. We observe transient pushing of chromosomes away from the spindle by as much as 10 microns in distance.

Fluctuation dynamics of exchange rates on Polish financial market

NASA Astrophysics Data System (ADS)

Orłowski, A.; Struzik, Z. R.; Syczewska, E.; Załuska-Kotur, M. A.

2004-12-01

We show results of local fluctuation analysis, probability distributions, and fractional integration analysis for nominal exchange rates of the Polish zloty versus two foreign currencies (US dollar and German mark/euro). The results confirm the rapid change of the volatility pattern in August 1997. We compare the type of the fluctuation behavior before and after this date.

Flagellar dynamics reveal the distribution of chemotactic signaling molecule CheY-P in E. coli

NASA Astrophysics Data System (ADS)

Bano, Roshni; Mears, Patrick; Chemla, Yann; Golding, Ido

E. colicells swim in a random walk consisting of ''runs'' - during which the flagella that propel the cell rotate counter-clockwise (CCW) - and ''tumbles''- during which one or more flagella rotate clockwise (CW). The tumbling frequency is modulated by the phosphorylation state of the signaling molecule CheY, which depends on the cell's environment. Phosphorylated CheY (CheY-P) binds to a flagellar motor and engenders a change in rotation state from CCW to CW. Despite advances in methods used to observe chemotactic signaling, it remains a challenge to measure the CheY-P level in cells directly. Here, we used an optical trap assay coupled with fluorescence microscopy to observe the dynamics of fluorescently labelled flagella in individual cells. By measuring the distribution of flagellar states in multi-flagellated cells and using our recent finding that each flagellar motor independently measures the cellular CheY-P concentration, we are able to extract the probability distribution of the CheY-P level in the cell. This analysis reveals the magnitude of fluctuations in chemotactic signaling in the live cell. We further investigate how this CheY-P distribution changes when cells encounter chemical gradients and perform chemotaxis. This work was supported by the National Science Foundation (NSF) through the Centre for Physics of Living Cells (CPLC).

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells.

PubMed

Regmi, Raju; Winkler, Pamina M; Flauraud, Valentin; Borgman, Kyra J E; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F

2017-10-11

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

NASA Astrophysics Data System (ADS)

Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

2017-10-01

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

Nonintrusive Measurements for High-Speed, Supersonic, and Hypersonic Flows

NASA Astrophysics Data System (ADS)

Bonnet, J. P.; Grésillon, D.; Taran, J. P.

The need to develop new diagnostics for turbulent flows at supersonic and hypersonic regimes is discussed. New experimental results can be obtained in supersonic flows by using the collective light scattering method. Typical results obtained by this method in a supersonic mixing layer are illustrated. The collective light scattering method is a directional densitometer (with a new type of spectral analysis of density fluctuations), a nonparticle anemometer, a Mach-meter (or thermometer), and a directional remote microphone. Various other optical techniques that can be applied for point, line-of-sight, or imaging measurements are reviewed. For point measurements, light-scattering methods such as Raman, Rayleigh, or electron beam fluorescence are discussed, but only briefly, since they are of little use, especially when enthalpy is very high and flow naturally bright. Emphasis is placed instead on nonlinear laser spectroscopy such as coherent anti-Stokes Raman scattering, which has recently been successful in determining temperature and density in high-enthalpy shocks. A description of diode laser absorption spectroscopy follows. A high data-rate instrument now routinely gives the static temperature and the velocity of the stream in the hot shot facility F4 of ONERA, at stagnation enthalpies in excess of 15 MJ/kg. Finally, electron beam fluorescence imaging in the same facility has made it possible to perform measurements of velocity across the external boundary layer into the flow core using a high-energy-pulsed electron gun.

Enhanced photoluminescence of corrugated Al2O3 film assisted by colloidal CdSe quantum dots.

PubMed

Bai, Zhongchen; Hao, Licai; Zhang, Zhengping; Huang, Zhaoling; Qin, Shuijie

2017-05-19

We present the enhanced photoluminescence (PL) of a corrugated Al 2 O 3 film enabled by colloidal CdSe quantum dots. The colloidal CdSe quantum dots are fabricated directly on a corrugated Al 2 O 3 substrate using an electrochemical deposition (ECD) method in a microfluidic system. The photoluminescence is excited by using a 150 nm diameter ultraviolet laser spot of a scanning near-field optical microscope. Owing to the electron transfer from the conduction band of the CdSe quantum dots to that of Al 2 O 3 , the enhanced photoluminescence effect is observed, which results from the increase in the recombination rate of electrons and holes on the Al 2 O 3 surface and the reduction in the fluorescence of the CdSe quantum dots. A periodically-fluctuating fluorescent spectrum was exhibited because of the periodical wire-like corrugated Al 2 O 3 surface serving as an optical grating. The spectral topographic map around the fluorescence peak from the Al 2 O 3 areas covered with CdSe quantum dots was unique and attributed to the uniform deposition of CdSe QDs on the corrugated Al 2 O 3 surface. We believe that the microfluidic ECD system and the surface enhanced fluorescence method described in this paper have potential applications in forming uniform optoelectronic films of colloidal quantum dots with controllable QD spacing and in boosting the fluorescent efficiency of weak PL devices.

Information Transfer Analysis of Spontaneous Low-frequency Fluctuations in Cerebral Hemodynamics and Cardiovascular Dynamics

NASA Astrophysics Data System (ADS)

Katura, Takusige; Tanaka, Naoki; Obata, Akiko; Sato, Hiroki; Maki, Atsushi

2005-08-01

In this study, from the information-theoretic viewpoint, we analyzed the interrelation between the spontaneous low-frequency fluctuations around 0.1Hz in the hemoglobin concentration in the cerebral cortex, mean arterial blood pressure and the heart rate. For this analysis, as measures of information transfer, we used transfer entropy (TE) proposed for two-factor systems by Schreiber and intrinsic transfer entropy (ITE) introduced for further analysis of three-factor systems by extending the original TE. In our analysis, information transfer analysis based on both TE and ITE suggests the systemic cardiovascular fluctuations alone cannot account for the cerebrovascular fluctuations, that is, the regulation of the regional cerebral energetic metabolism is important as a candidate of its generation mechanism Such an information transfer analysis seems useful to reveal the interrelation between the elements regulated each other in a complex manner.

Synchrony in broadband fluctuation and the 2008 financial crisis.

PubMed

Lin, Der Chyan

2013-01-01

We propose phase-like characteristics in scale-free broadband processes and consider fluctuation synchrony based on the temporal signature of significant amplitude fluctuation. Using wavelet transform, successful captures of similar fluctuation pattern between such broadband processes are demonstrated. The application to the financial data leading to the 2008 financial crisis reveals the transition towards a qualitatively different dynamical regime with many equity price in fluctuation synchrony. Further analysis suggests an underlying scale free "price fluctuation network" with large clustering coefficient.

Single Molecular Level Probing of Structure and Dynamics of Papain Under Denaturation.

PubMed

Sengupta, Bhaswati; Chaudhury, Apala; Das, Nilimesh; Sen, Pratik

2017-01-01

Papain is a cysteine protease enzyme present in papaya and known to help in digesting peptide. Thus the structure and function of the active site of papain is of interest. The objective of present study is to unveil the overall structural transformation and the local structural change around the active site of papain as a function of chemical denaturant. Papain has been tagged at Cys-25 with a thiol specific fluorescence probe N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA). Guanidine hydrochloride (GnHCl) has been used as the chemical denaturant. Steady state, time-resolved, and single molecular level fluorescence techniques was applied to map the change in the local environment. It is found that papain undergoes a two-step denaturation in the presence of GnHCl. Fluorescence correlation spectroscopic (FCS) data indicate that the size (hydrodynamic diameter) of native papain is ~36.8 Å, which steadily increases to ~53 Å in the presence of 6M GnHCl. FCS study also reveals that the conformational fluctuation time of papain is 6.3 µs in its native state, which decreased to 2.7 µs in the presence of 0.75 M GnHCl. Upon further increase in GnHCl concentration the conformational fluctuation time increase monotonically till 6 M GnHCl, where the time constant is measured as 14 µs. On the other hand, the measurement of ellipticity, hence the helical structure, by circular dichroism spectroscopy is found to be incapable to capture such structural transformation. It is concluded that in the presence of small amount of GnHCl the active site of papain takes up a more compact structure (although the overall size increases) than in the native state, which has been designated as the intermediate state. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Endogenous and exogenous dynamics in the fluctuations of capital fluxes. An empirical analysis of the Chinese stock market

NASA Astrophysics Data System (ADS)

Jiang, Z.-Q.; Guo, L.; Zhou, W.-X.

2007-06-01

A phenomenological investigation of the endogenous and exogenous dynamics in the fluctuations of capital fluxes is carried out on the Chinese stock market using mean-variance analysis, fluctuation analysis, and their generalizations to higher orders. Non-universal dynamics have been found not only in the scaling exponent α, which is different from the universal values 1/2 and 1, but also in the distributions of the ratio η= σexo / σendo of individual stocks. Both the scaling exponent α of fluctuations and the Hurst exponent Hi increase in logarithmic form with the time scale Δt and the mean traded value per minute , respectively. We find that the scaling exponent αendo of the endogenous fluctuations is independent of the time scale. Multiscaling and multifractal features are observed in the data as well. However, the inhomogeneous impact model is not verified.

Detrended fluctuation analysis of human brain electroencephalogram

NASA Astrophysics Data System (ADS)

Pan, C. P.; Zheng, B.; Wu, Y. Z.; Wang, Y.; Tang, X. W.

2004-08-01

With the detrended fluctuation analysis, we investigate dynamics of human brain electroencephalogram. Long-range temporal correlation and scaling behavior are observed, and certain characteristic of the Alzheimer's disease is revealed.

The Statistical Value of Raw Fluorescence Signal in Luminex xMAP Based Multiplex Immunoassays

PubMed Central

Breen, Edmond J.; Tan, Woei; Khan, Alamgir

2016-01-01

Tissue samples (plasma, saliva, serum or urine) from 169 patients classified as either normal or having one of seven possible diseases are analysed across three 96-well plates for the presences of 37 analytes using cytokine inflammation multiplexed immunoassay panels. Censoring for concentration data caused problems for analysis of the low abundant analytes. Using fluorescence analysis over concentration based analysis allowed analysis of these low abundant analytes. Mixed-effects analysis on the resulting fluorescence and concentration responses reveals a combination of censoring and mapping the fluorescence responses to concentration values, through a 5PL curve, changed observed analyte concentrations. Simulation verifies this, by showing a dependence on the mean florescence response and its distribution on the observed analyte concentration levels. Differences from normality, in the fluorescence responses, can lead to differences in concentration estimates and unreliable probabilities for treatment effects. It is seen that when fluorescence responses are normally distributed, probabilities of treatment effects for fluorescence based t-tests has greater statistical power than the same probabilities from concentration based t-tests. We add evidence that the fluorescence response, unlike concentration values, doesn’t require censoring and we show with respect to differential analysis on the fluorescence responses that background correction is not required. PMID:27243383

Detecting weak position fluctuations from encoder signal using singular spectrum analysis.

PubMed

Xu, Xiaoqiang; Zhao, Ming; Lin, Jing

2017-11-01

Mechanical fault or defect will cause some weak fluctuations to the position signal. Detection of such fluctuations via encoders can help determine the health condition and performance of the machine, and offer a promising alternative to the vibration-based monitoring scheme. However, besides the interested fluctuations, encoder signal also contains a large trend and some measurement noise. In applications, the trend is normally several orders larger than the concerned fluctuations in magnitude, which makes it difficult to detect the weak fluctuations without signal distortion. In addition, the fluctuations can be complicated and amplitude modulated under non-stationary working condition. To overcome this issue, singular spectrum analysis (SSA) is proposed for detecting weak position fluctuations from encoder signal in this paper. It enables complicated encode signal to be reduced into several interpretable components including a trend, a set of periodic fluctuations and noise. A numerical simulation is given to demonstrate the performance of the method, it shows that SSA outperforms empirical mode decomposition (EMD) in terms of capability and accuracy. Moreover, linear encoder signals from a CNC machine tool are analyzed to determine the magnitudes and sources of fluctuations during feed motion. The proposed method is proven to be feasible and reliable for machinery condition monitoring. Copyright © 2017 ISA. Published by Elsevier Ltd. All rights reserved.

Electrostatic Interactions at the Dimer Interface Stabilize the E. coli β Sliding Clamp.

PubMed

Purohit, Anirban; England, Jennifer K; Douma, Lauren G; Tondnevis, Farzaneh; Bloom, Linda B; Levitus, Marcia

2017-08-22

Sliding clamps are ring-shaped oligomeric proteins that encircle DNA and associate with DNA polymerases for processive DNA replication. The dimeric Escherichia coli β-clamp is closed in solution but must adopt an open conformation to be assembled onto DNA by a clamp loader. To determine what factors contribute to the stability of the dimer interfaces in the closed conformation and how clamp dynamics contribute to formation of the open conformation, we identified conditions that destabilized the dimer and measured the effects of these conditions on clamp dynamics. We characterized the role of electrostatic interactions in stabilizing the β-clamp interface. Increasing salt concentration results in decreased dimer stability and faster subunit dissociation kinetics. The equilibrium dissociation constant of the dimeric clamp varies with salt concentration as predicted by simple charge-screening models, indicating that charged amino acids contribute to the remarkable stability of the interface at physiological salt concentrations. Mutation of a charged residue at the interface (Arg-103) weakens the interface significantly, whereas effects are negligible when a hydrophilic (Ser-109) or a hydrophobic (Ile-305) amino acid is mutated instead. It has been suggested that clamp opening by the clamp loader takes advantage of spontaneous opening-closing fluctuations at the clamp's interface, but our time-resolved fluorescence and fluorescence correlation experiments rule out conformational fluctuations that lead to a significant fraction of open states. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Inertial picobalance reveals fast mass fluctuations in mammalian cells

NASA Astrophysics Data System (ADS)

Martínez-Martín, David; Fläschner, Gotthold; Gaub, Benjamin; Martin, Sascha; Newton, Richard; Beerli, Corina; Mercer, Jason; Gerber, Christoph; Müller, Daniel J.

2017-10-01

The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a ‘picobalance’), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

Atomistic observation and simulation analysis of spatio-temporal fluctuations during radiation-induced amorphization.

PubMed

Watanabe, Seiichi; Hoshino, Misaki; Koike, Takuto; Suda, Takanori; Ohnuki, Soumei; Takahashi, Heishichirou; Lam, Nighi Q

2003-01-01

We performed a dynamical-atomistic study of radiation-induced amorphization in the NiTi intermetallic compound using in situ high-resolution high-voltage electron microscopy and molecular dynamics simulations in connection with image simulation. Spatio-temporal fluctuations as non-equilibrium fluctuations in an energy-dissipative system, due to transient atom-cluster formation during amorphization, were revealed by the present spatial autocorrelation analysis.

Near real time, accurate, and sensitive microbiological safety monitoring using an all-fibre spectroscopic fluorescence system

NASA Astrophysics Data System (ADS)

Vanholsbeeck, F.; Swift, S.; Cheng, M.; Bogomolny, E.

2013-11-01

Enumeration of microorganisms is an essential microbiological task for many industrial sectors and research fields. Various tests for detection and counting of microorganisms are used today. However most of the current methods to enumerate bacteria require either long incubation time for limited accuracy, or use complicated protocols along with bulky equipment. We have developed an accurate, all-fibre spectroscopic system to measure fluorescence signal in-situ. In this paper, we examine the potential of this setup for near real time bacteria enumeration in aquatic environment. The concept is based on a well-known phenomenon that the fluorescence quantum yields of some nucleic acid stains significantly increase upon binding with nucleic acids of microorganisms. In addition we have used GFP labeled organisms. The fluorescence signal increase can be correlated to the amount of nucleic acid present in the sample. In addition we have used GFP labeled organisms. Our results show that we are able to detect a wide range of bacteria concentrations without dilution or filtration (1-108 CFU/ml) using different optical probes we designed. This high sensitivity is due to efficient light delivery with an appropriate collection volume and in situ fluorescence detection as well as the use of a sensitive CCD spectrometer. By monitoring the laser power, we can account for laser fluctuations while measuring the fluorescence signal which improves as well the system accuracy. A synchronized laser shutter allows us to achieve a high SNR with minimal integration time, thereby reducing the photobleaching effect. In summary, we conclude that our optical setup may offer a robust method for near real time bacterial detection in aquatic environment.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

PubMed Central

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology. PMID:29888200

Fluorescence intermittency originates from reclustering in two-dimensional organic semiconductors

NASA Astrophysics Data System (ADS)

Ruth, Anthony; Hayashi, Michitoshi; Zapol, Peter; Si, Jixin; McDonald, Matthew P.; Morozov, Yurii V.; Kuno, Masaru; Jankó, Boldizsár

2017-02-01

Fluorescence intermittency or blinking is observed in nearly all nanoscale fluorophores. It is characterized by universal power-law distributions in on- and off-times as well as 1/f behaviour in corresponding emission power spectral densities. Blinking, previously seen in confined zero- and one-dimensional systems has recently been documented in two-dimensional reduced graphene oxide. Here we show that unexpected blinking during graphene oxide-to-reduced graphene oxide photoreduction is attributed, in large part, to the redistribution of carbon sp2 domains. This reclustering generates fluctuations in the number/size of emissive graphenic nanoclusters wherein multiscale modelling captures essential experimental aspects of reduced graphene oxide's absorption/emission trajectories, while simultaneously connecting them to the underlying photochemistry responsible for graphene oxide's reduction. These simulations thus establish causality between currently unexplained, long timescale emission intermittency in a quantum mechanical fluorophore and identifiable chemical reactions that ultimately lead to switching between on and off states.

Rich stochastic dynamics of co-doped Er:Yb fluorescence upconversion nanoparticles in the presence of thermal, non-conservative, harmonic and optical forces

NASA Astrophysics Data System (ADS)

Nome, Rene A.; Sorbello, Cecilia; Jobbágy, Matías; Barja, Beatriz C.; Sanches, Vitor; Cruz, Joyce S.; Aguiar, Vinicius F.

2017-03-01

The stochastic dynamics of individual co-doped Er:Yb upconversion nanoparticles (UCNP) were investigated from experiments and simulations. The UCNP were characterized by high-resolution scanning electron microscopy, dynamic light scattering, and zeta potential measurements. Single UCNP measurements were performed by fluorescence upconversion micro-spectroscopy and optical trapping. The mean-square displacement (MSD) from single UCNP exhibited a time-dependent diffusion coefficient which was compared with Brownian dynamics simulations of a viscoelastic model of harmonically bound spheres. Experimental time-dependent two-dimensional trajectories of individual UCNP revealed correlated two-dimensional nanoparticle motion. The measurements were compared with stochastic trajectories calculated in the presence of a non-conservative rotational force field. Overall, the complex interplay of UCNP adhesion, thermal fluctuations and optical forces led to a rich stochastic behavior of these nanoparticles.

Real-time intravital imaging of pH variation associated with osteoclast activity.

PubMed

Maeda, Hiroki; Kowada, Toshiyuki; Kikuta, Junichi; Furuya, Masayuki; Shirazaki, Mai; Mizukami, Shin; Ishii, Masaru; Kikuchi, Kazuya

2016-08-01

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

Optical spectroscopy and photo modification of individual single-photon emitters in hexagonal boron nitride

NASA Astrophysics Data System (ADS)

Jayakumar, Harishankar; Shotan, Zav; Considine, Christopher; Mazkoit, Mažena; Fedder, Helmut; Wrachtrup, Joerg; Alkauskas, Audrius; Doherty, Marcus; Menon, Vinod; Meriles, Carlos

Fluorescent defects recently observed under ambient conditions in hexagonal boron nitride (h-BN) promise to open novel opportunities for the implementation of on-chip photonic devices that rely on identical photons from single emitters. Here we report on the room temperature photo-luminescence dynamics of individual emitters in multilayer h-BN flakes exposed to blue laser light. Comparison of optical spectra recorded at successive times reveals considerable spectral diffusion, possibly the result of slowly fluctuating, trapped-carrier-induced stark shifts. Large spectral jumps - reaching up to 100 nm - followed by bleaching are observed in most cases upon prolonged exposure to blue light, an indication of one-directional, photo-chemical changes likely taking place on the flake surface. Remarkably, only a fraction of the observed emitters also fluoresces on green illumination suggesting a more complex optical excitation dynamics than previously anticipated and raising questions on the physical nature of the atomic defect at play.

Heavy-Tailed Fluctuations in the Spiking Output Intensity of Semiconductor Lasers with Optical Feedback

PubMed Central

2016-01-01

Although heavy-tailed fluctuations are ubiquitous in complex systems, a good understanding of the mechanisms that generate them is still lacking. Optical complex systems are ideal candidates for investigating heavy-tailed fluctuations, as they allow recording large datasets under controllable experimental conditions. A dynamical regime that has attracted a lot of attention over the years is the so-called low-frequency fluctuations (LFFs) of semiconductor lasers with optical feedback. In this regime, the laser output intensity is characterized by abrupt and apparently random dropouts. The statistical analysis of the inter-dropout-intervals (IDIs) has provided many useful insights into the underlying dynamics. However, the presence of large temporal fluctuations in the IDI sequence has not yet been investigated. Here, by applying fluctuation analysis we show that the experimental distribution of IDI fluctuations is heavy-tailed, and specifically, is well-modeled by a non-Gaussian stable distribution. We find a good qualitative agreement with simulations of the Lang-Kobayashi model. Moreover, we uncover a transition from a less-heavy-tailed state at low pump current to a more-heavy-tailed state at higher pump current. Our results indicate that fluctuation analysis can be a useful tool for investigating the output signals of complex optical systems; it can be used for detecting underlying regime shifts, for model validation and parameter estimation. PMID:26901346

Interpreting the Holocene fluctuations of Quelccaya Ice Cap, Peru: using a combination of glacial and non-glacial lake records

NASA Astrophysics Data System (ADS)

Stroup, J. S.; Kelly, M. A.; Lowell, T. V.; Smith, C.; Beal, S. A., Jr.; Tapia, P. M.

2016-12-01

The past fluctuations of Quelccaya Ice Cap (QIC) are an indicator of tropical paleoclimate. At QIC, ice core and glacial geological records provide late Holocene climate constraints. However, early and middle Holocene QIC fluctuations are less well-known. To interpret past QIC fluctuations, we present Holocene-long lake sediment records from Challpacocha, a lake fed by QIC meltwater, and Yanacocha, a lake that has not received meltwater during the Holocene. To assess the clastic sediment delivered to Challpacocha by QIC meltwater, we compare visual stratigraphy, X-ray fluorescence chemistry, grainsize, loss on ignition and clastic flux records from both lakes (additional Yanacocha proxies are presented by Axford et al. (this meeting, abstract 157985)). We compare the meltwater derived clastic sediment record from Challpacocha with moraine and stratigraphic records of past ice extents during the late Holocene. This comparison indicates that clastic sediment flux in Challpacocha increased during QIC recession and decreased during QIC advance, or significantly reduced QIC extent. We then use the Challpacocha clastic sediment record to interpret early and middle Holocene QIC fluctuations. Based on the Challpacocha sediment record, combined with prior work, we suggest that from 11 to 6.5 ka QIC was similar to or smaller than its late Holocene extent. From 6.9 to 6.5 ka QIC may have been absent from the landscape. At 3-2.4 and 0.62-0.31 ka QIC experienced the most extensive Holocene fluctuations. We compare the clastic sediment fluxes from Challpacocha and Pacococha (a nearby lake fed by QIC; Rodbell et al., 2008) to infer QIC expansion between 6.5-5 ka. This is supported by 14C ages of in-situ subfossil plants which indicate ice advance at 6.3-4.7 ka (Thompson et al., 2006, 2013; Buffen et al., 2009). Our study highlights the value of using multiple datasets to improve lake sediment record interpretations.

Super-resolution study of polymer mobility fluctuations near c*.

PubMed

King, John T; Yu, Changqian; Wilson, William L; Granick, Steve

2014-09-23

Nanoscale dynamic heterogeneities in synthetic polymer solutions are detected using super-resolution optical microscopy. To this end, we map concentration fluctuations in polystyrene-toluene solutions with spatial resolution below the diffraction limit, focusing on critical fluctuations near the polymer overlap concentration, c*. Two-photon super-resolution microscopy was adapted to be applicable in an organic solvent, and a home-built STED-FCS system with stimulated emission depletion (STED) was used to perform fluorescence correlation spectroscopy (FCS). The polystyrene serving as the tracer probe (670 kg mol(-1), radius of gyration RG ≈ 35 nm, end-labeled with a bodipy derivative chromophore) was dissolved in toluene at room temperature (good solvent) and mixed with matrix polystyrene (3,840 kg mol(-1), RG ≈ 97 nm, Mw/Mn = 1.04) whose concentration was varied from dilute to more than 10c*. Whereas for dilute solutions the intensity-intensity correlation function follows a single diffusion process, it splits starting at c* to imply an additional relaxation process provided that the experimental focal area does not greatly exceed the polymer blob size. We identify the slower mode as self-diffusion and the increasingly rapid mode as correlated segment fluctuations that reflect the cooperative diffusion coefficient, Dcoop. These real-space measurements find quantitative agreement between correlation lengths inferred from dynamic measurements and those from determining the limit below which diffusion coefficients are independent of spot size. This study is considered to illustrate the potential of importing into polymer science the techniques of super-resolution imaging.

Barrancaceae: A new green algal lineage with structural and behavioral adaptations to a fluctuating environment.

PubMed

Caisová, Lenka; Reyes, Carolina Pérez; Álamo, Virginia Cruz; Quintana, Antera Martel; Surek, Barbara; Melkonian, Michael

2015-09-01

To enhance our knowledge of the diversity of microalgae, a phycological survey of the Canary Islands (Spain) was undertaken. Here we report the discovery of a (semi)terrestrial green filamentous alga isolated from a steep volcanic canyon on La Palma. This alga is continually exposed to changing weather conditions (floods vs. droughts) and thus provides a good opportunity to investigate possible adaptations to a semiterrestrial habitat with large fluctuations of environmental parameters. We used axenic cultures, simulated flood and drought stresses and studied their effect on the life history of the alga using light, confocal laser scanning and scanning electron microscopy including fluorescent staining. Furthermore, phylogenetic analyses using rDNA sequence comparisons were performed. Three specific life-history traits that likely represent adaptations to the fluctuating environment of the canyon were observed: (1) fragmentation through "filament splitting", a unique branching mechanism not reported before in algae and initiated by formation of oblique cross walls, (2) aplanospore formation, and (3) reproduction by multiflagellate zoospores with 4-24 flagella arranged in groups of four. Phylogenetic analyses identified the alga as Barranca multiflagellata gen. et sp. nov. (Barrancaceae fam. nov., Chaetophorales, Chlorophyceae). Moreover, the Chaetophoraceae Greville, 1824 was emended and a new family, Uronemataceae (fam. nov.) erected. The discovery of Barrancaceae fam. nov. highlights the importance of investigating nonconventional habitats to explore microalgal diversity. The reproductive versatility demonstrated by Barranca suggests adaptation to a semiterrestrial habitat with large fluctuations in water availability. © 2015 Botanical Society of America.

Fractal scaling analysis of groundwater dynamics in confined aquifers

NASA Astrophysics Data System (ADS)

Tu, Tongbi; Ercan, Ali; Kavvas, M. Levent

2017-10-01

Groundwater closely interacts with surface water and even climate systems in most hydroclimatic settings. Fractal scaling analysis of groundwater dynamics is of significance in modeling hydrological processes by considering potential temporal long-range dependence and scaling crossovers in the groundwater level fluctuations. In this study, it is demonstrated that the groundwater level fluctuations in confined aquifer wells with long observations exhibit site-specific fractal scaling behavior. Detrended fluctuation analysis (DFA) was utilized to quantify the monofractality, and multifractal detrended fluctuation analysis (MF-DFA) and multiscale multifractal analysis (MMA) were employed to examine the multifractal behavior. The DFA results indicated that fractals exist in groundwater level time series, and it was shown that the estimated Hurst exponent is closely dependent on the length and specific time interval of the time series. The MF-DFA and MMA analyses showed that different levels of multifractality exist, which may be partially due to a broad probability density distribution with infinite moments. Furthermore, it is demonstrated that the underlying distribution of groundwater level fluctuations exhibits either non-Gaussian characteristics, which may be fitted by the Lévy stable distribution, or Gaussian characteristics depending on the site characteristics. However, fractional Brownian motion (fBm), which has been identified as an appropriate model to characterize groundwater level fluctuation, is Gaussian with finite moments. Therefore, fBm may be inadequate for the description of physical processes with infinite moments, such as the groundwater level fluctuations in this study. It is concluded that there is a need for generalized governing equations of groundwater flow processes that can model both the long-memory behavior and the Brownian finite-memory behavior.

[Periodic fluctuation features of air temperature, precipitation, and aboveground net primary production of alpine meadow ecosystem on Qinghai-Tibetan Plateau].

PubMed

Zhang, Fa-wei; Li, Hong-qin; Li, Ying-nian; Li, Yi-kang; Lin, Li

2009-03-01

With Mexican Hat function as mother function, a wavelet analysis was conducted on the periodic fluctuation features of air temperature, precipitation, and aboveground net primary production (ANPP) in the Alpine Meadow Ecosystem Research Station, Chinese Academy of Sciences from 1980 to 2007. The results showed that there was a main period of 13 years for the annual fluctuations of air temperature, precipitation, and ANPP. A secondary period of 2 years for the annual fluctuations of air temperature and ANPP had lesser influence, whereas that of 4 years for the annual fluctuation of precipitation had greater effect. Lagged correlation analysis indicated that the annual fluctuation of ANNP was mainly controlled by the air temperature in a 20 years scale and had a weak 5-9 years lag effect, but there was a less correlation between ANPP and precipitation.

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices

PubMed Central

Abdelfattah, Ahmed S.; Farhi, Samouil L.; Zhao, Yongxin; Brinks, Daan; Zou, Peng; Ruangkittisakul, Araya; Platisa, Jelena; Pieribone, Vincent A.; Ballanyi, Klaus; Cohen, Adam E.

2016-01-01

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation. SIGNIFICANCE STATEMENT Fluorescent-protein-based voltage indicators enable imaging of the electrical activity of many genetically targeted neurons with high spatial and temporal resolution. Here, we describe the engineering of a bright red fluorescent protein-based voltage indicator designated as FlicR1 (fluorescent indicator for voltage imaging red). FlicR1 has sufficient speed and sensitivity to report single action potentials and voltage fluctuations at frequencies up to 100 Hz in single-trial recordings with wide-field microscopy. Because it is excitable with yellow light, FlicR1 can be used in conjunction with blue-light-activated optogenetic actuators. However, spatially distinct patterns of optogenetic activation and voltage imaging are required to avoid fluorescence artifacts due to photoactivation of the FlicR1 chromophore. PMID:26911693

Spectral and Temporal Laser Fluorescence Analysis Such as for Natural Aquatic Environments

NASA Technical Reports Server (NTRS)

Chekalyuk, Alexander (Inventor)

2015-01-01

An Advanced Laser Fluorometer (ALF) can combine spectrally and temporally resolved measurements of laser-stimulated emission (LSE) for characterization of dissolved and particulate matter, including fluorescence constituents, in liquids. Spectral deconvolution (SDC) analysis of LSE spectral measurements can accurately retrieve information about individual fluorescent bands, such as can be attributed to chlorophyll-a (Chl-a), phycobiliprotein (PBP) pigments, or chromophoric dissolved organic matter (CDOM), among others. Improved physiological assessments of photosynthesizing organisms can use SDC analysis and temporal LSE measurements to assess variable fluorescence corrected for SDC-retrieved background fluorescence. Fluorescence assessments of Chl-a concentration based on LSE spectral measurements can be improved using photo-physiological information from temporal measurements. Quantitative assessments of PBP pigments, CDOM, and other fluorescent constituents, as well as basic structural characterizations of photosynthesizing populations, can be performed using SDC analysis of LSE spectral measurements.

Probing Biomolecular Structures and Dynamics of Single Molecules Using In-Gel Alternating-Laser Excitation

PubMed Central

Santoso, Yusdi; Kapanidis, Achillefs N.

2009-01-01

Gel electrophoresis is a standard biochemical technique used for separating biomolecules on the basis of size and charge. Despite the use of gels in early single-molecule experiments, gel electrophoresis has not been widely adopted for single-molecule fluorescence spectroscopy. We present a novel method that combines gel electrophoresis and single-molecule fluorescence spectroscopy to simultaneously purify and analyze biomolecules in a gel matrix. Our method, in-gel ALEX, uses non-denaturing gels to purify biomolecular complexes of interest from free components, aggregates, and non-specific complexes. The gel matrix also slows down translational diffusion of molecules, giving rise to long, high-resolution time traces without surface immobilization, which allow extended observations of conformational dynamics in a biologically friendly environment. We demonstrated the compatibility of this method with different types of single molecule spectroscopy techniques, including confocal detection and fluorescence-correlation spectroscopy. We demonstrated that in-gel ALEX can be used to study conformational dynamics at the millisecond timescale; by studying a DNA hairpin in gels, we directly observed fluorescence fluctuations due to conformational interconversion between folded and unfolded states. Our method is amenable to the addition of small molecules that can alter the equilibrium and dynamic properties of the system. In-gel ALEX will be a versatile tool for studying structures and dynamics of complex biomolecules and their assemblies. PMID:19863108

Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells.

PubMed

Norcross, Stevie; Trull, Keelan J; Snaider, Jordan; Doan, Sara; Tat, Kiet; Huang, Libai; Tantama, Mathew

2017-11-22

Reactive oxygen species (ROS) mediate both intercellular and intraorganellar signaling, and ROS propagate oxidative stress between cellular compartments such as mitochondria and the cytosol. Each cellular compartment contains its own sources of ROS as well as antioxidant mechanisms, which contribute to dynamic fluctuations in ROS levels that occur during signaling, metabolism, and stress. However, the coupling of redox dynamics between cellular compartments has not been well studied because of the lack of available sensors to simultaneously measure more than one subcellular compartment in the same cell. Currently, the redox-sensitive green fluorescent protein, roGFP, has been used extensively to study compartment-specific redox dynamics because it provides a quantitative ratiometric readout and it is amenable to subcellular targeting as a genetically encoded sensor. Here, we report a new family of genetically encoded fluorescent protein sensors that extend the fluorescence emission of roGFP via Förster-type resonance energy transfer to an acceptor red fluorescent protein for dual-color live-cell microscopy. We characterize the redox and optical properties of the sensor proteins, and we demonstrate that they can be used to simultaneously measure cytosolic and mitochondrial ROS in living cells. Furthermore, we use these sensors to reveal cell-to-cell heterogeneity in redox coupling between the cytosol and mitochondria when neuroblastoma cells are exposed to reductive and metabolic stresses.

Glutathione-stabilized Cu nanoclusters as fluorescent probes for sensing pH and vitamin B1.

PubMed

Luo, Yawen; Miao, Hong; Yang, Xiaoming

2015-11-01

Glutathione (GSH), playing roles as both a reducing reagent and protecting ligand, has been successfully employed for synthesizing Cu nanoclusters (CuNCs@GSH) on the basis of a simple and facile approach. The as-prepared CuNCs exhibited a fluorescence emission at 600nm with a quantum yield (QY) of approximately 3.6%. Subsequently, the CuNCs described here was employed as a broad-range pH sensor by virtue of the fluorescence intensity of CuNCs responding sensitively to pH fluctuating in a linear range of 4.0-12.0. Meanwhile, these prepared CuNCs were applied for detections of vitamin B1 (VB1) on the basis of positively charged VB1 neutralizing the negative surface charge of CuNCs, thus leading to the instability and aggregations of CuNCs, and further facilitating to quench their fluorescence. In addition, the proposed analytical method permitted detecting VB1 with a linear range of 2.0×10(-8)-1.0×10(-4) mol L(-1) as well as a detection limit of 4.6×10(-9) mol L(-1). Eventually, the practicability of this sensing approach was validated by assaying VB1 in human urine samples and pharmaceutical tablets, confirming its potential to broaden avenues for assaying VB1. Copyright © 2015 Elsevier B.V. All rights reserved.

Amine-derived synthetic approach to color-tunable InP/ZnS quantum dots with high fluorescent qualities

NASA Astrophysics Data System (ADS)

Song, Woo-Seuk; Lee, Hye-Seung; Lee, Ju Chul; Jang, Dong Seon; Choi, Yoonyoung; Choi, Moongoo; Yang, Heesun

2013-06-01

High-quality, Cd-free InP quantum dots (QDs) have been conventionally synthesized by exclusively selecting tris(trimethylsilyl)phosphine (P(TMS)3) as a phosphorus (P) precursor, which is problematic from the standpoint of green and economic chemistry. Thus, other synthetic chemistries adopting alternative P sources to P(TMS)3 have been introduced, however, they could not guarantee the production of satisfactorily fluorescence-efficient, color-pure InP QDs. In this study, the unprecedented controlled synthesis of a series of band-gap-tuned InP QDs is demonstrated through a hot-injection of a far safer and cheaper tris(dimethylamino)phosphine in the presence of a key coordinating solvent of oleylamine that enables successful QD nucleation/growth. Effects of the co-existence of Zn additive, the core growth temperature, and the amount of P source injected on the growth behaviors of InP QD are investigated. After ZnS overcoating by a successive injection of 1-dodecanethiol only, high-fluorescence-quality, green-to-red color emission-tunable core/shell QDs of InP/ZnS are obtained. The fluorescent characteristics of different color-emitting QDs desirably exhibit little fluctuations in quantum yield and emission bandwidth, specifically ranging 51-53 % and 60-64 nm, respectively. Lastly, the utility of the introduction of a secondary shelling process in rendering the QDs are more bright, photostable is also proved.

Fluctuation Analysis of Redox Potential to Distinguish Microbial Fe(II) Oxidation.

PubMed

Enright, A M L; Ferris, F G

2016-11-01

We developed a novel method for distinguishing abiotic and biological iron oxidation in liquid media using oxidation-reduction (redox) potential time series data. The instrument and processing algorithm were tested by immersing the tip of a Pt electrode with an Ag-AgCl reference electrode into an active iron-oxidizing biofilm in a groundwater discharge zone, as well as in two abiotic systems: a killed sample and a chemical control from the same site. We used detrended fluctuation analysis to characterize average root mean square fluctuation behavior, which was distinct in the live system. The calculated α value scaling exponents determined by detrended fluctuation analysis were significantly different at p < 0.001. This indicates that time series of electrode response data may be used to distinguish live and abiotic chemical reaction pathways. Due to the simplicity, portability, and small size, it may be suitable for characterization of extraterrestrial environments where water has been observed, such as Mars and Europa. Key Words: Oxidation-reduction potential-Detrended fluctuation analysis-Iron-oxidizing bacteria. Astrobiology 16, 846-852.

Frequency domain analysis of noise in simple gene circuits

NASA Astrophysics Data System (ADS)

Cox, Chris D.; McCollum, James M.; Austin, Derek W.; Allen, Michael S.; Dar, Roy D.; Simpson, Michael L.

2006-06-01

Recent advances in single cell methods have spurred progress in quantifying and analyzing stochastic fluctuations, or noise, in genetic networks. Many of these studies have focused on identifying the sources of noise and quantifying its magnitude, and at the same time, paying less attention to the frequency content of the noise. We have developed a frequency domain approach to extract the information contained in the frequency content of the noise. In this article we review our work in this area and extend it to explicitly consider sources of extrinsic and intrinsic noise. First we review applications of the frequency domain approach to several simple circuits, including a constitutively expressed gene, a gene regulated by transitions in its operator state, and a negatively autoregulated gene. We then review our recent experimental study, in which time-lapse microscopy was used to measure noise in the expression of green fluorescent protein in individual cells. The results demonstrate how changes in rate constants within the gene circuit are reflected in the spectral content of the noise in a manner consistent with the predictions derived through frequency domain analysis. The experimental results confirm our earlier theoretical prediction that negative autoregulation not only reduces the magnitude of the noise but shifts its content out to higher frequency. Finally, we develop a frequency domain model of gene expression that explicitly accounts for extrinsic noise at the transcriptional and translational levels. We apply the model to interpret a shift in the autocorrelation function of green fluorescent protein induced by perturbations of the translational process as a shift in the frequency spectrum of extrinsic noise and a decrease in its weighting relative to intrinsic noise.

Particle Tracking Facilitates Real Time Capable Motion Correction in 2D or 3D Two-Photon Imaging of Neuronal Activity.

PubMed

Aghayee, Samira; Winkowski, Daniel E; Bowen, Zachary; Marshall, Erin E; Harrington, Matt J; Kanold, Patrick O; Losert, Wolfgang

2017-01-01

The application of 2-photon laser scanning microscopy (TPLSM) techniques to measure the dynamics of cellular calcium signals in populations of neurons is an extremely powerful technique for characterizing neural activity within the central nervous system. The use of TPLSM on awake and behaving subjects promises new insights into how neural circuit elements cooperatively interact to form sensory perceptions and generate behavior. A major challenge in imaging such preparations is unavoidable animal and tissue movement, which leads to shifts in the imaging location (jitter). The presence of image motion can lead to artifacts, especially since quantification of TPLSM images involves analysis of fluctuations in fluorescence intensities for each neuron, determined from small regions of interest (ROIs). Here, we validate a new motion correction approach to compensate for motion of TPLSM images in the superficial layers of auditory cortex of awake mice. We use a nominally uniform fluorescent signal as a secondary signal to complement the dynamic signals from genetically encoded calcium indicators. We tested motion correction for single plane time lapse imaging as well as multiplane (i.e., volume) time lapse imaging of cortical tissue. Our procedure of motion correction relies on locating the brightest neurons and tracking their positions over time using established techniques of particle finding and tracking. We show that our tracking based approach provides subpixel resolution without compromising speed. Unlike most established methods, our algorithm also captures deformations of the field of view and thus can compensate e.g., for rotations. Object tracking based motion correction thus offers an alternative approach for motion correction, one that is well suited for real time spike inference analysis and feedback control, and for correcting for tissue distortions.

Particle Tracking Facilitates Real Time Capable Motion Correction in 2D or 3D Two-Photon Imaging of Neuronal Activity

PubMed Central

Aghayee, Samira; Winkowski, Daniel E.; Bowen, Zachary; Marshall, Erin E.; Harrington, Matt J.; Kanold, Patrick O.; Losert, Wolfgang

2017-01-01

The application of 2-photon laser scanning microscopy (TPLSM) techniques to measure the dynamics of cellular calcium signals in populations of neurons is an extremely powerful technique for characterizing neural activity within the central nervous system. The use of TPLSM on awake and behaving subjects promises new insights into how neural circuit elements cooperatively interact to form sensory perceptions and generate behavior. A major challenge in imaging such preparations is unavoidable animal and tissue movement, which leads to shifts in the imaging location (jitter). The presence of image motion can lead to artifacts, especially since quantification of TPLSM images involves analysis of fluctuations in fluorescence intensities for each neuron, determined from small regions of interest (ROIs). Here, we validate a new motion correction approach to compensate for motion of TPLSM images in the superficial layers of auditory cortex of awake mice. We use a nominally uniform fluorescent signal as a secondary signal to complement the dynamic signals from genetically encoded calcium indicators. We tested motion correction for single plane time lapse imaging as well as multiplane (i.e., volume) time lapse imaging of cortical tissue. Our procedure of motion correction relies on locating the brightest neurons and tracking their positions over time using established techniques of particle finding and tracking. We show that our tracking based approach provides subpixel resolution without compromising speed. Unlike most established methods, our algorithm also captures deformations of the field of view and thus can compensate e.g., for rotations. Object tracking based motion correction thus offers an alternative approach for motion correction, one that is well suited for real time spike inference analysis and feedback control, and for correcting for tissue distortions. PMID:28860973

MicroRNA Detection by DNA-Mediated Liposome Fusion.

PubMed

Jumeaux, Coline; Wahlsten, Olov; Block, Stephan; Kim, Eunjung; Chandrawati, Rona; Howes, Philip D; Höök, Fredrik; Stevens, Molly M

2018-03-02

Membrane fusion is a process of fundamental importance in biological systems that involves highly selective recognition mechanisms for the trafficking of molecular and ionic cargos. Mimicking natural membrane fusion mechanisms for the purpose of biosensor development holds great potential for amplified detection because relatively few highly discriminating targets lead to fusion and an accompanied engagement of a large payload of signal-generating molecules. In this work, sequence-specific DNA-mediated liposome fusion is used for the highly selective detection of microRNA. The detection of miR-29a, a known flu biomarker, is demonstrated down to 18 nm within 30 min with high specificity by using a standard laboratory microplate reader. Furthermore, one order of magnitude improvement in the limit of detection is demonstrated by using a novel imaging technique combined with an intensity fluctuation analysis, which is coined two-color fluorescence correlation microscopy. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Microbial community structure in the gut of the New Zealand insect Auckland tree weta (Hemideina thoracica).

PubMed

Waite, David W; Dsouza, Melissa; Biswas, Kristi; Ward, Darren F; Deines, Peter; Taylor, Michael W

2015-05-01

The endemic New Zealand weta is an enigmatic insect. Although the insect is well known by its distinctive name, considerable size, and morphology, many basic aspects of weta biology remain unknown. Here, we employed cultivation-independent enumeration techniques and rRNA gene sequencing to investigate the gut microbiota of the Auckland tree weta (Hemideina thoracica). Fluorescence in situ hybridisation performed on different sections of the gut revealed a bacterial community of fluctuating density, while rRNA gene-targeted amplicon pyrosequencing revealed the presence of a microbial community containing high bacterial diversity, but an apparent absence of archaea. Bacteria were further studied using full-length 16S rRNA gene sequences, with statistical testing of bacterial community membership against publicly available termite- and cockroach-derived sequences, revealing that the weta gut microbiota is similar to that of cockroaches. These data represent the first analysis of the weta microbiota and provide initial insights into the potential function of these microorganisms.

Optimization of Fe2+ supplement in anaerobic digestion accounting for the Fe-bioavailability.

PubMed

Cai, Yafan; Zhao, Xiaoling; Zhao, Yubin; Wang, Hongliang; Yuan, Xufeng; Zhu, Wanbin; Cui, Zongjun; Wang, Xiaofen

2018-02-01

Fe is widely used as an additive in anaerobic digestion, but its bioavailability and the mechanism by which it enhances digestion are unclear. In this study, sequential extraction was used to measure Fe bioavailability, while biochemical parameters, kinetics model and Q-PCR (fluorescence quantitative PCR) were used to explore its mechanism of stimulation. The results showed that sequential extraction is a suitable method to assess the anaerobic system bioavailability of Fe, which is low and fluctuates to a limited extent (1.7 to -3.1wt%), indicating that it would be easy for Fe levels to be insufficient. Methane yield increased when the added Fe 2+ was 10-500mg/L. Appropriate amounts of Fe 2+ accelerated the decomposition of rice straw and facilitated methanogen metabolism, thereby improving reactor performance. The modified Gompertz model better fitted the results than the first-order kinetic model. Feasibility analysis showed that addition of Fe 2+ at ≤50mg/L was suitable. Copyright © 2017. Published by Elsevier Ltd.

Numerical Modeling of Fluorescence Emission Energy Dispersion in Luminescent Solar Concentrator

NASA Astrophysics Data System (ADS)

Li, Lanfang; Sheng, Xing; Rogers, John; Nuzzo, Ralph

2013-03-01

We present a numerical modeling method and the corresponding experimental results, to address fluorescence emission dispersion for applications such as luminescent solar concentrator and light emitting diode color correction. Previously established modeling methods utilized a statistic-thermodynamic theory (Kenard-Stepnov etc.) that required a thorough understanding of the free energy landscape of the fluorophores. Some more recent work used an empirical approximation of the measured emission energy dispersion profile without considering anti-Stokes shifting during absorption and emission. In this work we present a technique for modeling fluorescence absorption and emission that utilizes the experimentally measured spectrum and approximates the observable Frank-Condon vibronic states as a continuum and takes into account thermodynamic energy relaxation by allowing thermal fluctuations. This new approximation method relaxes the requirement for knowledge of the fluorophore system and reduces demand on computing resources while still capturing the essence of physical process. We present simulation results of the energy distribution of emitted photons and compare them with experimental results with good agreement in terms of peak red-shift and intensity attenuation in a luminescent solar concentrator. This work is supported by the DOE `Light-Material Interactions in Energy Conversion' Energy Frontier Research Center under grant DE-SC0001293.

Fluorescence correlation spectroscopy to measure the metabolism of high-density lipoprotein

NASA Astrophysics Data System (ADS)

Deitrick, Russell; Gibson, Emily; Razzaghi, Hamid

2009-10-01

High-density lipoprotein (HDL), referred to as the ``good cholesterol'', carries free cholesterol to the liver to be filtered from the bloodstream and is important to our understanding of atherosclerosis. HDL is metabolized in part by the enzyme Endothelial Lipase (EL). With this project we will use fluorescence correlation spectroscopy (FCS) to study the metabolism of HDL by EL comparing wild type with different genetic mutations. FCS is an advanced microscopy technique in which we record fluctuations in the fluorescence of dye-labeled molecules (in this case, HDL labeled with Nile Red) as they freely diffuse through a small focal volume. This data can be analyzed mathematically using the cross-correlation function, from which we can ultimately ascertain much information. In our case, we are interested in the diffusion coefficient which, via the Stokes-Einstein relation for a sphere, we can determine the size of HDL as it undergoes the process of metabolism. Preliminary results seem to indicate that the metabolic process occurs very quickly, that the final size of HDL depends primarily on the concentration of EL, and that the wild and mutant variants of EL have a similar effectiveness. In following experiments, we hope to investigate these relationships further.

Defining the Subcellular Interface of Nanoparticles by Live-Cell Imaging

PubMed Central

Hemmerich, Peter H.; von Mikecz, Anna H.

2013-01-01

Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes. PMID:23637951

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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,more » 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.« less

Cerebral energy metabolism in diving and non-diving birds during hypoxia and apnoeic asphyxia.

PubMed Central

Bryan, R M; Jones, D R

1980-01-01

1. Cerebral energy metabolism during apnoeic asphyxia and steady-state hypoxia was compared in ducks and chickens; ducks tolerate apnoeic asphyxia 3-8 times longer than chickens. 2. Fluctuations in the reduced form of respiratory chain nicotinamide adenine dinucleotide (NADH) were monitored from the left cerebral hemisphere by a noninvasive fluorometric technique and used as an indicator of mitochondrial hypoxia. NADH fluorescence was expressed in aribtrary units (a.u.) where 100 a.u. was defined as the fluorescence change from normoxia to anoxia. Electroencephalogram (e.e.g.) and surface Po2 were recorded from the right hemisphere. 3. After 1 min of asphyxia NADH fluorescence increased by 37 a.u.+/-3.60 S.E. of mean (n=54) in paralysed chickens and 8 a.u.+/-1.41 (n=55) in aralysed ducks. After 2 min the fluorescence increased by only 15 a.u.+/-1.95 in ducks. 4. Both species showed an isoelectric e.e.g. when fluorescence increased by approximately 35 a.u., indicating that anaerobic ATP production in ducks did not maintain brain function (e.e.g.) for a greater accumulation of respiratory chain NADH. 5. At a given decrease in tissue Po2 ducks and chickens showed the same level of NADH increase, indicating that both species are equally dependent on tissue Po2 for the maintenance of redox state. 6. We conclude that biochemical adjustment which enhance anaerobic ATP production and/or prolong oxidative phosphorylation during progressive hypoxia are not responsible for increased cerebral tolerance to apnoeic asphyxia in the duck. PMID:7381772

Single-pulse measurement of density and temperature in a turbulent, supersonic flow using UV laser spectroscopy

NASA Technical Reports Server (NTRS)

Fletcher, D. G.; Mckenzie, R. L.

1992-01-01

Nonintrusive measurements of density and temperature and their turbulent fluctuation levels have been obtained in the boundary layer of an unseeded, Mach 2 wind tunnel flow. The spectroscopic technique that was used to make the measurements is based on the combination of laser-induced oxygen fluorescence and Raman scattering by oxygen and nitrogen from the same laser pulse. Results from this demonstration experiment compare favorably with previous measurements obtained in the same facility from conventional probes and an earlier spectroscopic technique.

Cross-sectional fluctuation scaling in the high-frequency illiquidity of Chinese stocks

NASA Astrophysics Data System (ADS)

Cai, Qing; Gao, Xing-Lu; Zhou, Wei-Xing; Stanley, H. Eugene

2018-03-01

Taylor's law of temporal and ensemble fluctuation scaling has been ubiquitously observed in diverse complex systems including financial markets. Stock illiquidity is an important nonadditive financial quantity, which is found to comply with Taylor's temporal fluctuation scaling law. In this paper, we perform the cross-sectional analysis of the 1 min high-frequency illiquidity time series of Chinese stocks and unveil the presence of Taylor's law of ensemble fluctuation scaling. The estimated daily Taylor scaling exponent fluctuates around 1.442. We find that Taylor's scaling exponents of stock illiquidity do not relate to the ensemble mean and ensemble variety of returns. Our analysis uncovers a new scaling law of financial markets and might stimulate further investigations for a better understanding of financial markets' dynamics.

Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging

PubMed Central

Angert, Isaac; Cao, Sheng; Berk, Serkan; Zhang, Wei; Mueller, Joachim D.

2017-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500–2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag–Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag–Gag interactions that occur during virus particle assembly and in released immature particles. PMID:28753950

Chemometric analysis for extraction of individual fluorescence spectrum and lifetimes from a target mixture

NASA Technical Reports Server (NTRS)

Hallidy, William H. (Inventor); Chin, Robert C. (Inventor)

1999-01-01

The present invention is a system for chemometric analysis for the extraction of the individual component fluorescence spectra and fluorescence lifetimes from a target mixture. The present invention combines a processor with an apparatus for generating an excitation signal to transmit at a target mixture and an apparatus for detecting the emitted signal from the target mixture. The present invention extracts the individual fluorescence spectrum and fluorescence lifetime measurements from the frequency and wavelength data acquired from the emitted signal. The present invention uses an iterative solution that first requires the initialization of several decision variables and the initial approximation determinations of intermediate matrices. The iterative solution compares the decision variables for convergence to see if further approximation determinations are necessary. If the solution converges, the present invention then determines the reduced best fit error for the analysis of the individual fluorescence lifetime and the fluorescence spectrum before extracting the individual fluorescence lifetime and fluorescence spectrum from the emitted signal of the target mixture.

Detrended fluctuation analysis made flexible to detect range of cross-correlated fluctuations

NASA Astrophysics Data System (ADS)

Kwapień, Jarosław; Oświecimka, Paweł; DroŻdŻ, Stanisław

2015-11-01

The detrended cross-correlation coefficient ρDCCA has recently been proposed to quantify the strength of cross-correlations on different temporal scales in bivariate, nonstationary time series. It is based on the detrended cross-correlation and detrended fluctuation analyses (DCCA and DFA, respectively) and can be viewed as an analog of the Pearson coefficient in the case of the fluctuation analysis. The coefficient ρDCCA works well in many practical situations but by construction its applicability is limited to detection of whether two signals are generally cross-correlated, without the possibility to obtain information on the amplitude of fluctuations that are responsible for those cross-correlations. In order to introduce some related flexibility, here we propose an extension of ρDCCA that exploits the multifractal versions of DFA and DCCA: multifractal detrended fluctuation analysis and multifractal detrended cross-correlation analysis, respectively. The resulting new coefficient ρq not only is able to quantify the strength of correlations but also allows one to identify the range of detrended fluctuation amplitudes that are correlated in two signals under study. We show how the coefficient ρq works in practical situations by applying it to stochastic time series representing processes with long memory: autoregressive and multiplicative ones. Such processes are often used to model signals recorded from complex systems and complex physical phenomena like turbulence, so we are convinced that this new measure can successfully be applied in time-series analysis. In particular, we present an example of such application to highly complex empirical data from financial markets. The present formulation can straightforwardly be extended to multivariate data in terms of the q -dependent counterpart of the correlation matrices and then to the network representation.

A broad-host range dual-fluorescence reporter system for gene expression analysis in Gram-negative bacteria.

PubMed

Hennessy, Rosanna C; Christiansen, Line; Olsson, Stefan; Stougaard, Peter

2018-01-01

Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. Here we describe a dual-fluorescence reporter system carrying the red fluorescent marker mCherry and the blue fluorescent protein EBFP2 enabling the simultaneous analysis of two promoters in broad-host range autofluorescent Gram-negative bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Manual-control Analysis Applied to the Money-supply Control Task

NASA Technical Reports Server (NTRS)

Wingrove, R. C.

1984-01-01

The recent procedure implemented by the Federal Reserve Board to control the money supply is formulated in the form of a tracking model as used in the study of manual-control tasks. Using this model, an analysis is made to determine the effect of monetary control on the fluctuations in economic output. The results indicate that monetary control can reduce the amplitude of fluctuations at frequencies near the region of historic business cycles. However, with significant time lags in the control loop, monetary control tends to increase the amplitude of the fluctuations at the higher frequencies. How the investigator or student can use the tools developed in the field of manual-control analysis to study the nature of economic fluctuations and to examine different strategies for stabilization is examined.

Wavelets and Multifractal Analysis

DTIC Science & Technology

2004-07-01

distribution unlimited 13. SUPPLEMENTARY NOTES See also ADM001750, Wavelets and Multifractal Analysis (WAMA) Workshop held on 19-31 July 2004., The original...f)] . . . 16 2.5.4 Detrended Fluctuation Analysis [DFA(m)] . . . . . . . . . . . . . . . 17 2.6 Scale-Independent Measures...18 2.6.1 Detrended -Fluctuation- Analysis Power-Law Exponent (αD) . . . . . . 18 2.6.2 Wavelet-Transform Power-Law Exponent

Quantitative evaluation of malignant gliomas damage induced by photoactivation of IR700 dye

NASA Astrophysics Data System (ADS)

Sakuma, Morito; Kita, Sayaka; Higuchi, Hideo

2016-01-01

The processes involved in malignant gliomas damage were quantitatively evaluated by microscopy. The near-infrared fluorescent dye IR700 that is conjugated to an anti-CD133 antibody (IR700-CD133) specifically targets malignant gliomas (U87MG) and stem cells (BT142) and is endocytosed into the cells. The gliomas are then photodamaged by the release of reactive oxygen species (ROS) and the heat induced by illumination of IR700 by a red laser, and the motility of the vesicles within these cells is altered as a result of cellular damage. To investigate these changes in motility, we developed a new method that measures fluctuations in the intensity of phase-contrast images obtained from small areas within cells. The intensity fluctuation in U87MG cells gradually decreased as cell damage progressed, whereas the fluctuation in BT142 cells increased. The endocytosed IR700 dye was co-localized in acidic organelles such as endosomes and lysosomes. The pH in U87MG cells, as monitored by a pH indicator, was decreased and then gradually increased by the illumination of IR700, while the pH in BT142 cells increased monotonically. In these experiments, the processes of cell damage were quantitatively evaluated according to the motility of vesicles and changes in pH.

Theory and simulations of covariance mapping in multiple dimensions for data analysis in high-event-rate experiments

NASA Astrophysics Data System (ADS)

Zhaunerchyk, V.; Frasinski, L. J.; Eland, J. H. D.; Feifel, R.

2014-05-01

Multidimensional covariance analysis and its validity for correlation of processes leading to multiple products are investigated from a theoretical point of view. The need to correct for false correlations induced by experimental parameters which fluctuate from shot to shot, such as the intensity of self-amplified spontaneous emission x-ray free-electron laser pulses, is emphasized. Threefold covariance analysis based on simple extension of the two-variable formulation is shown to be valid for variables exhibiting Poisson statistics. In this case, false correlations arising from fluctuations in an unstable experimental parameter that scale linearly with signals can be eliminated by threefold partial covariance analysis, as defined here. Fourfold covariance based on the same simple extension is found to be invalid in general. Where fluctuations in an unstable parameter induce nonlinear signal variations, a technique of contingent covariance analysis is proposed here to suppress false correlations. In this paper we also show a method to eliminate false correlations associated with fluctuations of several unstable experimental parameters.

Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest.

PubMed

Libregts, S F W M; Arkesteijn, G J A; Németh, A; Nolte-'t Hoen, E N M; Wauben, M H M

2018-05-20

Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. Background Extracellular vesicles (EVs) in plasma are increasingly being recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets with a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria, as it allows multiparameter analysis of individual EVs. However, analysis of plasma EVs is challenging, because of their size and heterogeneity, and the presence of other submicrometer-sized particles in plasma that could interfere with EV analysis. Objectives To explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labeled or differently labeled EVs and particles. Methods Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry with fluorescence threshold triggering. Results We found that light scatter detection of low-abundance or rare EV subsets during fluorescence threshold triggering was severely affected by particles of non-interest, owing to coincidence and swarming. Importantly, we show that interfering particles labeled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. Conclusions We demonstrate how particles of non-interest in plasma can impact on the light scatter and fluorescence detection of low-abundance EVs of interest during fluorescence-based flow cytometric analysis, and provide a means to prevent erroneous data interpretation. © 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

An analysis of alternative conceptual models relating hyporheic exchange flow to diel fluctuations in discharge during baseflow recession

Treesearch

Steven M. Wondzell; Michael N. Gooseff; Brian L. McGlynn

2009-01-01

Diel fluctuations in streamflow during base flow have been observed in many streams and are typically attributed to water losses from evapotranspiration (ET). However, there is no widely transferable conceptual model that explains how ET results in diel fluctuations in streamflow at the watershed outlet. For fluctuations to occur, two factors must be present: (1) some...

Kinetic-Scale Electric and Magnetic Field Fluctuations in the Solar Wind at 1 AU: THEMIS/ARTEMIS Observations

NASA Astrophysics Data System (ADS)

Salem, C. S.; Hanson, E.; Bonnell, J. W.; Chaston, C. C.; Bale, S. D.; Mozer, F.

2017-12-01

We present here an analysis of kinetic-scale electromagnetic fluctuations in the solar wind using data from THEMIS and ARTEMIS spacecraft. We use high-time resolution electric and magnetic field measurements, as well as density fluctuations, up to 128 samples per second, as well as particle burst plasma data during carefully selected solar wind intervals. We focus our analysis on a few such intervals spanning different values of plasma beta and angles between the local magnetic field and the radial Sun-Earth direction. We discuss the careful analysis process of characterizing and removing the different instrumental effects and noise sources affecting the electric and magnetic field data at those scales, above 0.1 Hz or so, above the breakpoint marking the start of the so-called dissipation range of solar wind turbulence. We compute parameters such as the electric to magnetic field ratio, the magnetic compressibility, magnetic helicity, and other relevant quantities in order to diagnose the nature of the fluctuations at those scales between the ion and electron cyclotron frequencies, extracting information on the dominant modes composing the fluctuations. We also discuss the presence and role of coherent structures in the measured fluctuations. The nature of the fluctuations in the dissipation or dispersive scales of solar wind turbulence is still debated. This observational study is also highly relevant to the current Turbulent Dissipation Challenge.

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

PubMed Central

Hosny, Neveen A.; Song, Mingying; Connelly, John T.; Ameer-Beg, Simon; Knight, Martin M.; Wheeler, Ann P.

2013-01-01

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging. PMID:24130668

Temporal scaling and spatial statistical analyses of groundwater level fluctuations

NASA Astrophysics Data System (ADS)

Sun, H.; Yuan, L., Sr.; Zhang, Y.

2017-12-01

Natural dynamics such as groundwater level fluctuations can exhibit multifractionality and/or multifractality due likely to multi-scale aquifer heterogeneity and controlling factors, whose statistics requires efficient quantification methods. This study explores multifractionality and non-Gaussian properties in groundwater dynamics expressed by time series of daily level fluctuation at three wells located in the lower Mississippi valley, after removing the seasonal cycle in the temporal scaling and spatial statistical analysis. First, using the time-scale multifractional analysis, a systematic statistical method is developed to analyze groundwater level fluctuations quantified by the time-scale local Hurst exponent (TS-LHE). Results show that the TS-LHE does not remain constant, implying the fractal-scaling behavior changing with time and location. Hence, we can distinguish the potentially location-dependent scaling feature, which may characterize the hydrology dynamic system. Second, spatial statistical analysis shows that the increment of groundwater level fluctuations exhibits a heavy tailed, non-Gaussian distribution, which can be better quantified by a Lévy stable distribution. Monte Carlo simulations of the fluctuation process also show that the linear fractional stable motion model can well depict the transient dynamics (i.e., fractal non-Gaussian property) of groundwater level, while fractional Brownian motion is inadequate to describe natural processes with anomalous dynamics. Analysis of temporal scaling and spatial statistics therefore may provide useful information and quantification to understand further the nature of complex dynamics in hydrology.

Visualizing and quantifying dose distribution in a UV reactor using three-dimensional laser-induced fluorescence.

PubMed

Gandhi, Varun N; Roberts, Philip J W; Kim, Jae-Hong

2012-12-18

Evaluating the performance of typical water treatment UV reactors is challenging due to the complexity in assessing spatial and temporal variation of UV fluence, resulting from highly unsteady, turbulent nature of flow and variation in UV intensity. In this study, three-dimensional laser-induced fluorescence (3DLIF) was applied to visualize and quantitatively analyze a lab-scale UV reactor consisting of one lamp sleeve placed perpendicular to flow. Mapping the spatial and temporal fluence delivery and MS2 inactivation revealed the highest local fluence in the wake zone due to longer residence time and higher UV exposure, while the lowest local fluence occurred in a region near the walls due to short-circuiting flow and lower UV fluence rate. Comparing the tracer based decomposition between hydrodynamics and IT revealed similar coherent structures showing the dependency of fluence delivery on the reactor flow. The location of tracer injection, varying the height and upstream distance from the lamp center, was found to significantly affect the UV fluence received by the tracer. A Lagrangian-based analysis was also employed to predict the fluence along specific paths of travel, which agreed with the experiments. The 3DLIF technique developed in this study provides new insight on dose delivery that fluctuates both spatially and temporally and is expected to aid design and optimization of UV reactors as well as validate computational fluid dynamics models that are widely used to simulate UV reactor performances.

Evaluation of the indirect fluorescent antibody test as a diagnostic tool for East Coast fever in eastern Zambia.

PubMed

Billiouw, M; Brandt, J; Vercruysse, J; Speybroeck, N; Marcotty, T; Mulumba, M; Berkvens, D

2005-02-28

Serological surveys using the schizont indirect fluorescence antibody test (IFAt) are routinely carried out to monitor the Theileria parva infection prevalence. The present study evaluates the diagnostic accuracy of the IFAt in eastern Zambia, where the transmission of T. parva is highly seasonal. The data set resulted from a sentinel herd (n = 105 animals) study carried out between 1995 and 2000 and was split into an epidemic period, during which the majority of the cattle became infected, and an endemic period with seasonal disease incidence in calves. In the epidemic period the T. parva seroprevalence followed closely the build up of the herd immunity. In the endemic period the seroprevalence fluctuates considerably although most of the animals had been infected. Overall, the diagnostic sensitivity of the IFA test was 55% at cut-off titre 1:40 and 28% at cut-off 1:160. The specificity of the test was 86 and 95%, respectively. A logistic regression model demonstrates that the sensitivity is significantly lower when the T. parva transmission is low (p < 0.01). The analysis of receiver operator characteristic curves classifies the test as moderately accurate (area under the curve, AUC = 0.79) during the epidemic period and less accurate in the endemic period (AUC = 0.63). Neonatal serology surveys yield a better estimate of the infection prevalence. The sensitivity of the neonatal test was 73% at cut-off titre 1:40 and 24% at cut-off 1:160.

Total reflection X-ray fluorescence analysis of trace-elements in candies marketed in Mexico

NASA Astrophysics Data System (ADS)

Martinez, T.; Lartigue, J.; Zarazua, G.; Avila-Perez, P.; Navarrete, M.; Tejeda, S.

2010-06-01

Trace metals concentrations in food are significant for nutrition, due either to their nature or toxicity. Sweets, including chewing gum and candies, are not exactly a food, but they usually are unwearied consumed by children, the most vulnerable age-group to any kind of metal contamination in the food chain. The presence of relatively high concentrations of heavy metals such as Lead elicits concern since children are highly susceptible to heavy metals poisoning. Trace-metals concentrations were determined for six different flavors of a Mexican candy by means of Total X-ray Fluorescence Spectrometry. Triplicate samples of the various candy's flavours (strawberry, pineapple, lemon, blackberry, orange and chilli) were digested in 8 mL of a mix of supra-pure HNO 3 and H 2O 2 (6 mL: 2 mL) in a microwave oven MARS-X. Results show the presence of essential and toxic elements such as Ti, Cr, Mn, Fe, Ni, Cu, Zn, Br, Rb, Sr, and Pb. All metal concentrations were higher and significantly different ( α = 0.05) in chilli candy, compared to other candy flavours. Lead concentration fluctuated in the range of 0.102 to 0.342 μg g - 1 . A discussion about risk consumption and concentration allowed by Mexican and International Norms is made. As a part of the Quality Control Program, a NIST standard of "Citrus Leaves" and a blank were treated in the same way.

Detrended fluctuation analysis of non-stationary cardiac beat-to-beat interval of sick infants

NASA Astrophysics Data System (ADS)

Govindan, Rathinaswamy B.; Massaro, An N.; Al-Shargabi, Tareq; Niforatos Andescavage, Nickie; Chang, Taeun; Glass, Penny; du Plessis, Adre J.

2014-11-01

We performed detrended fluctuation analysis (DFA) of cardiac beat-to-beat intervals (RRis) collected from sick newborn infants over 1-4 day periods. We calculated four different metrics from the DFA fluctuation function: the DFA exponents αL (>40 beats up to one-fourth of the record length), αs (15-30 beats), root-mean-square (RMS) fluctuation on a short-time scale (20-50 beats), and RMS fluctuation on a long-time scale (110-150 beats). Except αL , all metrics clearly distinguished two groups of newborn infants (favourable vs. adverse) with well-characterized outcomes. However, the RMS fluctuations distinguished the two groups more consistently over time compared to αS . Furthermore, RMS distinguished the RRi of the two groups earlier compared to the DFA exponent. In all the three measures, the favourable outcome group displayed higher values, indicating a higher magnitude of (auto-)correlation and variability, thus normal physiology, compared to the adverse outcome group.

From force-fields to photons: MD simulations of dye-labeled nucleic acids and Monte Carlo modeling of FRET

NASA Astrophysics Data System (ADS)

Goldner, Lori

2012-02-01

Fluorescence resonance energy transfer (FRET) is a powerful technique for understanding the structural fluctuations and transformations of RNA, DNA and proteins. Molecular dynamics (MD) simulations provide a window into the nature of these fluctuations on a different, faster, time scale. We use Monte Carlo methods to model and compare FRET data from dye-labeled RNA with what might be predicted from the MD simulation. With a few notable exceptions, the contribution of fluorophore and linker dynamics to these FRET measurements has not been investigated. We include the dynamics of the ground state dyes and linkers in our study of a 16mer double-stranded RNA. Water is included explicitly in the simulation. Cyanine dyes are attached at either the 3' or 5' ends with a 3 carbon linker, and differences in labeling schemes are discussed.[4pt] Work done in collaboration with Peker Milas, Benjamin D. Gamari, and Louis Parrot.

DNA motion capture reveals the mechanical properties of DNA at the mesoscale.

PubMed

Price, Allen C; Pilkiewicz, Kevin R; Graham, Thomas G W; Song, Dan; Eaves, Joel D; Loparo, Joseph J

2015-05-19

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fluctuating exciton localization in giant π-conjugated spoked-wheel macrocycles.

PubMed

Aggarwal, A Vikas; Thiessen, Alexander; Idelson, Alissa; Kalle, Daniel; Würsch, Dominik; Stangl, Thomas; Steiner, Florian; Jester, Stefan-S; Vogelsang, Jan; Höger, Sigurd; Lupton, John M

2013-11-01

Conjugated polymers offer potential for many diverse applications, but we still lack a fundamental microscopic understanding of their electronic structure. Elementary photoexcitations (excitons) span only a few nanometres of a molecule, which itself can extend over microns, and how their behaviour is affected by molecular dimensions is not immediately obvious. For example, where is the exciton formed within a conjugated segment and is it always situated on the same repeat units? Here, we introduce structurally rigid molecular spoked wheels, 6 nm in diameter, as a model of extended π conjugation. Single-molecule fluorescence reveals random exciton localization, which leads to temporally varying emission polarization. Initially, this random localization arises after every photon absorption event because of temperature-independent spontaneous symmetry breaking. These fast fluctuations are slowed to millisecond timescales after prolonged illumination. Intramolecular heterogeneity is revealed in cryogenic spectroscopy by jumps in transition energy, but emission polarization can also switch without a spectral jump occurring, which implies long-range homogeneity in the local dielectric environment.

Fluctuating exciton localization in giant π-conjugated spoked-wheel macrocycles

NASA Astrophysics Data System (ADS)

Aggarwal, A. Vikas; Thiessen, Alexander; Idelson, Alissa; Kalle, Daniel; Würsch, Dominik; Stangl, Thomas; Steiner, Florian; Jester, Stefan-S.; Vogelsang, Jan; Höger, Sigurd; Lupton, John M.

2013-11-01

Conjugated polymers offer potential for many diverse applications, but we still lack a fundamental microscopic understanding of their electronic structure. Elementary photoexcitations (excitons) span only a few nanometres of a molecule, which itself can extend over microns, and how their behaviour is affected by molecular dimensions is not immediately obvious. For example, where is the exciton formed within a conjugated segment and is it always situated on the same repeat units? Here, we introduce structurally rigid molecular spoked wheels, 6 nm in diameter, as a model of extended π conjugation. Single-molecule fluorescence reveals random exciton localization, which leads to temporally varying emission polarization. Initially, this random localization arises after every photon absorption event because of temperature-independent spontaneous symmetry breaking. These fast fluctuations are slowed to millisecond timescales after prolonged illumination. Intramolecular heterogeneity is revealed in cryogenic spectroscopy by jumps in transition energy, but emission polarization can also switch without a spectral jump occurring, which implies long-range homogeneity in the local dielectric environment.

Exploring quantum thermodynamics in continuous measurement of superconducting qubits

NASA Astrophysics Data System (ADS)

Murch, Kater

The extension of thermodynamics into the realm of quantum mechanics, where quantum fluctuations dominate and systems need not occupy definite states, poses unique challenges. Superconducting quantum circuits offer exquisite control over the environment of simple quantum systems allowing the exploration of thermodynamics at the quantum level through measurement and feedback control. We use a superconducting transmon qubit that is resonantly coupled to a waveguide cavity as an effectively one-dimensional quantum emitter. By driving the emitter and detecting the fluorescence with a near-quantum-limited Josephson parametric amplifier, we track the evolution of the quantum state and characterize the work and heat along single quantum trajectories. By using quantum feedback control to compensate for heat exchanged with the emitter's environment we are able to extract the work statistics associated with the quantum evolution and examine fundamental fluctuation theorems in non-equilibrium thermodynamics. This work was supported by the Alfred P. Sloan Foundation, the National Science Foundation, and the Office of Naval Research.

Investigation of flameholding mechanisms in a kerosene-fueled scramjet combustor

NASA Astrophysics Data System (ADS)

Wang, Yu-hang; Song, Wen-yan; Shi, De-yong

2017-11-01

Laser-induced fluorescence and high-speed photography were employed to investigate the kerosene flame stabilization mechanism in a cavity-based scramjet combustor with an inlet condition corresponds to flight Mach number of 4. Pilot hydrogen was used to ignite the kerosene fuel. The PLIF results of kerosene distribution in the reacting cases showed that the mixing process was dramatically enhanced compared to the non-reacting cases. Sharp OH gradients were observed in the shear layer and the aft region of cavity, which indicated that the flame was located at these positions. A portion of hot products participated in the recirculation of the cavity and preheated the kerosene-air mixture in the leading edge. The heated mixture was ignited in the mid-cavity and the reaction zone spread into the mainstream flow. Due to the competition between the local flame speed and the local flow speed, the high-speed images showed that the spreading location was in fluctuation. This movement was observed to cause a low-frequency wall pressure fluctuation.

FSCS Reveals the Complexity of Lipid Domain Dynamics in the Plasma Membrane of Live Cells.

PubMed

Nicovich, Philip R; Kwiatek, Joanna M; Ma, Yuanqing; Benda, Aleš; Gaus, Katharina

2018-06-19

The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

NASA Astrophysics Data System (ADS)

Adio, Sarah; Senyushkina, Tamara; Peske, Frank; Fischer, Niels; Wintermeyer, Wolfgang; Rodnina, Marina V.

2015-06-01

The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging.

PubMed

Ellefsen, Kyle L; Settle, Brett; Parker, Ian; Smith, Ian F

2014-09-01

Local Ca(2+) transients such as puffs and sparks form the building blocks of cellular Ca(2+) signaling in numerous cell types. They have traditionally been studied by linescan confocal microscopy, but advances in TIRF microscopy together with improved electron-multiplied CCD (EMCCD) cameras now enable rapid (>500 frames s(-1)) imaging of subcellular Ca(2+) signals with high spatial resolution in two dimensions. This approach yields vastly more information (ca. 1 Gb min(-1)) than linescan imaging, rendering visual identification and analysis of local events imaged both laborious and subject to user bias. Here we describe a routine to rapidly automate identification and analysis of local Ca(2+) events. This features an intuitive graphical user-interfaces and runs under Matlab and the open-source Python software. The underlying algorithm features spatial and temporal noise filtering to reliably detect even small events in the presence of noisy and fluctuating baselines; localizes sites of Ca(2+) release with sub-pixel resolution; facilitates user review and editing of data; and outputs time-sequences of fluorescence ratio signals for identified event sites along with Excel-compatible tables listing amplitudes and kinetics of events. Copyright © 2014 Elsevier Ltd. All rights reserved.

Extracting physics of life at the molecular level: A review of single-molecule data analyses.

PubMed

Colomb, Warren; Sarkar, Susanta K

2015-06-01

Studying individual biomolecules at the single-molecule level has proved very insightful recently. Single-molecule experiments allow us to probe both the equilibrium and nonequilibrium properties as well as make quantitative connections with ensemble experiments and equilibrium thermodynamics. However, it is important to be careful about the analysis of single-molecule data because of the noise present and the lack of theoretical framework for processes far away from equilibrium. Biomolecular motion, whether it is free in solution, on a substrate, or under force, involves thermal fluctuations in varying degrees, which makes the motion noisy. In addition, the noise from the experimental setup makes it even more complex. The details of biologically relevant interactions, conformational dynamics, and activities are hidden in the noisy single-molecule data. As such, extracting biological insights from noisy data is still an active area of research. In this review, we will focus on analyzing both fluorescence-based and force-based single-molecule experiments and gaining biological insights at the single-molecule level. Inherently nonequilibrium nature of biological processes will be highlighted. Simulated trajectories of biomolecular diffusion will be used to compare and validate various analysis techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

Stochastic lumping analysis for linear kinetics and its application to the fluctuation relations between hierarchical kinetic networks.

PubMed

Deng, De-Ming; Chang, Cheng-Hung

2015-05-14

Conventional studies of biomolecular behaviors rely largely on the construction of kinetic schemes. Since the selection of these networks is not unique, a concern is raised whether and under which conditions hierarchical schemes can reveal the same experimentally measured fluctuating behaviors and unique fluctuation related physical properties. To clarify these questions, we introduce stochasticity into the traditional lumping analysis, generalize it from rate equations to chemical master equations and stochastic differential equations, and extract the fluctuation relations between kinetically and thermodynamically equivalent networks under intrinsic and extrinsic noises. The results provide a theoretical basis for the legitimate use of low-dimensional models in the studies of macromolecular fluctuations and, more generally, for exploring stochastic features in different levels of contracted networks in chemical and biological kinetic systems.

New approaches to the analysis of complex samples using fluorescence lifetime techniques and organized media

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hertz, P.R.

Fluorescence spectroscopy is a highly sensitive and selective tool for the analysis of complex systems. In order to investigate the efficacy of several steady state and dynamic techniques for the analysis of complex systems, this work focuses on two types of complex, multicomponent samples: petrolatums and coal liquids. It is shown in these studies dynamic, fluorescence lifetime-based measurements provide enhanced discrimination between complex petrolatum samples. Additionally, improved quantitative analysis of multicomponent systems is demonstrated via incorporation of organized media in coal liquid samples. This research provides the first systematic studies of (1) multifrequency phase-resolved fluorescence spectroscopy for dynamic fluorescence spectralmore » fingerprinting of complex samples, and (2) the incorporation of bile salt micellar media to improve accuracy and sensitivity for characterization of complex systems. In the petroleum studies, phase-resolved fluorescence spectroscopy is used to combine spectral and lifetime information through the measurement of phase-resolved fluorescence intensity. The intensity is collected as a function of excitation and emission wavelengths, angular modulation frequency, and detector phase angle. This multidimensional information enhances the ability to distinguish between complex samples with similar spectral characteristics. Examination of the eigenvalues and eigenvectors from factor analysis of phase-resolved and steady state excitation-emission matrices, using chemometric methods of data analysis, confirms that phase-resolved fluorescence techniques offer improved discrimination between complex samples as compared with conventional steady state methods.« less

Random Dopant Induced Threshold Voltage Lowering and Fluctuations in Sub-0.1 (micron)meter MOSFET's: A 3-D 'Atomistic' Simulation Study

NASA Technical Reports Server (NTRS)

Asenov, Asen

1998-01-01

A three-dimensional (3-D) "atomistic" simulation study of random dopant induced threshold voltage lowering and fluctuations in sub-0.1 microns MOSFET's is presented. For the first time a systematic analysis of random dopant effects down to an individual dopant level was carried out in 3-D on a scale sufficient to provide quantitative statistical predictions. Efficient algorithms based on a single multigrid solution of the Poisson equation followed by the solution of a simplified current continuity equation are used in the simulations. The effects of various MOSFET design parameters, including the channel length and width, oxide thickness and channel doping, on the threshold voltage lowering and fluctuations are studied using typical samples of 200 atomistically different MOSFET's. The atomistic results for the threshold voltage fluctuations were compared with two analytical models based on dopant number fluctuations. Although the analytical models predict the general trends in the threshold voltage fluctuations, they fail to describe quantitatively the magnitude of the fluctuations. The distribution of the atomistically calculated threshold voltage and its correlation with the number of dopants in the channel of the MOSFET's was analyzed based on a sample of 2500 microscopically different devices. The detailed analysis shows that the threshold voltage fluctuations are determined not only by the fluctuation in the dopant number, but also in the dopant position.

Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells.

PubMed

Pallavicini, Carla; Levi, Valeria; Wetzler, Diana E; Angiolini, Juan F; Benseñor, Lorena; Despósito, Marcelo A; Bruno, Luciana

2014-06-17

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Individual Test Point Fluctuations of Macular Sensitivity in Healthy Eyes and Eyes With Age-Related Macular Degeneration Measured With Microperimetry.

PubMed

Barboni, Mirella Telles Salgueiro; Szepessy, Zsuzsanna; Ventura, Dora Fix; Németh, János

2018-04-01

To establish fluctuation limits, it was considered that not only overall macular sensitivity but also fluctuations of individual test points in the macula might have clinical value. Three repeated measurements of microperimetry were performed using the Standard Expert test of Macular Integrity Assessment (MAIA) in healthy subjects ( N = 12, age = 23.8 ± 1.5 years old) and in patients with age-related macular degeneration (AMD) ( N = 11, age = 68.5 ± 7.4 years old). A total of 37 macular points arranged in four concentric rings and in four quadrants were analyzed individually and in groups. The data show low fluctuation of macular sensitivity of individual test points in healthy subjects (average = 1.38 ± 0.28 dB) and AMD patients (average = 2.12 ± 0.60 dB). Lower sensitivity points are more related to higher fluctuation than to the distance from the central point. Fixation stability showed no effect on the sensitivity fluctuation. The 95th percentile of the standard deviations of healthy subjects was, on average, 2.7 dB, ranging from 1.2 to 4 dB, depending on the point tested. Point analysis and regional analysis might be considered prior to evaluating macular sensitivity fluctuation in order to distinguish between normal variation and a clinical change. S tatistical methods were used to compare repeated microperimetry measurements and to establish fluctuation limits of the macular sensitivity. This analysis could add information regarding the integrity of different macular areas and provide new insights into fixation points prior to the biofeedback fixation training.

Fluctuation spectroscopy: From Rayleigh-Jeans waves to Abrikosov vortex clusters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varlamov, A. A.; Galda, A.; Glatz, A.

Superconducting (SC) fluctuations, discovered in the late 1960s, have constituted an important research area in superconductivity as they are manifest in a variety of phenomena. Indeed, the underlying physics of SC fluctuations makes it possible to elucidate the fundamental properties of the superconducting state. The interest in SC fluctuation phenomena was further enhanced with the discovery of cuprate high-temperature superconductors (HTSs). In these materials, superconducting fluctuations appear over a wide range of temperatures due to the superconductors extremely short coherence lengths and low effective dimensionality of the electron systems. These strong fluctuations lead to anomalous properties of the normal statemore » in some HTS materials. Within the framework of the phenomenological Ginzburg-Landau theory, and more extensively in the diagrammatic microscopic approach based on BCS theory, SC fluctuations as well as other quantum contributions (weak localization, etc.) enabled a new way to investigate and characterize disordered electron systems, granular metals, Josephson structures, artificial superlattices, and others. The characteristic feature of SC fluctuations is its strong dependence on temperature and magnetic field in the vicinity of the superconducting phase transition. This dependence allows the separation of fluctuation effects from other contributions and provides information about the microscopic parameters of a material, in particular, the critical temperature and the zero-temperature critical magnetic field. As such, SC fluctuations are very sensitive to the relaxation processes that break phase coherence and can be used as a versatile characterization instrument for SCs: Fluctuation spectroscopy has emerged as a powerful tool for studying the properties of superconducting systems on a quantitative level. Here the physics of SC fluctuations is reviewed, commencing from a qualitative description of thermodynamic fluctuations close to the critical temperature and quantum fluctuations at zero temperature in the vicinity of the second critical field. The analysis of the latter allows us to present fluctuation formation as a fragmentation of the Abrikosov lattice. Finally, this review highlights a series of experimental findings followed by microscopic description and numerical analysis of the effects of fluctuations on numerous properties of superconductors in the entire phase diagram and beyond the superconducting phase.« less

Fluctuation spectroscopy: From Rayleigh-Jeans waves to Abrikosov vortex clusters

DOE PAGES

Varlamov, A. A.; Galda, A.; Glatz, A.

2018-03-27

Superconducting (SC) fluctuations, discovered in the late 1960s, have constituted an important research area in superconductivity as they are manifest in a variety of phenomena. Indeed, the underlying physics of SC fluctuations makes it possible to elucidate the fundamental properties of the superconducting state. The interest in SC fluctuation phenomena was further enhanced with the discovery of cuprate high-temperature superconductors (HTSs). In these materials, superconducting fluctuations appear over a wide range of temperatures due to the superconductors extremely short coherence lengths and low effective dimensionality of the electron systems. These strong fluctuations lead to anomalous properties of the normal statemore » in some HTS materials. Within the framework of the phenomenological Ginzburg-Landau theory, and more extensively in the diagrammatic microscopic approach based on BCS theory, SC fluctuations as well as other quantum contributions (weak localization, etc.) enabled a new way to investigate and characterize disordered electron systems, granular metals, Josephson structures, artificial superlattices, and others. The characteristic feature of SC fluctuations is its strong dependence on temperature and magnetic field in the vicinity of the superconducting phase transition. This dependence allows the separation of fluctuation effects from other contributions and provides information about the microscopic parameters of a material, in particular, the critical temperature and the zero-temperature critical magnetic field. As such, SC fluctuations are very sensitive to the relaxation processes that break phase coherence and can be used as a versatile characterization instrument for SCs: Fluctuation spectroscopy has emerged as a powerful tool for studying the properties of superconducting systems on a quantitative level. Here the physics of SC fluctuations is reviewed, commencing from a qualitative description of thermodynamic fluctuations close to the critical temperature and quantum fluctuations at zero temperature in the vicinity of the second critical field. The analysis of the latter allows us to present fluctuation formation as a fragmentation of the Abrikosov lattice. Finally, this review highlights a series of experimental findings followed by microscopic description and numerical analysis of the effects of fluctuations on numerous properties of superconductors in the entire phase diagram and beyond the superconducting phase.« less

Fluctuation spectroscopy: From Rayleigh-Jeans waves to Abrikosov vortex clusters

NASA Astrophysics Data System (ADS)

Varlamov, A. A.; Galda, A.; Glatz, A.

2018-01-01

Superconducting (SC) fluctuations, discovered in the late 1960s, have constituted an important research area in superconductivity as they are manifest in a variety of phenomena. Indeed, the underlying physics of SC fluctuations makes it possible to elucidate the fundamental properties of the superconducting state. The interest in SC fluctuation phenomena was further enhanced with the discovery of cuprate high-temperature superconductors (HTSs). In these materials, superconducting fluctuations appear over a wide range of temperatures due to the superconductors extremely short coherence lengths and low effective dimensionality of the electron systems. These strong fluctuations lead to anomalous properties of the normal state in some HTS materials. Within the framework of the phenomenological Ginzburg-Landau theory, and more extensively in the diagrammatic microscopic approach based on BCS theory, SC fluctuations as well as other quantum contributions (weak localization, etc.) enabled a new way to investigate and characterize disordered electron systems, granular metals, Josephson structures, artificial superlattices, and others. The characteristic feature of SC fluctuations is its strong dependence on temperature and magnetic field in the vicinity of the superconducting phase transition. This dependence allows the separation of fluctuation effects from other contributions and provides information about the microscopic parameters of a material, in particular, the critical temperature and the zero-temperature critical magnetic field. As such, SC fluctuations are very sensitive to the relaxation processes that break phase coherence and can be used as a versatile characterization instrument for SCs: Fluctuation spectroscopy has emerged as a powerful tool for studying the properties of superconducting systems on a quantitative level. Here the physics of SC fluctuations is reviewed, commencing from a qualitative description of thermodynamic fluctuations close to the critical temperature and quantum fluctuations at zero temperature in the vicinity of the second critical field. The analysis of the latter allows us to present fluctuation formation as a fragmentation of the Abrikosov lattice. This review highlights a series of experimental findings followed by microscopic description and numerical analysis of the effects of fluctuations on numerous properties of superconductors in the entire phase diagram and beyond the superconducting phase.

On-line capillary electrophoresis/laser-induced fluorescence/mass spectrometry analysis of glycans labeled with Teal™ fluorescent dye using an electrokinetic sheath liquid pump-based nanospray ion source.

PubMed

Khan, Shaheer; Liu, Jenkuei; Szabo, Zoltan; Kunnummal, Baburaj; Han, Xiaorui; Ouyang, Yilan; Linhardt, Robert J; Xia, Qiangwei

2018-06-15

N-linked glycan analysis of recombinant therapeutic proteins, such as monoclonal antibodies, Fc-fusion proteins, and antibody-drug conjugates, provides valuable information regarding protein therapeutics glycosylation profile. Both qualitative identification and quantitative analysis of N-linked glycans on recombinant therapeutic proteins are critical analytical tasks in the biopharma industry during the development of a biotherapeutic. Currently, such analyses are mainly carried out using capillary electrophoresis/laser-induced fluorescence (CE/LIF), liquid chromatography/fluorescence (LC/FLR), and liquid chromatography/fluorescence/mass spectrometry (LC/FLR/MS) technologies. N-linked glycans are first released from glycoproteins by enzymatic digestion, then labeled with fluorescence dyes for subsequent CE or LC separation, and LIF or MS detection. Here we present an on-line CE/LIF/MS N-glycan analysis workflow that incorporates the fluorescent Teal™ dye and an electrokinetic pump-based nanospray sheath liquid capillary electrophoresis/mass spectrometry (CE/MS) ion source. Electrophoresis running buffer systems using ammonium acetate and ammonium hydroxide were developed for the negative ion mode CE/MS analysis of fluorescence-labeled N-linked glycans. Results show that on-line CE/LIF/MS analysis can be readily achieved using this versatile CE/MS ion source on common CE/MS instrument platforms. This on-line CE/LIF/MS method using Teal™ fluorescent dye and electrokinetic pump-based nanospray sheath liquid CE/MS coupling technology holds promise for on-line quantitation and identification of N-linked glycans on recombinant therapeutic proteins. Copyright © 2018 John Wiley & Sons, Ltd.

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

PubMed Central

Wang, Li; Carnegie, Graeme K.

2013-01-01

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction. PMID:23979513

Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.

PubMed

Wang, Li; Carnegie, Graeme K

2013-08-15

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Correcting for surface topography in X-ray fluorescence imaging

PubMed Central

Geil, E. C.; Thorne, R. E.

2014-01-01

Samples with non-planar surfaces present challenges for X-ray fluorescence imaging analysis. Here, approximations are derived to describe the modulation of fluorescence signals by surface angles and topography, and suggestions are made for reducing this effect. A correction procedure is developed that is effective for trace element analysis of samples having a uniform matrix, and requires only a fluorescence map from a single detector. This procedure is applied to fluorescence maps from an incised gypsum tablet. PMID:25343805

Fluctuation solution theory of pure fluids

NASA Astrophysics Data System (ADS)

Ploetz, Elizabeth A.; Pallewela, Gayani N.; Smith, Paul E.

2017-03-01

Fluctuation Solution Theory (FST) provides an alternative view of fluid thermodynamics in terms of pair fluctuations in the particle number and excess energy observed for an equivalent open system. Here we extend the FST approach to provide a series of triplet and quadruplet particle and excess energy fluctuations that can also be used to help understand the behavior of fluids. The fluctuations for the gas, liquid, and supercritical regions of three fluids (H2O, CO2, and SF6) are then determined from accurate equations of state. Many of the fluctuating quantities change sign on moving from the gas to liquid phase and, therefore, we argue that the fluctuations can be used to characterize gas and liquid behavior. Further analysis provides an approach to isolate contributions to the excess energy fluctuations arising from just the intermolecular interactions and also indicates that the triplet and quadruplet particle fluctuations are related to the pair particle fluctuations by a simple power law for large regions of the phase diagram away from the critical point.

Total ozone trend over Cairo

NASA Technical Reports Server (NTRS)

Hassan, G. K. Y.

1994-01-01

A world wide interest in protecting ozone layer against manmade effects is now increasing. Assessment of the ozone depletion due to these activities depends on how successfully we can separate the natural variabilities from the data. The monthly mean values of total ozone over Cairo (30 05N) for the period 1968-1988, have been analyzed using the power spectral analysis technique. The technique used in this analysis does not depend on a pre-understanding of the natural fluctuations in the ozone data. The method depends on increasing the resolution of the spectral peaks in order to obtain the more accurate sinusoidal fluctuations with wavelength equal to or less than record length. Also it handles the possible sinusoidal fluctuations with wavelength equal to or less than record length. The results show that it is possible to detect some of the well known national fluctuations in the ozone record such as annual, semiannual, quasi-biennial and quasi-quadrennial oscillations. After separating the natural fluctuations from the ozone record, the trend analysis of total ozone over Cairo showed that a decrease of about -1.2% per decade has occurred since 1979.

Dynamical glucometry: Use of multiscale entropy analysis in diabetes

NASA Astrophysics Data System (ADS)

Costa, Madalena D.; Henriques, Teresa; Munshi, Medha N.; Segal, Alissa R.; Goldberger, Ary L.

2014-09-01

Diabetes mellitus (DM) is one of the world's most prevalent medical conditions. Contemporary management focuses on lowering mean blood glucose values toward a normal range, but largely ignores the dynamics of glucose fluctuations. We probed analyte time series obtained from continuous glucose monitor (CGM) sensors. We show that the fluctuations in CGM values sampled every 5 min are not uncorrelated noise. Next, using multiscale entropy analysis, we quantified the complexity of the temporal structure of the CGM time series from a group of elderly subjects with type 2 DM and age-matched controls. We further probed the structure of these CGM time series using detrended fluctuation analysis. Our findings indicate that the dynamics of glucose fluctuations from control subjects are more complex than those of subjects with type 2 DM over time scales ranging from about 5 min to 5 h. These findings support consideration of a new framework, dynamical glucometry, to guide mechanistic research and to help assess and compare therapeutic interventions, which should enhance complexity of glucose fluctuations and not just lower mean and variance of blood glucose levels.

Meaningful interpretation of subdiffusive measurements in living cells (crowded environment) by fluorescence fluctuation microscopy.

PubMed

Baumann, Gerd; Place, Robert F; Földes-Papp, Zeno

2010-08-01

In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times tau(D)(tau) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent alpha the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d(f)/d(w) and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of alpha # 1 but increases for a larger time scale tau at a given value of alpha # 1. The effect of translational anomalous motion is substantially greater if alpha differs much from 1. An alpha value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales tau must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent alpha also determines the resolution of differences in diffusion times between two components in addition to photophysical parameters well-known for normal motion in dilute solution. The resolution limit between two different kinds of single molecule species is also analyzed under translational anomalous motion with broken ergodicity. We apply our theoretical predictions of diffusion times and lower limits for the time resolution of two components to fluorescence images in human prostate cancer cells transfected with GFP-Ago2 and GFP-Ago1. In order to mimic heterogeneous behavior in crowded environments of living cells, we need to introduce so-called continuous time random walks (CTRW). CTRWs were originally performed on regular lattice. This purely stochastic molecule behavior leads to subdiffusive motion with broken ergodicity in our simulations. For the first time, we are able to quantitatively differentiate between anomalous motion without broken ergodicity and anomalous motion with broken ergodicity in time-dependent fluorescence microscopy data sets of living cells. Since the experimental conditions to measure a selfsame molecule over an extended period of time, at which biology is taken place, in living cells or even in dilute solution are very restrictive, we need to perform the time average over a subpopulation of different single molecules of the same kind. For time averages over subpopulations of single molecules, the temporal auto- and crosscorrelation functions are first found. Knowing the crowding parameter alpha for the cell type and cellular compartment type, respectively, the heterogeneous parameter gamma can be obtained from the measurements in the presence of the interacting reaction partner, e.g. ligand, with the same alpha value. The product alpha x gamma = gamma is not a simple fitting parameter in the temporal auto- and two-color crosscorrelation functions because it is related to the proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in cellular systems.We have already derived an analytical solution gamma for in the special case of gamma = 3/2. In the case of two-color crosscorrelation or/and two-color fluorescence imaging (co-localization experiments), the second component is also a two-color species gr, for example a different molecular complex with an additional ligand. Here, we first show that plausible biological mechanisms from FCS/ FCCS and fluorescence imaging in living cells are highly questionable without proper quantitative physical models of subdiffusive motion and temporal randomness. At best, such quantitative FCS/ FCCS and fluorescence imaging data are difficult to interpret under crowding and heterogeneous conditions. It is challenging to translate proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in living cells and their cellular compartments like the nucleus into biological models of the cell biological process under study testable by single-molecule approaches. Otherwise, quantitative FCS/FCCS and fluorescence imaging measurements in living cells are not well described and cannot be interpreted in a meaningful way.

Fractal Dynamics of Heartbeat Interval Fluctuations in Health and Disease

NASA Astrophysics Data System (ADS)

Meyer, M.; Marconi, C.; Rahmel, A.; Grassi, B.; Ferretti, G.; Skinner, J. E.; Cerretelli, P.

The dynamics of heartbeat interval time series were studied by a modified random walk analysis recently introduced as Detrended Fluctuation Analysis. In this analysis, the intrinsic fractal long-range power-law correlation properties of beat-to-beat fluctuations generated by the dynamical system (i.e. cardiac rhythm generator), after decomposition from extrinsic uncorrelated sources, can be quantified by the scaling exponent which, in healthy subjects, is about 1.0. The finding of a scaling coefficient of 1.0, indicating scale-invariant long-range power-law correlations (1/ƒnoise) of heartbeat fluctuations, would reflect a genuinely self-similar fractal process that typically generates fluctuations on a wide range of time scales. Lack of a characteristic time scale suggests that the neuroautonomic system underlying the control of heart rate dynamics helps prevent excessive mode-locking (error tolerance) that would restrict its functional responsiveness (plasticity) to environmental stimuli. The 1/ƒ dynamics of heartbeat interval fluctuations are unaffected by exposure to chronic hypoxia suggesting that the neuroautonomic cardiac control system is preadapted to hypoxia. Functional (hypothermia, cardiac disease) and/or structural (cardiac transplantation, early cardiac development) inactivation of neuroautonomic control is associated with the breakdown or absence of fractal complexity reflected by anticorrelated random walk-like dynamics, indicating that in these conditions the heart is unadapted to its environment.

Effects of the lateral amplitude and regularity of upper body fluctuation on step time variability evaluated using return map analysis

PubMed Central

2017-01-01

The aim of this study was to evaluate the effects of the lateral amplitude and regularity of upper body fluctuation on step time variability. Return map analysis was used to clarify the relationship between step time variability and a history of falling. Eleven healthy, community-dwelling older adults and twelve younger adults participated in the study. All of the subjects walked 25 m at a comfortable speed. Trunk acceleration was measured using triaxial accelerometers attached to the third lumbar vertebrae (L3) and the seventh cervical vertebrae (C7). The normalized average magnitude of acceleration, the coefficient of determination ($R^2$) of the return map, and the step time variabilities, were calculated. Cluster analysis using the average fluctuation and the regularity of C7 fluctuation identified four walking patterns in the mediolateral (ML) direction. The participants with higher fluctuation and lower regularity showed significantly greater step time variability compared with the others. Additionally, elderly participants who had fallen in the past year had higher amplitude and a lower regularity of fluctuation during walking. In conclusion, by focusing on the time evolution of each step, it is possible to understand the cause of stride and/or step time variability that is associated with a risk of falls. PMID:28700633

Studies on the latitudinal distribution of ground-based geomagnetic pulsations and fluctuations in the interplanetary medium using discrete mathematical analysis methods

NASA Astrophysics Data System (ADS)

Zelinsky, N. R.; Kleimenova, N. G.; Malysheva, L. M.

2014-07-01

Ground-based geomagnetic Pc5 (2-7 mHz) pulsations, caused by the passage of dense transients (density disturbances) in the solar wind, were analyzed. It was shown that intensive bursts can appear in the density of the solar wind and its fluctuations, up to Np ˜ 30-50 cm3, even during the most magnetically calm year in the past decades (2009). The analysis, performed using one of the latest methods of discrete mathematical analysis (DMA), is presented. The energy functional of a time-series fragment (called "anomaly rectification" in DMA terms) of two such events was calculated. It was established that fluctuations in the dynamic pressure (density) of the solar wind (SW) cause the global excitation of Pc5 geomagnetic pulsations in the daytime sector of the Earth's magnetosphere, i.e., from polar to equatorial latitudes. Such pulsations started and ended suddenly and simultaneously at all latitudes. Fluctuations in the interplanetary magnetic field (IMF) have turned up to be less geoeffective in exciting geomagnetic pulsations than fluctuations in the SW density. The pulsation generation mechanisms in various structural regions of the magnetosphere were probably different. It was therefore concluded that the most probable source of ground-based pulsations are fluctuations of the corresponding periods in the SW density.

Effects of the lateral amplitude and regularity of upper body fluctuation on step time variability evaluated using return map analysis.

PubMed

Chidori, Kazuhiro; Yamamoto, Yuji

2017-01-01

The aim of this study was to evaluate the effects of the lateral amplitude and regularity of upper body fluctuation on step time variability. Return map analysis was used to clarify the relationship between step time variability and a history of falling. Eleven healthy, community-dwelling older adults and twelve younger adults participated in the study. All of the subjects walked 25 m at a comfortable speed. Trunk acceleration was measured using triaxial accelerometers attached to the third lumbar vertebrae (L3) and the seventh cervical vertebrae (C7). The normalized average magnitude of acceleration, the coefficient of determination ($R^2$) of the return map, and the step time variabilities, were calculated. Cluster analysis using the average fluctuation and the regularity of C7 fluctuation identified four walking patterns in the mediolateral (ML) direction. The participants with higher fluctuation and lower regularity showed significantly greater step time variability compared with the others. Additionally, elderly participants who had fallen in the past year had higher amplitude and a lower regularity of fluctuation during walking. In conclusion, by focusing on the time evolution of each step, it is possible to understand the cause of stride and/or step time variability that is associated with a risk of falls.

Tracking individual membrane proteins and their biochemistry: The power of direct observation.

PubMed

Barden, Adam O; Goler, Adam S; Humphreys, Sara C; Tabatabaei, Samaneh; Lochner, Martin; Ruepp, Marc-David; Jack, Thomas; Simonin, Jonathan; Thompson, Andrew J; Jones, Jeffrey P; Brozik, James A

2015-11-01

The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fully Quantum Fluctuation Theorems

NASA Astrophysics Data System (ADS)

Åberg, Johan

2018-02-01

Systems that are driven out of thermal equilibrium typically dissipate random quantities of energy on microscopic scales. Crooks fluctuation theorem relates the distribution of these random work costs to the corresponding distribution for the reverse process. By an analysis that explicitly incorporates the energy reservoir that donates the energy and the control system that implements the dynamic, we obtain a quantum generalization of Crooks theorem that not only includes the energy changes in the reservoir but also the full description of its evolution, including coherences. Moreover, this approach opens up the possibility for generalizations of the concept of fluctuation relations. Here, we introduce "conditional" fluctuation relations that are applicable to nonequilibrium systems, as well as approximate fluctuation relations that allow for the analysis of autonomous evolution generated by global time-independent Hamiltonians. We furthermore extend these notions to Markovian master equations, implicitly modeling the influence of the heat bath.

Comparisons of Photosynthetic Responses of Xanthium strumarium and Helianthus annuus to Chronic and Acute Water Stress in Sun and Shade 1

PubMed Central

Ben, Gui-Ying; Osmond, C. Barry; Sharkey, Thomas D.

1987-01-01

We have examined the effects of mild, chronic water stress and acute water stress on two water stress sensitive plants, Xanthium strumarium and Helianthus annuus. Using a combination of the leaf disc O2 electrode to measure the light responses of photosynthesis and 77 K fluorescence to monitor damage to the primary photochemistry, we have found the following: (a) The CO2 saturated rate of photosynthesis at high light is the most water stress sensitive parameter measured. (b) The apparent quantum yield (moles O2 per mole photons) was slightly, if at all, affected by mild water stress (>−1.5 megapascals). (c) Severe water stress (<−1.5 megapascals) reduced the quantum yield of photosynthesis regardless of whether the stress was applied in sun or shade. The light independent reduction of quantum yield was not associated with a reduction in 77 K fluorescence (Fv/Fm) indicating that the quantum yield reduction was not the result of damage to primary photochemistry. (d) The diel fluctuation in 77 K fluorescence seen in sun-exposed control leaves was greatly exaggerated in water stressed leaves because of enhanced decline in 77 K fluorescence in the morning. The rate of recovery was similar in both control and water stressed leaves. Shaded leaves showed no change in 77 K fluorescence regardless of whether water stress was imposed or not. (e) The water stress sensitive plants used in these experiments did not recover from acute water stress severe enough to reduce the quantum yield or chronic water stress which lasted long enough that light dependent damage to primary photochemistry occurred. PMID:16665465

Comparisons of Photosynthetic Responses of Xanthium strumarium and Helianthus annuus to Chronic and Acute Water Stress in Sun and Shade.

PubMed

Ben, G Y; Osmond, C B; Sharkey, T D

1987-06-01

We have examined the effects of mild, chronic water stress and acute water stress on two water stress sensitive plants, Xanthium strumarium and Helianthus annuus. Using a combination of the leaf disc O(2) electrode to measure the light responses of photosynthesis and 77 K fluorescence to monitor damage to the primary photochemistry, we have found the following: (a) The CO(2) saturated rate of photosynthesis at high light is the most water stress sensitive parameter measured. (b) The apparent quantum yield (moles O(2) per mole photons) was slightly, if at all, affected by mild water stress (>-1.5 megapascals). (c) Severe water stress (<-1.5 megapascals) reduced the quantum yield of photosynthesis regardless of whether the stress was applied in sun or shade. The light independent reduction of quantum yield was not associated with a reduction in 77 K fluorescence (F(v)/F(m)) indicating that the quantum yield reduction was not the result of damage to primary photochemistry. (d) The diel fluctuation in 77 K fluorescence seen in sun-exposed control leaves was greatly exaggerated in water stressed leaves because of enhanced decline in 77 K fluorescence in the morning. The rate of recovery was similar in both control and water stressed leaves. Shaded leaves showed no change in 77 K fluorescence regardless of whether water stress was imposed or not. (e) The water stress sensitive plants used in these experiments did not recover from acute water stress severe enough to reduce the quantum yield or chronic water stress which lasted long enough that light dependent damage to primary photochemistry occurred.

Transition between Two Regimes Describing Internal Fluctuation of DNA in a Nanochannel

PubMed Central

Su, Tianxiang; Das, Somes K.; Xiao, Ming; Purohit, Prashant K.

2011-01-01

We measure the thermal fluctuation of the internal segments of a piece of DNA confined in a nanochannel about 50100 nm wide. This local thermodynamic property is key to accurate measurement of distances in genomic analysis. For DNA in 100 nm channels, we observe a critical length scale 10 m for the mean extension of internal segments, below which the de Gennes' theory describes the fluctuations with no fitting parameters, and above which the fluctuation data falls into Odijk's deflection theory regime. By analyzing the probability distributions of the extensions of the internal segments, we infer that folded structures of length 150250 nm, separated by 10 m exist in the confined DNA during the transition between the two regimes. For 50 nm channels we find that the fluctuation is significantly reduced since the Odijk regime appears earlier. This is critical for genomic analysis. We further propose a more detailed theory based on small fluctuations and incorporating the effects of confinement to explicitly calculate the statistical properties of the internal fluctuations. Our theory is applicable to polymers with heterogeneous mechanical properties confined in non-uniform channels. We show that existing theories for the end-to-end extension/fluctuation of polymers can be used to study the internal fluctuations only when the contour length of the polymer is many times larger than its persistence length. Finally, our results suggest that introducing nicks in the DNA will not change its fluctuation behavior when the nick density is below 1 nick per kbp DNA. PMID:21423606

Chemical characterization of dissolved organic matter in an alpine stream from thawing and collapsing permafrost to Qinghai Lake

NASA Astrophysics Data System (ADS)

Wang, Y.; Xu, Y.

2016-12-01

The Tibetan Plateau is the world's largest and highest plateau, approximately two thirds of which is covered by permafrost. Due to recent climate warming, large organic carbon stored in the permafrost is thawing and becomes available for transport to aquatic ecosystems (i.e., stream and lake) as dissolved organic matter (DOM) and fine particulate organic matter (POM). These DOM and POM are not only important food sources for the aquatic food web, but also a significant feedback if remineralized during transport. In this work, we collected water samples along a stream from the headwater in the Permafrost region to the downstream in the Qinghai Lake. The concentration and composition of DOM were determined using high temperature combustion analysis, UV- Vis absorption spectroscopy and fluorescence spectroscopy. The concentration of dissolved organic carbon decreased sharply from 13.87 mg/L to 4.32 mg/L from collapsing permafrost area (3850 m a.s.l.) to the foot of the mountain (3200 m a.s.l.), and then fluctuated in a narrow range between 3.00 mg/L and 4.50 mg/L. The DOM with high humic-like fluorescence, specific UV absorbance (SUVA254), and low spectral slope ratio (S275-295) and fluorescence index (FI) was observed in the headwater, which was distinct difference from that at the middle and downstream area where the DOM are less aromatic and low molecular weight. Meanwhile, the freshness index (β/α) increased slightly in mid and down-stream. This increasing trend for FI and β/α indicated a contribution of recently in situ produced DOM by aquatic bacteria and algae in the stream. We speculate that the biological process is an important way to cause the chemical change of DOM composition and concentration, and therefore the thawing and transport of permafrost carbon may play a key role in sustaining the alpine stream ecosystem.

The Phasor Approach to Fluorescence Lifetime Imaging Analysis

PubMed Central

Digman, Michelle A.; Caiolfa, Valeria R.; Zamai, Moreno; Gratton, Enrico

2008-01-01

Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of fluorescence resonance energy transfer. The analysis of the fluorescence lifetime imaging microscopy (FLIM) data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or nonlinear fitting. The phasor approach has the potential to simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the nonexpert in spectroscopy and data analysis. PMID:17981902

Fluorescence intermittency originates from reclustering in two-dimensional organic semiconductors

DOE PAGES

Ruth, Anthony; Hayashi, Michitoshi; Zapol, Peter; ...

2017-02-22

Fluorescence intermittency or blinking is observed in nearly all nanoscale fluorophores. It is characterized by universal power-law distributions in on- and off-times as well as 1/f behaviour in corresponding emission power spectral densities. Blinking, previously seen in confined zero- and one-dimensional systems has recently been documented in two-dimensional reduced graphene oxide. Here we show that unexpected blinking during graphene oxide-to-reduced graphene oxide photoreduction is attributed, in large part, to the redistribution of carbon sp 2 domains. This reclustering generates fluctuations in the number/size of emissive graphenic nanoclusters wherein multiscale modelling captures essential experimental aspects of reduced graphene oxide’s absorption/emission trajectories,more » while simultaneously connecting them to the underlying photochemistry responsible for graphene oxide’s reduction. These simulations thus establish causality between currently unexplained, long timescale emission intermittency in a quantum mechanical fluorophore and identifiable chemical reactions that ultimately lead to switching between on and off states.« less

Single-Molecule Spectroscopy Unmasks the Lowest Exciton State of the B850 Assembly in LH2 from Rps. acidophila

PubMed Central

Kunz, Ralf; Timpmann, Kõu; Southall, June; Cogdell, Richard J.; Freiberg, Arvi; Köhler, Jürgen

2014-01-01

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy. PMID:24806933

Benzothiazole-Based AIEgen with Tunable Excited-State Intramolecular Proton Transfer and Restricted Intramolecular Rotation Processes for Highly Sensitive Physiological pH Sensing.

PubMed

Li, Kai; Feng, Qi; Niu, Guangle; Zhang, Weijie; Li, Yuanyuan; Kang, Miaomiao; Xu, Kui; He, Juan; Hou, Hongwei; Tang, Ben Zhong

2018-04-23

In this work, a benzothiazole-based aggregation-induced emission luminogen (AIEgen) of 2-(5-(4-carboxyphenyl)-2-hydroxyphenyl)benzothiazole (3) was designed and synthesized, which exhibited multifluorescence emissions in different dispersed or aggregated states based on tunable excited-state intramolecular proton transfer (ESIPT) and restricted intramolecular rotation (RIR) processes. 3 was successfully used as a ratiometric fluorescent chemosensor for the detection of pH, which exhibited reversible acid/base-switched yellow/cyan emission transition. More importantly, the pH jump of 3 was very precipitous from 7.0 to 8.0 with a midpoint of 7.5, which was well matched with the physiological pH. This feature makes 3 very suitable for the highly sensitive detection of pH fluctuation in biosamples and neutral water samples. 3 was also successfully used as a ratiometric fluorescence chemosensor for the detection of acidic and basic organic vapors in test papers.

Bioinspired design of a polymer gel sensor for the realization of extracellular Ca2+ imaging

NASA Astrophysics Data System (ADS)

Ishiwari, Fumitaka; Hasebe, Hanako; Matsumura, Satoko; Hajjaj, Fatin; Horii-Hayashi, Noriko; Nishi, Mayumi; Someya, Takao; Fukushima, Takanori

2016-04-01

Although the role of extracellular Ca2+ draws increasing attention as a messenger in intercellular communications, there is currently no tool available for imaging Ca2+ dynamics in extracellular regions. Here we report the first solid-state fluorescent Ca2+ sensor that fulfills the essential requirements for realizing extracellular Ca2+ imaging. Inspired by natural extracellular Ca2+-sensing receptors, we designed a particular type of chemically-crosslinked polyacrylic acid gel, which can undergo single-chain aggregation in the presence of Ca2+. By attaching aggregation-induced emission luminogen to the polyacrylic acid as a pendant, the conformational state of the main chain at a given Ca2+ concentration is successfully translated into fluorescence property. The Ca2+ sensor has a millimolar-order apparent dissociation constant compatible with extracellular Ca2+ concentrations, and exhibits sufficient dynamic range and excellent selectivity in the presence of physiological concentrations of biologically relevant ions, thus enabling monitoring of submillimolar fluctuations of Ca2+ in flowing analytes containing millimolar Ca2+ concentrations.

Measurement of density and temperature in a hypersonic turbulent boundary layer using the electron beam fluorescence technique. Ph.D. Thesis. Final Report, 1 Oct. 1969 - 1 Sep. 1972

NASA Technical Reports Server (NTRS)

Mcronald, A. D.

1975-01-01

Mean density and temperature fluctuations were measured across the turbulent, cooled-wall boundary layer in a continuous hypersonic (Mach 9.4) wind tunnel in air, using the nitrogen fluorescence excited by a 50 kV electron beam. Data were taken at three values of the tunnel stagnation pressure, the corresponding free stream densities being equivalent to 1.2, 4.0, and 7.4 torr at room temperature, and the boundary layer thicknesses about 4.0, 4.5, and 6.0 inches. The mean temperature and density profiles were similar to those previously determined in the same facility by conventional probes (static and pitot pressure, total temperature). A static pressure variation of about 50% across the boundary layer was found, the shape of the variation changing somewhat for the three stagnation pressure levels. The quadrupole model for rotational temperature spectra gave closer agreement with the free stream isentropic level (approximately 44 K) than the dipole model.

Subcycle quantum physics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Leitenstorfer, Alfred

2017-02-01

A time-domain approach to quantum electrodynamics is presented, covering the entire mid-infrared and terahertz frequency ranges. Ultrabroadband electro-optic sampling with few-femtosecond laser pulses allows direct detection of the vacuum fluctuations of the electric field in free space [1,2]. Besides the Planck and electric field fundamental constants, the variance of the ground state is determined solely by the inverse of the four-dimensional space-time volume over which a measurement or physical process integrates. Therefore, we can vary the contribution of multi-terahertz vacuum fluctuations and discriminate against the trivial shot noise due to the constant flux of near-infrared probe photons. Subcycle temporal resolution based on a nonlinear phase shift provides signals from purely virtual photons for accessing the ground-state wave function without amplification to finite intensity. Recently, we have succeeded in generation and analysis of mid-infrared squeezed transients with quantum noise patterns that are time-locked to the intensity envelope of the probe pulses. We find subcycle temporal positions with a noise level distinctly below the bare vacuum which serves as a direct reference. Delay times with increased differential noise indicate generation of highly correlated quantum fields by spontaneous parametric fluorescence. Our time-domain approach offers a generalized understanding of spontaneous emission processes as a consequence of local anomalies in the co-propagating reference frame modulating the quantum vacuum, in combination with the boundary conditions set by Heisenberg's uncertainty principle. [1] C. Riek et al., Science 350, 420 (2015) [2] A. S. Moskalenko et al., Phys. Rev. Lett. 115, 263601 (2015)

Mass sensitive observables of the Pierre Auger Observatory

NASA Astrophysics Data System (ADS)

Unger, M.

2013-06-01

In this article we will discuss measurements of the longitudinal development of air showers at the Pierre Auger Observatory. The longitudinal development of the electromagnetic component can be directly observed by the fluorescence telescopes of the Auger Observatory and we will present the results on the evolution of the average shower maximum and its fluctuations as a function of energy. Moreover, two observables from the surface detector, the asymmetry of the rise time of the station signals and the muon production depth, will be discussed and the measurements will be compared to predictions from air shower simulations for different primary particle types.

Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations

PubMed Central

Mlodzianoski, Michael J.; Curthoys, Nikki M.; Gunewardene, Mudalige S.; Carter, Sean; Hess, Samuel T.

2016-01-01

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample. PMID:27002724

Analysis of Handwriting based on Rhythm Perception

NASA Astrophysics Data System (ADS)

Saito, Kazuya; Uchida, Masafumi; Nozawa, Akio

Humanity fluctuation was reported in some fields. In handwriting process, fluctuation appears on handwriting-velocity. In this report, we focused attention on human rhythm perception and analyzed fluctuation in handwriting process. As a result, 1/f noise related to rhythm perception and features may caused by Kahneman's capacity model were measured on handwriting process.

Life cycle analysis of greenhouse gas emissions for fluorescent lamps in mainland China.

PubMed

Chen, Sha; Zhang, Jiaxing; Kim, Junbeum

2017-01-01

China is the world's largest emitter of carbon dioxide, and it is also one of the largest fluorescent lamp consuming and producing country in the world. However, there are few studies evaluating greenhouse gas (GHG) emissions of fluorescent lamps in China. This analysis compared GHG emissions of compact fluorescent lamps with linear fluorescent lamps using life cycle assessment method in China's national conditions. The GHG emissions of fluorescent lamps from their manufacture to the final disposal phase on the national level of China were also quantified. The results indicate that the use phase dominates the GHG emissions for both lamps. Linear fluorescent lamp is a better source of light compared to compact fluorescent lamp with respect to GHG emissions. The analysis found that in 2011, China generated around 710.90milliontons CO 2 -eq associated with fluorescent lamps. The raw material production and use phases accounted for major GHG emissions. More than half of GHG emissions during the domestic production were embodied in the exported lamps in recent years. This urges the government to take necessary measures that lead to more environmental friendly production, consumption and trade patterns. Copyright © 2016 Elsevier B.V. All rights reserved.

Investigation of Membrane Receptors' Oligomers Using Fluorescence Resonance Energy Transfer and Multiphoton Microscopy in Living Cells

NASA Astrophysics Data System (ADS)

Mishra, Ashish K.

Investigating quaternary structure (oligomerization) of macromolecules (such as proteins and nucleic acids) in living systems (in vivo) has been a great challenge in biophysics, due to molecular diffusion, fluctuations in several biochemical parameters such as pH, quenching of fluorescence by oxygen (when fluorescence methods are used), etc. We studied oligomerization of membrane receptors in living cells by means of Fluorescence (Forster) Resonance Energy Transfer (FRET) using fluorescent markers and two photon excitation fluorescence micro-spectroscopy. Using suitable FRET models, we determined the stoichiometry and quaternary structure of various macromolecular complexes. The proteins of interest for this work are : (1) sigma-1 receptor and (2) rhodopsin, are described as below. (1) Sigma-1 receptors are molecular chaperone proteins, which also regulate ion channels. S1R seems to be involved in substance abuse, as well as several diseases such as Alzheimer's. We studied S1R in the presence and absence of its ligands haloperidol (an antagonist) and pentazocine +/- (an agonist), and found that at low concentration they reside as a mixture of monomers and dimers and that they may form higher order oligomers at higher concentrations. (2) Rhodopsin is a prototypical G protein coupled receptor (GPCR) and is directly involved in vision. GPCRs form a large family of receptors that participate in cell signaling by responding to external stimuli such as drugs, thus being a major drug target (more than 40% drugs target GPCRs). Their oligomerization has been largely controversial. Understanding this may help to understand the functional role of GPCRs oligomerization, and may lead to the discovery of more drugs targeting GPCR oligomers. It may also contribute toward finding a cure for Retinitis Pigmentosa, which is caused by a mutation (G188R) in rhodopsin, a disease which causes blindness and has no cure so far. Comparing healthy rhodopsin's oligomeric structure with that of the mutant may give clues to find the cure.

Lettuce flavonoids screening and phenotyping by chlorophyll fluorescence excitation ratio.

PubMed

Zivcak, Marek; Brückova, Klaudia; Sytar, Oksana; Brestic, Marian; Olsovska, Katarina; Allakhverdiev, Suleyman I

2017-06-01

Environmentally induced variation and the genotypic differences in flavonoid and phenolic content in lettuce can be reliably detected using the appropriate parameters derived from the records of rapid non-invasive fluorescence technique. The chlorophyll fluorescence excitation ratio method was designed as a rapid and non-invasive tool to estimate the content of UV-absorbing phenolic compounds in plants. Using this technique, we have assessed the dynamics of accumulation of flavonoids related to developmental changes and environmental effects. Moreover, we have tested appropriateness of the method to identify the genotypic differences and fluctuations in total phenolics and flavonoid content in lettuce. Six green and two red genotypes of lettuce (Lactuca sativa L.) grown in pots were exposed to two different environments for 50 days: direct sunlight (UV-exposed) and greenhouse conditions (low UV). The indices based on the measurements of chlorophyll fluorescence after red, green and UV excitation indicated increase of the content of UV-absorbing compounds and anthocyanins in the epidermis of lettuce leaves. In similar, the biochemical analyses performed at the end of the experiment confirmed significantly higher total phenolic and flavonoid content in lettuce plants exposed to direct sun compared to greenhouse conditions and in red compared to green genotypes. As the correlation between the standard fluorescence indices and the biochemical records was negatively influenced by the presence of red genotypes, we proposed the use of a new parameter named Modified Flavonoid Index (MFI) taking into an account both absorbance changes due to flavonol and anthocyanin content, for which the correlation with flavonoid and phenolic content was relatively good. Thus, our results confirmed that the fluorescence excitation ratio method is useful for identifying the major differences in phenolic and flavonoid content in lettuce plants and it can be used for high-throughput pre-screening and phenotyping of leafy vegetables in research and breeding applications towards improvement of vegetable health effects.

Quasi-linear theory of electron density and temperature fluctuations with application to MHD generators and MPD arc thrusters

NASA Technical Reports Server (NTRS)

Smith, M.

1972-01-01

Fluctuations in electron density and temperature coupled through Ohm's law are studied for an ionizable medium. The nonlinear effects are considered in the limit of a third order quasi-linear treatment. Equations are derived for the amplitude of the fluctuation. Conditions under which a steady state can exist in the presence of the fluctuation are examined and effective transport properties are determined. A comparison is made to previously considered second order theory. The effect of third order terms indicates the possibility of fluctuations existing in regions predicted stable by previous analysis.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

PubMed

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun; Chang, Po-Ling

2017-09-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target ( HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

Mixedness determination of rare earth-doped ceramics

NASA Astrophysics Data System (ADS)

Czerepinski, Jennifer H.

The lack of chemical uniformity in a powder mixture, such as clustering of a minor component, can lead to deterioration of materials properties. A method to determine powder mixture quality is to correlate the chemical homogeneity of a multi-component mixture with its particle size distribution and mixing method. This is applicable to rare earth-doped ceramics, which require at least 1-2 nm dopant ion spacing to optimize optical properties. Mixedness simulations were conducted for random heterogeneous mixtures of Nd-doped LaF3 mixtures using the Concentric Shell Model of Mixedness (CSMM). Results indicate that when the host to dopant particle size ratio is 100, multi-scale concentration variance is optimized. In order to verify results from the model, experimental methods that probe a mixture at the micro, meso, and macro scales are needed. To directly compare CSMM results experimentally, an image processing method was developed to calculate variance profiles from electron images. An in-lens (IL) secondary electron image is subtracted from the corresponding Everhart-Thornley (ET) secondary electron image in a Field-Emission Scanning Electron Microscope (FESEM) to produce two phases and pores that can be quantified with 50 nm spatial resolution. A macro was developed to quickly analyze multi-scale compositional variance from these images. Results for a 50:50 mixture of NdF3 and LaF3 agree with the computational model. The method has proven to be applicable only for mixtures with major components and specific particle morphologies, but the macro is useful for any type of imaging that produces excellent phase contrast, such as confocal microscopy. Fluorescence spectroscopy was used as an indirect method to confirm computational results for Nd-doped LaF3 mixtures. Fluorescence lifetime can be used as a quantitative method to indirectly measure chemical homogeneity when the limits of electron microscopy have been reached. Fluorescence lifetime represents the compositional fluctuations of a dopant on the nanoscale while accounting for billions of particles in a fast, non-destructive manner. The significance of this study will show how small-scale fluctuations in homogeneity limit the optimization of optical properties, which can be improved by the proper selection of particle size and mixing method.

Optically detected X-ray absorption spectroscopy measurements as a means of monitoring corrosion layers on copper.

PubMed

Dowsett, Mark G; Adriaens, Annemie; Jones, Gareth K C; Poolton, Nigel; Fiddy, Steven; Nikitenko, Sergé

2008-11-15

XANES and EXAFS information is conventionally measured in transmission through the energy-dependent absorption of X-rays or by observing X-ray fluorescence, but secondary fluorescence processes, such as the emission of electrons and optical photons (e.g., 200-1000 nm), can also be used as a carrier of the XAS signatures, providing complementary information such as improved surface specificity. Where the near-visible photons have a shorter range in a material, the data will be more surface specific. Moreover, optical radiation may escape more readily than X-rays through liquid in an environmental cell. Here, we describe a first test of optically detected X-ray absorption spectroscopy (ODXAS) for monitoring electrochemical treatments on copper-based alloys, for example, heritage metals. Artificially made corrosion products deposited on a copper substrate were analyzed in air and in a 1% (w/v) sodium sesquicarbonate solution to simulate typical conservation methods for copper-based objects recovered from marine environments. The measurements were made on stations 7.1 and 9.2 MF (SRS Daresbury, UK) using the mobile luminescence end station (MoLES), supplemented by XAS measurements taken on DUBBLE (BM26 A) at the ESRF. The ODXAS spectra usually contain fine structure similar to that of XAS spectra measured in X-ray fluorescence. Importantly, for the compounds examined, the ODXAS is significantly more surface specific, and >98% characteristic of thin surface layers of 0.5-1.5-microm thickness in cases where X-ray measurements are dominated by the substrate. However, EXAFS and XANES from broadband optical measurements are superimposed on a high background due to other optical emission modes. This produces statistical fluctuations up to double what would be expected from normal counting statistics because the data retain the absolute statistical fluctuation in the original raw count, while losing up to 70% of their magnitude when background is removed. The problem may be solved in future through optical filtering to isolate the information-containing band, combined with the use of higher input X-ray fluxes available on third-generation light sources.

The impact of fluctuating light on the dinoflagellate Prorocentrum micans depends on NO3(-) and CO2 availability.

PubMed

Zheng, Ying; Giordano, Mario; Gao, Kunshan

2015-05-15

Increasing atmospheric pCO2 and its dissolution into oceans leads to ocean acidification and warming, which reduces the thickness of upper mixing layer (UML) and upward nutrient supply from deeper layers. These events may alter the nutritional conditions and the light regime to which primary producers are exposed in the UML. In order to better understand the physiology behind the responses to the concomitant climate changes factors, we examined the impact of light fluctuation on the dinoflagellate Prorocentrum micans grown at low (1 μmol L(-1)) or high (800 μmol L(-1)) [NO3(-)] and at high (1000 μatm) or low (390 μatm, ambient) pCO2. The light regimes to which the algal cells were subjected were (1) constant light at a photon flux density (PFD) of either 100 (C100) or 500 (C500) μmol m(-2) s(-1) or (2) fluctuating light between 100 or 500 μmol photons m(-2) s(-1) with a frequency of either 15 (F15) or 60 (F60) min. Under continuous light, the initial portion of the light phase required the concomitant presence of high CO2 and NO3(-) concentrations for maximum growth. After exposure to light for 3h, high CO2 exerted a negative effect on growth and effective quantum yield of photosystem II (F'(v)/F'(m)). Fluctuating light ameliorated growth in the first period of illumination. In the second 3h of treatment, higher frequency (F15) of fluctuations afforded high growth rates, whereas the F60 treatment had detrimental consequences, especially when NO3(-) concentration was lower. F'(v)/F'(m) respondent differently from growth to fluctuating light: the fluorescence yield was always lower than at continuous light at 100 μmol m(-2) s(-1), and always higher at 500 μmol m(-2) s(-1). Our data show that the impact of atmospheric pCO2 increase on primary production of dinoflagellate depends on the availability of nitrate and the irradiance (intensity and the frequency of irradiance fluctuations) to which the cells are exposed. The impact of global change on oceanic primary producers would therefore be different in waters with different chemical and physical (mixing) properties. Copyright © 2015 Elsevier GmbH. All rights reserved.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

NASA Astrophysics Data System (ADS)

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-09-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

PubMed Central

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-01-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology. PMID:25252596

Comparison of fluorescence probes for intracellular sodium imaging in prostate cancer cell lines.

PubMed

Iamshanova, Oksana; Mariot, Pascal; Lehen'kyi, V'yacheslav; Prevarskaya, Natalia

2016-10-01

Sodium (Na + ) ions are known to regulate many signaling pathways involved in both physiological and pathological conditions. In particular, alterations in intracellular concentrations of Na + and corresponding changes in membrane potential are known to be major actors of cancer progression to metastatic phenotype. Though the functionality of Na + channels and the corresponding Na + currents can be investigated using the patch-clamp technique, the latter is rather invasive and a technically difficult method to study intracellular Na + transients compared to Na + fluorescence imaging. Despite the fact that Na + signaling is considered an important controller of cancer progression, only few data using Na + imaging approaches are available so far, suggesting the persisting challenge within the scientific community. In this study, we describe in detail the approach for application of Na + imaging technique to measure intracellular Na + variations in human prostate cancer cells. Accordingly, we used three Na + -specific fluorescent dyes-Na + -binding benzofuran isophthalate (SBFI), CoroNa™ Green (Corona) and Asante NaTRIUM Green-2 (ANG-2). These dyes have been assessed for optimal loading conditions, dissociation constant and working range after different calibration methods, and intracellular Na + sensitivity, in order to determine which probe can be considered as the most reliable to visualize Na + fluctuations in vitro.

Studying ion exchange in solution and at biological membranes by FCS.

PubMed

Widengren, Jerker

2013-01-01

By FCS, a wide range of processes can be studied, covering time ranges from subnanoseconds to seconds. In principle, any process at equilibrium conditions can be measured, which reflects itself by a change in the detected fluorescence intensity. In this review, it is described how FCS and variants thereof can be used to monitor ion exchange, in solution and along biological membranes. Analyzing fluorescence fluctuations of ion-sensitive fluorophores by FCS offers selective advantages over other techniques for measuring local ion concentrations, and, in particular, for studying exchange kinetics of ions on a very local scale. This opens for several areas of application. The FCS approach was used to investigate fundamental aspects of proton exchange at and along biological membranes. The protonation relaxation rate, as measured by FCS for a pH-sensitive dye, can also provide information about local accessibility/interaction of a particular labeling site and conformational states of biomolecules, in a similar fashion as in a fluorescence quenching experiment. The same FCS concept can also be applied to ion exchange studies using other ion-sensitive fluorophores, and by use of dyes sensitive to other ambient conditions the concept can be extended also beyond ion exchange studies. Copyright © 2013 Elsevier Inc. All rights reserved.

Time-resolved fluorescence decay measurements for flowing particles

DOEpatents

Deka, C.; Steinkamp, J.A.

1999-06-01

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

Time-resolved fluorescence decay measurements for flowing particles

DOEpatents

Deka, Chiranjit; Steinkamp, John A.

1999-01-01

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

5-ALA induced fluorescent image analysis of actinic keratosis

NASA Astrophysics Data System (ADS)

Cho, Yong-Jin; Bae, Youngwoo; Choi, Eung-Ho; Jung, Byungjo

2010-02-01

In this study, we quantitatively analyzed 5-ALA induced fluorescent images of actinic keratosis using digital fluorescent color and hyperspectral imaging modalities. UV-A was utilized to induce fluorescent images and actinic keratosis (AK) lesions were demarcated from surrounding the normal region with different methods. Eight subjects with AK lesion were participated in this study. In the hyperspectral imaging modality, spectral analysis method was utilized for hyperspectral cube image and AK lesions were demarcated from the normal region. Before image acquisition, we designated biopsy position for histopathology of AK lesion and surrounding normal region. Erythema index (E.I.) values on both regions were calculated from the spectral cube data. Image analysis of subjects resulted in two different groups: the first group with the higher fluorescence signal and E.I. on AK lesion than the normal region; the second group with lower fluorescence signal and without big difference in E.I. between two regions. In fluorescent color image analysis of facial AK, E.I. images were calculated on both normal and AK lesions and compared with the results of hyperspectral imaging modality. The results might indicate that the different intensity of fluorescence and E.I. among the subjects with AK might be interpreted as different phases of morphological and metabolic changes of AK lesions.

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

PubMed

Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

Critical analysis of commonly used fluorescence metrics to characterize dissolved organic matter.

PubMed

Korak, Julie A; Dotson, Aaron D; Summers, R Scott; Rosario-Ortiz, Fernando L

2014-02-01

The use of fluorescence spectroscopy for the analysis and characterization of dissolved organic matter (DOM) has gained widespread interest over the past decade, in part because of its ease of use and ability to provide bulk DOM chemical characteristics. However, the lack of standard approaches for analysis and data evaluation has complicated its use. This study utilized comparative statistics to systematically evaluate commonly used fluorescence metrics for DOM characterization to provide insight into the implications for data analysis and interpretation such as peak picking methods, carbon-normalized metrics and the fluorescence index (FI). The uncertainty associated with peak picking methods was evaluated, including the reporting of peak intensity and peak position. The linear relationship between fluorescence intensity and dissolved organic carbon (DOC) concentration was found to deviate from linearity at environmentally relevant concentrations and simultaneously across all peak regions. Comparative analysis suggests that the loss of linearity is composition specific and likely due to non-ideal intermolecular interactions of the DOM rather than the inner filter effects. For some DOM sources, Peak A deviated from linearity at optical densities a factor of 2 higher than that of Peak C. For carbon-normalized fluorescence intensities, the error associated with DOC measurements significantly decreases the ability to distinguish compositional differences. An in-depth analysis of FI determined that the metric is mostly driven by peak emission wavelength and less by emission spectra slope. This study also demonstrates that fluorescence intensity follows property balance principles, but the fluorescence index does not. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fluctuations of pol I and fibrillarin contents of the nucleoli.

PubMed

Hornáček, M; Kováčik, L; Mazel, T; Cmarko, D; Bártová, E; Raška, I; Smirnov, E

2017-07-04

Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

Distribution of transverse chain fluctuations in harmonically confined semiflexible polymers

NASA Astrophysics Data System (ADS)

Sharma, Rati; Cherayil, Binny J.

2012-05-01

Two different experimental studies of polymer dynamics based on single-molecule fluorescence imaging have recently found evidence of heterogeneities in the widths of the putative tubes that surround filaments of F-actin during their motion in concentrated solution. In one [J. Glaser, D. Chakraborty, K. Kroy, I. Lauter, M. Degawa, N. Kirchesner, B. Hoffmann, R. Merkel, and M. Giesen, Phys. Rev. Lett. 105, 037801 (2010)], 10.1103/PhysRevLett.105.037801, the observations were explained in terms of the statistics of a worm-like chain confined to a potential determined self-consistently by a binary collision approximation, and in the other [B. Wang, J. Guan, S. M. Anthony, S. C. Bae, K. S. Schweizer, and S. Granick, Phys. Rev. Lett. 104, 118301 (2010)], 10.1103/PhysRevLett.104.118301, they were explained in terms of the scaling properties of a random fluid of thin rods. In this paper, we show, using an exact path integral calculation, that the distribution of the length-averaged transverse fluctuations of a harmonically confined weakly bendable rod (one possible realization of a semiflexible chain in a tube), is in good qualitative agreement with the experimental data, although it is qualitatively different in analytic structure from the earlier theoretical predictions. We also show that similar path integral techniques can be used to obtain an exact expression for the time correlation function of fluctuations in the tube cross section.

Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin.

PubMed

Nakashima, Kenichi; Yuda, Kazuki; Ozaki, Yukihiro; Noda, Isao

2003-11-01

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.

Analysis of fluctuations in semiconductor devices

NASA Astrophysics Data System (ADS)

Andrei, Petru

The random nature of ion implantation and diffusion processes as well as inevitable tolerances in fabrication result in random fluctuations of doping concentrations and oxide thickness in semiconductor devices. These fluctuations are especially pronounced in ultrasmall (nanoscale) semiconductor devices when the spatial scale of doping and oxide thickness variations become comparable with the geometric dimensions of devices. In the dissertation, the effects of these fluctuations on device characteristics are analyzed by using a new technique for the analysis of random doping and oxide thickness induced fluctuations. This technique is universal in nature in the sense that it is applicable to any transport model (drift-diffusion, semiclassical transport, quantum transport etc.) and it can be naturally extended to take into account random fluctuations of the oxide (trapped) charges and channel length. The technique is based on linearization of the transport equations with respect to the fluctuating quantities. It is computationally much (a few orders of magnitude) more efficient than the traditional Monte-Carlo approach and it yields information on the sensitivity of fluctuations of parameters of interest (e.g. threshold voltage, small-signal parameters, cut-off frequencies, etc.) to the locations of doping and oxide thickness fluctuations. For this reason, it can be very instrumental in the design of fluctuation-resistant structures of semiconductor devices. Quantum mechanical effects are taken into account by using the density-gradient model as well as through self-consistent Poisson-Schrodinger computations. Special attention is paid to the presenting of the technique in a form that is suitable for implementation on commercial device simulators. The numerical implementation of the technique is discussed in detail and numerous computational results are presented and compared with those previously published in literature.

Fluorescence characterization of fractionated dissolved organic matter in the five tributaries of Poyang Lake, China.

PubMed

Yan, Caixia; Liu, Huihui; Sheng, Yanru; Huang, Xian; Nie, Minghua; Huang, Qi; Baalousha, Mohammed

2018-10-01

Characterization of natural colloids is the key to understand pollutant fate and transport in the environment. The present study investigates the relationship between size and fluorescence properties of colloidal organic matter (COM) from five tributaries of Poyang Lake. Colloids were size-fractionated using cross-flow ultrafiltration and their fluorescence properties were measured by three-dimensional excitation-emission matrix fluorescence spectroscopy (3D-EEM). Parallel factor analysis (PARAFAC) and/or Self-organizing map (SOM) were applied to assess fluorescence properties as proxy indicators for the different size of colloids. PARAFAC analysis identified four fluorescence components including three humic-like components (C1-C3) and a protein-like component (C4). These four fluorescence components, and in particular the protein-like component, are primarily present in <1 kDa phase. For the colloidal fractions (1-10 kDa, 10-100 kDa, and 100 kDa-0.7 μm), the majority of fluorophores are associated with the smallest size fraction. SOM analysis demonstrated that relatively high fluorescence intensity and aromaticity occur primarily in <1 kDa phase, followed by 1-10 kDa colloids. Coupling PARAFAC and SOM facilitate the visualization and interpretation of the relationship between colloidal size and fluorescence properties with fewer input variables, shorter running time, higher reliability, and nondestructive results. Fluorescence indices analysis reveals that the smallest colloidal fraction (1-10 kDa) was dominated by higher humified and less autochthonous COM. Copyright © 2018 Elsevier B.V. All rights reserved.

Receiver operating characteristic analysis. Application to the study of quantum fluctuation effects in optic nerve of Rana pipiens

PubMed Central

1975-01-01

Receiver operating characteristic (ROC) analysis of nerve messages is described. The hypothesis that quantum fluctuations provide the only limit to the ability of frog ganglion cells to signal luminance change information is examined using ROC analysis. In the context of ROC analysis, the quantum fluctuation hypothesis predicts (a) the detectability of a luminance change signal should rise proportionally to the size of the change, (b) detectability should decrease as the square root of background, an implication of which is the deVries-Rose law, and (c) ROC curves should exhibit a shape particular to underlying Poisson distributions. Each of these predictions is confirmed for the responses of dimming ganglion cells to brief luminance decrements at scotopic levels, but none could have been tested using classical nerve message analysis procedures. PMID:172597

Correlation electron cyclotron emission diagnostic and improved calculation of turbulent temperature fluctuation levels on ASDEX Upgrade

NASA Astrophysics Data System (ADS)

Creely, A. J.; Freethy, S. J.; Burke, W. M.; Conway, G. D.; Leccacorvi, R.; Parkin, W. C.; Terry, D. R.; White, A. E.

2018-05-01

A newly upgraded correlation electron cyclotron emission (CECE) diagnostic has been installed on the ASDEX Upgrade tokamak and has begun to perform experimental measurements of electron temperature fluctuations. CECE diagnostics measure small amplitude electron temperature fluctuations by correlating closely spaced heterodyne radiometer channels. This upgrade expanded the system from six channels to thirty, allowing simultaneous measurement of fluctuation level radial profiles without repeat discharges, as well as opening up the possibility of measuring radial turbulent correlation lengths. Newly refined statistical techniques have been developed in order to accurately analyze the fluctuation data collected from the CECE system. This paper presents the hardware upgrades for this system and the analysis techniques used to interpret the raw data, as well as measurements of fluctuation spectra and fluctuation level radial profiles.

Molecular dynamics simulations of domain motions of substrate-free S-adenosyl- L-homocysteine hydrolase in solution.

PubMed

Hu, Chen; Fang, Jianwen; Borchardt, Ronald T; Schowen, Richard L; Kuczera, Krzysztof

2008-04-01

S-Adenosyl-L-homocysteine hydrolase (SAHH) is an enzyme regulating intracellular methylation reactions. The homotetrameric SAHH exists in an open conformation in absence of substrate, while enzyme:inhibitor complexes crystallize in the closed conformation, in which the ligands are engulfed by the protein due to an 18 degrees domain reorientation within each of the four subunits. We present a microscopic description of the structure and dynamics of the substrate-free, NAD(+)-bound SAHH in solution, based on a 15-ns molecular dynamics simulation in explicit solvent. In the trajectory, the four cofactor-binding domains formed a relatively rigid core with structure very similar to the crystal conformation. The four substrate-binding domains, located at the protein exterior, also retained internal structures similar to the crystal, while undergoing large amplitude rigid-body reorientations. The trajectory domain motions exhibited two interesting properties. First, within each subunit the domains fluctuated between open and closed conformations, while at the tetramer level 80% of the domain motions were perpendicular to the direction of the open-to-closed structural transition. Second, the domain reorientations in solution could be represented as a sum of two components, faster, with 20-50 ps correlation time and 3-4 degrees amplitude, and slower, with 8-23 ns correlation time and amplitude of 14-22 degrees . The faster motion is similar to the 1.5 cm(-1) frequency hinge-bending vibrations found in our recent normal mode analysis (Wang et al., Biochemistry 2005;44:7228-7239). The slower motion agrees with fluorescence anisotropy decay measurements, which detected a 10-20 ns domain reorientation of ca. 26 degrees amplitude in the substrate-free enzyme (Wang et al., Biochemistry 2006;45:7778-7786). Our simulations are thus in excellent agreement with experimental data. The simulations allow us to assign the observed nanosecond fluorescence anisotropy signal to fluctuations in domain orientations, and indicate that the microscopic mechanism of the motion involves rotational diffusion within a cone of 10-20 degrees . Overall, our simulation results complement the existing experimental data and provide important new insights into SAHH domain motions in solution, which play a crucial role in the catalytic mechanism of SAHH. (c) 2007 Wiley-Liss, Inc.

Can we understand, control and use the blinking of quantum dots in biological surroundings?

NASA Astrophysics Data System (ADS)

Durisic, Nela

Semiconductor nanocrystals, also known as quantum dots (QDs), are evoking remarkable technological and scientific interest due to their fascinating size-dependent electronic and optical properties. Many biophysical studies to date have used effectively as brighter and more photostable replacement of organic dyes. In this thesis we focus on the most prominent feature of their photophysical properties, a random switching between emitting and non-emitting states, also known as fluorescence intermittency, or "blinking," in order to better understand the mechanism of quantum dot emission and how it reflects interaction with their immediate environment. We first designed and built a total internal reflection florescent microscope (TIRFM) with single molecule detection capabilities and determined optimal conditions for single QD studies. We then used image correlation techniques to show that the change in blinking dynamics could be detected and that it could complicate interpretation of the commonly used analytical techniques that rely on intensity fluctuations as reporters of particle mobility. In particular, we demonstrated that the transport coefficients recovered from fluorescence fluctuation analysis of diffusional mobility using temporal image correlation spectroscopy (TICS) had significant systematic errors due to blinking of the nanoparticles. We then performed a thorough, systematic study of the effects of protons on QDs' photochemical stability by varying the pH of their aqueous environment and related the single particle properties to properties of an ensemble of QDs. As pH was lowered, shorter "on" times and longer "off" times were observed, brightness of single QDs decreased and the number of permanently non-emitting QDs (dark fraction) increased. Based on these results, we proposed a coupled role for H+ ions by which they first reduced the intensity of the emitting state as well as affected probabilities of the QD to switch between the "on" and "off" states and eventually trapped the QD in a permanent dark state. We discussed and extended theoretical blinking models to account for the effect of the H+ ions as well as to highlight a common mechanism governing blinking and the dark fraction formation. Results presented in this thesis are particularly important for developing a more complete theory of QD blinking, but are also relevant in various applications of QDs, especially for quantitative biological imaging applications where pH variations exist between the cytoplasmic and extra cellular spaces and within cellular organelles.

Scaling characteristics of one-dimensional fractional diffusion processes in the presence of power-law distributed random noise

NASA Astrophysics Data System (ADS)

Nezhadhaghighi, Mohsen Ghasemi

2017-08-01

Here, we present results of numerical simulations and the scaling characteristics of one-dimensional random fluctuations with heavy-tailed probability distribution functions. Assuming that the distribution function of the random fluctuations obeys Lévy statistics with a power-law scaling exponent, we investigate the fractional diffusion equation in the presence of μ -stable Lévy noise. We study the scaling properties of the global width and two-point correlation functions and then compare the analytical and numerical results for the growth exponent β and the roughness exponent α . We also investigate the fractional Fokker-Planck equation for heavy-tailed random fluctuations. We show that the fractional diffusion processes in the presence of μ -stable Lévy noise display special scaling properties in the probability distribution function (PDF). Finally, we numerically study the scaling properties of the heavy-tailed random fluctuations by using the diffusion entropy analysis. This method is based on the evaluation of the Shannon entropy of the PDF generated by the random fluctuations, rather than on the measurement of the global width of the process. We apply the diffusion entropy analysis to extract the growth exponent β and to confirm the validity of our numerical analysis.

Modeling and statistical analysis of non-Gaussian random fields with heavy-tailed distributions.

PubMed

Nezhadhaghighi, Mohsen Ghasemi; Nakhlband, Abbas

2017-04-01

In this paper, we investigate and develop an alternative approach to the numerical analysis and characterization of random fluctuations with the heavy-tailed probability distribution function (PDF), such as turbulent heat flow and solar flare fluctuations. We identify the heavy-tailed random fluctuations based on the scaling properties of the tail exponent of the PDF, power-law growth of qth order correlation function, and the self-similar properties of the contour lines in two-dimensional random fields. Moreover, this work leads to a substitution for the fractional Edwards-Wilkinson (EW) equation that works in the presence of μ-stable Lévy noise. Our proposed model explains the configuration dynamics of the systems with heavy-tailed correlated random fluctuations. We also present an alternative solution to the fractional EW equation in the presence of μ-stable Lévy noise in the steady state, which is implemented numerically, using the μ-stable fractional Lévy motion. Based on the analysis of the self-similar properties of contour loops, we numerically show that the scaling properties of contour loop ensembles can qualitatively and quantitatively distinguish non-Gaussian random fields from Gaussian random fluctuations.

Scaling characteristics of one-dimensional fractional diffusion processes in the presence of power-law distributed random noise.

PubMed

Nezhadhaghighi, Mohsen Ghasemi

2017-08-01

Here, we present results of numerical simulations and the scaling characteristics of one-dimensional random fluctuations with heavy-tailed probability distribution functions. Assuming that the distribution function of the random fluctuations obeys Lévy statistics with a power-law scaling exponent, we investigate the fractional diffusion equation in the presence of μ-stable Lévy noise. We study the scaling properties of the global width and two-point correlation functions and then compare the analytical and numerical results for the growth exponent β and the roughness exponent α. We also investigate the fractional Fokker-Planck equation for heavy-tailed random fluctuations. We show that the fractional diffusion processes in the presence of μ-stable Lévy noise display special scaling properties in the probability distribution function (PDF). Finally, we numerically study the scaling properties of the heavy-tailed random fluctuations by using the diffusion entropy analysis. This method is based on the evaluation of the Shannon entropy of the PDF generated by the random fluctuations, rather than on the measurement of the global width of the process. We apply the diffusion entropy analysis to extract the growth exponent β and to confirm the validity of our numerical analysis.

Mean-field analysis of an inductive reasoning game: Application to influenza vaccination

NASA Astrophysics Data System (ADS)

Breban, Romulus; Vardavas, Raffaele; Blower, Sally

2007-09-01

Recently we have introduced an inductive reasoning game of voluntary yearly vaccination to establish whether or not a population of individuals acting in their own self-interest would be able to prevent influenza epidemics. Here, we analyze our model to describe the dynamics of the collective yearly vaccination uptake. We discuss the mean-field equations of our model and first order effects of fluctuations. We explain why our model predicts that severe epidemics are periodically expected even without the introduction of pandemic strains. We find that fluctuations in the collective yearly vaccination uptake induce severe epidemics with an expected periodicity that depends on the number of independent decision makers in the population. The mean-field dynamics also reveal that there are conditions for which the dynamics become robust to the fluctuations. However, the transition between fluctuation-sensitive and fluctuation-robust dynamics occurs for biologically implausible parameters. We also analyze our model when incentive-based vaccination programs are offered. When a family-based incentive is offered, the expected periodicity of severe epidemics is increased. This results from the fact that the number of independent decision makers is reduced, increasing the effect of the fluctuations. However, incentives based on the number of years of prepayment of vaccination may yield fluctuation-robust dynamics where severe epidemics are prevented. In this case, depending on prepayment, the transition between fluctuation-sensitive and fluctuation-robust dynamics may occur for biologically plausible parameters. Our analysis provides a practical method for identifying how many years of free vaccination should be provided in order to successfully ameliorate influenza epidemics.

Mean-field analysis of an inductive reasoning game: application to influenza vaccination.

PubMed

Breban, Romulus; Vardavas, Raffaele; Blower, Sally

2007-09-01

Recently we have introduced an inductive reasoning game of voluntary yearly vaccination to establish whether or not a population of individuals acting in their own self-interest would be able to prevent influenza epidemics. Here, we analyze our model to describe the dynamics of the collective yearly vaccination uptake. We discuss the mean-field equations of our model and first order effects of fluctuations. We explain why our model predicts that severe epidemics are periodically expected even without the introduction of pandemic strains. We find that fluctuations in the collective yearly vaccination uptake induce severe epidemics with an expected periodicity that depends on the number of independent decision makers in the population. The mean-field dynamics also reveal that there are conditions for which the dynamics become robust to the fluctuations. However, the transition between fluctuation-sensitive and fluctuation-robust dynamics occurs for biologically implausible parameters. We also analyze our model when incentive-based vaccination programs are offered. When a family-based incentive is offered, the expected periodicity of severe epidemics is increased. This results from the fact that the number of independent decision makers is reduced, increasing the effect of the fluctuations. However, incentives based on the number of years of prepayment of vaccination may yield fluctuation-robust dynamics where severe epidemics are prevented. In this case, depending on prepayment, the transition between fluctuation-sensitive and fluctuation-robust dynamics may occur for biologically plausible parameters. Our analysis provides a practical method for identifying how many years of free vaccination should be provided in order to successfully ameliorate influenza epidemics.

Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10*

PubMed Central

Ho, Derek; Lugo, Miguel R.; Merrill, A. Rod

2013-01-01

The pre-channel state of helices 6, 7, and 10 (Val447–Gly475 and Ile508–Ile522) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain. PMID:23264635

Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation.

PubMed

He, Huaizhen; Zhan, Yingzhuan; Zhang, Yanmin; Zhang, Jie; He, Langchong

2012-01-01

Two novel taspine diphenyl derivatives (Ta-dD) were designed and synthesized by introducing different coumarin fluorescent groups into the basic structure of Ta-dD. The main advantage of these two compounds is that they can be used as fluorescence probes and inhibitors simultaneously. In the present study, the fluorescent properties of the probes were measured and their inhibition of four breast cancer cell lines was tested. Different concentrations of the fluorescence probe were added to MCF-7 breast cancer cells for fluorescence imaging analysis under normal conditions. The results suggested that both of the new compounds have not only fluorescence but also the ability to inhibit effects on different breast cancer cell lines, which indicates their possible further use as dual functional fluorescence probes in tracer analysis. Copyright © 2011 John Wiley & Sons, Ltd.

An investigation of combustion and entropy noise

NASA Technical Reports Server (NTRS)

Strahle, W. C.

1977-01-01

The relative importance of entropy and direct combustion noise in turbopropulsion systems and the parameters upon which these noise sources depend were studied. Theory and experiment were employed to determine that at least with the apparatus used here, entropy noise can dominate combustion noise if there is a sufficient pressure gradient terminating the combustor. Measurements included combustor interior fluctuating pressure, near and far field fluctuating pressure, and combustor exit plane fluctuating temperatures, as well as mean pressures and temperatures. Analysis techniques included spectral, cross-correlation, cross power spectra, and ordinary and partial coherence analysis. Also conducted were combustor liner modification experiments to investigate the origin of the frequency content of combustion noise. Techniques were developed to extract nonpropagational pseudo-sound and the heat release fluctuation spectra from the data.

Wavenumber-frequency Spectra of Pressure Fluctuations Measured via Fast Response Pressure Sensitive Paint

NASA Technical Reports Server (NTRS)

Panda, J.; Roozeboom, N. H.; Ross, J. C.

2016-01-01

The recent advancement in fast-response Pressure-Sensitive Paint (PSP) allows time-resolved measurements of unsteady pressure fluctuations from a dense grid of spatial points on a wind tunnel model. This capability allows for direct calculations of the wavenumber-frequency (k-?) spectrum of pressure fluctuations. Such data, useful for the vibro-acoustics analysis of aerospace vehicles, are difficult to obtain otherwise. For the present work, time histories of pressure fluctuations on a flat plate subjected to vortex shedding from a rectangular bluff-body were measured using PSP. The light intensity levels in the photographic images were then converted to instantaneous pressure histories by applying calibration constants, which were calculated from a few dynamic pressure sensors placed at selective points on the plate. Fourier transform of the time-histories from a large number of spatial points provided k-? spectra for pressure fluctuations. The data provides first glimpse into the possibility of creating detailed forcing functions for vibro-acoustics analysis of aerospace vehicles, albeit for a limited frequency range.

How to resolve microsecond current fluctuations in single ion channels: The power of beta distributions

PubMed Central

Schroeder, Indra

2015-01-01

Abstract A main ingredient for the understanding of structure/function correlates of ion channels is the quantitative description of single-channel gating and conductance. However, a wealth of information provided from fast current fluctuations beyond the temporal resolution of the recording system is often ignored, even though it is close to the time window accessible to molecular dynamics simulations. This kind of current fluctuations provide a special technical challenge, because individual opening/closing or blocking/unblocking events cannot be resolved, and the resulting averaging over undetected events decreases the single-channel current. Here, I briefly summarize the history of fast-current fluctuation analysis and focus on the so-called “beta distributions.” This tool exploits characteristics of current fluctuation-induced excess noise on the current amplitude histograms to reconstruct the true single-channel current and kinetic parameters. A guideline for the analysis and recent applications demonstrate that a construction of theoretical beta distributions by Markov Model simulations offers maximum flexibility as compared to analytical solutions. PMID:26368656

Universality of Electron Distributions in Extensive Air Showers

NASA Astrophysics Data System (ADS)

Śmiałkowski, Andrzej; Giller, Maria

2018-02-01

Based on extensive air shower simulations, it is shown that electron distributions with respect to two angles determining the electron direction at a given shower age, for a fixed electron energy and lateral distance, are universal. This means that the distributions do not depend on the primary particle energy or mass (thus, neither on the interaction model), shower zenith angle, or shower to shower fluctuations, if they are taken at the same shower age. Together with previous work showing the universality of the distributions of the electron energy, lateral distance (integrated over angles), and angle (integrated over lateral distance) for fixed electron energy, this paper completes a full universal description of the electron states at various shower ages. Analytical parametrizations of the full electron states are given. It is also shown that some distributions can be described by a number of variables smaller than five, with the new ones being products of old ones raised to some power. The accuracy of the present parametrization is sufficiently good to apply to showers with a primary energy uncertainty of 14% (as is the case at the Pierre Auger Observatory). The shower fluctuations in the chosen bins of the multidimensional variable space are about 6%, determining the minimum uncertainty needed for the parametrization of the universal distributions. An analytical way of estimating the effect of the geomagnetic field is given. Thanks to the universality of the electron distribution in any shower, a new method of shower reconstruction can be worked out from the data from observatories using the fluorescence technique. The light fluxes (both fluorescence and Cherenkov) for any shower age can be exactly predicted for a shower with any primary energy and shower maximum depth, so that the two quantities can be obtained by best fitting the predictions to the measurements.

Fluorescence analysis of ubiquinone and its application in quality control of medical supplies

NASA Astrophysics Data System (ADS)

Timofeeva, Elvira O.; Gorbunova, Elena V.; Chertov, Aleksandr N.

2017-02-01

The presence of antioxidant issues such as redox potential imbalance in human body is a very important question for modern clinical diagnostics. Implementation of fluorescence analysis into optical diagnostics of such wide distributed in a human body antioxidant as ubiquinone is one of the steps for development of the device with a view to clinical diagnostics of redox potential. Recording of fluorescence was carried out with spectrometer using UV irradiation source with thin band (max at 287 and 330 nm) as a background radiation. Concentrations of ubiquinone from 0.25 to 2.5 mmol/l in explored samples were used for investigation. Recording data was processed using correlation analysis and differential analytical technique. The fourth derivative spectrum of fluorescence spectrum provided the basis for a multicomponent analysis of the solutions. As a technique in clinical diagnostics fluorescence analysis with processing method including differential spectrophotometry, it is step forward towards redox potential calculation and quality control in pharmacy for better health care.

Maximum entropy analysis of polarized fluorescence decay of (E)GFP in aqueous solution

NASA Astrophysics Data System (ADS)

Novikov, Eugene G.; Skakun, Victor V.; Borst, Jan Willem; Visser, Antonie J. W. G.

2018-01-01

The maximum entropy method (MEM) was used for the analysis of polarized fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc. 4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total fluorescence decay and fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions.

Prognostic value of residual fluorescent tissue in glioblastoma patients after gross total resection in 5-aminolevulinic Acid-guided surgery.

PubMed

Aldave, Guillermo; Tejada, Sonia; Pay, Eva; Marigil, Miguel; Bejarano, Bartolomé; Idoate, Miguel A; Díez-Valle, Ricardo

2013-06-01

There is evidence in the literature supporting that fluorescent tissue signal in fluorescence-guided surgery extends farther than tissue highlighted in gadolinium in T1 sequence magnetic resonance imaging (MRI), which is the standard to quantify the extent of resection. To study whether the presence of residual fluorescent tissue after surgery carries a different prognosis for glioblastoma (GBM) cases with complete resection confirmed by MRI. A retrospective review in our center found 118 consecutive patients with high-grade gliomas operated on with the use of fluorescence-guided surgery with 5-aminolevulinic acid. Within that series, the 52 patients with newly diagnosed GBM and complete resection of enhancing tumor (CRET) in early MRI were selected for analysis. We studied the influence of residual fluorescence in the surgical field on overall survival and neurological complication rate. Multivariate analysis included potential relevant factors: age, Karnofsky Performance Scale, O-methylguanine methyltransferase methylation promoter status, tumor eloquent location, preoperative tumor volume, and adjuvant therapy. The median overall survival was 27.0 months (confidence interval = 22.4-31.6) in patients with nonresidual fluorescence (n = 25) and 17.5 months (confidence interval = 12.5-22.5) for the group with residual fluorescence (n = 27) (P = .015). The influence of residual fluorescence was maintained in the multivariate analysis with all covariables, hazard ratio = 2.5 (P = .041). The neurological complication rate was 18.5% in patients with nonresidual fluorescence and 8% for the group with residual fluorescence (P = .267). GBM patients with CRET in early MRI and no fluorescent residual tissue had longer overall survival than patients with CRET and residual fluorescent tissue.

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 fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry PMID:25523156

Endogenous synchronous fluorescence spectroscopy (SFS) of basal cell carcinoma-initial study

NASA Astrophysics Data System (ADS)

Borisova, E.; Zhelyazkova, Al.; Keremedchiev, M.; Penkov, N.; Semyachkina-Glushkovskaya, O.; Avramov, L.

2016-01-01

The human skin is a complex, multilayered and inhomogeneous organ with spatially varying optical properties. Analysis of cutaneous fluorescence spectra could be a very complicated task; therefore researchers apply complex mathematical tools for data evaluation, or try to find some specific approaches, that would simplify the spectral analysis. Synchronous fluorescence spectroscopy (SFS) allows improving the spectral resolution, which could be useful for the biological tissue fluorescence characterization and could increase the tumour detection diagnostic accuracy.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements

PubMed Central

Fuertes, Gustavo; Banterle, Niccolò; Ruff, Kiersten M.; Chowdhury, Aritra; Mercadante, Davide; Koehler, Christine; Kachala, Michael; Estrada Girona, Gemma; Milles, Sigrid; Mishra, Ankur; Onck, Patrick R.; Gräter, Frauke; Esteban-Martín, Santiago; Pappu, Rohit V.; Svergun, Dmitri I.; Lemke, Edward A.

2017-01-01

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE. For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE, whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles. PMID:28716919

[Applications of three-dimensional fluorescence spectrum of dissolved organic matter to identification of red tide algae].

PubMed

Lü, Gui-Cai; Zhao, Wei-Hong; Wang, Jiang-Tao

2011-01-01

The identification techniques for 10 species of red tide algae often found in the coastal areas of China were developed by combining the three-dimensional fluorescence spectra of fluorescence dissolved organic matter (FDOM) from the cultured red tide algae with principal component analysis. Based on the results of principal component analysis, the first principal component loading spectrum of three-dimensional fluorescence spectrum was chosen as the identification characteristic spectrum for red tide algae, and the phytoplankton fluorescence characteristic spectrum band was established. Then the 10 algae species were tested using Bayesian discriminant analysis with a correct identification rate of more than 92% for Pyrrophyta on the level of species, and that of more than 75% for Bacillariophyta on the level of genus in which the correct identification rates were more than 90% for the phaeodactylum and chaetoceros. The results showed that the identification techniques for 10 species of red tide algae based on the three-dimensional fluorescence spectra of FDOM from the cultured red tide algae and principal component analysis could work well.

Macroscopic Spatial Complexity of the Game of Life Cellular Automaton: A Simple Data Analysis

NASA Astrophysics Data System (ADS)

Hernández-Montoya, A. R.; Coronel-Brizio, H. F.; Rodríguez-Achach, M. E.

In this chapter we present a simple data analysis of an ensemble of 20 time series, generated by averaging the spatial positions of the living cells for each state of the Game of Life Cellular Automaton (GoL). We show that at the macroscopic level described by these time series, complexity properties of GoL are also presented and the following emergent properties, typical of data extracted complex systems such as financial or economical come out: variations of the generated time series following an asymptotic power law distribution, large fluctuations tending to be followed by large fluctuations, and small fluctuations tending to be followed by small ones, and fast decay of linear correlations, however, the correlations associated to their absolute variations exhibit a long range memory. Finally, a Detrended Fluctuation Analysis (DFA) of the generated time series, indicates that the GoL spatial macro states described by the time series are not either completely ordered or random, in a measurable and very interesting way.

ERK reinforces actin polymerization to power persistent edge protrusion during motility.

PubMed

Mendoza, Michelle C; Vilela, Marco; Juarez, Jesus E; Blenis, John; Danuser, Gaudenz

2015-05-19

Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. We tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular signal-regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell surface receptors, and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility. Copyright © 2015, American Association for the Advancement of Science.

ERK reinforces actin polymerization to power persistent edge protrusion during motility

PubMed Central

Mendoza, Michelle C.; Vilela, Marco; Juarez, Jesus E.; Blenis, John; Danuser, Gaudenz

2016-01-01

Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. Here, we tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell-surface receptors and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy (qFSM) and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. Arp2/3 activity generates branched actin networks that can produce pushing force. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility. PMID:25990957

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

PubMed

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Preliminary Analysis of Fluctuations in the Received Uplink-Beacon-Power Data Obtained From the GOLD Experiments

NASA Technical Reports Server (NTRS)

Jeganathan, M.; Wilson, K. E.; Lesh, J. R.

1996-01-01

Uplink data from recent free-space optical communication experiments carried out between the Table Mountain Facility and the Japanese Engineering Test Satellite are used to study fluctuations caused by beam propagation through the atmosphere. The influence of atmospheric scintillation, beam wander and jitter, and multiple uplink beams on the statistics of power received by the satellite is analyzed and compared to experimental data. Preliminary analysis indicates the received signal obeys an approximate lognormal distribution, as predicted by the weak-turbulence model, but further characterization of other sources of fluctuations is necessary for accurate link predictions.

Preliminary analysis of fluctuations in the received uplink-beacon-power data obtained from the GOLD experiments

NASA Technical Reports Server (NTRS)

Jeganathan, M.; Wilson, K. E.; Lesh, J. R.

1996-01-01

Uplink data from recent free-space optical communication experiments carried out between the Table Mountain Facility and the Japanese Engineering Test Satellite are used to study fluctuations caused by beam propagation through the atmosphere. The influence of atmospheric scintillation, beam wander and jitter, and multiple uplink beams on the statistics of power received by the satellite is analyzed and compared to experimental data. Preliminary analysis indicates the received signal obeys an approximate lognormal distribution, as predicted by the weak-turbulence model, but further characterization of other sources of fluctuations is necessary for accurate link predictions.

Comparison of detrending methods for fluctuation analysis in hydrology

NASA Astrophysics Data System (ADS)

Zhang, Qiang; Zhou, Yu; Singh, Vijay P.; Chen, Yongqin David

2011-03-01

SummaryTrends within a hydrologic time series can significantly influence the scaling results of fluctuation analysis, such as rescaled range (RS) analysis and (multifractal) detrended fluctuation analysis (MF-DFA). Therefore, removal of trends is important in the study of scaling properties of the time series. In this study, three detrending methods, including adaptive detrending algorithm (ADA), Fourier-based method, and average removing technique, were evaluated by analyzing numerically generated series and observed streamflow series with obvious relative regular periodic trend. Results indicated that: (1) the Fourier-based detrending method and ADA were similar in detrending practices, and given proper parameters, these two methods can produce similarly satisfactory results; (2) detrended series by Fourier-based detrending method and ADA lose the fluctuation information at larger time scales, and the location of crossover points is heavily impacted by the chosen parameters of these two methods; and (3) the average removing method has an advantage over the other two methods, i.e., the fluctuation information at larger time scales is kept well-an indication of relatively reliable performance in detrending. In addition, the average removing method performed reasonably well in detrending a time series with regular periods or trends. In this sense, the average removing method should be preferred in the study of scaling properties of the hydrometeorolgical series with relative regular periodic trend using MF-DFA.

Stochastic analysis of concentration field in a wake region.

PubMed

Yassin, Mohamed F; Elmi, Abdirashid A

2011-02-01

Identifying geographic locations in urban areas from which air pollutants enter the atmosphere is one of the most important information needed to develop effective mitigation strategies for pollution control. Stochastic analysis is a powerful tool that can be used for estimating concentration fluctuation in plume dispersion in a wake region around buildings. Only few studies have been devoted to evaluate applications of stochastic analysis to pollutant dispersion in an urban area. This study was designed to investigate the concentration fields in the wake region using obstacle model such as an isolated building model. We measured concentration fluctuations at centerline of various downwind distances from the source, and different heights with the frequency of 1 KHz. Concentration fields were analyzed stochastically, using the probability density functions (pdf). Stochastic analysis was performed on the concentration fluctuation and the pdf of mean concentration, fluctuation intensity, and crosswind mean-plume dispersion. The pdf of the concentration fluctuation data have shown a significant non-Gaussian behavior. The lognormal distribution appeared to be the best fit to the shape of concentration measured in the boundary layer. We observed that the plume dispersion pdf near the source was shorter than the plume dispersion far from the source. Our findings suggest that the use of stochastic technique in complex building environment can be a powerful tool to help understand the distribution and location of air pollutants.

Surface and finite size effect on fluctuations dynamics in nanoparticles with long-range order

NASA Astrophysics Data System (ADS)

Morozovska, A. N.; Eliseev, E. A.

2010-02-01

The influence of surface and finite size on the dynamics of the order parameter fluctuations and critical phenomena in the three-dimensional (3D)-confined systems with long-range order was not considered theoretically. In this paper, we study the influence of surface and finite size on the dynamics of the order parameter fluctuations in the particles of arbitrary shape. We consider concrete examples of the spherical and cylindrical ferroic nanoparticles within Landau-Ginzburg-Devonshire phenomenological approach. Allowing for the strong surface energy contribution in micro and nanoparticles, the analytical expressions derived for the Ornstein-Zernike correlator of the long-range order parameter spatial-temporal fluctuations, dynamic generalized susceptibility, relaxation times, and correlation radii discrete spectra are different from those known for bulk systems. Obtained analytical expressions for the correlation function of the order parameter spatial-temporal fluctuations in micro and nanosized systems can be useful for the quantitative analysis of the dynamical structural factors determined from magnetic resonance diffraction and scattering spectra. Besides the practical importance of the correlation function for the analysis of the experimental data, derived expressions for the fluctuations strength determine the fundamental limits of phenomenological theories applicability for 3D-confined systems.

Nonequilibrium fluctuations during diffusion in liquid layers

NASA Astrophysics Data System (ADS)

Brogioli, Doriano; Vailati, Alberto

2017-07-01

Theoretical analysis and experiments have provided compelling evidence of the presence of long-range nonequilibrium concentration fluctuations during diffusion processes in fluids. In this paper, we investigate the dependence of the features of the fluctuations from the dimensionality of the system. In three-dimensional fluids the amplitude of nonequilibrium fluctuations can become several orders of magnitude larger than that of equilibrium fluctuations. Notwithstanding that, the amplitude of nonequilibrium fluctuations remains small with respect to the concentration difference driving the diffusion process. By extending the theory to two-dimensional systems, such as liquid monolayers and bilayers, we show that the amplitude of the fluctuations becomes much stronger than in three-dimensional systems. We investigate the properties of the fronts of diffusion and show that they have a self-affine structure characterized by a Hurst exponent H =1 . We discuss the implications of these results for diffusion in liquid crystals and in cellular membranes of living organisms.

Nonequilibrium fluctuations during diffusion in liquid layers.

PubMed

Brogioli, Doriano; Vailati, Alberto

2017-07-01

Theoretical analysis and experiments have provided compelling evidence of the presence of long-range nonequilibrium concentration fluctuations during diffusion processes in fluids. In this paper, we investigate the dependence of the features of the fluctuations from the dimensionality of the system. In three-dimensional fluids the amplitude of nonequilibrium fluctuations can become several orders of magnitude larger than that of equilibrium fluctuations. Notwithstanding that, the amplitude of nonequilibrium fluctuations remains small with respect to the concentration difference driving the diffusion process. By extending the theory to two-dimensional systems, such as liquid monolayers and bilayers, we show that the amplitude of the fluctuations becomes much stronger than in three-dimensional systems. We investigate the properties of the fronts of diffusion and show that they have a self-affine structure characterized by a Hurst exponent H=1. We discuss the implications of these results for diffusion in liquid crystals and in cellular membranes of living organisms.

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

Photoinduction of cyclosis-mediated interactions between distant chloroplasts.

PubMed

Bulychev, Alexander A; Komarova, Anna V

2015-01-01

Communications between chloroplasts and other organelles based on the exchange of metabolites, including redox active substances, are recognized as a part of intracellular regulation, chlororespiration, and defense against oxidative stress. Similar communications may operate between spatially distant chloroplasts in large cells where photosynthetic and respiratory activities are distributed unevenly under fluctuating patterned illumination. Microfluorometry of chlorophyll fluorescence in vivo in internodal cells of the alga Chara corallina revealed that a 30-s pulse of localized light induces a transient increase (~25%) in F' fluorescence of remote cell parts exposed to dim background light at a 1.5-mm distance on the downstream side from the illuminated spot in the plane of unilateral cytoplasmic streaming but has no effect on F' at equal distance on the upstream side. An abrupt arrest of cytoplasmic streaming for about 30s by triggering the action potential extended either the ascending or descending fronts of the F' fluorescence response, depending on the exact moment of streaming cessation. The response of F' fluorescence to localized illumination of a distant cell region was absent in dark-adapted internodes, when the localized light was applied within the first minute after switching on continuous background illumination of the whole cell, but it appeared in full after longer exposures to continuous background light. These results and the elimination of the F' response by methyl viologen known to redirect electron transport pathways beyond photosystem I indicate the importance of photosynthetic induction and the stromal redox state for long-distance communications of chloroplasts in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.

PubMed

Vedula, Pavan; Cruz, Lissette A; Gutierrez, Natasha; Davis, Justin; Ayee, Brian; Abramczyk, Rachel; Rodriguez, Alexis J

2016-06-30

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.

Electronic zero-point fluctuation forces inside circuit components

PubMed Central

Leonhardt, Ulf

2018-01-01

One of the most intriguing manifestations of quantum zero-point fluctuations are the van der Waals and Casimir forces, often associated with vacuum fluctuations of the electromagnetic field. We study generalized fluctuation potentials acting on internal degrees of freedom of components in electrical circuits. These electronic Casimir-like potentials are induced by the zero-point current fluctuations of any general conductive circuit. For realistic examples of an electromechanical capacitor and a superconducting qubit, our results reveal the possibility of tunable forces between the capacitor plates, or the level shifts of the qubit, respectively. Our analysis suggests an alternative route toward the exploration of Casimir-like fluctuation potentials, namely, by characterizing and measuring them as a function of parameters of the environment. These tunable potentials may be useful for future nanoelectromechanical and quantum technologies. PMID:29719863

Fluctuations of conserved charges in relativistic heavy ion collisions: An introduction

NASA Astrophysics Data System (ADS)

Asakawa, Masayuki; Kitazawa, Masakiyo

2016-09-01

Bulk fluctuations of conserved charges measured by event-by-event analysis in relativistic heavy ion collisions are observables which are believed to carry significant amount of information on the hot medium created by the collisions. Active studies have been done recently experimentally, theoretically, and on the lattice. In particular, non-Gaussianity of the fluctuations has acquired much attention recently. In this review, we give a pedagogical introduction to these issues, and survey recent developments in this field of research. Starting from the definition of cumulants, basic concepts in fluctuation physics, such as thermal fluctuations in statistical mechanics and time evolution of fluctuations in diffusive systems, are described. Phenomena which are expected to occur in finite temperature and/or density QCD matter and their measurement by event-by-event analyses are also elucidated.

Pathological changes in Alzheimer"s brain evaluated with fluorescence emission analysis (FEA)

NASA Astrophysics Data System (ADS)

Christov, Alexander; Ottman, Todd; Grammas, Paula

2004-07-01

Development of AD is associated with cerebrovascular deposition of amyloid beta (Aβ) as well as a progressive increase in vasular collagen content. Both AΒ and collagen are naturally fluorescent compounds when exposed to UV light. We analyzed autofluorescence emitted from brain tissue samples and isolated brain resistance vessels harvested postmortem from patients with Alzheimer's disease (AD) and age-matched controls. Fluorescence emission, excited at 355 nm with an Nd:YAG laser, was measured using a fiber-optic based fluorescence spectroscopic system for tissue analysis. Significantly higher values of fluorescence emission intensity (P<0.001) in the spectral region from 465 to 490 nm were detected in brain resistance vessel samples from AD patients compared to the normal individuals. Results from western blot analysis showed elevated levels of type I and type III collagen, and reduced levels of type IV collagen in resistance vessels from AD patients, compared to control samples. In addition, using direct scanning of the cortical suface for fluoresxcence emission by the laser-induced fluorescence spectroscopy system we detected a significantly (P<0.05) higher level of apoptosis in AD brain tissue compared to age-matched controls. Fluorescence emission analysis (FEA) appears to be a sensitive technique for detecting structural changes in AD brain tissue.

Laser-induced fluorescence microscopic system using an optical parametric oscillator for tunable detection in microchip analysis.

PubMed

Kumemura, Momoko; Odake, Tamao; Korenaga, Takashi

2005-06-01

A laser-induced fluorescence microscopic system based on optical parametric oscillation has been constructed as a tunable detector for microchip analysis. The detection limit of sulforhodamine B (Ex. 520 nm, Em. 570 nm) was 0.2 mumol, which was approximately eight orders of magnitude better than with a conventional fluorophotometer. The system was applied to the determination of fluorescence-labeled DNA (Ex. 494 nm, Em. 519 nm) in a microchannel and the detection limit reached a single molecule. These results showed the feasibility of this system as a highly sensitive and tunable fluorescence detector for microchip analysis.

Studies of the interaction between FNC and human hemoglobin: a spectroscopic analysis and molecular docking.

PubMed

Li, Huiyi; Dou, Huanjing; Zhang, Yuhai; Li, Zhigang; Wang, Ruiyong; Chang, Junbiao

2015-02-05

FNC (2'-deoxy-2'-bfluoro-4'-azidocytidine) is a novel nucleoside analogue with pharmacologic effects on several human diseases. In this work, the binding of FNC to human hemoglobin (HHb) have been investigated by absorption spectroscopy, fluorescence quenching technique, synchronous fluorescence, three-dimensional fluorescence and molecular modeling methods. Analysis of fluorescence data showed that the binding of FNC to HHb occurred via a static quenching mechanism. Thermodynamic analysis and molecular modeling suggest that hydrogen bond and van der Waals force are the mainly binding force in the binding of FNC to HHb. Copyright © 2014 Elsevier B.V. All rights reserved.

Physiological noise in murine solid tumours using T2*-weighted gradient-echo imaging: a marker of tumour acute hypoxia?

PubMed

Baudelet, Christine; Ansiaux, Réginald; Jordan, Bénédicte F; Havaux, Xavier; Macq, Benoit; Gallez, Bernard

2004-08-07

T2*-weighted gradient-echo magnetic resonance imaging (T2*-weighted GRE MRI) was used to investigate spontaneous fluctuations in tumour vasculature non-invasively. FSa fibrosarcomas, implanted intramuscularly (i.m.) in the legs of mice, were imaged at 4.7 T, over a 30 min or 1 h sampling period. On a voxel-by-voxel basis, time courses of signal intensity were analysed using a power spectrum density (PSD) analysis to isolate voxels for which signal changes did not originate from Gaussian white noise or linear drift. Under baseline conditions, the tumours exhibited spontaneous signal fluctuations showing spatial and temporal heterogeneity over the tumour. Statistically significant fluctuations occurred at frequencies ranging from 1 cycle/3 min to 1 cycle/h. The fluctuations were independent of the scanner instabilities. Two categories of signal fluctuations were reported: (i) true fluctuations (TFV), i.e., sequential signal increase and decrease, and (ii) profound drop in signal intensity with no apparent signal recovery (SDV). No temporal correlation between tumour and contralateral muscle fluctuations was observed. Furthermore, treatments aimed at decreasing perfusion-limited hypoxia, such as carbogen combined with nicotinamide and flunarizine, decreased the incidence of tumour T2*-weighted GRE fluctuations. We also tracked dynamic changes in T2* using multiple GRE imaging. Fluctuations of T2* were observed; however, fluctuation maps using PSD analysis could not be generated reliably. An echo-time dependency of the signal fluctuations was observed, which is typical to physiological noise. Finally, at the end of T2*-weighted GRE MRI acquisition, a dynamic contrast-enhanced MRI was performed to characterize the microenvironment in which tumour signal fluctuations occurred in terms of vessel functionality, vascularity and microvascular permeability. Our data showed that TFV were predominantly located in regions with functional vessels, whereas SDV occurred in regions with no contrast enhancement as the result of vessel functional impairment. Furthermore, transient fluctuations appeared to occur preferentially in neoangiogenic hyperpermeable vessels. The present study suggests that spontaneous T2*-weighted GRE fluctuations are very likely to be related to the spontaneous fluctuations in blood flow and oxygenation associated with the pathophysiology of acute hypoxia in tumours. The disadvantage of the T2*-weighted GRE MRI technique is the complexity of signal interpretation with regard to pO2 changes. Compared to established techniques such as intravital microscopy or histological assessments, the major advantage of the MRI technique lies in its capacity to provide simultaneously both temporal and detailed spatial information on spontaneous fluctuations throughout the tumour.

Fluctuations and symmetry energy in nuclear fragmentation dynamics.

PubMed

Colonna, M

2013-01-25

Within a dynamical description of nuclear fragmentation, based on the liquid-gas phase transition scenario, we explore the relation between neutron-proton density fluctuations and nuclear symmetry energy. We show that, along the fragmentation path, isovector fluctuations follow the evolution of the local density and approach an equilibrium value connected to the local symmetry energy. Higher-density regions are characterized by smaller average asymmetry and narrower isotopic distributions. This dynamical analysis points out that fragment final state isospin fluctuations can probe the symmetry energy of the density domains from which fragments originate.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy† †Electronic supplementary information (ESI) available: The LED device for the sample photobleaching, a schematic presentation of HILO microscopy, fluorescence spectra and hybridization curves of the molecular beacons, the linear correlation between the miRNA fluorescence intensity and the miRNA copy number, a validation of the miRNA adsorption and miRNA target gene expression via RT-qPCR, a validation of RT-qPCR using capillary electrophoresis, the reproducibility of RT-qPCR and Poisson distribution of the miRNA pipetting as well as a complete list of the oligonucleotides used in this study. See DOI: 10.1039/c7sc02701j Click here for additional data file.

PubMed Central

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun

2017-01-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target (HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells. PMID:28989695

High-speed laser diagnostics for the study of flame dynamics in a lean premixed gas turbine model combustor

NASA Astrophysics Data System (ADS)

Boxx, Isaac; Arndt, Christoph M.; Carter, Campbell D.; Meier, Wolfgang

2012-03-01

A series of measurements was taken on two technically premixed, swirl-stabilized methane-air flames (at overall equivalence ratios of ϕ = 0.73 and 0.83) in an optically accessible gas turbine model combustor. The primary diagnostics used were combined planar laser-induced fluorescence of the OH radical and stereoscopic particle image velocimetry (PIV) with simultaneous repetition rates of 10 kHz and a measurement duration of 0.8 s. Also measured were acoustic pulsations and OH chemiluminescence. Analysis revealed strong local periodicity in the thermoacoustically self-excited (or ` noisy') flame (ϕ = 0.73) in the regions of the flow corresponding to the inner shear layer and the jet-inflow. This periodicity appears to be the result of a helical precessing vortex core (PVC) present in that region of the combustor. The PVC has a precession frequency double (at 570 Hz) that of the thermo-acoustic pulsation (at 288 Hz). A comparison of the various data sets and analysis techniques applied to each flame suggests a strong coupling between the PVC and the thermo-acoustic pulsation in the noisy flame. Measurements of the stable (` quiet') flame (ϕ = 0.83) revealed a global fluctuation in both velocity and heat-release around 364 Hz, but no clear evidence of a PVC.

rSalvador: An R Package for the Fluctuation Experiment

PubMed Central

Zheng, Qi

2017-01-01

The past few years have seen a surge of novel applications of the Luria-Delbrück fluctuation assay protocol in bacterial research. Appropriate analysis of fluctuation assay data often requires computational methods that are unavailable in the popular web tool FALCOR. This paper introduces an R package named rSalvador to bring improvements to the field. The paper focuses on rSalvador’s capabilities to alleviate three kinds of problems found in recent investigations: (i) resorting to partial plating without properly accounting for the effects of partial plating; (ii) conducting attendant fitness assays without incorporating mutants’ relative fitness in subsequent data analysis; and (iii) comparing mutation rates using methods that are in general inapplicable to fluctuation assay data. In addition, the paper touches on rSalvador’s capabilities to estimate sample size and the difficulties related to parameter nonidentifiability. PMID:29084818

AKARI OBSERVATION OF THE SUB-DEGREE SCALE FLUCTUATION OF THE NEAR-INFRARED BACKGROUND

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, H. J.; Lee, Hyung Mok; Lee, Myung Gyoon

2015-07-10

We report spatial fluctuation analysis of the sky brightness in the near-infrared from observations toward the north ecliptic pole (NEP) by the AKARI at 2.4 and 3.2 μm. As a follow-up study of our previous work on the Monitor field of AKARI, we used NEP deep survey data, which covered a circular area of about 0.4 square degrees, in order to extend fluctuation analysis at angular scales up to 1000″. We found residual fluctuation over the estimated shot noise at larger angles than the angular scale of the Monitor field. The excess fluctuation of the NEP deep field smoothly connectsmore » with that of the Monitor field at angular scales of a few hundred arcseconds and extends without any significant variation to larger angular scales up to 1000″. By comparing excess fluctuations at two wavelengths, we confirm a blue spectral feature similar to the result of the Monitor field. We find that the result of this study is consistent with Spitzer Space Telescope observations at 3.6 μm. The origin of the excess fluctuation in the near-infrared background remains to be determined, but we could exclude zodiacal light, diffuse Galactic light, and unresolved faint galaxies at low redshift based on the comparison with mid- and far-infrared brightness, ground-based near-infrared images.« less

Decomposition of spontaneous fluctuations in tumour oxygenation using BOLD MRI and independent component analysis

PubMed Central

Gonçalves, Miguel R; Johnson, S Peter; Ramasawmy, Rajiv; Pedley, R Barbara; Lythgoe, Mark F; Walker-Samuel, Simon

2015-01-01

Background: Solid tumours can undergo cycles of hypoxia, followed by reoxygenation, which can have significant implications for the success of anticancer therapies. A need therefore exists to develop methods to aid its detection and to further characterise its biological basis. We present here a novel method for decomposing systemic and tumour-specific contributions to fluctuations in tumour deoxyhaemoglobin concentration, based on magnetic resonance imaging measurements. Methods: Fluctuations in deoxyhaemoglobin concentration in two tumour xenograft models of colorectal carcinoma were decomposed into distinct contributions using independent component analysis. These components were then correlated with systemic pulse oximetry measurements to assess the influence of systemic variations in blood oxygenation in tumours, compared with those that arise within the tumour itself (tumour-specific). Immunohistochemical staining was used to assess the physiological basis of each source of fluctuation. Results: Systemic fluctuations in blood oxygenation were found to contribute to cycling hypoxia in tumours, but tumour-specific fluctuations were also evident. Moreover, the size of the tumours was found to influence the degree of systemic, but not tumour-specific, oscillations. The degree of vessel maturation was related to the amplitude of tumour-specific, but not systemic, oscillations. Conclusions: Our results provide further insights into the complexity of spontaneous fluctuations in tumour oxygenation and its relationship with tumour pathophysiology. These observations could be used to develop improved drug delivery strategies. PMID:26484634

The study of RMB exchange rate complex networks based on fluctuation mode

NASA Astrophysics Data System (ADS)

Yao, Can-Zhong; Lin, Ji-Nan; Zheng, Xu-Zhou; Liu, Xiao-Feng

2015-10-01

In the paper, we research on the characteristics of RMB exchange rate time series fluctuation with methods of symbolization and coarse gaining. First, based on fluctuation features of RMB exchange rate, we define the first type of fluctuation mode as one specific foreign currency against RMB in four days' fluctuating situations, and the second type as four different foreign currencies against RMB in one day's fluctuating situation. With the transforming method, we construct the unique-currency and multi-currency complex networks. Further, through analyzing the topological features including out-degree, betweenness centrality and clustering coefficient of fluctuation-mode complex networks, we find that the out-degree distribution of both types of fluctuation mode basically follows power-law distributions with exponents between 1 and 2. The further analysis reveals that the out-degree and the clustering coefficient generally obey the approximated negative correlation. With this result, we confirm previous observations showing that the RMB exchange rate exhibits a characteristic of long-range memory. Finally, we analyze the most probable transmission route of fluctuation modes, and provide probability prediction matrix. The transmission route for RMB exchange rate fluctuation modes exhibits the characteristics of partially closed loop, repeat and reversibility, which lays a solid foundation for predicting RMB exchange rate fluctuation patterns with large volume of data.

Time series analysis of ozone data in Isfahan

NASA Astrophysics Data System (ADS)

Omidvari, M.; Hassanzadeh, S.; Hosseinibalam, F.

2008-07-01

Time series analysis used to investigate the stratospheric ozone formation and decomposition processes. Different time series methods are applied to detect the reason for extreme high ozone concentrations for each season. Data was convert into seasonal component and frequency domain, the latter has been evaluated by using the Fast Fourier Transform (FFT), spectral analysis. The power density spectrum estimated from the ozone data showed peaks at cycle duration of 22, 20, 36, 186, 365 and 40 days. According to seasonal component analysis most fluctuation was in 1999 and 2000, but the least fluctuation was in 2003. The best correlation between ozone and sun radiation was found in 2000. Other variables which are not available cause to this fluctuation in the 1999 and 2001. The trend of ozone is increasing in 1999 and is decreasing in other years.

Investigation of the complexity of streamflow fluctuations in a large heterogeneous lake catchment in China

NASA Astrophysics Data System (ADS)

Ye, Xuchun; Xu, Chong-Yu; Li, Xianghu; Zhang, Qi

2018-05-01

The occurrence of flood and drought frequency is highly correlated with the temporal fluctuations of streamflow series; understanding of these fluctuations is essential for the improved modeling and statistical prediction of extreme changes in river basins. In this study, the complexity of daily streamflow fluctuations was investigated by using multifractal detrended fluctuation analysis (MF-DFA) in a large heterogeneous lake basin, the Poyang Lake basin in China, and the potential impacts of human activities were also explored. Major results indicate that the multifractality of streamflow fluctuations shows significant regional characteristics. In the study catchment, all the daily streamflow series present a strong long-range correlation with Hurst exponents bigger than 0.8. The q-order Hurst exponent h( q) of all the hydrostations can be characterized well by only two parameters: a (0.354 ≤ a ≤ 0.384) and b (0.627 ≤ b ≤ 0.677), with no pronounced differences. Singularity spectrum analysis pointed out that small fluctuations play a dominant role in all daily streamflow series. Our research also revealed that both the correlation properties and the broad probability density function (PDF) of hydrological series can be responsible for the multifractality of streamflow series that depends on watershed areas. In addition, we emphasized the relationship between watershed area and the estimated multifractal parameters, such as the Hurst exponent and fitted parameters a and b from the q-order Hurst exponent h( q). However, the relationship between the width of the singularity spectrum (Δ α) and watershed area is not clear. Further investigation revealed that increasing forest coverage and reservoir storage can effectively enhance the persistence of daily streamflow, decrease the hydrological complexity of large fluctuations, and increase the small fluctuations.

Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dong-Shan; Zhou, Guo-Hua; Luo, Ming; Ji, Xing-Hu; He, Zhi-Ke

2012-08-21

We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.

Highly Sensitive Ratiometric Fluorescent Sensor for Trinitrotoluene Based on the Inner Filter Effect between Gold Nanoparticles and Fluorescent Nanoparticles.

PubMed

Lu, Hongzhi; Quan, Shuai; Xu, Shoufang

2017-11-08

In this work, we developed a simple and sensitive ratiometric fluorescent assay for sensing trinitrotoluene (TNT) based on the inner filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent nanoparticles (RFNs), which was designed by hybridizing green emissive carbon dots (CDs) and red emissive quantum dots (QDs) into a silica sphere as a fluorophore pair. AuNPs in their dispersion state can be a powerful absorber to quench CDs, while the aggregated AuNPs can quench QDs in the IFE-based fluorescent assays as a result of complementary overlap between the absorption spectrum of AuNPs and emission spectrum of RFNs. As a result of the fact that TNT can induce the aggregation of AuNPs, with the addition of TNT, the fluorescent of QDs can be quenched, while the fluorescent of CDs would be recovered. Then, ratiometric fluorescent detection of TNT is feasible. The present IFE-based ratiometric fluorescent sensor can detect TNT ranging from 0.1 to 270 nM, with a detection limit of 0.029 nM. In addition, the developed method was successfully applied to investigate TNT in water and soil samples with satisfactory recoveries ranging from 95 to 103%, with precision below 4.5%. The simple sensing approach proposed here could improve the sensitivity of colorimetric analysis by changing the ultraviolet analysis to ratiometric fluorescent analysis and promote the development of a dual-mode detection system.

Critical fluctuations of the proton density in A+A collisions at 158A GeV

DOE PAGES

Anticic, T.; Baatar, B.; Bartke, J.; ...

2015-12-12

Here, we look for fluctuations expected for the QCD critical point using an intermittency analysis in the transverse momentum phase space of protons produced around midrapidity in the 12.5 % most central C+C, Si+Si and Pb+Pb collisions at the maximum SPS energy of 158A GeV. We find evidence of power-law fluctuations for the Si+Si data. The fitted power-law exponent Φ 2=0.96 +0.38 –0.25 (stat.) ± 0.16 (syst.) is consistent with the value expected for critical fluctuations. Power-law fluctuations had previously also been observed in low-mass π +π – pairs in the same Si+Si collisions.

Global and system-specific resting-state fMRI fluctuations are uncorrelated: principal component analysis reveals anti-correlated networks.

PubMed

Carbonell, Felix; Bellec, Pierre; Shmuel, Amir

2011-01-01

The influence of the global average signal (GAS) on functional-magnetic resonance imaging (fMRI)-based resting-state functional connectivity is a matter of ongoing debate. The global average fluctuations increase the correlation between functional systems beyond the correlation that reflects their specific functional connectivity. Hence, removal of the GAS is a common practice for facilitating the observation of network-specific functional connectivity. This strategy relies on the implicit assumption of a linear-additive model according to which global fluctuations, irrespective of their origin, and network-specific fluctuations are super-positioned. However, removal of the GAS introduces spurious negative correlations between functional systems, bringing into question the validity of previous findings of negative correlations between fluctuations in the default-mode and the task-positive networks. Here we present an alternative method for estimating global fluctuations, immune to the complications associated with the GAS. Principal components analysis was applied to resting-state fMRI time-series. A global-signal effect estimator was defined as the principal component (PC) that correlated best with the GAS. The mean correlation coefficient between our proposed PC-based global effect estimator and the GAS was 0.97±0.05, demonstrating that our estimator successfully approximated the GAS. In 66 out of 68 runs, the PC that showed the highest correlation with the GAS was the first PC. Since PCs are orthogonal, our method provides an estimator of the global fluctuations, which is uncorrelated to the remaining, network-specific fluctuations. Moreover, unlike the regression of the GAS, the regression of the PC-based global effect estimator does not introduce spurious anti-correlations beyond the decrease in seed-based correlation values allowed by the assumed additive model. After regressing this PC-based estimator out of the original time-series, we observed robust anti-correlations between resting-state fluctuations in the default-mode and the task-positive networks. We conclude that resting-state global fluctuations and network-specific fluctuations are uncorrelated, supporting a Resting-State Linear-Additive Model. In addition, we conclude that the network-specific resting-state fluctuations of the default-mode and task-positive networks show artifact-free anti-correlations.

Fluorescence lifetime imaging and Fourier transform infrared spectroscopy of Michelangelo's David.

PubMed

Comelli, Daniela; Valentini, Gianluca; Cubeddu, Rinaldo; Toniolo, Lucia

2005-09-01

We developed a combined procedure for the analysis of works of art based on a portable system for fluorescence imaging integrated with analytical measurements on microsamples. The method allows us to localize and identify organic and inorganic compounds present on the surface of artworks. The fluorescence apparatus measures the temporal and spectral features of the fluorescence emission, excited by ultraviolet (UV) laser pulses. The kinetic of the emission is studied through a fluorescence lifetime imaging system, while an optical multichannel analyzer measures the fluorescence spectra of selected points. The chemical characterization of the compounds present on the artistic surfaces is then performed by means of analytical measurements on microsamples collected with the assistance of the fluorescence maps. The previous concepts have been successfully applied to study the contaminants on the surface of Michelangelo's David. The fluorescence analysis combined with Fourier transform infrared (FT-IR) measurements revealed the presence of beeswax, which permeates most of the statue surface, and calcium oxalate deposits mainly arranged in vertical patterns and related to rain washing.

Detection of individual atoms in helium buffer gas and observation of their real-time motion

NASA Technical Reports Server (NTRS)

Pan, C. L.; Prodan, J. V.; Fairbank, W. M., Jr.; She, C. Y.

1980-01-01

Single atoms are detected and their motion measured for the first time to our knowledge by the fluorescence photon-burst method in the presence of large quantities of buffer gas. A single-clipped digital correlator records the photon burst in real time and displays the atom's transit time across the laser beam. A comparison is made of the special requirements for single-atom detection in vacuum and in a buffer gas. Finally, the probability distribution of the bursts from many atoms is measured. It further proves that the bursts observed on resonance are due to single atoms and not simply to noise fluctuations.

The Onset of the 1997-1998 El Nino and its Impact on the Phytoplankton Community of the Central Equatorial Pacific

NASA Technical Reports Server (NTRS)

Chavez, F. P.; Strutton, P. G.; McPhaden, M. J.

1996-01-01

Using physical and bio-optical data from moorings in the central equatorial Pacific, the perturbations to phytoplankton biomass and productivity associated with the onset of the 1997-98 El Nino event were investigated. The data presented depict the physical progression of El Nino onset, from reversal of the trade winds in the western equatorial Pacific, through eastward propagation of equatorially trapped Kelvin waves and advection of waters from the nutrient-poor western equatorial warm pool. The physical perturbations led to fluctuations in phytoplankton biomass, quantum yield of fluorescence and a 50% reduction in primary productivity.

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions.

PubMed

Žoldák, Gabriel; Jancura, Daniel; Sedlák, Erik

2017-06-01

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady-state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high-throughput drug screening campaigns. © 2017 The Protein Society.

Statistical physics and physiology: monofractal and multifractal approaches

NASA Technical Reports Server (NTRS)

Stanley, H. E.; Amaral, L. A.; Goldberger, A. L.; Havlin, S.; Peng, C. K.

1999-01-01

Even under healthy, basal conditions, physiologic systems show erratic fluctuations resembling those found in dynamical systems driven away from a single equilibrium state. Do such "nonequilibrium" fluctuations simply reflect the fact that physiologic systems are being constantly perturbed by external and intrinsic noise? Or, do these fluctuations actually, contain useful, "hidden" information about the underlying nonequilibrium control mechanisms? We report some recent attempts to understand the dynamics of complex physiologic fluctuations by adapting and extending concepts and methods developed very recently in statistical physics. Specifically, we focus on interbeat interval variability as an important quantity to help elucidate possibly non-homeostatic physiologic variability because (i) the heart rate is under direct neuroautonomic control, (ii) interbeat interval variability is readily measured by noninvasive means, and (iii) analysis of these heart rate dynamics may provide important practical diagnostic and prognostic information not obtainable with current approaches. The analytic tools we discuss may be used on a wider range of physiologic signals. We first review recent progress using two analysis methods--detrended fluctuation analysis and wavelets--sufficient for quantifying monofractual structures. We then describe recent work that quantifies multifractal features of interbeat interval series, and the discovery that the multifractal structure of healthy subjects is different than that of diseased subjects.

Irregular-regular mode oscillations inside plasma bubble and its fractal analysis in glow discharge magnetized plasma

NASA Astrophysics Data System (ADS)

Megalingam, Mariammal; Hari Prakash, N.; Solomon, Infant; Sarma, Arun; Sarma, Bornali

2017-04-01

Experimental evidence of different kinds of oscillations in floating potential fluctuations of glow discharge magnetized plasma is being reported. A spherical gridded cage is inserted into the ambient plasma volume for creating plasma bubbles. Plasma is produced between a spherical mesh grid and chamber. The spherical mesh grid of 80% optical transparency is connected to the positive terminal of power supply and considered as anode. Two Langmuir probes are kept in the ambient plasma to measure the floating potential fluctuations in different positions within the system, viz., inside and outside the spherical mesh grid. At certain conditions of discharge voltage (Vd) and magnetic field, irregular to regular mode appears, and it shows chronological changes with respect to magnetic field. Further various nonlinear analyses such as Recurrence Plot, Hurst exponent, and Lyapunov exponent have been carried out to investigate the dynamics of oscillation at a range of discharge voltages and external magnetic fields. Determinism, entropy, and Lmax are important measures of Recurrence Quantification Analysis which indicate an irregular to regular transition in the dynamics of the fluctuations. Furthermore, behavior of the plasma oscillation is characterized by the technique called multifractal detrended fluctuation analysis to explore the nature of the fluctuations. It reveals that it has a multifractal nature and behaves as a long range correlated process.

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

PubMed

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

Fluorescence spectroscopy for the detection of potentially malignant disorders of the oral cavity: analysis of 30 cases

NASA Astrophysics Data System (ADS)

Francisco, A. L. N.; Correr, W. R.; Azevedo, L. H.; Galletta, V. K.; Pinto, C. A. L.; Kowalski, L. P.; Kurachi, C.

2014-01-01

Oral cancer is a major health problem worldwide and although early diagnosis of potentially malignant and malignant diseases is associated with better treatment results, a large number of cancers are initially misdiagnosed, with unfortunate consequences for long-term survival. Fluorescence spectroscopy is a noninvasive modality of diagnostic approach using induced fluorescence emission in tumors that can improve diagnostic accuracy. The objective of this study was to determine the ability to discriminate between normal oral mucosa and potentially malignant disorders by fluorescence spectroscopy. Fluorescence investigation under 408 and 532 nm excitation wavelengths was performed on 60 subjects, 30 with potentially malignant disorders and 30 volunteers with normal mucosa. Data was analyzed to correlate fluorescence patterns with clinical and histopathological diagnostics. Fluorescence spectroscopy used as a point measurement technique resulted in a great variety of spectral information. In a qualitative analysis of the fluorescence spectral characteristics of each type of injury evaluated, it was possible to discriminate between normal and abnormal oral mucosa. The results show the potential use of fluorescence spectroscopy for an improved discrimination of oral disorders.

Crystallization Kinetics of an Amorphous Pharmaceutical Compound Using Fluorescence-Lifetime-Imaging Microscopy.

PubMed

Rautaniemi, Kaisa; Vuorimaa-Laukkanen, Elina; Strachan, Clare J; Laaksonen, Timo

2018-05-07

Pharmaceutical scientists are increasingly interested in amorphous drug formulations especially because of their higher dissolution rates. Consequently, the thorough characterization and analysis of these formulations are becoming more and more important for the pharmaceutical industry. Here, fluorescence-lifetime-imaging microscopy (FLIM) was used to monitor the crystallization of an amorphous pharmaceutical compound, indomethacin. Initially, we identified different solid indomethacin forms, amorphous and γ- and α-crystalline, on the basis of their time-resolved fluorescence. All of the studied indomethacin forms showed biexponential decays with characteristic fluorescence lifetimes and amplitudes. Using this information, the crystallization of amorphous indomethacin upon storage in 60 °C was monitored for 10 days with FLIM. The progress of crystallization was detected as lifetime changes both in the FLIM images and in the fluorescence-decay curves extracted from the images. The fluorescence-lifetime amplitudes were used for quantitative analysis of the crystallization process. We also demonstrated that the fluorescence-lifetime distribution of the sample changed during crystallization, and when the sample was not moved between measuring times, the lifetime distribution could also be used for the analysis of the reaction kinetics. Our results clearly show that FLIM is a sensitive and nondestructive method for monitoring solid-state transformations on the surfaces of fluorescent samples.

Research of the fluorescence detection apparatus for nutrients

NASA Astrophysics Data System (ADS)

Wang, Yu; Yan, Huimin; Ni, Xuxiang; Xu, Xiaoyi; Chen, Shibing

2015-10-01

The research of the multifunctional analyzer of Clinical Nutrition, which integrates the absorbance, luminescence, fluorescence and other optical detection methods, can overcome the functional limitations of a single technology on human nutrition analysis, and realize a rapid and accurate analysis of the nutrients. This article focuses on the design of fluorescence detection module that uses a photomultiplier tube(PMT) to detect weak fluorescence, and utilizes the single photon counting method to measure the fluorescence intensity, and then according to the relationship between the fluorescent marker and fluorescence intensity, the concentration of the analyte can be derived. Using fluorescein isothiocyanate(FITC, the most widely used fluorescein currently)to mark antibodies in the experiment, therefore, according to the maximum absorption wavelength and the maximum emission wavelength of the fluorescein isothiocyanate, to select the appropriate filters to set up the optical path. In addition, the fluorescence detection apparatus proposed in this paper uses an aspherical lens with large numerical aperture, in order to improve the capacity of signal acquisition more effectively, and the selective adoption of flexible optical fiber can realize a compact opto-mechanical structure, which is also conducive to the miniaturization of the device. The experimental results show that this apparatus has a high sensitivity, can be used for the detection and analysis of human nutrition.

Ultratrace analysis of transuranic actinides by laser-induced fluorescence

DOEpatents

Miller, S.M.

1983-10-31

Ultratrace quantities of transuranic actinides are detected indirectly by their effect on the fluorescent emissions of a preselected fluorescent species. Transuranic actinides in a sample are coprecipitated with a host lattice material containing at least one preselected fluorescent species. The actinide either quenches or enhances the laser-induced fluorescence of the preselected fluorescent species. The degree of enhancement or quenching is quantitatively related to the concentration of actinide in the sample.

Fluorescence recovery after photo-bleaching as a method to determine local diffusion coefficient in the stratum corneum.

PubMed

Anissimov, Yuri G; Zhao, Xin; Roberts, Michael S; Zvyagin, Andrei V

2012-10-01

Fluorescence recovery after photo-bleaching experiments were performed in human stratum corneum in vitro. Fluorescence multiphoton tomography was used, which allowed the dimensions of the photobleached volume to be at the micron scale and located fully within the lipid phase of the stratum corneum. Analysis of the fluorescence recovery data with simplified mathematical models yielded the diffusion coefficient of small molecular weight organic fluorescent dye Rhodamine B in the stratum corneum lipid phase of about (3-6) × 10(-9)cm(2) s(-1). It was concluded that the presented method can be used for detailed analysis of localised diffusion coefficients in the stratum corneum phases for various fluorescent probes. Copyright © 2012 Elsevier B.V. All rights reserved.

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis

PubMed Central

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-01-01

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots. PMID:28714873

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis.

PubMed

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-07-15

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.

A Compact, Tunable Near-UV Source for Quantitative Microgravity Combustion Diagnostics

NASA Technical Reports Server (NTRS)

Peterson, K. A.; Oh, D. B.

1999-01-01

There is a need for improved optical diagnostic methods for use in microgravity combustion research. Spectroscopic methods with fast time response that can provide absolute concentrations and concentration profiles of important chemical species in flames are needed to facilitate the understanding of combustion kinetics in microgravity. Although a variety of sophisticated laser-based diagnostics (such as planar laser induced fluorescence, degenerate four wave mixing and coherent Raman methods) have been applied to the study of combustion in laboratory flames, the instrumentation associated with these methods is not well suited to microgravity drop tower or space station platforms. Important attributes of diagnostic systems for such applications include compact size, low power consumption, ruggedness, and reliability. We describe a diode laser-based near-UV source designed with the constraints of microgravity research in mind. Coherent light near 420 nm is generated by frequency doubling in a nonlinear crystal. This light source is single mode with a very narrow bandwidth suitable for gas phase diagnostics, can be tuned over several 1/cm and can be wavelength modulated at up to MHz frequencies. We demonstrate the usefulness of this source for combustion diagnostics by measuring CH radical concentration profiles in an atmospheric pressure laboratory flame. The radical concentrations are measured using wavelength modulation spectroscopy (WMS) to obtain the line-of-sight integrated absorption for different paths through the flame. Laser induced fluorescence (LIF) measurements are also demonstrated with this instrument, showing the feasibility of simultaneous WMS absorption and LIF measurements with the same light source. LIF detection perpendicular to the laser beam can be used to map relative species densities along the line-of-sight while the integrated absorption available through WMS provides a mathematical constraint on the extraction of quantitative information from the LIF data. Combining absorption with LIF - especially if the measurements are made simultaneously with the same excitation beam - may allow elimination of geometrical factors and effects of intensity fluctuations (common difficulties with the analysis of LIF data) from the analysis.

Cosmic Infrared Background Fluctuations in Deep Spitzer Infrared Array Camera Images: Data Processing and Analysis

NASA Astrophysics Data System (ADS)

Arendt, Richard G.; Kashlinsky, A.; Moseley, S. H.; Mather, J.

2010-01-01

This paper provides a detailed description of the data reduction and analysis procedures that have been employed in our previous studies of spatial fluctuation of the cosmic infrared background (CIB) using deep Spitzer Infrared Array Camera observations. The self-calibration we apply removes a strong instrumental signal from the fluctuations that would otherwise corrupt the results. The procedures and results for masking bright sources and modeling faint sources down to levels set by the instrumental noise are presented. Various tests are performed to demonstrate that the resulting power spectra of these fields are not dominated by instrumental or procedural effects. These tests indicate that the large-scale (gsim30') fluctuations that remain in the deepest fields are not directly related to the galaxies that are bright enough to be individually detected. We provide the parameterization of these power spectra in terms of separate instrument noise, shot noise, and power-law components. We discuss the relationship between fluctuations measured at different wavelengths and depths, and the relations between constraints on the mean intensity of the CIB and its fluctuation spectrum. Consistent with growing evidence that the ~1-5 μm mean intensity of the CIB may not be as far above the integrated emission of resolved galaxies as has been reported in some analyses of DIRBE and IRTS observations, our measurements of spatial fluctuations of the CIB intensity indicate the mean emission from the objects producing the fluctuations is quite low (gsim1 nW m-2 sr-1 at 3-5 μm), and thus consistent with current γ-ray absorption constraints. The source of the fluctuations may be high-z Population III objects, or a more local component of very low luminosity objects with clustering properties that differ from the resolved galaxies. Finally, we discuss the prospects of the upcoming space-based surveys to directly measure the epochs inhabited by the populations producing these source-subtracted CIB fluctuations, and to isolate the individual fluxes of these populations.

Trip-Induced Transition Measurements in a Hypersonic Boundary Layer Using Molecular Tagging Velocimetry

NASA Technical Reports Server (NTRS)

Bathel, Brett F.; Danehy, Paul M.; Jones, Stephen B.; Johansen, Craig T.; Goyne, Christopher P.

2013-01-01

Measurements of mean streamwise velocity, fluctuating streamwise velocity, and instantaneous streamwise velocity profiles in a hypersonic boundary layer were obtained over a 10-degree half-angle wedge model. A laser-induced fluorescence-based molecular tagging velocimetry technique was used to make the measurements. The nominal edge Mach number was 4.2. Velocity profiles were measured both in an untripped boundary layer and in the wake of a 4-mm diameter cylindrical tripping element centered 75.4 mm downstream of the sharp leading edge. Three different trip heights were investigated: k = 0.53 mm, k = 1.0 mm and k = 2.0 mm. The laminar boundary layer thickness at the position of the measurements was approximately 1 mm, though the exact thickness was dependent on Reynolds number and wall temperature. All of the measurements were made starting from a streamwise location approximately 18 mm downstream of the tripping element. This measurement region continued approximately 30 mm in the streamwise direction. Additionally, measurements were made at several spanwise locations. An analysis of flow features show how the magnitude, spatial location, and spatial growth of streamwise velocity instabilities are affected by parameters such as the ratio of trip height to boundary layer thickness and roughness Reynolds number. The fluctuating component of streamwise velocity measured along the centerline of the model increased from approximately 75 m/s with no trip to +/-225 m/s with a 0.53-mm trip, and to +/-240 m/s with a 1-mm trip, while holding the freestream Reynolds number constant. These measurements were performed in the 31-inch Mach 10 Air Tunnel at the NASA Langley Research Center.

Rate kernel theory for pseudo-first-order kinetics of diffusion-influenced reactions and application to fluorescence quenching kinetics.

PubMed

Yang, Mino

2007-06-07

Theoretical foundation of rate kernel equation approaches for diffusion-influenced chemical reactions is presented and applied to explain the kinetics of fluorescence quenching reactions. A many-body master equation is constructed by introducing stochastic terms, which characterize the rates of chemical reactions, into the many-body Smoluchowski equation. A Langevin-type of memory equation for the density fields of reactants evolving under the influence of time-independent perturbation is derived. This equation should be useful in predicting the time evolution of reactant concentrations approaching the steady state attained by the perturbation as well as the steady-state concentrations. The dynamics of fluctuation occurring in equilibrium state can be predicted by the memory equation by turning the perturbation off and consequently may be useful in obtaining the linear response to a time-dependent perturbation. It is found that unimolecular decay processes including the time-independent perturbation can be incorporated into bimolecular reaction kinetics as a Laplace transform variable. As a result, a theory for bimolecular reactions along with the unimolecular process turned off is sufficient to predict overall reaction kinetics including the effects of unimolecular reactions and perturbation. As the present formulation is applied to steady-state kinetics of fluorescence quenching reactions, the exact relation between fluorophore concentrations and the intensity of excitation light is derived.

A polarized view on DNA under tension

NASA Astrophysics Data System (ADS)

van Mameren, Joost; Vermeulen, Karen; Wuite, Gijs J. L.; Peterman, Erwin J. G.

2018-03-01

In the past decades, sensitive fluorescence microscopy techniques have contributed significantly to our understanding of the dynamics of DNA. The specific labeling of DNA using intercalating dyes has allowed for quantitative measurement of the thermal fluctuations the polymers undergo. On the other hand, recent advances in single-molecule manipulation techniques have unraveled the mechanical and elastic properties of this intricate polymer. Here, we have combined these two approaches to study the conformational dynamics of DNA under a wide range of tensions. Using polarized fluorescence microscopy in conjunction with optical-tweezers-based manipulation of YOYO-intercalated DNA, we controllably align the YOYO dyes using DNA tension, enabling us to disentangle the rapid dynamics of the dyes from that of the DNA itself. With unprecedented control of the DNA alignment, we resolve an inconsistency in reports about the tilted orientation of intercalated dyes. We find that intercalated dyes are on average oriented perpendicular to the long axis of the DNA, yet undergo fast dynamics on the time scale of absorption and fluorescence emission. In the overstretching transition of double-stranded DNA, we do not observe changes in orientation or orientational dynamics of the dyes. Only beyond the overstretching transition, a considerable depolarization is observed, presumably caused by an average tilting of the DNA base pairs. Our combined approach thus contributes to the elucidation of unique features of the molecular dynamics of DNA.

Characterizing chlorine oxidation of dissolved organic matter and disinfection by-product formation with fluorescence spectroscopy and parallel factor analysis

NASA Astrophysics Data System (ADS)

Beggs, Katherine M. H.; Summers, R. Scott; McKnight, Diane M.

2009-12-01

Relationships between chlorine demand and disinfection by-product (DBP) formation during chlorination and fluorescence of dissolved organic matter (DOM) were developed. Fluorescence excitation and emission (EEM) spectroscopy was employed, and parameters including fluorescence index, redox index, and overall fluorescence intensity (OFI) were correlated to chlorine demand and DBP formation. The EEMs were also analyzed using a well established global parallel factor analysis (PARAFAC) model which resolves the fluorescence signal into 13 components, including quinone-like and protein-like components. Over an 8-day chlorination period the OFI and sum of the 13 PARAFAC loadings decreased by more than 70%. The remaining identified quinone-like compounds within the DOM were shifted to a more oxidized state. Quinone fluorescence was strongly correlated to both reduced fluorescence intensity and to chlorine demand which indicates that fluorescence may be used to track the chlorine oxidation of DOM. Quinone fluorescence was also correlated strongly with both classes of regulated DBPs: total trihalomethanes and haloacetic acids. Quinone-like components were found to be strongly correlated to overall, short-term, and long-term specific DBP formation. The results of this study show that fluorescence is a useful tool in tracking both DOM oxidation and DBP formation during chlorination.

A spatial length scale analysis of turbulent temperature and velocity fluctuations within and above an orchard canopy

USGS Publications Warehouse

Wang, Y.S.; Miller, D.R.; Anderson, D.E.; Cionco, R.M.; Lin, J.D.

1992-01-01

Turbulent flow within and above an almond orchard was measured with three-dimensional wind sensors and fine-wire thermocouple sensors arranged in a horizontal array. The data showed organized turbulent structures as indicated by coherent asymmetric ramp patterns in the time series traces across the sensor array. Space-time correlation analysis indicated that velocity and temperature fluctuations were significantly correlated over a transverse distance more than 4m. Integral length scales of velocity and temperature fluctuations were substantially greater in unstable conditions than those in stable conditions. The coherence spectral analysis indicated that Davenport's geometric similarity hypothesis was satisfied in the lower frequency region. From the geometric similarity hypothesis, the spatial extents of large ramp structures were also estimated with the coherence functions.

Multifractal Detrended Fluctuation Analysis of Self-Potential Field Prior to the M 6.5, October 24, 1993 Earthquake in MÉXICO

NASA Astrophysics Data System (ADS)

Cervantes, F.; González-Trejo, J. I.; Real-Ramírez, C. A.; Hoyos-Reyes, L. F.; Area de Sistemas Computacionales

2013-05-01

In the current literature on seismo electromagnetic, it has been reported many earthquakes which present electromagnetic anomalies as probable precursors of their occurrences. Although this methodology remains yet under discussion, is relevant to study many particular cases. In this work, we report a multifractal detrended fluctuation analysis (MFDFA) of electroseismic signals recorded in the Acapulco station during 1993. In October 24, 1993, occurred and earthquake (EQ) with M 6.5, with epicenter at (16.54 N, 98.98 W), 100Km away from the mentioned station. The multifractal spectrum identifies the deviations in fractal structure within time periods with large and small fluctuations. We discuss the dynamical meaning of this analysis and its possible relation with the mentioned EQ.

Deviations from uniform power law scaling in nonstationary time series

NASA Technical Reports Server (NTRS)

Viswanathan, G. M.; Peng, C. K.; Stanley, H. E.; Goldberger, A. L.

1997-01-01

A classic problem in physics is the analysis of highly nonstationary time series that typically exhibit long-range correlations. Here we test the hypothesis that the scaling properties of the dynamics of healthy physiological systems are more stable than those of pathological systems by studying beat-to-beat fluctuations in the human heart rate. We develop techniques based on the Fano factor and Allan factor functions, as well as on detrended fluctuation analysis, for quantifying deviations from uniform power-law scaling in nonstationary time series. By analyzing extremely long data sets of up to N = 10(5) beats for 11 healthy subjects, we find that the fluctuations in the heart rate scale approximately uniformly over several temporal orders of magnitude. By contrast, we find that in data sets of comparable length for 14 subjects with heart disease, the fluctuations grow erratically, indicating a loss of scaling stability.

TESTING THE PROPAGATING FLUCTUATIONS MODEL WITH A LONG, GLOBAL ACCRETION DISK SIMULATION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogg, J Drew; Reynolds, Christopher S.

2016-07-20

The broadband variability of many accreting systems displays characteristic structures; log-normal flux distributions, root-mean square (rms)-flux relations, and long inter-band lags. These characteristics are usually interpreted as inward propagating fluctuations of the mass accretion rate in an accretion disk driven by stochasticity of the angular momentum transport mechanism. We present the first analysis of propagating fluctuations in a long-duration, high-resolution, global three-dimensional magnetohydrodynamic (MHD) simulation of a geometrically thin ( h / r ≈ 0.1) accretion disk around a black hole. While the dynamical-timescale turbulent fluctuations in the Maxwell stresses are too rapid to drive radially coherent fluctuations in themore » accretion rate, we find that the low-frequency quasi-periodic dynamo action introduces low-frequency fluctuations in the Maxwell stresses, which then drive the propagating fluctuations. Examining both the mass accretion rate and emission proxies, we recover log-normality, linear rms-flux relations, and radial coherence that would produce inter-band lags. Hence, we successfully relate and connect the phenomenology of propagating fluctuations to modern MHD accretion disk theory.« less

Random fluctuations of optical signal path delay in the atmosphere

NASA Astrophysics Data System (ADS)

Kral, L.; Prochazka, I.; Hamal, K.

2006-09-01

Atmospheric turbulence induces random delay fluctuations to any optical signal transmitted through the air. These fluctuations can influence for example the measurement precision of laser rangefinders. We have found an appropriate theoretical model based on geometrical optics that allows us to predict the amplitude of the random delay fluctuations for different observing conditions. We have successfully proved the applicability of this model by a series of experiments, directly determining the amplitude of the turbulence-induced pulse delay fluctuations by analysis of a high precision laser ranging data. Moreover, we have also shown that a standard theoretical approach based on diffractive propagation of light through inhomogeneous media and implemented using the GLAD software is not suitable for modeling of the optical signal delay fluctuations caused by the atmosphere. These models based on diffractive propagation predict the turbulence-induced optical path length fluctuations of the order of micrometers, whereas the fluctuations predicted by the geometrical optics model (in agreement with our experimental data) are generally larger by two orders of magnitude, i.e. in the submillimeter range. The reason of this discrepancy is a subject to discussion.

Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry

PubMed Central

Houston, Jessica P.; Naivar, Mark A.; Freyer, James P.

2010-01-01

Frequency-domain flow cytometry techniques are combined with modifications to the digital signal processing capabilities of the Open Reconfigurable Cytometric Acquisition System (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency modulated detector signals, implementing Fourier analysis programming with ORCAS’ digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5–25 ns simulated lifetime), pulse widths ranging from 2 to 15 µs, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142° to 1.6°. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 µs and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a radiofrequency modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to ~98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will both allow better dissemination of this technology and better exploit the traditionally underutilized parameter of fluorescence lifetime. PMID:20662090

Characterization of dissolved organic matter in a submerged membrane bioreactor by using three-dimensional excitation and emission matrix fluorescence spectroscopy.

PubMed

Wang, Zhiwei; Wu, Zhichao; Tang, Shujuan

2009-04-01

Three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy was employed to characterize dissolved organic matter (DOM) in a submerged membrane bioreactor (MBR). Three fluorescence peaks could be identified from the EEM fluorescence spectra of the DOM samples in the MBR. Two peaks were associated with the protein-like fluorophores, and the third was related to the visible humic acid-like fluorophores. Only two main peaks were observed in the EEM fluorescence spectra of the extracellular polymeric substance (EPS) samples, which were due to the fluorescence of protein-like and humic acid-like matters, respectively. However, the EEM fluorescence spectra of membrane foulants were observed to have three peaks. It was also found that the dominant fluorescence substances in membrane foulants were protein-like substances, which might be due to the retention of proteins in the DOM and/or EPS in the MBR by the fine pores of the membrane. Quantitative analysis of the fluorescence spectra including peak locations, fluorescence intensity, and different peak intensity ratios and the fluorescence regional integration (FRI) analysis were also carried out in order to better understand the similarities and differences among the EEM spectra of the DOM, EPS, and membrane foulant samples and to further provide an insight into membrane fouling caused by the fluorescence substances in the DOM in submerged MBRs.

[Discrimination of Red Tide algae by fluorescence spectra and principle component analysis].

PubMed

Su, Rong-guo; Hu, Xu-peng; Zhang, Chuan-song; Wang, Xiu-lin

2007-07-01

Fluorescence discrimination technology for 11 species of the Red Tide algae at genus level was constructed by principle component analysis and non-negative least squares. Rayleigh and Raman scattering peaks of 3D fluorescence spectra were eliminated by Delaunay triangulation method. According to the results of Fisher linear discrimination, the first principle component score and the second component score of 3D fluorescence spectra were chosen as discriminant feature and the feature base was established. The 11 algae species were tested, and more than 85% samples were accurately determinated, especially for Prorocentrum donghaiense, Skeletonema costatum, Gymnodinium sp., which have frequently brought Red tide in the East China Sea. More than 95% samples were right discriminated. The results showed that the genus discriminant feature of 3D fluorescence spectra of Red Tide algae given by principle component analysis could work well.

Characterization of DOM adsorption of CNTs by using excitation-emission matrix fluorescence spectroscopy and multiway analysis.

PubMed

Peng, Mingguo; Li, Huajie; Li, Dongdong; Du, Erdeng; Li, Zhihong

2017-06-01

Carbon nanotubes (CNTs) were utilized to adsorb DOM in micro-polluted water. The characteristics of DOM adsorption on CNTs were investigated based on UV 254 , TOC, and fluorescence spectrum measurements. Based on PARAFAC (parallel factor) analysis, four fluorescent components were extracted, including one protein-like component (C4) and three humic acid-like components (C1, C2, and C3). The adsorption isotherms, kinetics, and thermodynamics of DOM adsorption on CNTs were further investigated. A Freundlich isotherm model fit the adsorption data well with high values of correlation. As a type of macro-porous and meso-porous adsorbent, CNTs preferably adsorb humic acid-like substances rather than protein-like substances. The increasing temperature will speed up the adsorption process. The self-organizing map (SOM) analysis further explains the fluorescent properties of water samples. The results provide a new insight into the adsorption behaviour of DOM fluorescent components on CNTs.

Spontaneous fluctuation of the resting membrane potential in Paramecium: amplification caused by intracellular Ca2+.

PubMed

Nakaoka, Yasuo; Imaji, Takafumi; Hara, Masahiro; Hashimoto, Noboru

2009-01-01

The ciliated protozoan Paramecium spontaneously changes its swimming direction in the absence of external stimuli. Such behavior is based on resting potential fluctuations, the amplitudes of which reach a few mV. When the resting potential fluctuation is positive and large, a spike-like depolarization is frequently elicited that reverses the beating of the cilia associated with directional changes during swimming. We aimed to study how the resting potential fluctuation is amplified. Simultaneous measurements of the resting potential and intracellular Ca(2+) ([Ca(2+)](i)) from a deciliated cell showed that positive potential fluctuations were frequently accompanied by a small increase in [Ca(2+)](i). This result suggests that Ca(2+) influx through the somatic membrane occurs during the resting state. The mean amplitude of the resting potential fluctuation was largely decreased by either an intracellular injection of a calcium chelater (BAPTA) or by an extracellular addition of Ba(2+). Hence, a small increase in [Ca(2+)](i) amplifies the resting potential fluctuation. Simulation analysis of the potential fluctuation was made by assuming that Ca(2+) and K(+) channels of surface membrane are fluctuating between open and closed states. The simulated fluctuation increased to exhibit almost the same amplitude as the measured fluctuation using the assumption that a small Ca(2+) influx activates Ca(2+) channels in a positive feedback manner.

Visualizing substructure of Ca2+ waves by total internal reflection fluorescence microscopy

NASA Astrophysics Data System (ADS)

Bai, Yongqiang; Tang, Aihui; Wang, Shiqiang; Zhu, Xing

2005-02-01

Total internal reflection fluorescence microscope is a new optical microscopic system based on near-field optical theory. Its character of illumination by evanescent wave, together with the great signal-to-noise ratio and temporal resolution achieved by high quality CCD, allows us to analyze the spatiotemporal details of local Ca2+ dynamics within the nanoscale microdomain surrounding different Ca2+ channels. We have recently constructed a versatile objective TIRFM equipped with a high numerical aperture (NA=1.45) objective. Using fluo-4 as the Ca2+ indicator, we visualized the near-membrane profiles of Ca2+ waves and elementary Ca2+ sparks generated by Ca2+ release channels in rat ventricular myocytes. Different from those detected using conventional and confocal microscopy, Ca2+ waves observed with TIRFM exhibited fine inhomogenous substructures composed of fluctuating Ca2+ sparks. The anfractuous routes of spark recruitment suggested that the propagation of Ca2+ waves is much more complicated than previously imagined. We believe that TIRFM will provide a unique tool for dissecting the microscopic mechanisms of intracellular Ca2+ signaling.

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

PubMed

Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

2015-01-13

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

Manipulating freely diffusing single 20-nm particles in an Anti-Brownian Electrokinetic Trap (ABELtrap)

NASA Astrophysics Data System (ADS)

Zarrabi, Nawid; Clausen, Caterina; Düser, Monika G.; Börsch, Michael

2013-02-01

Conformational changes of individual fluorescently labeled proteins can be followed in solution using a confocal microscope. Two fluorophores attached to selected domains of the protein report fluctuating conformations. Based on Förster resonance energy transfer (FRET) between these fluorophores on a single protein, sequential distance changes between the dyes provide the real time trajectories of protein conformations. However, observation times are limited for freely diffusing biomolecules by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices with 4 electrodes for an Anti-Brownian Electrokinetic Trap (ABELtrap). Here we present an ABELtrap based on a laser focus pattern generated by a pair of acousto-optical beam deflectors and controlled by a programmable FPGA chip. Fluorescent 20-nm beads in solution were used to mimic freely diffusing large proteins like solubilized FoF1-ATP synthase. The ABELtrap could hold these nanobeads for about 10 seconds at the given position. Thereby, observation times of a single particle were increased by a factor of 1000.

A novel ''donor-π-acceptor'' type fluorescence probe for sensing pH: mechanism and application in vivo.

PubMed

Chao, Jianbin; Wang, Huijuan; Zhang, Yongbin; Yin, Caixia; Huo, Fangjun; Song, Kailun; Li, Zhiqing; Zhang, Ting; Zhao, Yaqin

2017-11-01

A novel pH fluorescent probe 1-(pyren-1-yl)-3-(6-methoxypridin-3-yl)-acrylketone, (PMPA), which had a pyrene structure attached to methoxypyridine, was synthesized for monitoring extremely acidic and alkaline pH. The pH titrations indicated that PMPA displayed a remarkable emission enhancement with a pK a of 2.70 and responded linearly to minor pH fluctuations within the extremely acidic range of 1.26-3.97. Interestingly, PMPA also exhibited strong pH-dependent characteristics with pK a 9.32 and linear response to extreme-alkalinity range of 8.54-10.36. In addition, PMPA displayed a good selectivity, excellent photostability and large Stokes shift (167nm). Furthermore, the probe PMPA had excellent cell membrane permeability and was applied successfully to rapidly detect pH in living cells. pH value in these organs was closely related to many diseases, so these findings suggested that the probe had potential application in pH detecting for disease diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Single-molecule spectroscopy unmasks the lowest exciton state of the B850 assembly in LH2 from Rps. acidophila.

PubMed

Kunz, Ralf; Timpmann, Kõu; Southall, June; Cogdell, Richard J; Freiberg, Arvi; Köhler, Jürgen

2014-05-06

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Instantaneous velocity measurement of AC electroosmotic flows by laser induced fluorescence photobleaching anemometer with high temporal resolution

NASA Astrophysics Data System (ADS)

Zhao, Wei; Yang, Fang; Qiao, Rui; Wang, Guiren; Rui Qiao Collaboration

2015-11-01

Understanding the instantaneous response of flows to applied AC electric fields may help understand some unsolved issues in induced-charge electrokinetics and enhance performance of microfluidic devices. Since currently available velocimeters have difficulty in measuring velocity fluctuations with frequency higher than 1 kHz, most experimental studies so far focus only on the average velocity measurement in AC electrokinetic flows. Here, we present measurements of AC electroosmotic flow (AC-EOF) response time in microchannels by a novel velocimeter with submicrometer spatial resolution and microsecond temporal resolution, i.e. laser-induced fluorescence photobleaching anemometer (LIFPA). Several parameters affecting the AC-EOF response time to the applied electric signal were investigated, i.e. channel length, transverse position and solution conductivity. The experimental results show that the EOF response time under a pulsed electric field decreases with the reduction of the microchannel length, distance between the detection position to the wall and the conductivity of the solution. This work could provide a new powerful tool to measure AC electrokinetics and enhance our understanding of AC electrokinetic flows.

Two-photon voltage imaging using a genetically encoded voltage indicator

PubMed Central

Akemann, Walther; Sasaki, Mari; Mutoh, Hiroki; Imamura, Takeshi; Honkura, Naoki; Knöpfel, Thomas

2013-01-01

Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2–3 larger ΔR/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex. PMID:23868559

Direct measurements of classical and enhanced gradient-aligned cross-field ion flows in a helicon plasma source using laser-induced fluorescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siddiqui, M. Umair, E-mail: musiddiqui@mail.wvu.edu; Thompson, Derek S.; McIlvain, Julianne M.

2015-12-15

Direct laser induced fluorescence measurements are shown of cross-field ion flows normal to an absorbing boundary that is aligned parallel to the axial magnetic field in a helicon plasma. We show Langmuir and emissive probe measurements of local density and plasma potential in the same region, as well as floating probe spectra near the boundary. With these measurements, we investigate the influence of ion-neutral collisionality on radial ion transport by varying the ratio of the ion gyro-radius, ρ{sub i}, to the ion-neutral collision length, λ, over the range 0.34 ≤ ρ{sub i}λ{sup −1} ≤ 1.60. Classical drift-diffusion transport along density and potential gradients ismore » sufficient to describe flow profiles for most cases. For two parameter regimes (ρ{sub i}λ{sup −1} = 0.65 and 0.44), low-frequency electrostatic fluctuations (f < 10 kHz) and enhanced cross-field bulk ion flow to the boundary are observed.« less

Range-dependence of acoustic channel with traveling sinusoidal surface wave.

PubMed

Choo, Youngmin; Seong, Woojae; Lee, Keunhwa

2014-04-01

Range-dependence of time-varying acoustic channels caused by a traveling surface wave is investigated through water tank experiments and acoustic propagation analysis schemes. As the surface wave travels, surface reflected signals fluctuate and the fluctuation varies with source-receiver horizontal range. Amplitude fluctuations of surface reflected signals increase with increasing horizontal range whereas the opposite occurs in delay fluctuations. The scattered pressure field at a fixed time shows strong dependence on the receiver position because of caustics and shadow zones formed by the surface. The Doppler shifts of surface reflected signals also depend on the horizontal range. Comparison between measurement data and model results indicates the Doppler shift relies on the delay fluctuation under current experimental conditions.

Dynamic fluctuations in single-molecule biophysics experiments. Comment on "Extracting physics of life at the molecular level: A review of single-molecule data analyses" by W. Colomb and S.K. Sarkar

NASA Astrophysics Data System (ADS)

Krapf, Diego

2015-06-01

Single-molecule biophysics includes the study of isolated molecules and that of individual molecules within living cells. In both cases, dynamic fluctuations at the nanoscale play a critical role. Colomb and Sarkar emphasize how different noise sources affect the analysis of single molecule data [1]. Fluctuations in biomolecular systems arise from two very different mechanisms. On one hand thermal fluctuations are a predominant feature in the behavior of individual molecules. On the other hand, non-Gaussian fluctuations can arise from inter- and intramolecular interactions [2], spatial heterogeneities [3], non-Poisson external perturbations [4] and complex non-linear dynamics in general [5,6].

Asymmetric multiscale detrended fluctuation analysis of California electricity spot price

NASA Astrophysics Data System (ADS)

Fan, Qingju

2016-01-01

In this paper, we develop a new method called asymmetric multiscale detrended fluctuation analysis, which is an extension of asymmetric detrended fluctuation analysis (A-DFA) and can assess the asymmetry correlation properties of series with a variable scale range. We investigate the asymmetric correlations in California 1999-2000 power market after filtering some periodic trends by empirical mode decomposition (EMD). Our findings show the coexistence of symmetric and asymmetric correlations in the price series of 1999 and strong asymmetric correlations in 2000. What is more, we detect subtle correlation properties of the upward and downward price series for most larger scale intervals in 2000. Meanwhile, the fluctuations of Δα(s) (asymmetry) and | Δα(s) | (absolute asymmetry) are more significant in 2000 than that in 1999 for larger scale intervals, and they have similar characteristics for smaller scale intervals. We conclude that the strong asymmetry property and different correlation properties of upward and downward price series for larger scale intervals in 2000 have important implications on the collapse of California power market, and our findings shed a new light on the underlying mechanisms of power price.

Direct comparison of Fe-Cr unmixing characterization by atom probe tomography and small angle scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Couturier, Laurent, E-mail: laurent.couturier55@ho

The fine microstructure obtained by unmixing of a solid solution either by classical precipitation or spinodal decomposition is often characterized either by small angle scattering or atom probe tomography. This article shows that a common data analysis framework can be used to analyze data obtained from these two techniques. An example of the application of this common analysis is given for characterization of the unmixing of the Fe-Cr matrix of a 15-5 PH stainless steel during long-term ageing at 350 °C and 400 °C. A direct comparison of the Cr composition fluctuations amplitudes and characteristic lengths obtained with both techniquesmore » is made showing a quantitative agreement for the fluctuation amplitudes. The origin of the discrepancy remaining for the characteristic lengths is discussed. - Highlights: •Common analysis framework for atom probe tomography and small angle scattering •Comparison of same microstructural characteristics obtained using both techniques •Good correlation of Cr composition fluctuations amplitudes from both techniques •Good correlation of Cr composition fluctuations amplitudes with classic V parameter.« less

Anti-correlation and multifractal features of Spain electricity spot market

NASA Astrophysics Data System (ADS)

Norouzzadeh, P.; Dullaert, W.; Rahmani, B.

2007-07-01

We use multifractal detrended fluctuation analysis (MF-DFA) to numerically investigate correlation, persistence, multifractal properties and scaling behavior of the hourly spot prices for the Spain electricity exchange-Compania O Peradora del Mercado de Electricidad (OMEL). Through multifractal analysis, fluctuations behavior, the scaling exponents and generalized Hurst exponents are studied. Moreover, contribution of fat-tailed probability distributions and nonlinear temporal correlations to multifractality is studied.

Anchoring effect on first passage process in Taiwan financial market

NASA Astrophysics Data System (ADS)

Liu, Hsing; Liao, Chi-Yo; Ko, Jing-Yuan; Lih, Jiann-Shing

2017-07-01

Empirical analysis of the price fluctuations of financial markets has received extensive attention because a substantial amount of financial market data has been collected and because of advances in data-mining techniques. Price fluctuation trends can help investors to make informed trading decisions, but such decisions may also be affected by a psychological factors-the anchoring effect. This study explores the intraday price time series of Taiwan futures, and applies diffusion model and quantitative methods to analyze the relationship between the anchoring effect and price fluctuations during first passage process. Our results indicate that power-law scaling and anomalous diffusion for stock price fluctuations are related to the anchoring effect. Moreover, microscopic price fluctuations before switching point in first passage process correspond with long-term price fluctuations of Taiwan's stock market. We find that microscopic trends could provide useful information for understanding macroscopic trends in stock markets.

Extreme values in the Chinese and American stock markets based on detrended fluctuation analysis

NASA Astrophysics Data System (ADS)

Cao, Guangxi; Zhang, Minjia

2015-10-01

This paper focuses on the comparative analysis of extreme values in the Chinese and American stock markets based on the detrended fluctuation analysis (DFA) algorithm using the daily data of Shanghai composite index and Dow Jones Industrial Average. The empirical results indicate that the multifractal detrended fluctuation analysis (MF-DFA) method is more objective than the traditional percentile method. The range of extreme value of Dow Jones Industrial Average is smaller than that of Shanghai composite index, and the extreme value of Dow Jones Industrial Average is more time clustering. The extreme value of the Chinese or American stock markets is concentrated in 2008, which is consistent with the financial crisis in 2008. Moreover, we investigate whether extreme events affect the cross-correlation between the Chinese and American stock markets using multifractal detrended cross-correlation analysis algorithm. The results show that extreme events have nothing to do with the cross-correlation between the Chinese and American stock markets.

Synchronous fluorescence spectroscopy for analysis of wine and wine distillates

NASA Astrophysics Data System (ADS)

Andreeva, Ya.; Borisova, E.; Genova, Ts.; Zhelyazkova, Al.; Avramov, L.

2015-01-01

Wine and brandies are multicomponent systems and conventional fluorescence techniques, relying on recording of single emission or excitation spectra, are often insufficient. In such cases synchronous fluorescence spectra can be used for revealing the potential of the fluorescence techniques. The technique is based on simultaneously scanning of the excitation and emission wavelength with constant difference (Δλ) maintained between them. In this study the measurements were made using FluoroLog3 spectrofluorimeter (HORIBA Jobin Yvon, France) and collected for excitation and emission in the wavelength region 220 - 700 nm using wavelength interval Δλ from 10 to 100 nm in 10 nm steps. This research includes the results obtained for brandy and red wine samples. Fluorescence analysis takes advantage in the presence of natural fluorophores in wines and brandies, such as gallic, vanillic, p-coumaric, syringic, ferulic acid, umbelliferone, scopoletin and etc. Applying of synchronous fluorescence spectroscopy for analysis of these types of alcohols allows us to estimate the quality of wines and also to detect adulteration of brandies like adding of a caramel to wine distillates for imitating the quality of the original product aged in oak casks.