Comparison of red autofluorescing plaque and disclosed plaque-a cross-sectional study.

PubMed

Volgenant, Catherine M C; Fernandez Y Mostajo, Mercedes; Rosema, Nanning A M; van der Weijden, Fridus A; Ten Cate, Jacob M; van der Veen, Monique H

2016-12-01

The aim of this cross-sectional study was to assess the correlation between dental plaque scores determined by the measurement of red autofluorescence or by visualization with a two-tone solution. Clinical photographs were used for this study. Overnight plaque from the anterior teeth of 48 participants was assessed for red fluorescence on photographs (taken with a QLF-camera) using a modified Quigley & Hein (mQH) index. A two-tone disclosing solution was applied. Total disclosed plaque was clinically assessed using the mQH index. In addition, total and blue disclosed plaque was scored on clinical photographs using the mQH index. A strong correlation was observed between the total disclosed plaque scored on photographs and the clinical scores (r = 0.70 at site level; r = 0.88 at subject level). The correlation between red fluorescent plaque and total plaque, as assessed on the photographs, was moderate to strong and significant (r = 0.50 at the site level; r = 0.70 at the subject level), with the total plaque scores consistently higher than the red fluorescent plaque scores. The correlation between red fluorescent plaque and blue disclosed plaque was weak to moderate and significant (r = 0.30 at the site level; r = 0.50 at the subject level). Plaque, as scored on white-light photographs, corresponds well with clinically assessed plaque. A weak to moderate correlation between red fluorescing plaque and total disclosed plaque or blue disclosed plaque was found. What at present is considered to be matured dental plaque, which appears blue following the application of a two-tone disclosing solution, is not in agreement with red fluorescent dental plaque assessment.

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.

Dual-color two-photon fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Berland, Keith M.

2001-04-01

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a research tool in biological and biophysical research. Under favorable conditions, FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. However, interpretation of FCS data quickly becomes complicated as the heterogeneity of a molecular system increases, as well as when there is significant non-stationery fluorescence background (e.g. intracellular autofluorescence). Use of multi-parameter correlation measurements is one promising approach that can improve the fidelity of FCS measurements in complex systems. In particular, the use of dual-color fluorescence assays, in which different interacting molecular species are labeled with unique fluorescent indicators, can "tune" the sensitivity of FCS measurements in favor of particular molecular species of interest, while simultaneously minimizing the contribution of other molecular species to the overall fluorescence correlation signal. Here we introduce the combined application of two-photon fluorescence excitation and dual-color cross-correlation analysis for detecting molecular interactions in solution. The use of two-photon excitation is particularly advantageous for dual-color FCS applications due to the uncomplicated optical alignment and the superior capabilities for intracellular applications. The theory of two-photon dual-color FCS is introduced, and initial results quantifying hybridization reactions between three independent single stranded DNA molecules are presented.

Red fluorescence of dental plaque in children -A cross-sectional study.

PubMed

Volgenant, Catherine M C; Zaura, Egija; Brandt, Bernd W; Buijs, Mark J; Tellez, Marisol; Malik, Gayatri; Ismail, Amid I; Ten Cate, Jacob M; van der Veen, Monique H

2017-03-01

The relation between the presence of red fluorescent plaque and the caries status in children was studied. In addition, the microbial composition of dental plaque from sites with red fluorescent plaque (RFP) and from sites with no red fluorescent plaque (NFP) was assessed. Fluorescence photographs were taken from fifty children (6-14 years old) with overnight plaque. Full-mouth caries scores (ICDAS II) were obtained. The composition of a saliva sample and two plaque samples (RFP and NFP) was assessed using 16S rDNA sequencing. At the site level, no clinically relevant correlations were found between the presence of RFP and the caries status. At the subject level, a weak correlation was found between RFP and the caries status when non-cavitated lesions were included (r s =0.37, p=0.007). The microbial composition of RFP differed significantly from NFP. RFP had more anaerobes and more Gram-negative bacterial taxa. The most discriminative operational taxonomic units (OTUs) for RFP were Corynebacterium, Leptotrichia, Porphyromonas and Selenomonas, while the most discriminative OTUs for NFP were Neisseria, Actinomyces, Streptococcus and Rothia. There were no clinical relevant correlations in this cross-sectional study between the presence of RFP and (early) caries lesions. There were differences in the composition of these phenotypically different plaque samples: RFP contained more Gram-negative, anaerobic taxa and was more diverse than NFP. The study outcomes provide more insight in the possibilities to use plaque fluorescence in oral health risk assessments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations.

PubMed

Wenger, Jérôme; Gérard, Davy; Lenne, Pierre-François; Rigneault, Hervé; Dintinger, José; Ebbesen, Thomas W; Boned, Annie; Conchonaud, Fabien; Marguet, Didier

2006-12-11

Single nanometric apertures in a metallic film are used to develop a simple and robust setup for dual-color fluorescence cross-correlation spectroscopy (FCCS) at high concentrations. If the nanoaperture concept has already proven to be useful for single-species analysis, its extension to the dual-color case brings new interesting specificities. The alignment and overlap of the two excitation beams are greatly simplified. No confocal pinhole is used, relaxing the requirement for accurate correction of chromatic aberrations. Compared to two-photon excitation, nanoapertures have the advantage to work with standard fluorophore constructions having high absorption cross-section and well-known absorption/emission spectra. Thanks to the ultra-low volume analysed within one single aperture, fluorescence correlation analysis can be performed with single molecule resolution at micromolar concentrations, resulting in 3 orders of magnitude gain compared to conventional setups. As applications of this technique, we follow the kinetics of an enzymatic cleavage reaction at 2 muM DNA oligonucleotide concentration.We also demonstrate that FCCS in nanoaper-tures can be applied to the fast screening of a sample for dual-labeled species within 1 s acquisition time. This offers new possibilities for rapid screening applications in biotechnology at high concentrations.

Iodine imaging in thyroid by fluorescent X-ray CT with 0.05 mm spatial resolution

NASA Astrophysics Data System (ADS)

Takeda, T.; Yu, Q.; Yashiro, T.; Zeniya, T.; Wu, J.; Hasegawa, Y.; Thet-Thet-Lwin; Hyodo, K.; Yuasa, T.; Dilmanian, F. A.; Akatsuka, T.; Itai, Y.

2001-07-01

Fluorescent X-ray computed tomography (FXCT) at a 0.05 mm in-plane spatial resolution and 0.05 mm slice thickness depicted the cross sectional distribution of endogenous iodine within thyroid. The distribution obtained from the FXCT image correlated closely to that obtained from the pathological pictures.

Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

PubMed Central

Kohl, Tobias; Haustein, Elke; Schwille, Petra

2005-01-01

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

Surface Transient Binding-Based Fluorescence Correlation Spectroscopy (STB-FCS), a Simple and Easy-to-Implement Method to Extend the Upper Limit of the Time Window to Seconds.

PubMed

Peng, Sijia; Wang, Wenjuan; Chen, Chunlai

2018-05-10

Fluorescence correlation spectroscopy is a powerful single-molecule tool that is able to capture kinetic processes occurring at the nanosecond time scale. However, the upper limit of its time window is restricted by the dwell time of the molecule of interest in the confocal detection volume, which is usually around submilliseconds for a freely diffusing biomolecule. Here, we present a simple and easy-to-implement method, named surface transient binding-based fluorescence correlation spectroscopy (STB-FCS), which extends the upper limit of the time window to seconds. We further demonstrated that STB-FCS enables capture of both intramolecular and intermolecular kinetic processes whose time scales cross several orders of magnitude.

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

NASA Astrophysics Data System (ADS)

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

2015-07-01

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

Studying flow close to an interface by total internal reflection fluorescence cross-correlation spectroscopy: Quantitative data analysis

NASA Astrophysics Data System (ADS)

Schmitz, R.; Yordanov, S.; Butt, H. J.; Koynov, K.; Dünweg, B.

2011-12-01

Total internal reflection fluorescence cross-correlation spectroscopy (TIR-FCCS) has recently [S. Yordanov , Optics ExpressOPEXFF1094-408710.1364/OE.17.021149 17, 21149 (2009)] been established as an experimental method to probe hydrodynamic flows near surfaces, on length scales of tens of nanometers. Its main advantage is that fluorescence occurs only for tracer particles close to the surface, thus resulting in high sensitivity. However, the measured correlation functions provide only rather indirect information about the flow parameters of interest, such as the shear rate and the slip length. In the present paper, we show how to combine detailed and fairly realistic theoretical modeling of the phenomena by Brownian dynamics simulations with accurate measurements of the correlation functions, in order to establish a quantitative method to retrieve the flow properties from the experiments. First, Brownian dynamics is used to sample highly accurate correlation functions for a fixed set of model parameters. Second, these parameters are varied systematically by means of an importance-sampling Monte Carlo procedure in order to fit the experiments. This provides the optimum parameter values together with their statistical error bars. The approach is well suited for massively parallel computers, which allows us to do the data analysis within moderate computing times. The method is applied to flow near a hydrophilic surface, where the slip length is observed to be smaller than 10nm, and, within the limitations of the experiments and the model, indistinguishable from zero.

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

PubMed Central

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki

2014-01-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)–Ras–extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. PMID:24958104

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

PubMed

Smith, Adam W

2015-01-01

Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508-3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842-2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833-10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543-556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.

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

Hirvonen, Liisa M.; Le Marois, Alix; Suhling, Klaus, E-mail: klaus.suhling@kcl.ac.uk

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

Correlated expression of gfp and Bt cry1Ac gene facilitates quantification of transgenic hybridization between Brassicas.

PubMed

Shen, B-C; Stewart, C N; Zhang, M-Q; Le, Y-T; Tang, Z-X; Mi, X-C; Wei, W; Ma, K-P

2006-09-01

Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.

Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

PubMed Central

Angiolini, Juan; Plachta, Nicolas; Mocskos, Esteban; Levi, Valeria

2015-01-01

Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments. PMID:26039162

Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.

PubMed

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki; Aoki, Kazuhiro

2014-09-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Correlation between cross-bridge kinetics obtained from Trp fluorescence of myofibril suspensions and mechanical studies of single muscle fibers in rabbit psoas.

PubMed

Candau, Robin; Kawai, Masataka

2011-12-01

Our goal is to correlate kinetic constants obtained from fluorescence studies of myofibril suspension with those from mechanical studies of skinned muscle fibers from rabbit psoas. In myofibril studies, the stopped-flow technique with tryptophan fluorescence was used; in muscle fiber studies, tension transients with small amplitude sinusoidal length perturbations were used. All experiments were performed using the equivalent solution conditions (200 mM ionic strength, pH 7.00) at 10°C. The concentration of MgATP was varied to characterize kinetic constants of the ATP binding step 1 (K (1): dissociation constant), the binding induced cross-bridge detachment step 2 (k (2), k (-2): rate constants), and the ATP cleavage step 3 (k (3), k (-3)). In myofibrils we found that K (1) = 0.52 ± 0.08 mM (±95% confidence limits), k (2) = 242 ± 24 s(-1), and k (-2) ≈ 0; in muscle fibers, K (1) = 0.46 ± 0.06 mM, k (2) = 286 ± 32 s(-1), and k (-2) = 57 ± 21 s(-1). From these results, we conclude that myofibrils and muscle fibers exhibit nearly equal ATP binding step, and nearly equal ATP binding induced cross-bridge detachment step. Consequently, there is a good correlation between process C (phase 2 of step analysis) and the cross-bridge detachment step. The reverse detachment step is finite in fibers, but almost absent in myofibrils. We further studied partially cross-linked myofibrils and found little change in steps 2 and 3, indicating that cross-linking does not affect these steps. However, we found that K (1) is 2.5× of native myofibrils, indicating that MgATP binding is weakened by the presence of the extra load. We further studied the phosphate (Pi) effect in myofibrils, and found that Pi is a competitive inhibitor of MgATP, with the inhibitory dissociation constant of ~9 mM. Similar results were also deduced from fiber studies. To characterize the ATP cleavage step in myofibrils, we measured the slow rate constant in fluorescence, and found that k (3) + k (-3) = 16 ± 1 s(-1).

Two fluorescent wavelengths, 440(ex)/520(em) nm and 370(ex)/440(em) nm, reflect advanced glycation and oxidation end products in human skin without diabetes.

PubMed

Beisswenger, Paul J; Howell, Scott; Mackenzie, Todd; Corstjens, Hugo; Muizzuddin, Neelam; Matsui, Mary S

2012-03-01

Advanced glycation end products (AGEs) and oxidation products (OPs) play an important role in diabetes complications, aging, and damage from sun exposure. Measurement of skin autofluorescence (SAF) has been promoted as a noninvasive technique to measure skin AGEs, but the actual products quantified are uncertain. We have compared specific SAF measurements with analytically determined AGEs and oxidative biomarkers in skin collagen and determined if these measurements can be correlated with chronological aging and actinic exposure. SAF at four excitation (ex)/emission (em) intensities was measured on the upper inner arm ("sun protected") and dorsal forearm ("sun exposed") in 40 subjects without diabetes 20-60 years old. Skin collagen from the same sites was analyzed by liquid chromatography-tandem mass spectrometry for three AGEs-pentosidine, carboxymethyllysine (CML), and carboxyethyllysine (CEL)-and the OP methionine sulfoxide (MetSO). There was poor correlation of AGE-associated fluorescence spectra with AGEs and OP in collagen, with only pentosidine correlating with fluorescence at 370(ex)/440(em) nm. A little-studied SAF (440(ex)/520(em) nm), possibly reflecting elastin cross-links, correlated with all AGEs and OPs. Levels of CML, pentosidine, and MetSO, but not SAF, were significantly higher in sun-exposed skin. These AGEs and OPs, as well as SAF at 370(ex)/440(em) nm and 440(ex)/520(em) nm, increased with chronological aging. SAF measurements at 370(ex)/440(em) nm and 335(ex)/385(em) nm, except for pentosidine, which correlated with fluorescence at 370(ex)/440(em), correlate poorly with glycated and oxidatively modified protein in human skin and do not reflect actinic modification. A new fluorescence measurement (440(ex)/520(em) nm) appears to reflect AGEs and OPs in skin.

Two-dimensional fluorescence lifetime correlation spectroscopy. 2. Application.

PubMed

Ishii, Kunihiko; Tahara, Tahei

2013-10-03

In the preceding article, we introduced the theoretical framework of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). In this article, we report the experimental implementation of 2D FLCS. In this method, two-dimensional emission-delay correlation maps are constructed from the photon data obtained with the time-correlated single photon counting (TCSPC), and then they are converted to 2D lifetime correlation maps by the inverse Laplace transform. We develop a numerical method to realize reliable transformation, employing the maximum entropy method (MEM). We apply the developed actual 2D FLCS to two real systems, a dye mixture and a DNA hairpin. For the dye mixture, we show that 2D FLCS is experimentally feasible and that it can identify different species in an inhomogeneous sample without any prior knowledge. The application to the DNA hairpin demonstrates that 2D FLCS can disclose microsecond spontaneous dynamics of biological molecules in a visually comprehensible manner, through identifying species as unique lifetime distributions. A FRET pair is attached to the both ends of the DNA hairpin, and the different structures of the DNA hairpin are distinguished as different fluorescence lifetimes in 2D FLCS. By constructing the 2D correlation maps of the fluorescence lifetime of the FRET donor, the equilibrium dynamics between the open and the closed forms of the DNA hairpin is clearly observed as the appearance of the cross peaks between the corresponding fluorescence lifetimes. This equilibrium dynamics of the DNA hairpin is clearly separated from the acceptor-missing DNA that appears as an isolated diagonal peak in the 2D maps. The present study clearly shows that newly developed 2D FLCS can disclose spontaneous structural dynamics of biological molecules with microsecond time resolution.

Note: a 4 ns hardware photon correlator based on a general-purpose field-programmable gate array development board implemented in a compact setup for fluorescence correlation spectroscopy.

PubMed

Kalinin, Stanislav; Kühnemuth, Ralf; Vardanyan, Hayk; Seidel, Claus A M

2012-09-01

We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

Note: A 4 ns hardware photon correlator based on a general-purpose field-programmable gate array development board implemented in a compact setup for fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Kalinin, Stanislav; Kühnemuth, Ralf; Vardanyan, Hayk; Seidel, Claus A. M.

2012-09-01

We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

Tracking quasi-stationary flow of weak fluorescent signals by adaptive multi-frame correlation.

PubMed

Ji, L; Danuser, G

2005-12-01

We have developed a novel cross-correlation technique to probe quasi-stationary flow of fluorescent signals in live cells at a spatial resolution that is close to single particle tracking. By correlating image blocks between pairs of consecutive frames and integrating their correlation scores over multiple frame pairs, uncertainty in identifying a globally significant maximum in the correlation score function has been greatly reduced as compared with conventional correlation-based tracking using the signal of only two consecutive frames. This approach proves robust and very effective in analysing images with a weak, noise-perturbed signal contrast where texture characteristics cannot be matched between only a pair of frames. It can also be applied to images that lack prominent features that could be utilized for particle tracking or feature-based template matching. Furthermore, owing to the integration of correlation scores over multiple frames, the method can handle signals with substantial frame-to-frame intensity variation where conventional correlation-based tracking fails. We tested the performance of the method by tracking polymer flow in actin and microtubule cytoskeleton structures labelled at various fluorophore densities providing imagery with a broad range of signal modulation and noise. In applications to fluorescent speckle microscopy (FSM), where the fluorophore density is sufficiently low to reveal patterns of discrete fluorescent marks referred to as speckles, we combined the multi-frame correlation approach proposed above with particle tracking. This hybrid approach allowed us to follow single speckles robustly in areas of high speckle density and fast flow, where previously published FSM analysis methods were unsuccessful. Thus, we can now probe cytoskeleton polymer dynamics in living cells at an entirely new level of complexity and with unprecedented detail.

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

PubMed

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

2016-06-01

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Investigating the effect of poly-l-lactic acid nanoparticles carrying hypericin on the flow-biased diffusive motion of HeLa cell organelles.

PubMed

Penjweini, Rozhin; Deville, Sarah; Haji Maghsoudi, Omid; Notelaers, Kristof; Ethirajan, Anitha; Ameloot, Marcel

2017-07-19

In this study, we investigate in human cervical epithelial HeLa cells the intracellular dynamics and the mutual interaction with the organelles of the poly-l-lactic acid nanoparticles (PLLA NPs) carrying the naturally occurring hydrophobic photosensitizer hypericin. Temporal and spatiotemporal image correlation spectroscopy was used for the assessment of the intracellular diffusion and directed motion of the nanocarriers by tracking the hypericin fluorescence. Using image cross-correlation spectroscopy and specific fluorescent labelling of endosomes, lysosomes and mitochondria, the NPs dynamics in association with the cell organelles was studied. Static colocalization experiments were interpreted according to the Manders' overlap coefficient. Nanoparticles associate with a small fraction of the whole-organelle population. The organelles moving with NPs exhibit higher directed motion compared to those moving without them. The rate of the directed motion drops substantially after the application of nocodazole. The random component of the organelle motions is not influenced by the NPs. Image correlation and cross-correlation spectroscopy are most appropriate to unravel the motion of the PLLA nanocarrier and to demonstrate that the rate of the directed motion of organelles is influenced by their interaction with the nanocarriers. Not all PLLA-hypericin NPs are associated with organelles. © 2017 Royal Pharmaceutical Society.

The Application of Fluorescent Quantum Dots to Confocal, Multiphoton, and Electron Microscopic Imaging

PubMed Central

Deerinck, Thomas J.

2009-01-01

Fluorescent quantum dots are emerging as an important tool for imaging cells and tissues, and their unique optical and physical properties have captured the attention of the research community. The most common types of commercially available quantum dots consist of a nanocrystalline semiconductor core composed of cadmium selenide with a zinc sulfide capping layer and an outer polymer layer to facilitate conjugation to targeting biomolecules such as immunoglobulins. They exhibit high fluorescent quantum yields and have large absorption cross-sections, possess excellent photostability, and can be synthesized so that their narrow-band fluorescence emission can occur in a wide spectrum of colors. These properties make them excellent candidates for serving as multiplexing molecular beacons using a variety of imaging modalities including highly correlated microscopies. Whereas much attention has been focused on quantum-dot applications for live-cell imaging, we have sought to characterize and exploit their utility for enabling simultaneous multiprotein immunolabeling in fixed cells and tissues. Considerations for their application to immunolabeling for correlated light and electron microscopic analysis are discussed. PMID:18337229

Reflection-induced source correlation in spontaneous emission

NASA Astrophysics Data System (ADS)

Drabe, Karel E.; Cnossen, Gerard; Wiersma, Douwe A.; Ferwerda, H. A.; Hoenders, B. J.

1990-09-01

We present fluorescence spectra showing that the spontaneous-emission spectrum of a molecule in front of a mirror is not invariant on propagation. We also calculate the cross-spectral density of this light source and show that it does not obey Wolf's scaling law [Phys. Rev. Lett. 56, 1370 (1986)].

Fluorescence detection of the movement of single KcsA subunits reveals cooperativity

PubMed Central

Blunck, Rikard; McGuire, Hugo; Hyde, H. Clark; Bezanilla, Francisco

2008-01-01

The prokaryotic KcsA channel is gated at the helical bundle crossing by intracellular protons and inactivates at the extracellular selectivity filter. The C-terminal transmembrane helix has to undergo a conformational change for potassium ions to access the central cavity. Whereas a partial opening of the tetrameric channel is suggested to be responsible for subconductance levels of ion channels, including KcsA, a cooperative opening of the 4 subunits is postulated as the final opening step. In this study, we used single-channel fluorescence spectroscopy of KcsA to directly observe the movement of each subunit and the temporal correlation between subunits. Purified KcsA channels labeled at the C terminus near the bundle crossing have been inserted into supported lipid bilayer, and the fluorescence traces analyzed by means of a cooperative or independent Markov model. The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step. PMID:19074286

The endogenous fluorescence of fibroblast in collagen gels as indicator of stiffness of the extracellular matrix

NASA Astrophysics Data System (ADS)

Padilla-Martinez, J. P.; Ortega-Martinez, A.; Franco, W.

2016-03-01

The stiffness or rigidity of the extracellular matrix (ECM) regulates cell response. Established mechanical tests to measure stiffness, such as indentation and tensile tests, are invasive and destructive to the sample. Endogenous or native molecules to cells and ECM components, like tryptophan and cross-links of collagen, display fluorescence upon irradiation with ultraviolet light. Most likely, the concentration of these endogenous fluorophores changes as the stiffness of the ECM changes. In this work we investigate the endogenous fluorescence of collagen gels containing fibroblasts as a non-invasive non-destructive method to measure stiffness of the ECM. Human fibroblast cells were cultured in three-dimensional gels of type I collagen (50,000 cells/ml). This construct is a simple model of tissue contraction. During contraction, changes in the excitation-emission matrix (a fluorescence map in the 240-520/290-530 nm range) of constructs were measured with a spectrofluoremeter, and changes in stiffness were measured with a standard indentation test over 16 days. Results show that a progressive increase in fluorescence of the 290/340 nm excitation-emission pair correlates with a progressive increase in stiffness (r=0.9, α=0.5). The fluorescence of this excitation-emission pair is ascribed to tryptophan and variations in the fluorescence of this pair correlate with cellular proliferation. In this tissue model, the endogenous functional fluorescence of proliferating fibroblast cells is a biomechanical marker of stiffness of the ECM.

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

NASA Astrophysics Data System (ADS)

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

1992-01-01

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

Kinetics of a single cross-bridge in familial hypertrophic cardiomyopathy heart muscle measured by reverse Kretschmann fluorescence

PubMed Central

Mettikolla, Prasad; Calander, Nils; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian

2010-01-01

Familial hypertrophic cardiomyopathy (FHC) is a serious heart disease that often leads to a sudden cardiac death of young athletes. It is believed that the alteration of the kinetics of interaction between actin and myosin causes FHC by making the heart to pump blood inefficiently. We set out to check this hypothesis ex vivo. During contraction of heart muscle, a myosin cross-bridge imparts periodic force impulses to actin. The impulses are analyzed by fluorescence correlation spectroscopy (FCS) of fluorescently labeled actin. To minimize observation volume and background fluorescence, we carry out FCS measurements in surface plasmon coupled emission mode in a reverse Kretschmann configuration. Fluorescence is a result of near-field coupling of fluorophores excited in the vicinity of the metal-coated surface of a coverslip with the surface plasmons propagating in the metal. Surface plasmons decouple on opposite sides of the metal film and emit in a directional manner as far-field p-polarized radiation. We show that the rate of changes of orientation is significantly faster in contracting cardiac myofibrils of transgenic mice than wild type. These results are consistent with the fact that mutated heart muscle myosin translates actin faster in in vitro motility assays. PMID:20210485

Polarization effects in cutaneous autofluorescent spectra

NASA Astrophysics Data System (ADS)

Borisova, E.; Angelova, L.; Jeliazkova, Al.; Genova, Ts.; Pavlova, E.; Troyanova, P.; Avramov, L.

2014-05-01

Used polarized light for fluorescence excitation one could obtain response related to the anisotropy features of extracellular matrix. The fluorophore anisotropy is attenuated during lesions' growth and level of such decrease could be correlated with the stage of tumor development. Our preliminary investigations are based on in vivo point-by-point measurements of excitation-emission matrices (EEM) from healthy volunteers skin on different ages and from different anatomical places using linear polarizer and analyzer for excitation and emission light detected. Measurements were made using spectrofluorimeter FluoroLog 3 (HORIBA Jobin Yvon, France) with fiber-optic probe in steady-state regime using excitation in the region of 280-440 nm. Three different situations were evaluated and corresponding excitation-emission matrices were developed - with parallel and perpendicular positions for linear polarizer and analyzer, and without polarization of excitation and fluorescence light detected from a forearm skin surface. The fluorescence spectra obtained reveal differences in spectral intensity, related to general attenuation, due to filtering effects of used polarizer/analyzer couple. Significant spectral shape changes were observed for the complex autofluorescence signal detected, which correlated with collagen and protein cross-links fluorescence, that could be addressed to the tissue extracellular matrix and general condition of the skin investigated, due to morphological destruction during lesions' growth. A correlation between volunteers' age and the fluorescence spectra detected was observed during our measurements. Our next step is to increase developed initial database and to evaluate all sources of intrinsic fluorescent polarization effects and found if they are significantly altered from normal skin to cancerous state of the tissue, this way to develop a non-invasive diagnostic tool for dermatological practice.

Red fluorescence imaging for dental plaque detection and quantification: pilot study

NASA Astrophysics Data System (ADS)

Liu, Zhao; Gomez, Juliana; Khan, Soniya; Peru, Debbie; Ellwood, Roger

2017-09-01

The red fluorescence of dental plaque originating from porphyrins in oral bacteria may allow visualization, detection, and scoring of plaque without disclosing agents. Two studies were conducted. The first included 24 healthy participants who abstained from oral hygiene for 24 h. Dental plaque was collected from tooth surfaces, and a 10% solution was prepared. These were scanned by a molecular spectrometer to identify the optimum excitation and emission wavelengths of plaque for developing a red fluorescence imaging system. Fourteen healthy subjects completed the second study. After a washout period (1 week), participants had a prophylaxis at baseline and abstained from oral hygiene during the study. They were monitored using the fluorescence imaging system at baseline, 24 h, and 48 h. A dentist clinically assessed plaque after disclosing and on red fluorescence images. Three descriptors were extracted from images and a RUSBoost classifier derived computer fluorescence scores through cross-validation. Red fluorescence plaque levels increased during the 48-h accumulation. Plaque progression was identified by dentist assessment and computer analysis, presenting significant differences between visits at tooth and subject levels (p<0.05). Moderate correlations showed between clinical plaque and red fluorescence plaque (r=0.62 dentist, r=0.55 computer). The best agreement was observed when disclosing plaque threshold at level 2, for both dentist evaluation (sensitivity 71.1%, specificity 67.7%, accuracy 70.2%) and computer classification (sensitivity 68.4%, specificity 62.9%, accuracy 67.1%). Given the correlation with clinical diagnosis, red fluorescence imaging shows its potential for providing an objective and promising method for proper oral hygiene assessment.

A novel approach to dual excitation ratiometric optical mapping of cardiac action potentials with di-4-ANEPPS using pulsed LED excitation.

PubMed

Bachtel, Andrew D; Gray, Richard A; Stohlman, Jayna M; Bourgeois, Elliot B; Pollard, Andrew E; Rogers, Jack M

2011-07-01

We developed a new method for ratiometric optical mapping of transmembrane potential (V(m)) in cardiac preparations stained with di-4-ANEPPS. V(m)-dependent shifts of excitation and emission spectra establish two excitation bands (<481 and >481 nm) that produce fluorescence changes of opposite polarity within a single emission band (575-620 nm). The ratio of these positive and negative fluorescence signals (excitation ratiometry) increases V(m) sensitivity and removes artifacts common to both signals. We pulsed blue (450 ± 10 nm) and cyan (505 ± 15 nm) light emitting diodes (LEDs) at 375 Hz in alternating phase synchronized to a camera (750 frames-per-second). Fluorescence was bandpass filtered (585 ± 20 nm). This produced signals with upright (blue) and inverted (cyan) action potentials (APs) interleaved in sequential frames. In four whole swine hearts with motion chemically arrested, fractional fluorescence for blue, cyan, and ratio signals was 1.2 ± 0.3%, 1.2 ± 0.3%, and 2.4 ± 0.6%, respectively. Signal-to-noise ratios were 4.3 ± 1.4, 4.0 ± 1.2, and 5.8 ± 1.9, respectively. After washing out the electromechanical uncoupling agent, we characterized motion artifact by cross-correlating blue, cyan, and ratio signals with a signal with normal AP morphology. Ratiometry improved cross-correlation coefficients from 0.50 ± 0.48 to 0.81 ± 0.25, but did not cancel all motion artifacts. These findings demonstrate the feasibility of pulsed LED excitation ratiometry in myocardium. © 2011 IEEE

A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials with Di-4-ANEPPS using Pulsed LED Excitation

PubMed Central

Bachtel, Andrew D.; Gray, Richard A.; Stohlman, Jayna M.; Bourgeois, Elliot B.; Pollard, Andrew E.

2011-01-01

We developed a new method for ratiometric optical mapping of transmembrane potential (Vm) in cardiac preparations stained with di-4-ANEPPS. Vm-dependent shifts of excitation and emission spectra establish two excitation bands (<481 and >481 nm) that produce fluorescence changes of opposite polarity within a single emission band (575–620 nm). The ratio of these positive and negative fluorescence signals (excitation ratiometry) increases Vm sensitivity and removes artifacts common to both signals. We pulsed blue (450±10 nm) and cyan (505±15 nm) light emitting diodes (LEDs) at 375 Hz in alternating phase synchronized to a camera (750 frames-per-second). Fluorescence was bandpass filtered (585±20 nm). This produced signals with upright (blue) and inverted (cyan) action potentials (APs) interleaved in sequential frames. In 4 whole swine hearts with motion chemically arrested, fractional fluorescence for blue, cyan, and ratio signals was 1.2±0.3%, 1.2±0.3%, and 2.4±0.6%, respectively. Signal-to-noise ratios were 4.3±1.4, 4.0±1.2, and 5.8±1.9, respectively. After washing out the electromechanical uncoupling agent, we characterized motion artifact by cross-correlating blue, cyan, and ratio signals with a signal with normal AP morphology. Ratiometry improved cross-correlation coefficients from 0.50±0.48 to 0.81±0.25, but did not cancel all motion artifacts. These findings demonstrate the feasibility of pulsed LED excitation ratiometry in myocardium. PMID:21536528

A new serotyping method for Klebsiella species: evaluation of the technique.

PubMed Central

Riser, E; Noone, P; Bonnet, M L

1976-01-01

A new indirect fluorescent typing method for Klebsiella species is compared with an established method, capsular swelling. The fluorescent antibody (FA) technique was tested with standards and unknowns, and the results were checked by capsular swelling. Several unknowns were sent away for confirmation of typing, by capsular swelling. The FA method was also tried by a technician in the routine department for blind identification of standards. Fluorescence typing gives close correlation with the established capsular swelling technique but has greater sensitivity; allows more econimical use of expensive antisera; possesses greater objectivity as it requires less operator skill in the reading of results; resolves most of the cross reactions observed with capsular swelling; and has a higher per cent success rate in identification. PMID:777043

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

PubMed Central

Lagerkvist, Ann Catrin; Földes-Papp, Zeno; Persson, Mats A.A.; Rigler, Rudolf

2001-01-01

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage–antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system. PMID:11468349

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.

Intracellular localization and dynamics of Hypericin loaded PLLA nanocarriers by image correlation spectroscopy.

PubMed

Penjweini, Rozhin; Deville, Sarah; D'Olieslaeger, Lien; Berden, Mandy; Ameloot, Marcel; Ethirajan, Anitha

2015-11-28

The study of cell-nanoparticle interactions is an important aspect for understanding drug delivery using nanocarriers. In this regard, advances in fluorescence based microscopy are useful for the investigation of temporal and spatial behavior of nanoparticles (NPs) within the intracellular environment. In this work, we focus on the delivery of the naturally-occurring hydrophobic photosensitizer Hypericin in human lung carcinoma A549 cells by using biodegradable poly L-lactic acid NPs. For the first time, Hypericin containing NPs are prepared by combining the miniemulsion technique with the solvent evaporation method. This approach yields an efficient loading of the NPs with Hypericin and allows for additional cargo molecules. To monitor the release of Hypercin from the NPs, an additional fluorescent lipophilic dye Coumarin-6 is incorporated in the NPs. Temporal and spatiotemporal image correlation spectroscopy is used to determine the fate of the NPs carrying the potential cargo. Both directed and non-directed motions are detected. By using image cross-correlation spectroscopy and specific fluorescent labeling of endosomes, lysosomes and mitochondria, the dynamics of the cargo loaded NPs in association with the organelles is studied. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of essential oil distribution in the root cross-section of Valeriana officinalis L. s.l. by using histological imaging techniques.

PubMed

Penzkofer, Michael; Baron, Andrea; Naumann, Annette; Krähmer, Andrea; Schulz, Hartwig; Heuberger, Heidi

2018-01-01

The essential oil is an important compound of the root and rhizome of medicinally used valerian ( Valeriana officinalis L. s.l.), with a stated minimum content in the European pharmacopoeia. The essential oil is located in droplets, of which the position and distribution in the total root cross-section of different valerian varieties, root thicknesses and root horizons are determined in this study using an adapted fluorescence-microscopy and automatic imaging analysis method. The study was initiated by the following facts:A probable negative correlation between essential oil content and root thickness in selected single plants (elites), observed during the breeding of coarsely rooted valerian with high oil content.Higher essential oil content after careful hand-harvest and processing of the roots. In preliminary tests, the existence of oil containing droplets in the outer and inner regions of the valerian roots was confirmed by histological techniques and light-microscopy, as well as Fourier-transform infrared spectroscopy. Based on this, fluorescence-microscopy followed by image analysis of entire root cross-sections, showed that a large number of oil droplets (on average 43% of total oil droplets) are located close to the root surface. The remaining oil droplets are located in the inner regions (parenchyma) and showed varying density gradients from the inner to the outer regions depending on genotype, root thickness and harvesting depth. Fluorescence-microscopy is suitable to evaluate prevalence and distribution of essential oil droplets of valerian in entire root cross-sections. The oil droplet density gradient varies among genotypes. Genotypes with a linear rather than an exponential increase of oil droplet density from the inner to the outer parenchyma can be chosen for better stability during post-harvest processing. The negative correlation of essential oil content and root thickness as observed in our breeding material can be counteracted through a selection towards generally high oil droplet density levels, and large oil droplet sizes independent of root thickness.

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

NASA Astrophysics Data System (ADS)

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

2009-08-01

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

Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

NASA Astrophysics Data System (ADS)

Swift, Kerry M.; Matayoshi, Edmund D.

2008-02-01

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

Confocal microscopy evaluation of stromal fluorescence intensity after standard and accelerated iontophoresis-assisted corneal cross-linking.

PubMed

Lanzini, Manuela; Curcio, Claudia; Spoerl, Eberhard; Calienno, Roberta; Mastropasqua, Alessandra; Colasante, Martina; Mastropasqua, Rodolfo; Nubile, Mario; Mastropasqua, Leonardo

2017-02-01

The aim of this study is to determine modifications in stromal fluorescence intensity after different corneal cross-linking (CXL) procedures and to correlate stromal fluorescence to corneal biomechanical resistance. For confocal microscopy study, 15 human cadaver corneas were examined. Three served as control (group 1), three were just soaked with iontophoresis procedure (group 2), three were treated with standard epi-off technique (group 3), and six underwent iontophoresis imbibition. Three of later six were irradiated for 30 min with 3 mW/cm 2 UVA (group 4) and three for 9 min at 10 mW/cm 2 UVA (group 5). Confocal microscopy was performed to quantify the fluorescence intensity in the cornea at different stromal depths. For biomechanical study, 30 human cadaver corneas were randomly divided into five groups and treated as previously described. Static stress-strain measurements of the corneas were performed. Iontophoresis imbibition followed by 10mW/cm 2 irradiation proved to increase stromal fluorescence into the corneal stroma and significant differences were revealed between group 3 and 5 both at 100 (p = 0.0171) and 250 µm (p = 0.0024), respectively. Biomechanical analysis showed an improvement of corneal resistance in group 5. Iontophoresis imbibition followed by accelerated irradiation increased the stromal fluorescence and is related to an improvement of biomechanical resistance. This approach may represent a new strategy to achieve greater concentrations of riboflavin without removing corneal epithelium and improve clinical results while reducing the side effects of CXL.

Particle formation in the emulsion-solvent evaporation process.

PubMed

Staff, Roland H; Schaeffel, David; Turshatov, Andrey; Donadio, Davide; Butt, Hans-Jürgen; Landfester, Katharina; Koynov, Kaloian; Crespy, Daniel

2013-10-25

The mechanism of particle formation from submicrometer emulsion droplets by solvent evaporation is revisited. A combination of dynamic light scattering, fluorescence resonance energy transfer, zeta potential measurements, and fluorescence cross-correlation spectroscopy is used to analyze the colloids during the evaporation process. It is shown that a combination of different methods yields reliable and quantitative data for describing the fate of the droplets during the process. The results indicate that coalescence plays a minor role during the process; the relatively large size distribution of the obtained polymer colloids can be explained by the droplet distribution after their formation. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Degradable cationic nanohydrogel particles for stimuli-responsive release of siRNA.

PubMed

Nuhn, Lutz; Braun, Lydia; Overhoff, Iris; Kelsch, Annette; Schaeffel, David; Koynov, Kaloian; Zentel, Rudolf

2014-12-01

Well-defined nanogels have become quite attractive as safe and stable carriers for siRNA delivery. However, to avoid nanoparticle accumulation, they need to provide a stimuli-responsive degradation mechanism that can be activated at the payload's site of action. In this work, the synthetic concept for generating well-defined nanohydrogel particles is extended to incorporate disulfide cross-linkers into a cationic nanonetwork for redox-triggered release of oligonucleotide payload as well as nanoparticle degradation under reductive conditions of the cytoplasm. Therefore, a novel disulfide-modified spermine cross-linker is designed that both allows disassembly of the nanogel as well as removal of cationic charge from residual polymer fragments. The degradation process is monitored by scanning electron microscopy (SEM) and fluorescence correlation spectroscopy (FCS). Moreover, siRNA release is analyzed by agarose gel electrophoresis and a fluorescent RNA detection assay. The results exemplify the versatility of the applied nanogel manufacturing process, which allows alternative stimuli-responsive core cross-linkers to be integrated for triggered oligonucleotide release as well as effective biodegradation for reduced nanotoxicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermally activated delayed fluorescence of fluorescein derivative for time-resolved and confocal fluorescence imaging.

PubMed

Xiong, Xiaoqing; Song, Fengling; Wang, Jingyun; Zhang, Yukang; Xue, Yingying; Sun, Liangliang; Jiang, Na; Gao, Pan; Tian, Lu; Peng, Xiaojun

2014-07-09

Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

Fluorescence correlation spectroscopy directly monitors coalescence during nanoparticle preparation.

PubMed

Schaeffel, David; Staff, Roland Hinrich; Butt, Hans-Juergen; Landfester, Katharina; Crespy, Daniel; Koynov, Kaloian

2012-11-14

Dual color fluorescence cross-correlation spectroscopy (DC FCCS) experiments were conducted to study the coalescence and aggregation during the formation of nanoparticles. To assess the generality of the method, three completely different processes were selected to prepare the nanoparticles. Polymeric nanoparticles were formed either by solvent evaporation from emulsion nanodroplets of polymer solutions or by miniemulsion polymerization. Inorganic nanocapsules were formed by polycondensation of alkoxysilanes at the interface of nanodroplets. In all cases, DC FCCS provided fast and unambiguous information about the occurrence of coalescence and thus a deeper insight into the mechanism of nanoparticle formation. In particular, it was found that coalescence played a minor role for the emulsion-solvent evaporation process and the miniemulsion polymerization, whereas substantial coalescence was detected during the formation of the inorganic nanocapsules. These findings demonstrate that DC FCCS is a powerful tool for monitoring nanoparticles genesis.

Correlation Functions Quantify Super-Resolution Images and Estimate Apparent Clustering Due to Over-Counting

PubMed Central

Veatch, Sarah L.; Machta, Benjamin B.; Shelby, Sarah A.; Chiang, Ethan N.; Holowka, David A.; Baird, Barbara A.

2012-01-01

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins. PMID:22384026

Determination of rice syrup adulterant concentration in honey using three-dimensional fluorescence spectra and multivariate calibrations

NASA Astrophysics Data System (ADS)

Chen, Quansheng; Qi, Shuai; Li, Huanhuan; Han, Xiaoyan; Ouyang, Qin; Zhao, Jiewen

2014-10-01

To rapidly and efficiently detect the presence of adulterants in honey, three-dimensional fluorescence spectroscopy (3DFS) technique was employed with the help of multivariate calibration. The data of 3D fluorescence spectra were compressed using characteristic extraction and the principal component analysis (PCA). Then, partial least squares (PLS) and back propagation neural network (BP-ANN) algorithms were used for modeling. The model was optimized by cross validation, and its performance was evaluated according to root mean square error of prediction (RMSEP) and correlation coefficient (R) in prediction set. The results showed that BP-ANN model was superior to PLS models, and the optimum prediction results of the mixed group (sunflower ± longan ± buckwheat ± rape) model were achieved as follow: RMSEP = 0.0235 and R = 0.9787 in the prediction set. The study demonstrated that the 3D fluorescence spectroscopy technique combined with multivariate calibration has high potential in rapid, nondestructive, and accurate quantitative analysis of honey adulteration.

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

PubMed Central

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

2012-01-01

The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods. PMID:22869699

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

PubMed

Molteni, Matteo; Ferri, Fabio

2016-11-01

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

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

NASA Astrophysics Data System (ADS)

Molteni, Matteo; Ferri, Fabio

2016-11-01

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

pH-Mediated Fluorescent Polymer Particles and Gel from Hyperbranched Polyethylenimine and the Mechanism of Intrinsic Fluorescence.

PubMed

Liu, Shi Gang; Li, Na; Ling, Yu; Kang, Bei Hua; Geng, Shuo; Li, Nian Bing; Luo, Hong Qun

2016-02-23

We report that fluorescence properties and morphology of hyperbranched polyethylenimine (hPEI) cross-linked with formaldehyde are highly dependent on the pH values of the cross-linking reaction. Under acidic and neutral conditions, water-soluble fluorescent copolymer particles (CPs) were produced. However, under basic conditions, white gels with weak fluorescence emission would be obtained. The water-soluble hPEI-formaldehyde (hPEI-F) CPs show strong intrinsic fluorescence without the conjugation to any classical fluorescent agents. By the combination of spectroscopy and microscopy techniques, the mechanism of fluorescence emission was discussed. We propose that the intrinsic fluorescence originates from the formation of a Schiff base in the cross-linking process between hPEI and formaldehyde. Schiff base bonds are the fluorescence-emitting moieties, and the compact structure of hPEI-F CPs plays an important role in their strong fluorescence emission. The exploration on fluorescence mechanism may provide a new strategy to prepare fluorescent polymer particles. In addition, the investigation shows that the hPEI-F CPs hold potential as a fluorescent probe for the detection of copper ions in aqueous media.

HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

A portable fluorescence detector for fast ultra trace detection of explosive vapors

NASA Astrophysics Data System (ADS)

Xin, Yunhong; He, Gang; Wang, Qi; Fang, Yu

2011-10-01

This paper developed a portable detector based on a specific material-based fluorescent sensing film for an ultra trace detection of explosives, such as 2,4,6-trinitrotoluene (TNT) or its derivate 2,4-dinitrotoluene (DNT), in ambient air or on objects tainted by explosives. The fluorescent sensing films are based on single-layer chemistry and the signal amplification effect of conjugated polymers, which exhibited higher sensitivity and shorter response time to TNT or DNT at their vapor pressures. Due to application of the light emitting diode and the solid state photomultiplier and the cross-correlation-based circuit design technology, the device has the advantages of low-power, low-cost, small size, and an improved signal to noise ratio. The results of the experiments showed that the detector can real-time detect and identify of explosive vapors at extremely low levels; it is suitable for the identification of suspect luggage, forensic analyses, or battlefields clearing.

A portable fluorescence detector for fast ultra trace detection of explosive vapors.

PubMed

Xin, Yunhong; He, Gang; Wang, Qi; Fang, Yu

2011-10-01

This paper developed a portable detector based on a specific material-based fluorescent sensing film for an ultra trace detection of explosives, such as 2,4,6-trinitrotoluene (TNT) or its derivate 2,4-dinitrotoluene (DNT), in ambient air or on objects tainted by explosives. The fluorescent sensing films are based on single-layer chemistry and the signal amplification effect of conjugated polymers, which exhibited higher sensitivity and shorter response time to TNT or DNT at their vapor pressures. Due to application of the light emitting diode and the solid state photomultiplier and the cross-correlation-based circuit design technology, the device has the advantages of low-power, low-cost, small size, and an improved signal to noise ratio. The results of the experiments showed that the detector can real-time detect and identify of explosive vapors at extremely low levels; it is suitable for the identification of suspect luggage, forensic analyses, or battlefields clearing.

X-ray fluorescence cross sections for K and L x rays of the elements

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

Krause, M.O.; Nestor, C.W. Jr.; Sparks, C.J. Jr.

1978-06-01

X-ray fluorescence cross sections are calculated for the major x rays of the K series 5 less than or equal to Z less than or equal to 101, and the three L series 12 less than or equal to Z less than or equal to 101 in the energy range 1 to 200 keV. This calculation uses Scofield's theoretical partical photoionization cross sections, Krause's evaluation of fluorescence and Coster-Kronig yields, and Scofield's theoretical radiative rates. Values are presented in table and graph format, and an estimate of their accuracy is made. The following x rays are considered: K..cap alpha../sub 1/,more » K..cap alpha../sub 1/,/sub 2/, K..beta../sub 1/, K..beta../sub 1/,/sub 3/, L..cap alpha../sub 1/, L..cap alpha../sub 1/,/sub 2/, L..beta../sub 1/, L..beta../sub 2/,/sub 15/, L..beta../sub 3/, Ll, L..gamma../sub 1/, L..gamma../sub 4/, and L/sub 1/ ..-->.. L/sub 2/,/sub 3/. For use in x-ray fluorescence analysis, K..cap alpha.. and L..cap alpha.. fluorescence cross sections are presented at specific energies: TiK identical with 4.55 keV, CrK identical with 5.46 keV, CoK identical with 7.00 keV, CuK identical with 8.13 keV, MoK..cap alpha.. identical with 17.44 keV, AgK identical with 22.5 keV, DyK identical with 47.0 keV, and /sup 241/Am identical with 59.54 keV. Supplementary material includes fluorescence and Coster--Kronig yields, fractional radiative rates, fractional fluorescence yields, total L-shell fluorescence cross sections, fluorescence and Coster-Kronig yields in condensed matter, effective fluorescence yields, average L-shell fluorescence yield, L-subshell photoionization cross section ratios, and conversion factors from barns per atom to square centimeters per gram.« less

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

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

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

Mitigating fluorescence spectral overlap in wide-field endoscopic imaging

PubMed Central

Hou, Vivian; Nelson, Leonard Y.; Seibel, Eric J.

2013-01-01

Abstract. The number of molecular species suitable for multispectral fluorescence imaging is limited due to the overlap of the emission spectra of indicator fluorophores, e.g., dyes and nanoparticles. To remove fluorophore emission cross-talk in wide-field multispectral fluorescence molecular imaging, we evaluate three different solutions: (1) image stitching, (2) concurrent imaging with cross-talk ratio subtraction algorithm, and (3) frame-sequential imaging. A phantom with fluorophore emission cross-talk is fabricated, and a 1.2-mm ultrathin scanning fiber endoscope (SFE) is used to test and compare these approaches. Results show that fluorophore emission cross-talk could be successfully avoided or significantly reduced. Near term, the concurrent imaging method of wide-field multispectral fluorescence SFE is viable for early stage cancer detection and localization in vivo. Furthermore, a means to enhance exogenous fluorescence target-to-background ratio by the reduction of tissue autofluorescence background is demonstrated. PMID:23966226

Development and evaluation of a novel high-throughput image-based fluorescent neutralization test for detection of Zika virus infection.

PubMed

Koishi, Andrea Cristine; Suzukawa, Andréia Akemi; Zanluca, Camila; Camacho, Daria Elena; Comach, Guillermo; Duarte Dos Santos, Claudia Nunes

2018-03-01

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus flavivirus that comprises other important public health viruses, such as dengue (DENV) and yellow fever (YFV). In general, ZIKV infection is a self-limiting disease, however cases of Guillain-Barré syndrome and congenital brain abnormalities in newborn infants have been reported. Diagnosing ZIKV infection remains a challenge, as viral RNA detection is only applicable until a few days after the onset of symptoms. After that, serological tests must be applied, and, as expected, high cross-reactivity between ZIKV and other flavivirus serology is observed. Plaque reduction neutralization test (PRNT) is indicated to confirm positive samples for being more specific, however it is laborious intensive and time consuming, representing a major bottleneck for patient diagnosis. To overcome this limitation, we developed a high-throughput image-based fluorescent neutralization test for ZIKV infection by serological detection. Using 226 human specimens, we showed that the new test presented higher throughput than traditional PRNT, maintaining the correlation between results. Furthermore, when tested with dengue virus samples, it showed 50.53% less cross reactivity than MAC-ELISA. This fluorescent neutralization test could be used for clinical diagnosis confirmation of ZIKV infection, as well as for vaccine clinical trials and seroprevalence studies.

Carbon dioxide UV laser-induced fluorescence in high-pressure flames

NASA Astrophysics Data System (ADS)

Bessler, W. G.; Schulz, C.; Lee, T.; Jeffries, J. B.; Hanson, R. K.

2003-07-01

Laser-induced fluorescence (LIF) of carbon dioxide is investigated with excitation between 215 and 255 nm with spectrally resolved detection in 5-40 bar premixed CH 4/O 2/Ar and CH 4/air flat-flames at fuel/air ratios between 0.8 and 1.9. The LIF signal consists of a broad (200-450 nm) continuum with a faint superimposed structure, and this signal is absent in similar H 2/O 2/Ar flames. There is strong evidence this signal arises from CO 2, as the signal variations with excitation wavelength, equivalence ratio and flame temperature all correlate with CO 2 absorption cross-sections. The signal is linear with pressure and laser fluence within the investigated ranges.

Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy.

PubMed

Vasconcelos, Luís; Lehto, Tõnis; Madani, Fatemeh; Radoi, Vlad; Hällbrink, Mattias; Vukojević, Vladana; Langel, Ülo

2018-02-01

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

A Multi-Functional Imaging Approach to High-Content Protein Interaction Screening

PubMed Central

Matthews, Daniel R.; Fruhwirth, Gilbert O.; Weitsman, Gregory; Carlin, Leo M.; Ofo, Enyinnaya; Keppler, Melanie; Barber, Paul R.; Tullis, Iain D. C.; Vojnovic, Borivoj; Ng, Tony; Ameer-Beg, Simon M.

2012-01-01

Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging. PMID:22506000

Search for Cross-Correlations of Ultrahigh-Energy Cosmic Rays with BL Lacertae Objects

NASA Astrophysics Data System (ADS)

Abbasi, R. U.; Abu-Zayyad, T.; Amann, J. F.; Archbold, G.; Belov, K.; Belz, J. W.; BenZvi, S.; Bergman, D. R.; Blake, S. A.; Boyer, J. H.; Burt, G. W.; Cao, Z.; Connolly, B. M.; Deng, W.; Fedorova, Y.; Findlay, J.; Finley, C. B.; Hanlon, W. F.; Hoffman, C. M.; Holzscheiter, M. H.; Hughes, G. A.; Hüntemeyer, P.; Jui, C. C. H.; Kim, K.; Kirn, M. A.; Knapp, B. C.; Loh, E. C.; Maestas, M. M.; Manago, N.; Mannel, E. J.; Marek, L. J.; Martens, K.; Matthews, J. A. J.; Matthews, J. N.; O'Neill, A.; Painter, C. A.; Perera, L.; Reil, K.; Riehle, R.; Roberts, M. D.; Rodriguez, D.; Sasaki, M.; Schnetzer, S. R.; Seman, M.; Sinnis, G.; Smith, J. D.; Snow, R.; Sokolsky, P.; Springer, R. W.; Stokes, B. T.; Thomas, J. R.; Thomas, S. B.; Thomson, G. B.; Tupa, D.; Westerhoff, S.; Wiencke, L. R.; Zech, A.; HIRES Collaboration

2006-01-01

Data taken in stereo mode by the High Resolution Fly's Eye (HiRes) air fluorescence experiment are analyzed to search for correlations between the arrival directions of ultrahigh-energy cosmic rays with the positions of BL Lacertae objects. Several previous claims of significant correlations between BL Lac objects and cosmic rays observed by other experiments are tested. These claims are not supported by the HiRes data. However, we verify a recent analysis of correlations between HiRes events and a subset of confirmed BL Lac objects from the 10th Veron Catalog, and we study this correlation in detail. Due to the a posteriori nature of the search, the significance level cannot be reliably estimated and the correlation must be tested independently before any claim can be made. We identify the precise hypotheses that will be tested with statistically independent data.

Hydrodynamic boundary condition of water on hydrophobic surfaces.

PubMed

Schaeffel, David; Yordanov, Stoyan; Schmelzeisen, Marcus; Yamamoto, Tetsuya; Kappl, Michael; Schmitz, Roman; Dünweg, Burkhard; Butt, Hans-Jürgen; Koynov, Kaloian

2013-05-01

By combining total internal reflection fluorescence cross-correlation spectroscopy with Brownian dynamics simulations, we were able to measure the hydrodynamic boundary condition of water flowing over a smooth solid surface with exceptional accuracy. We analyzed the flow of aqueous electrolytes over glass coated with a layer of poly(dimethylsiloxane) (advancing contact angle Θ = 108°) or perfluorosilane (Θ = 113°). Within an error of better than 10 nm the slip length was indistinguishable from zero on all surfaces.

Fluorescence Cross-Correlation Spectroscopy Reveals Mechanistic Insights into the Effect of 2′-O-Methyl Modified siRNAs in Living Cells

PubMed Central

Ohrt, Thomas; Staroske, Wolfgang; Mütze, Jörg; Crell, Karin; Landthaler, Markus; Schwille, Petra

2011-01-01

RNA interference (RNAi) offers a powerful tool to specifically direct the degradation of complementary RNAs, and thus has great therapeutic potential for targeting diseases. Despite the reported preferences of RNAi, there is still a need for new techniques that will allow for a detailed mechanistic characterization of RNA-induced silencing complex (RISC) assembly and activity to further improve the biocompatibility of modified siRNAs. In contrast to previous reports, we investigated the effects of 2′-O-methyl (2′OMe) modifications introduced at specific positions within the siRNA at the early and late stages of RISC assembly, as well as their influence on target recognition and cleavage directly in living cells. We found that six to 10 2′OMe nucleotides on the 3′-end inhibit passenger-strand release as well as target-RNA cleavage without changing the affinity, strand asymmetry, or target recognition. 2′OMe modifications introduced at the 5′-end reduced activated RISC stability, whereas incorporations at the cleavage site showed only minor effects on passenger-strand release when present on the passenger strand. Our new fluorescence cross-correlation spectroscopy assays resolve different steps and stages of RISC assembly and target recognition with heretofore unresolved detail in living cells, which is needed to develop therapeutic siRNAs with optimized in vivo properties. PMID:21689532

Antiphase dual-color correlation in a reactant-product pair imparts ultrasensitivity in reaction-linked double-photoswitching fluorescence imaging.

PubMed

Wan, Wei; Zhu, Ming-Qiang; Tian, Zhiyuan; Li, Alexander D Q

2015-04-08

A pair of reversible photochemical reactions correlates their reactant and product specifically, and such a correlation uniquely distinguishes their correlated signal from others that are not linked by this reversible reaction. Here a nanoparticle-shielded fluorophore is photodriven to undergo structural dynamics, alternating between a green-fluorescence state and a red-fluorescence state. As time elapses, the fluorophore can be in either state but not both at the same time. Thus, the red fluorescence is maximized while the green fluorescence is minimized and vice versa. Such an antiphase dual-color (AD) corelationship between the red and green fluorescence maxima as well as between their minima can be exploited to greatly improve the signal-to-noise ratio, thus enhancing the ultimate detection limit. Potential benefits of this correlation include elimination of all interferences originating from single-color dyes and signal amplification of AD photoswitching molecules by orders of magnitude.

Effectiveness of fluorescence-based methods to detect in situ demineralization and remineralization on smooth surfaces.

PubMed

Moriyama, C M; Rodrigues, J A; Lussi, A; Diniz, M B

2014-01-01

This study aimed to evaluate the effectiveness of fluorescence-based methods (DIAGNOdent, LF; DIAGNOdent pen, LFpen, and VistaProof fluorescence camera, FC) in detecting demineralization and remineralization on smooth surfaces in situ. Ten volunteers wore acrylic palatal appliances, each containing 6 enamel blocks that were demineralized for 14 days by exposure to a 20% sucrose solution and 3 of them were remineralized for 7 days with fluoride dentifrice. Sixty enamel blocks were evaluated at baseline, after demineralization and 30 blocks after remineralization by two examiners using LF, LFpen and FC. They were submitted to surface microhardness (SMH) and cross-sectional microhardness analysis. The integrated loss of surface hardness (ΔKHN) was calculated. The intraclass correlation coefficient for interexaminer reproducibility ranged from 0.21 (FC) to 0.86 (LFpen). SMH, LF and LFpen values presented significant differences among the three phases. However, FC fluorescence values showed no significant differences between the demineralization and remineralization phases. Fluorescence values for baseline, demineralized and remineralized enamel were, respectively, 5.4 ± 1.0, 9.2 ± 2.2 and 7.0 ± 1.5 for LF; 10.5 ± 2.0, 15.0 ± 3.2 and 12.5 ± 2.9 for LFpen, and 1.0 ± 0.0, 1.0 ± 0.1 and 1.0 ± 0.1 for FC. SMH and ΔKHN showed significant differences between demineralization and remineralization phases. There was a negative and significant correlation between SMH and LF and LFpen in the remineralization phase. In conclusion, LF and LFpen devices were effective in detecting demineralization and remineralization on smooth surfaces provoked in situ.

Multichannel processes of H2O in the 18 eV region

NASA Technical Reports Server (NTRS)

Wu, C. Y. Robert; Judge, D. L.

1988-01-01

Measurements were made of: (1) the fluorescence cross sections of OH(A 2Sigma+) fragments; (2) the absolute cross sections producing H atoms in the n = 2, 3, and 4 states; (3) the cross section for producing excited O atoms which has an upper limit of 5 x 10 to the -21 sq cm; and (4) the fluorescence cross section for producing H2(a 3Sigma g +) fragments. It is shown that, in the 16-20 eV region, there are excellent correspondences in the peak positions and spacings among the photoabsorption, photoionization spectra, and fluorescence functions of OH(A) and H(n).

Quantitative photoabsorption and fluorescence spectroscopy of benzene, naphthalene, and some derivatives at 106-295 nm

NASA Technical Reports Server (NTRS)

Suto, Masako; Wang, Xiuyan; Shan, Jun; Lee, L. C.

1992-01-01

Photoabsorption and fluorescence cross sections of benzene, (o-, m-, p-) xylenes, naphthalene, 1-methylnaphthalene, and 2-ethylnaphthalene in the gas phase are measured at 106-295 nm using synchrotron radiation as a light source. Fluorescences are observed from the photoexcitation of benzene and xylenes at 230-280 nm and from naphthalene and its derivatives at 190-295 nm. The absolute fluorescence cross section is determined by calibration with respect to the emission intensity of the NO(A-X) system, for which the fluorescence quantum yield is equal to 1. To cross-check the current calibration method, the quantum yield of the SO2(C-X) system at 220-230 nm was measured since it is about equal to 1. The current quantum-yield data are compared with previously published values measured by different methods.

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.

In situ phytoplankton absorption, fluorescence emission, and particulate backscattering spectra determined from reflectance

NASA Technical Reports Server (NTRS)

Roesler, Collin S.; Pery, Mary Jane

1995-01-01

An inverse model was developed to extract the absortion and scattering (elastic and inelastic) properties of oceanic constituents from surface spectral reflectance measurements. In particular, phytoplankton spectral absorption coefficients, solar-stimulated chlorophyll a fluorescence spectra, and particle backscattering spectra were modeled. The model was tested on 35 reflectance spectra obtained from irradiance measurements in optically diverse ocean waters (0.07 to 25.35 mg/cu m range in surface chlorophyll a concentrations). The universality of the model was demonstrated by the accurate estimation of the spectral phytoplankton absorption coefficents over a range of 3 orders of magnitude (rho = 0.94 at 500 nm). Under most oceanic conditions (chlorophyll a less than 3 mg/cu m) the percent difference between measured and modeled phytoplankton absorption coefficents was less than 35%. Spectral variations in measured phytoplankton absorption spectra were well predicted by the inverse model. Modeled volume fluorescence was weakly correlated with measured chl a; fluorescence quantum yield varied from 0.008 to 0.09 as a function of environment and incident irradiance. Modeled particle backscattering coefficients were linearly related to total particle cross section over a twentyfold range in backscattering coefficents (rho = 0.996, n = 12).

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.

An Evaluation of Lysyl Oxidase–Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry

PubMed Central

Takaoka, Anna; Babar, Natasha; Hogan, Julia; Kim, MiJung; Price, Marianne O.; Price, Francis W.; Trokel, Stephen L.; Paik, David C.

2016-01-01

Purpose Current literature contains scant information regarding the extent of enzymatic collagen cross-linking in the keratoconus (KC) cornea. The aim of the present study was to examine levels of enzymatic lysyl oxidase–derived cross-links in stromal collagen in KC tissue, and to correlate the cross-link levels with collagen fibril stability as determined by thermal denaturation temperature (Tm). Methods Surgical KC samples (n = 17) and Eye-Bank control (n = 11) corneas of age 18 to 68 years were analyzed. The samples were defatted, reduced (NaBH4), hydrolyzed (6N HCl at 110°C for 18 hours), and cellulose enriched before analysis by C8 high-performance liquid chromatography equipped with parallel fluorescent and mass detectors in selective ion monitoring mode (20 mM heptafluorobutyric acid/methanol 70:30 isocratic at 1 mL/min). Nine different cross-links were measured, and the cross-link density was determined relative to collagen content (determined colorimetrically). The Tm was determined by differential scanning calorimetry. Results Cross-links detected were dihydroxylysinonorleucine (DHLNL), hydroxylysinonorleucine, lysinonorleucine (LNL), and histidinohydroxylysinonorleucine in both control and KC samples. Higher DHLNL levels were detected in KC, whereas the dominant cross-link, LNL, was decreased in KC samples. Decreased LNL levels were observed among KC ≤ 40 corneas. There was no difference in total cross-link density between KC samples and the controls. Pyridinolines, desmosines, and pentosidine were not detected. There was no notable correlation between cross-link levels with fibril instability as determined by Tm. Conclusions Lower levels of LNL in the KC cornea suggest that there might be a cross-linking defect either in fibrillar collagen or the microfibrillar elastic network composed of fibrillin. PMID:26780316

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

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

Tiwari, Manisha; Mikuni, Shintaro; Muto, Hideki

Highlights: •We used two-laser-beam FCCS to determine the dissociation constant (K{sub d}) of IPT domain of p50/p65 heterodimer in living cell. •Interaction of p50 and p65 was analyzed in the cytoplasm and nucleus of single living cell. •Binding affinity of p50/p65 heterodimer is higher in cytoplasm than that of nucleus. -- Abstract: Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K{sub d}, for the heterodimer inmore » living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K{sub d} values of mCherry{sub 2}- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.« less

Fluorescence correlation spectroscopy of diffusion probed with a Gaussian Lorentzian spatial distribution

NASA Astrophysics Data System (ADS)

Marrocco, Michele

2007-11-01

Fluorescence correlation spectroscopy is fundamental in many physical, chemical and biological studies of molecular diffusion. However, the concept of fluorescence correlation is founded on the assumption that the analytical description of the correlation decay of diffusion can be achieved if the spatial profile of the detected volume obeys a three-dimensional Gaussian distribution. In the present Letter, the analytical result is instead proven for the fundamental Gaussian-Lorentzian profile.

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

PubMed

Yang, Yang; Qin, Xiaodong; Sun, Yingjun; Chen, Ting; Zhang, Zhidong

2016-12-01

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

Monitoring the Behavior of Emerging Contaminants in Wastewater-Impacted Rivers Based on the Use of Fluorescence Excitation Emission Matrixes (EEM).

PubMed

Sgroi, Massimiliano; Roccaro, Paolo; Korshin, Gregory V; Vagliasindi, Federico G A

2017-04-18

This study investigated the applicability of fluorescence indexes based on the interpretation of excitation emission matrices (EEMs) by PARAFAC analysis and by selecting fluorescence intensities at a priori defined excitation/emission pairs as surrogates for monitoring the behavior of emerging organic compounds (EOCs) in two catchment basins impacted by wastewater discharges. Relevant EOC and EEM data were obtained for a 90 km stretch of the Simeto River, the main river in Sicily, and the smaller San Leonardo River, which was investigated for a 17 km stretch. The use of fluorescence indexes developed by these two different approaches resulted in similar observations. Changes of the fluorescence indexes that correspond to a group of humic-like fluorescing species were determined to be highly correlated with the concentrations of recalcitrant contaminants such as sucralose, sulfamethoxazole and carbamazepine, which are typical wastewater markers in river water. Changes of the fluorescence indexes related to tyrosine-like substances were well correlated with the concentrations of ibuprofen and caffeine, anthropogenic indicators of untreated wastewater discharges. Chemical oxygen demand and dissolved organic carbon concentrations were correlated with humic-like fluorescence indexes. The observed correlations were site-specific and characterized by different regression parameters for every collection event. Caffeine and carbamazepine showed correlations with florescence indexes in the San Leonardo River and in the alluvial plain stretch of the Simeto River, whereas sucralose, sulfamethoxazole and ibuprofen have always been well correlated in all the investigated river stretches. However, when data of different collection events from river stretches where correlations were observed were combined, good linear correlations were obtained for data sets generated via the normalization of the measured concentrations by the average value for the corresponding collection event. These results show that fluorescence based indexes can be used to monitor the behavior of some trace organic contaminants in wastewater impacted rivers and to track wastewater discharges in streams and rivers.

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

Correlation fluorescence method of amine detection

NASA Astrophysics Data System (ADS)

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

1997-12-01

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

Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

PubMed

Richard, S; Tamas, C; Sell, D R; Monnier, V M

1991-08-01

Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

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

PubMed Central

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

2009-01-01

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

Laboratory tank studies of a single species of phytoplankton using a remote sensing fluorosensor

NASA Technical Reports Server (NTRS)

Brown, C. A., Jr.; Jarrett, O., Jr.; Farmer, F. H.

1981-01-01

Phytoplankton were grown in the laboratory for the purpose of testing a remote fluorosensor. The fluorosensor uses a unique four-wavelength dye laser system to excite phytoplankton bearing chlorophyll and to measure the chlorophyll fluorescence generated by this excitation. Six different species were tested, one at a time, and each was grown two to four times. Fluorescence measured by the fluorosensor provides good quantitative measurement of chlorophyll concentrations for all species tested while the cultures were in log phase growth. Fluorescene cross section ratios obtained in the single species tank tests support the hypothesis that the shape of the fluorescence cross section curve remains constant with the species (differences in fluorescence cross section ratios are a basis for determining composition of phytoplankton according to color group when a multiwavelength source of excitation is used. Linear relationships exist between extracted chlorophyll concentration and fluorescence measured by the remote fluorosensor during the log phase growth of phytoplankton cultures tested.

M shell X-ray production cross sections and fluorescence yields for the elements with 71 <= Z <= 92 using 5.96 keV photons

NASA Astrophysics Data System (ADS)

Puri, S.; Mehta, D.; Chand, B.; Singh, Nirmal; Mangal, P. C.; Trehan, P. N.

1993-03-01

Total M X-ray production (XRP) cross sections for ten elements in the atomic number region 71 ≤ Z ≤ 92 were measured at 5.96 keV incident photon energy. The average M shell fluorescence yields < overlineωM> have also been computed using the present measured cross section values and the theoretical M shell photoionisation cross sections. The results are compared with theoretical values.

Signatures of Hong-Ou-Mandel interference at microwave frequencies

NASA Astrophysics Data System (ADS)

Woolley, M. J.; Lang, C.; Eichler, C.; Wallraff, A.; Blais, A.

2013-10-01

Two-photon quantum interference at a beam splitter, commonly known as Hong-Ou-Mandel interference, is a fundamental demonstration of the quantum mechanical nature of electromagnetic fields and a key component of various quantum information processing protocols. The phenomenon was recently demonstrated with microwave-frequency photons by Lang et al (2013 Nature Phys. 9 345-8). This experiment employed circuit QED systems as sources of microwave photons, and was based on the measurement of second-order cross-correlation and auto-correlation functions of the microwave fields at the outputs of the beam splitter using linear detectors. Here we present the calculation of these correlation functions for the cases of inputs corresponding to: (i) trains of pulsed Gaussian or Lorentzian single microwave photons and (ii) resonant fluorescent microwave fields from continuously driven circuit QED systems. In both cases, the signature of two-photon quantum interference is a suppression of the second-order cross-correlation function for small delays. The experiment described in Lang et al (2013) was performed with trains of Lorentzian single photons, and very good agreement with experimental data is obtained. The results are relevant not only to interference experiments using circuit QED systems, but any such setup with highly controllable sources and time-resolved detection.

L X-ray fluorescence cross sections of some rare earth elements ( Z = 62, 64, 66, 68 and 70) at 17.8, 22.6 and 25.8 keV

NASA Astrophysics Data System (ADS)

Mandal, A. C.; Santra, S.; Mitra, D.; Sarkar, M.; Bhattacharya, D.

2005-06-01

L X-ray fluorescence cross sections of the elements with Z = 62, 64, 66, 68 and 70 have been measured at 17.8, 22.6 and 25.8 keV using an X-ray tube and secondary exciters of Mo, Ag and Sn. The measured cross sections have been compared with the theoretical predictions and with the data of others. Theoretical values calculated using photoionisation cross sections from Scofield [Lawrence Livermore Laboratory, UCRL-51326, 1973], fluorescence yields and Coster-Kronig transition probabilities from Puri et al. [X-ray Spectrom. 22 (1993) 358] and radiative widths from Campbell and Wang [At. Data Nucl. Data Tables 43 (1989) 281] show good agreement with our data. Except two sets of data on Lγ cross sections, all the data of other groups agree well with those of ours.

Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections

NASA Astrophysics Data System (ADS)

DeArmond, Fredrick Michael

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

Fluorescence lifetime correlation spectroscopy for precise concentration detection in vivo by background subtraction

NASA Astrophysics Data System (ADS)

Gärtner, Maria; Mütze, Jörg; Ohrt, Thomas; Schwille, Petra

2009-07-01

In vivo studies of single molecule dynamics by means of Fluorescence correlation spectroscopy can suffer from high background. Fluorescence lifetime correlation spectroscopy provides a tool to distinguish between signal and unwanted contributions via lifetime separation. By studying the motion of the RNA-induced silencing complex (RISC) within two compartments of a human cell, the nucleus and the cytoplasm, we observed clear differences in concentration as well as mobility of the protein complex between those two locations. Especially in the nucleus, where the fluorescence signal is very weak, a correction for background is crucial to provide reliable results of the particle number. Utilizing the fluorescent lifetime of the different contributions, we show that it is possible to distinguish between the fluorescent signal and the autofluorescent background in vivo in a single measurement.

A practical implementation of multi-frequency widefield frequency-domain FLIM

PubMed Central

Chen, Hongtao

2013-01-01

Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime, especially in kinetic studies in biomedical researches. However, the small range of modulation frequencies available in commercial instruments makes this technique limited in its applications. Here we describe a practical implementation of multi-frequency widefield FD-FLIM using a pulsed supercontinuum laser and a direct digital synthesizer. In this instrument we use a pulse to modulate the image intensifier rather than the more conventional sine wave modulation. This allows parallel multi-frequency FLIM measurement using the Fast Fourier Transform and the cross-correlation technique, which permits precise and simultaneous isolation of individual frequencies. In addition, the pulse modulation at the cathode of image intensifier restored the loss of optical resolution caused by the defocusing effect when the voltage at the cathode is sinusoidally modulated. Furthermore, in our implementation of this technique, data can be graphically analyzed by the phasor method while data are acquired, which allows easy fit-free lifetime analysis of FLIM images. Here our measurements of standard fluorescent samples and a Föster resonance energy transfer pair demonstrate that the widefield multi-frequency FLIM system is a valuable and simple tool in fluorescence imaging studies. PMID:23296945

Multiple Velocity Profile Measurements in Hypersonic Flows using Sequentially-Imaged Fluorescence Tagging

NASA Technical Reports Server (NTRS)

Bathel, Brett F.; Danehy, Paul M.; Inmian, Jennifer A.; Jones, Stephen B.; Ivey, Christopher B.; Goyne, Christopher P.

2010-01-01

Nitric-oxide planar laser-induced fluorescence (NO PLIF) was used to perform velocity measurements in hypersonic flows by generating multiple tagged lines which fluoresce as they convect downstream. For each laser pulse, a single interline, progressive scan intensified CCD camera was used to obtain separate images of the initial undelayed and delayed NO molecules that had been tagged by the laser. The CCD configuration allowed for sub-microsecond acquisition of both images, resulting in sub-microsecond temporal resolution as well as sub-mm spatial resolution (0.5-mm x 0.7-mm). Determination of axial velocity was made by application of a cross-correlation analysis of the horizontal shift of individual tagged lines. Quantification of systematic errors, the contribution of gating/exposure duration errors, and influence of collision rate on fluorescence to temporal uncertainty were made. Quantification of the spatial uncertainty depended upon the analysis technique and signal-to-noise of the acquired profiles. This investigation focused on two hypersonic flow experiments: (1) a reaction control system (RCS) jet on an Orion Crew Exploration Vehicle (CEV) wind tunnel model and (2) a 10-degree half-angle wedge containing a 2-mm tall, 4-mm wide cylindrical boundary layer trip. The experiments were performed at the NASA Langley Research Center's 31-inch Mach 10 wind tunnel.

Albumin-stabilized fluorescent silver nanodots

NASA Astrophysics Data System (ADS)

Sych, Tomash; Polyanichko, Alexander; Kononov, Alexei

2017-07-01

Ligand-stabilized Ag nanoclusters (NCs) possess many attractive features including high fluorescence quantum yield, large absorption cross-section, good photostability, large Stokes shift and two-photon absorption cross sections. While plenty of fluorescent clusters have been synthesized on various polymer templates, only a few studies have been reported on the fluorescent Ag clusters on peptides and proteins. We study silver NCs synthesized on different protein matrices, including bovine serum albumin, human serum albumin, egg albumin, equine serum albumin, and lysozyme. Our results show that red-emitting Ag NCs can effectively be stabilized by the disulfide bonds in proteins and that the looser structure of the denatured protein favors formation of the clusters.

The repeatability of three diagnostic methods (visual using ICDAS II, laser fluorescence, and radiographic) for early caries detection

NASA Astrophysics Data System (ADS)

Sukmasari, S.; Lestari, W.; Ko, B. B.; Noh, Z.; Asmail, N.; Yaacob, N.

2017-08-01

Newly introduced ICDAS II as a visual method, laser fluorescence as another technique that have ability to quantify early mineral loss of tooth structure and intra oral radiograph, are methods can be used in the clinic. To provide standardization for comprehensive caries management at an early stage, all methods supposed to be tested between users. The objective of this research is to evaluate the repeatability of each system. It is a comparative cross sectional study using 100 extracted permanent teeth without obvious cavitation (premolar & molar) that were collected and stored in thymol solution. The teeth were embedded on the wax block and labeled with numbers. All 5 surfaces were examined by 5 examiners using visual (ICDAS II), laser fluorescence (LF) and radiographic examination. The data were then analyzed to measure intra and inter examiner repeatability using Cronbach’s alpha and inter-item correlation matrix. Intra-examiner repeatability for all examiners was >0.7. Chronbach’s a value for inter-examiner repeatability for ICDAS II was >0.8 on 3 surfaces except on buccal and lingual. LF exhibit repeatability of >0.8 on all surfaces. Radiograph shows a low value of inter examiner repeatability (<0.7). Lecturer examiners showed the highest agreement followed by undergraduate students for inter-item correlation while the 2nd and 3rd reading of LF displays the best agreement. ICDAS II score favors more non-invasive treatment compared to LF. ICDAS II showed good repeatability except on buccal and lingual surfaces. In line with some of the previous study, ICDAS II is applicable for caries detection in daily clinical basis. Laser fluorescence exhibits the highest repeatability while the radiograph showed weak inter-examiner repeatability. Treatment decisions of ICDAS II propose more preventive treatment for early caries lesions compared to laser fluorescence.

Complementary studies of lipid membrane dynamics using iSCAT and super-resolved fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Reina, Francesco; Galiani, Silvia; Shrestha, Dilip; Sezgin, Erdinc; de Wit, Gabrielle; Cole, Daniel; Lagerholm, B. Christoffer; Kukura, Philipp; Eggeling, Christian

2018-06-01

Observation techniques with high spatial and temporal resolution, such as single-particle tracking based on interferometric scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work, we have used lipid analogues tagged with a hybrid fluorescent tag–gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous supported lipid bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50  ⩽  t  ⩽  100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye tagged lipid analogues. These FCS measurements of hybrid fluorescent tag–gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2–3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially cross-linking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

Fluorescent biological aerosol particles measured with the Waveband Integrated Bioaerosol Sensor WIBS-4: laboratory tests combined with a one year field study

NASA Astrophysics Data System (ADS)

Toprak, E.; Schnaiter, M.

2013-01-01

In this paper bioaerosol measurements conducted with the Waveband Integrated Bioaerosol Sensor mark 4 (WIBS-4) are presented. The measurements comprise aerosol chamber characterization experiments and a one-year ambient measurement period at a semi-rural site in South Western Germany. This study aims to investigate the sensitivity of WIBS-4 to biological and non-biological aerosols and detection of biological particles in the ambient aerosol. Several types of biological and non-biological aerosol samples, including fungal spores, bacteria, mineral dust, ammonium sulphate, combustion soot, and fluorescent polystyrene spheres, were analyzed by WIBS-4 in the laboratory. The results confirm the sensitivity of the ultraviolet light-induced fluorescence (UV-LIF) method to biological fluorophores and show the good discrimination capabilities of the two excitation wavelengths/detection wavebands method applied in WIBS-4. However, a weak cross-sensitivity to non-biological fluorescent interferers remains and is discussed in this paper. All the laboratory studies have been undertaken in order to prepare WIBS-4 for ambient aerosol measurements. According to the one-year ambient aerosol study, number concentration of fluorescent biological aerosol particles (FBAP) show strong seasonal and diurnal variability. The highest number concentration of FBAP was measured during the summer term and decreased towards the winter period when colder and drier conditions prevail. Diurnal FBAP concentrations start to increase after sunset and reach maximum values during the late night and early morning hours. On the other hand, the total aerosol number concentration was almost always higher during daytime than during nighttime and a sharp decrease after sunset was observed. There was no correlation observed between the FBAP concentration and the meteorological parameters temperature, precipitation, wind direction and wind speed. However, a clear correlation was identified between the FBAP number concentration and the relative humidity. Humidity-controlled release mechanisms of some fungal spore species are discussed as a possible explanation.

Feasibility of the simultaneous determination of polycyclic aromatic hydrocarbons based on two-dimensional fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Renjie; Dong, Guimei; Sun, Xueshan; Yang, Yanrong; Yu, Yaping; Liu, Haixue; Zhang, Weiyu

2018-02-01

A new approach for quantitative determination of polycyclic aromatic hydrocarbons (PAHs) in environment was proposed based on two-dimensional (2D) fluorescence correlation spectroscopy in conjunction with multivariate method. 40 mixture solutions of anthracene and pyrene were prepared in the laboratory. Excitation-emission matrix (EEM) fluorescence spectra of all samples were collected. And 2D fluorescence correlation spectra were calculated under the excitation perturbation. The N-way partial least squares (N-PLS) models were developed based on 2D fluorescence correlation spectra, showing a root mean square error of calibration (RMSEC) of 3.50 μg L- 1 and root mean square error of prediction (RMSEP) of 4.42 μg L- 1 for anthracene and of 3.61 μg L- 1 and 4.29 μg L- 1 for pyrene, respectively. Also, the N-PLS models were developed for quantitative analysis of anthracene and pyrene using EEM fluorescence spectra. The RMSEC and RMSEP were 3.97 μg L- 1 and 4.63 μg L- 1 for anthracene, 4.46 μg L- 1 and 4.52 μg L- 1 for pyrene, respectively. It was found that the N-PLS model using 2D fluorescence correlation spectra could provide better results comparing with EEM fluorescence spectra because of its low RMSEC and RMSEP. The methodology proposed has the potential to be an alternative method for detection of PAHs in environment.

Electron Detachment Dissociation (EDD) of Fluorescently Labeled Sialylated Oligosaccharides

PubMed Central

Zhou, Wen; Håkansson, Kristina

2012-01-01

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared to IRMPD. Neutral losses and satellite ions such as C – 2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared to 2-AA labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. PMID:22120881

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.

Fluorescence-based multi-parameter approach to characterize dynamics of organic carbon, faecal bacteria and particles at alpine karst springs.

PubMed

Frank, Simon; Goeppert, Nadine; Goldscheider, Nico

2018-02-15

Karst springs, especially in alpine regions, are important for drinking water supply but also vulnerable to contamination, especially after rainfall events. This high variability of water quality requires rapid quantification of contamination parameters. Here, we used a fluorescence-based multi-parameter approach to characterize the dynamics of organic carbon, faecal bacteria, and particles at three alpine karst springs. We used excitation emission matrices (EEMs) to identify fluorescent dissolved organic material (FDOM). At the first system, peak A fluorescence and total organic carbon (TOC) were strongly correlated (Spearman's r s of 0.949), indicating that a large part of the organic matter is related to humic-like substances. Protein-like fluorescence and cultivation-based determination of coliform bacteria also had a significant correlation with r s =0.734, indicating that protein-like fluorescence is directly related to faecal pollution. At the second system, which has two spring outlets, the absolute values of all measured water-quality parameters were lower; there was a significant correlation between TOC and humic-like fluorescence (r s =0.588-0.689) but coliform bacteria and protein-like fluorescence at these two springs were not correlated. Additionally, there was a strong correlation (r s =0.571-0.647) between small particle fractions (1.0 and 2.0μm), a secondary turbidity peak and bacteria. At one of these springs, discharge was constant despite the reaction of all other parameters to the rainfall event. Our results demonstrated that i) all three springs showed fast and marked responses of all investigated water-quality parameters after rain events; ii) a constant discharge does not necessarily mean constant water quality; iii) at high contamination levels, protein-like fluorescence is a good indicator of bacterial contamination, while at low contamination levels no correlation between protein-like fluorescence and bacterial values was detected; and iv) a combination of fluorescence measurements and particle-size analysis is a promising approach for a rapid assessment of organic contamination, especially relative to time-consuming conventional bacterial determination methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope.

PubMed

Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D

2013-02-01

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0-400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument.

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope

PubMed Central

Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J.; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D.

2013-01-01

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0–400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument. PMID:23412564

Live Cell Imaging and Measurements of Molecular Dynamics

PubMed Central

Frigault, M.; Lacoste, J.; Swift, J.; Brown, C.

2010-01-01

w3-2 Live cell microscopy is becoming widespread across all fields of the life sciences, as well as, many areas of the physical sciences. In order to accurately obtain live cell microscopy data, the live specimens must be properly maintained on the imaging platform. In addition, the fluorescence light path must be optimized for efficient light transmission in order to reduce the intensity of excitation light impacting the living sample. With low incident light intensities the processes under study should not be altered due to phototoxic effects from the light allowing for the long term visualization of viable living samples. Aspects for maintaining a suitable environment for the living sample, minimizing incident light and maximizing detection efficiency will be presented for various fluorescence based live cell instruments. Raster Image Correlation Spectroscopy (RICS) is a technique that uses the intensity fluctuations within laser scanning confocal images, as well as the well characterized scanning dynamics of the laser beam, to extract the dynamics, concentrations and clustering of fluorescent molecules within the cell. In addition, two color cross-correlation RICS can be used to determine protein-protein interactions in living cells without the many technical difficulties encountered in FRET based measurements. RICS is an ideal live cell technique for measuring cellular dynamics because the potentially damaging high intensity laser bursts required for photobleaching recovery measurements are not required, rather low laser powers, suitable for imaging, can be used. The RICS theory will be presented along with examples of live cell applications.

Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

NASA Astrophysics Data System (ADS)

Sun, Yuansheng; Wang, Lifu; Jyothikumar, Vinod; Brautigan, David L.; Periasamy, Ammasi

2012-03-01

Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

Photo-triggered fluorescent theranostic prodrugs as DNA alkylating agents for mechlorethamine release and spatiotemporal monitoring.

PubMed

Cao, Yanting; Pan, Rong; Xuan, Weimin; Wei, Yongyi; Liu, Kejian; Zhou, Jiahong; Wang, Wei

2015-06-28

We describe a new theranostic strategy for selective delivery and spatiotemporal monitoring of mechlorethamine, a DNA alkylating agent. A photo-responsive prodrug is designed and composed of a photolabile o-nitrophenylethyl group, a DNA alkylating mechlorethamine drug and a coumarin fluorophore. Masking of the "N" in mechlorethamine in a positively charged state in the prodrug renders it inactive, non-toxic, selective and non-fluorescent. Indeed, the stable prodrug shows negligible cytotoxicity towards normal cells with and without UV activation and is completely non-fluorescent. However, upon photo-irradiation, the active mechlorethamine is released and induces efficient DNA cross-links, accompanied by a strong fluorescence enhancement (152 fold). Furthermore, DNA cross-linking activity from the release can be transformed into anticancer activity observed in in vitro studies of tumor cells. Importantly, the drug release progress and the movement can be conveniently monitored by fluorescence spectroscopy. The mechanistic study proves that the DNA cross-linking activity is mainly due to the release of DNA alkylating mechlorethamine. Altogether, the studies show the power of the theranostic strategy for efficient therapy in cancer treatment.

TCSPC based approaches for multiparameter detection in living cells

NASA Astrophysics Data System (ADS)

Jahn, Karolina; Buschmann, Volker; Koberling, Felix; Hille, Carsten

2014-03-01

In living cells a manifold of processes take place simultaneously. This implies a precise regulation of intracellular ion homeostasis. In order to understand their spatio-temporal pattern comprehensively, the development of multiplexing concepts is essential. Due to the multidimensional characteristics of fluorescence dyes (absorption and emission spectra, decay time, anisotropy), the highly sensitive and non-invasive fluorescence microscopy is a versatile tool for realising multiplexing concepts. A prerequisite are analyte-specific fluorescence dyes with low cross-sensitivity to other dyes and analytes, respectively. Here, two approaches for multiparameter detection in living cells are presented. Insect salivary glands are well characterised secretory active tissues which were used as model systems to evaluate multiplexing concepts. Salivary glands secrete a KCl-rich or NaCl-rich fluid upon stimulation which is mainly regulated by intracellular Ca2+ as second messenger. Thus, pairwise detection of intracellular Na+, Cl- and Ca2+ with the fluorescent dyes ANG2, MQAE and ACR were tested. Therefore, the dyes were excited simultaneously (2-photon excitation) and their corresponding fluorescence decay times were recorded within two spectral ranges using time-correlated singlephoton counting (TCSPC). A second approach presented here is based on a new TCSPC-platform covering decay time detection from picoseconds to milliseconds. Thereby, nanosecond decaying cellular fluorescence and microsecond decaying phosphorescence of Ruthenium-complexes, which is quenched by oxygen, were recorded simultaneously. In both cases changes in luminescence decay times can be linked to changes in analyte concentrations. In consequence of simultaneous excitation as well as detection, it is possible to get a deeper insight into spatio-temporal pattern in living tissues.

Fluorescent porous film modified polymer optical fiber via "click" chemistry: stable dye dispersion and trace explosive detection.

PubMed

Ma, Jiajun; Lv, Ling; Zou, Gang; Zhang, Qijin

2015-01-14

In this paper, we report a facile strategy to fabricate fluorescent porous thin film on the surface of U-bent poly(methyl methacrylate) optical fiber (U-bent POF) in situ via "click" polymerization for vapor phase sensing of explosives. Upon irradiation of evanescent UV light transmitting within the fiber under ambient condition, a porous film (POSS-thiol cross-linking film, PTCF) is synthesized on the side surface of the fiber by a thiol-ene "click" reaction of vinyl-functionalized polyhedral oligomeric silsesquioxanes (POSS-V8) and alkane dithiols. When vinyl-functionalized porphyrin, containing four allyl substituents at the periphery, is added into precursors for the polymerization, fluorescence porphyrin can be covalently bonded into the cross-linked network of PTCF. This "fastened" way reduces the aggregation-induced fluorescence self-quenching of porphyrin and enhances the physicochemical stability of the porous film on the surface of U-bent POF. Fluorescent signals of the PTCF/U-bent POF probe made by this method exhibit high fluorescence quenching toward trace TNT and DNT vapor and the highest fluorescence quenching efficiency is observed for 1, 6-hexanedimercaptan-based film. In addition, because of the presence of POSS-V8 with multi cross-linkable groups, PTCF exhibits well-organized pore network and stable dye dispersion, which not only causes fast and sensitive fluorescence quenching against vapors of nitroaromatic compounds, but also provides a repeatability of the probing performance.

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.

Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

PubMed Central

Lidke, Diane S.; Lidke, Keith A.

2015-01-01

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique. PMID:25860558

Dynamic interaction between actin and nesprin2 maintain the cell nucleus in a prestressed state

NASA Astrophysics Data System (ADS)

Kumar, Abhishek; Shivashankar, G. V.

2016-12-01

Mechanical coupling between the nucleus and the cytoskeleton is indispensable for direct force transduction from the extra cellular matrix (ECM) to the chromatin. Although this physical coupling has been shown to be crucial for nuclear positioning and its function, the quantification of nuclear-cytoskeleton interaction has been lacking. In this paper, using various quantitative fluorescence spectroscopy techniques, we investigate the nature of this connection. High-resolution 3D imaging shows that nesprin2G forms short linear structures along actin stress fibers (ASFs) in the apical region of the nucleus. Fluorescence recovery after photobleaching (FRAP) revealed that the alignment of nesprin2G becomes heterogeneous when cell shape is engineered from elongated rectangular shape to square using micropatterned substrates. Further, fluorescence cross-correlation spectroscopy (FCCS) revealed that actin interacts transiently with outer nuclear membrane protein nesprin2G with a time scale of 12 ms. In addition, fluorescence resonance energy transfer (FRET) experiments show that the apical ASFs and nesprin2G are in close physical proximity. This interaction is spatially heterogeneous with high FRET along the ASFs. Lastly, we show that the disruption of actin to nuclear connection by over-expression of Dominant Negative Klarsicht, ANC-1, Syne Homology (DNKASH) leads to an increase in nuclear height. These results not only reveal the characteristics of actin-nesprin2G interaction and its significance in regulating nuclear morphology, but also validate the utility of quantitative fluorescence techniques in deciphering physical connections that are essential for mechanotransduction.

Development of a fluorescent immnunochromatographic assay for the procalcitonin detection of clinical patients in China.

PubMed

Wang, Haiying; Wang, Hong; Chen, Shaopei; Dzakah, Emmanuel E; Kang, Keren; Wang, Jihua; Wang, Jufang

2015-04-15

Procalcitonin (PCT) has been recognized as a biomarker in severe inflammation, infection and sepsis. PCT detection in serum requires sensitive and specific antibodies. In this study, we generated monoclonal antibodies (mAbs) and developed fluorescent immunochromatographic assay for PCT detection. Human recombinant PCT was used as immunogen. mAbs against PCT were developed and applied to fluorescent immunochromatographic assay for PCT detection in clinical samples. Out of 35 hybridoma cell lines secreting antibodies against the recombinant PCT, five sensitive and specific cell lines were selected and designated as F6, G2, C2, D2 and E5. All these antibodies have no cross reaction with calcitonin or calcitonin gene-related peptides (CGRP). After screening for pairing, mAb F6 was labeled with fluorescent microspheres and C2 was coated on a nitrocellulose membrane for immunochromatographic test. All 35 clinical samples were detected by the mAb F6-C2 test strips and the bioMérieux PCT assay. The test strips showed high specificity and sensitivity for PCT. Good correlation was observed between our immunochromatographic test strips and the bioMérieux PCT assay (R(2):0.986). These newly developed anti-PCT mAbs and fluorescent immunochromatographic assay can serve as important diagnostic tools for a fast, reliable and point-of-care testing for easy determination of PCT in serum and diagnosis of bacterial infection, inflammation or sepsis. Copyright © 2015 Elsevier B.V. All rights reserved.

An AIEE fluorescent supramolecular cross-linked polymer network based on pillar[5]arene host-guest recognition: construction and application in explosive detection.

PubMed

Shao, Li; Sun, Jifu; Hua, Bin; Huang, Feihe

2018-05-08

Here a novel fluorescent supramolecular cross-linked polymer network with aggregation induced enhanced emission (AIEE) properties was constructed via pillar[5]arene-based host-guest recognition. Furthermore, the supramolecular polymer network can be used for explosive detection in both solution and thin films.

An intramolecular crossed-benzoin reaction based KCN fluorescent probe in aqueous and biological environments.

PubMed

Lee, Jae Hong; Jang, Joo Hee; Velusamy, Nithya; Jung, Hyo Sung; Bhuniya, Sankarprasad; Kim, Jong Seung

2015-05-04

A turn-on fluorescent probe was designed for selective cyanide anion sensing in aqueous and biological environments. The probe underwent an intramolecular crossed-benzoin reaction in the presence of KCN to expel the fluorophore resorufin. This probe was sensitive to KCN concentrations as low as 4 nM in aqueous media.

Real-time Fluorescence Polarization Microscopy of the Moving Boundary in Cross-Gradient SDS-PAGE

NASA Astrophysics Data System (ADS)

Hwang, Jeeseong; Giulian, Gary

2003-03-01

Real-time Fluorescence Polarization Microscopy of the Moving Boundary in Cross-Gradient SDS-PAGE Jeeseong Hwang, Jeffrey R. Krogmeier, Angela M. Bardo, Scott N. Goldie, Lori S. Goldner; Optical Technology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899 Gary G. Giulian, Carl R. Merril; National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a popular method to separate proteins by their apparent molecular weight. However, it is a limited technique due, in part, to its low spatial resolution. In order to improve the resolution and to enhance the detection sensitivity of proteins separated by SDS-PAGE we are studying the detergent properties at the moving boundary of precast Tris-Tricine-Acetate cross-gradient gels using fluorescent cationic and pH indicating dyes. We have developed real-time full-field fluorescence polarization microscopy to monitor the dynamic fluorescence anisotropy from the cationic tetramethylindocarbocyanine dyes localized in the "extended stack", a concentrated detergent zone. We will present quantitative results of the fluorescence anisotropy. Our system is capable of analyzing local structures of the detergent molecules in the moving boundary of SDS-PAGE and the microenvironment(s) near the boundary. We will discuss the significance of these results and their potential role in enhanced protein separation.

Measurement of Kα and Kβ fluorescence cross sections for elements in the range 44<=Z<=68 at 59.5 keV

NASA Astrophysics Data System (ADS)

Budak, G.; Karabulut, A.; Demir, L.; Sahin, Y.

1999-09-01

The Kα and Kβ x-ray fluorescence cross sections have been measured for elements in the range 44<=Z<=68 at an excitation energy of 59.5-keV γ ray from 241Am radioisotope with a Si(Li) detector. A reasonable agreement is found between the present experimental results and the theoretically calculated values based on photoionization cross sections by Scofield using Hartree-Slater and Hartree-Fock central potential theory.

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

Spectrally-resolved fluorescence cross sections of aerosolized biological live agents and simulants using five excitation wavelengths in a BSL-3 laboratory.

PubMed

Pan, Yong-Le; Hill, Steven C; Santarpia, Joshua L; Brinkley, Kelly; Sickler, Todd; Coleman, Mark; Williamson, Chatt; Gurton, Kris; Felton, Melvin; Pinnick, Ronald G; Baker, Neal; Eshbaugh, Jonathan; Hahn, Jerry; Smith, Emily; Alvarez, Ben; Prugh, Amber; Gardner, Warren

2014-04-07

A system for measuring spectrally-resolved fluorescence cross sections of single bioaerosol particles has been developed and employed in a biological safety level 3 (BSL-3) facility at Edgewood Chemical and Biological Center (ECBC). It is used to aerosolize the slurry or solution of live agents and surrogates into dried micron-size particles, and to measure the fluorescence spectra and sizes of the particles one at a time. Spectrally-resolved fluorescence cross sections were measured for (1) bacterial spores: Bacillus anthracis Ames (BaA), B. atrophaeus var. globigii (BG) (formerly known as Bacillus globigii), B. thuringiensis israelensis (Bti), B. thuringiensis kurstaki (Btk), B. anthracis Sterne (BaS); (2) vegetative bacteria: Escherichia coli (E. coli), Pantoea agglomerans (Eh) (formerly known as Erwinia herbicola), Yersinia rohdei (Yr), Yersinia pestis CO92 (Yp); and (3) virus preparations: Venezuelan equine encephalitis TC83 (VEE) and the bacteriophage MS2. The excitation wavelengths were 266 nm, 273 nm, 280 nm, 365 nm and 405 nm.

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides.

PubMed

Zhou, Wen; Håkansson, Kristina

2011-12-01

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Widefield High Frame Rate Single-Photon SPAD Imagers for SPIM-FCS.

PubMed

Buchholz, Jan; Krieger, Jan; Bruschini, Claudio; Burri, Samuel; Ardelean, Andrei; Charbon, Edoardo; Langowski, Jörg

2018-05-22

Photon-counting sensors based on standard complementary metal-oxide-semiconductor single-photon avalanche diodes (SPADs) represent an emerging class of imagers that enable the counting and/or timing of single photons at zero readout noise (better than high-speed electron-multiplying charge-coupling devices) and over large arrays. They have seen substantial progress over the last 15 years, increasing their spatial resolution, timing accuracy, and sensitivity while reducing spurious signals such as afterpulsing and dark counts. They are increasingly being applied for time-resolved applications with the added advantage of enabling real-time options such as autocorrelation. We report in this study on the use of such a state-of-the-art 512 × 128 SPAD array, capable of a time resolution of 10 -5 -10 -6 s for full frames while retaining acceptable photosensitivity thanks to the use of dedicated microlenses, in a selective plane illumination-fluorescence correlation spectroscopy setup. The latter allows us to perform thousands of fluorescence-correlation spectroscopy measurements simultaneously in a two-dimensional slice of the sample. This high-speed SPAD imager enables the measurement of molecular motion of small fluorescent particles such as single chemical dye molecules. Inhomogeneities in the molecular detection efficiency were compensated for by means of a global fit of the auto- and cross-correlation curves, which also made a calibration-free measurement of various samples possible. The afterpulsing effect could also be mitigated, making the measurement of the diffusion of Alexa-488 possible, and the overall result quality was further improved by spatial binning. The particle concentrations in the focus tend to be overestimated by a factor of 1.7 compared to a confocal setup; a calibration is thus required if absolute concentrations need to be measured. The first high-speed selective plane illumination-fluorescence correlation spectroscopy in vivo measurements to our knowledge were also recorded: although two-component fit models could not be employed because of noise, the diffusion of eGFP oligomers in HeLa cells could be measured. Sensitivity and noise will be further improved in the next generation of SPAD-based widefield sensors, which are currently under testing. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fiber optic immunosensor for cross-linked fibrin concentration

NASA Astrophysics Data System (ADS)

Moskowitz, Samuel E.

2000-08-01

Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels <0.1 ng/ml. A capture monoclonal antibody to the antigen soluble cross-linked fibrin is employed. Immobilized at the tip of an optical fiber by avidin-biotin, the captured antigen is detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.

NLO properties of ester containing fluorescent carbazole based styryl dyes - Consolidated spectroscopic and DFT approach

NASA Astrophysics Data System (ADS)

Rajeshirke, Manali; Sekar, Nagaiyan

2018-02-01

The linear and nonlinear optical (NLO) properties of new fluorescent styryl dyes based on anchoring ester containing carbazole as donor appended to different acceptor groups to have a conjugated π-system with push-pull geometry are studied. The NLO properties have been determined using solvatochromic and computational methods. Three different TD-DFT functional are used namely, B3LYP, BHandHLYP, and CAM-B3LYP, with aim of elucidating better functional for NLOphores. Further, the two photon properties (σ2PA) have been described theoretically by two level model considering the dipole moment difference between the ground and the final electronic states and bypassing the intermediated resonance state. The compounds with a high charge transfer from the acceptor group to the carbazole ring have relatively high two-photon absorption cross-sections (60-317 GM). The linear polarizability (αCT), first order hyperpolarizability (β) and second order hyperpolarizability (ɣ) for 4c dye was the highest among the studied dyes which is attributed to the lesser energy gap evident by both the methods. But in contrary, the σ2PA cross-section value was low for dye 4c which is due to the presence of freely rotatable twisted phenyl ring in the conjugation path, pulling the electron density towards itself and thus lead to decrease in σ2PA cross-section. Structure-property relationship is better understood by the correlation of bond length alternation/bond order alternation (BLA/BOA) with NLO properties of dyes. Thus by simple solvatochromic method and computational method, we have screened the carbazole styryls as NLO candidates with good first order hyperpolarizability and good two photon cross-section.

Temperature dependence of laser-induced fluorescence of Tb3+Tb3+ in molten LiCl-KCl eutectic

NASA Astrophysics Data System (ADS)

C., E.; -E., Jung | S.; | W., Bae; Cha | I., A.; Bae | Y., J.; | K., Park; Song

2011-01-01

Fluorescence spectra and lifetimes originated from both 5D3 →7FJ and 5D4 →7FJ transitions of Tb3+ were measured using time-resolved laser fluorescence spectroscopy in order to investigate the excited state relaxation in a molten salt medium. A cross-relaxation energy transfer of 5D3 →5D4 resulted in rise and decay behaviors in fluorescence signal waveforms of 5D4 →7FJ transitions. The fluorescence intensity ratios of 5D4 →7F5 to 5D3 →7F4 decreased drastically when the temperature of molten salt increased. This result suggests that the cross-relaxation effect becomes weakened with increasing temperature. In addition, a strong increase of the 5D4 emission over the 5D3 emission was observed at high Tb3+ concentration.

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates.

PubMed

Chen, Qiuying; Hirsch, Rhoda Elison

2006-03-01

Fluorescence emission of free protoporphyrin IX (PPIX, em. approximately 626 nm), zinc protoporphyrin IX (ZPP, em. approximately 594 nm) and fluorescent heme degradation product (FHDP, em. approximately 466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F(365/594)) correlates with the ZPP fluorescence obtained from acetone/water extraction (R(2) = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R(2) = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.

Depth-resolved fluorescence of human ectocervical tissue

NASA Astrophysics Data System (ADS)

Wu, Yicong; Xi, Peng; Cheung, Tak-Hong; Yim, So Fan; Yu, Mei-Yung; Qu, Jianan Y.

2005-04-01

The depth-resolved autofluorescence of normal and dysplastic human ectocervical tissue within 120um depth were investigated utilizing a portable confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of all ectocervical tissue samples, strong keratin fluorescence with the spectral characteristics similar to collagen was observed, which created serious interference in seeking the correlation between tissue fluorescence and tissue pathology. While from the underlying non-keratinizing epithelial layer, the measured NADH fluorescence induced by 355nm excitation and FAD fluorescence induced by 457nm excitation were strongly correlated to the tissue pathology. The ratios between NADH over FAD fluorescence increased statistically in the CIN epithelial relative to the normal and HPV epithelia, which indicated increased metabolic activity in precancerous tissue. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

Fluorescence decay time imaging using an imaging photon detector with a radio frequency photon correlation system

NASA Astrophysics Data System (ADS)

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

1990-05-01

An imaging photon detector has been modified to incorporate fast timing electronics coupled to a custom built photon correlator interfaced to a RISC computer. Using excitation with intensity- muodulated light, fluorescence images can be readily obtained where contrast is determined by the decay time of emission, rather than by intensity. This technology is readily extended to multifrequency phase/demodulation fluorescence imaging or to differential polarised phase fluorometry. The potential use of the correlator for confocal imaging with a laser scanner is also briefly discussed.

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

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

2013-01-01

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

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

PubMed Central

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

2012-01-01

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

Broadband two-photon absorption cross sections of benzothiazole derivatives and benzobisthiazolium salts

NASA Astrophysics Data System (ADS)

Noskovičova, Eva; Lorenc, Dušan; Magdolen, Peter; Sigmundová, Ivica; Zahradník, Pavol; Velič, Dušan

2018-05-01

Two-photon absorption (TPA) cross sections of conjugated donor-π-acceptor dipolar structures containing benzothiazole or benzobisthiazolium moieties are determined in a broad spectral range from 700 nm to 1000 nm using two-photon induced fluorescence technique. The TPA cross section values range from 150 GM to 4600 GM. The largest values are observed in near-infrared region. The dipolar derivative of benzothiazole has the largest TPA cross section of 4600 GM at wavelength of 890 nm. A combination of the large TPA in the near-infrared region and the high emission quantum yield makes these compounds excellent candidates for two-photon fluorescence microscopy.

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.

Theory of third-order spectroscopic methods to extract detailed molecular orientational dynamics for planar surfaces and other uniaxial systems

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

Nishida, Jun; Fayer, Michael D., E-mail: fayer@stanford.edu

Functionalized organic monolayers deposited on planar two-dimensional surfaces are important systems for studying ultrafast orientational motions and structures of interfacial molecules. Several studies have successfully observed the orientational relaxation of functionalized monolayers by fluorescence depolarization experiments and recently by polarization-resolved heterodyne detected vibrational transient grating (HDTG) experiments. In this article we provide a model-independent theory to extract orientational correlation functions unique to interfacial molecules and other uniaxial systems based on polarization-resolved resonant third-order spectroscopies, such as pump-probe spectroscopy, HDTG spectroscopy, and fluorescence depolarization experiment. It will be shown (in the small beam-crossing angle limit) that five measurements are necessary tomore » completely characterize the monolayer's motions: I{sub ∥}(t) and I{sub ⊥}(t) with the incident beams normal to the surface, I{sub ∥}(t) and I{sub ⊥}(t) with a non-zero incident angle, and a time averaged linear dichroism measurement. Once these measurements are performed, two orientational correlation functions corresponding to in-plane and out-of-plane motions are obtained. The procedure is applicable not only for monolayers on flat surfaces, but any samples with uniaxial symmetry such as uniaxial liquid crystals and aligned planar bilayers. The theory is valid regardless of the nature of the actual molecular motions on interface. We then apply the general results to wobbling-in-a-cone model, in which molecular motions are restricted to a limited range of angles. Within the context of the model, the cone angle, the tilt of the cone relative to the surface normal, and the orientational diffusion constant can be determined. The results are extended to describe analysis of experiments where the beams are not crossing in the small angle limit.« less

Temporal Effects on Internal Fluorescence Emissions Associated with Aflatoxin Contamination from Corn Kernel Cross-Sections Inoculated with Toxigenic and Atoxigenic Aspergillus flavus.

PubMed

Hruska, Zuzana; Yao, Haibo; Kincaid, Russell; Brown, Robert L; Bhatnagar, Deepak; Cleveland, Thomas E

2017-01-01

Non-invasive, easy to use and cost-effective technology offers a valuable alternative for rapid detection of carcinogenic fungal metabolites, namely aflatoxins, in commodities. One relatively recent development in this area is the use of spectral technology. Fluorescence hyperspectral imaging, in particular, offers a potential rapid and non-invasive method for detecting the presence of aflatoxins in maize infected with the toxigenic fungus Aspergillus flavus . Earlier studies have shown that whole maize kernels contaminated with aflatoxins exhibit different spectral signatures from uncontaminated kernels based on the external fluorescence emission of the whole kernels. Here, the effect of time on the internal fluorescence spectral emissions from cross-sections of kernels infected with toxigenic and atoxigenic A. flavus , were examined in order to elucidate the interaction between the fluorescence signals emitted by some aflatoxin contaminated maize kernels and the fungal invasion resulting in the production of aflatoxins. First, the difference in internal fluorescence emissions between cross-sections of kernels incubated in toxigenic and atoxigenic inoculum was assessed. Kernels were inoculated with each strain for 5, 7, and 9 days before cross-sectioning and imaging. There were 270 kernels (540 halves) imaged, including controls. Second, in a different set of kernels (15 kernels/group; 135 total), the germ of each kernel was separated from the endosperm to determine the major areas of aflatoxin accumulation and progression over nine growth days. Kernels were inoculated with toxigenic and atoxigenic fungal strains for 5, 7, and 9 days before the endosperm and germ were separated, followed by fluorescence hyperspectral imaging and chemical aflatoxin determination. A marked difference in fluorescence intensity was shown between the toxigenic and atoxigenic strains on day nine post-inoculation, which may be a useful indicator of the location of aflatoxin contamination. This finding suggests that both, the fluorescence peak shift and intensity as well as timing, may be essential in distinguishing toxigenic and atoxigenic fungi based on spectral features. Results also reveal a possible preferential difference in the internal colonization of maize kernels between the toxigenic and atoxigenic strains of A. flavus suggesting a potential window for differentiating the strains based on fluorescence spectra at specific time points.

Temporal Effects on Internal Fluorescence Emissions Associated with Aflatoxin Contamination from Corn Kernel Cross-Sections Inoculated with Toxigenic and Atoxigenic Aspergillus flavus

PubMed Central

Hruska, Zuzana; Yao, Haibo; Kincaid, Russell; Brown, Robert L.; Bhatnagar, Deepak; Cleveland, Thomas E.

2017-01-01

Non-invasive, easy to use and cost-effective technology offers a valuable alternative for rapid detection of carcinogenic fungal metabolites, namely aflatoxins, in commodities. One relatively recent development in this area is the use of spectral technology. Fluorescence hyperspectral imaging, in particular, offers a potential rapid and non-invasive method for detecting the presence of aflatoxins in maize infected with the toxigenic fungus Aspergillus flavus. Earlier studies have shown that whole maize kernels contaminated with aflatoxins exhibit different spectral signatures from uncontaminated kernels based on the external fluorescence emission of the whole kernels. Here, the effect of time on the internal fluorescence spectral emissions from cross-sections of kernels infected with toxigenic and atoxigenic A. flavus, were examined in order to elucidate the interaction between the fluorescence signals emitted by some aflatoxin contaminated maize kernels and the fungal invasion resulting in the production of aflatoxins. First, the difference in internal fluorescence emissions between cross-sections of kernels incubated in toxigenic and atoxigenic inoculum was assessed. Kernels were inoculated with each strain for 5, 7, and 9 days before cross-sectioning and imaging. There were 270 kernels (540 halves) imaged, including controls. Second, in a different set of kernels (15 kernels/group; 135 total), the germ of each kernel was separated from the endosperm to determine the major areas of aflatoxin accumulation and progression over nine growth days. Kernels were inoculated with toxigenic and atoxigenic fungal strains for 5, 7, and 9 days before the endosperm and germ were separated, followed by fluorescence hyperspectral imaging and chemical aflatoxin determination. A marked difference in fluorescence intensity was shown between the toxigenic and atoxigenic strains on day nine post-inoculation, which may be a useful indicator of the location of aflatoxin contamination. This finding suggests that both, the fluorescence peak shift and intensity as well as timing, may be essential in distinguishing toxigenic and atoxigenic fungi based on spectral features. Results also reveal a possible preferential difference in the internal colonization of maize kernels between the toxigenic and atoxigenic strains of A. flavus suggesting a potential window for differentiating the strains based on fluorescence spectra at specific time points. PMID:28966606

Photoresponsive cross-linked polymeric particles for phototriggered burst release.

PubMed

Wang, Zhen; Yu, Lili; Lv, Cong; Wang, Peng; Chen, Yedong; Tang, Xinjing

2013-01-01

We synthesized a series of cross-linked photoresponsive polymeric particles with photolabile monomers and cross-linkers through miniemulsion polymerization. These particles are quite stable in dark, while light irradiation caused the breakage of particles and the efficient release of encapsulated contents up to 95% based on Nile red fluorescence. Photoswitches of particle systems were confirmed by fluorescence spectroscopy, SEM and colorimetry. Particle uptake and triggered release in RAW264.7 cells were confirmed by fluorescein diacetate loaded particles. © 2013 The Authors. Photochemistry and Photobiology © 2013 The American Society of Photobiology.

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

PubMed

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

2010-07-27

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

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

PubMed Central

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

2010-01-01

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

UV fluorescence excitation imaging of healing of wounds in skin: Evaluation of wound closure in organ culture model.

PubMed

Wang, Ying; Gutierrez-Herrera, Enoch; Ortega-Martinez, Antonio; Anderson, Richard Rox; Franco, Walfre

2016-09-01

Molecules native to tissue that fluoresce upon light excitation can serve as reporters of cellular activity and protein structure. In skin, the fluorescence ascribed to tryptophan is a marker of cellular proliferation, whereas the fluorescence ascribed to cross-links of collagen is a structural marker. In this work, we introduce and demonstrate a simple but robust optical method to image the functional process of epithelialization and the exposed dermal collagen in wound healing of human skin in an organ culture model. Non-closing non-grafted, partial closing non-grafted, and grafted wounds were created in ex vivo human skin and kept in culture. A wide-field UV fluorescence excitation imaging system was used to visualize epithelialization of the exposed dermis and quantitate wound area, closure, and gap. Histology (H&E staining) was also used to evaluate epithelialization. The endogenous fluorescence excitation of cross-links of collagen at 335 nm clearly shows the dermis missing epithelium, while the endogenous fluorescence excitation of tryptophan at 295 nm shows keratinocytes in higher proliferating state. The size of the non-closing wound was 11.4 ± 1.8 mm and remained constant during the observation period, while the partial-close wound reached 65.5 ± 4.9% closure by day 16. Evaluations of wound gaps using fluorescence excitation images and histology images are in agreement. We have established a fluorescence imaging method for studying epithelialization processes, evaluating keratinocyte proliferation, and quantitating closure during wound healing of skin in an organ culture model: the dermal fluorescence of pepsin-digestible collagen cross-links can be used to quantitate wound size, closure extents, and gaps; and, the epidermal fluorescence ascribed to tryptophan can be used to monitor and quantitate functional states of epithelialization. UV fluorescence excitation imaging has the potential to become a valuable tool for research, diagnostic and educational purposes on evaluating the healing of wounds. Lasers Surg. Med. 48:678-685, 2016. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Development of a homogeneous assay format for p53 antibodies using fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

2005-08-01

The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.

An orange fluorescent protein tagging system for real-time pollen tracking.

PubMed

Rice, J Hollis; Millwood, Reginald J; Mundell, Richard E; Chambers, Orlando D; Abercrombie, Laura L; Davies, H Maelor; Stewart, C Neal

2013-09-27

Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum × Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification. We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum 'TN 90' and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression. The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.

[Fluorescence characterization of dissolved organic matter in the East China Sea after diatom red tide dispersion].

PubMed

Zhuo, Peng-ji; Zhao, Wei-hong

2009-05-01

Fluorescence excitation-emission spectroscopy (EEMS) was employed to analyze the 3-dimensional fluorescence of dissolved organic matter in the East China Sea after diatom red tide dispersion. The relationships between fluorescence peak intensity, and salinity and chlorophyll-a were discussed. The centers of protein-like fluorescence peaks dispersed at Exmax/Exmax = 270-280/290-315 nm (Peak B), 220-230/290-305 nm (Peak D), 230-240/335-350 nm (Peak S) and 280/320 nm (Peak T). Two humic-like peaks appeared at 255-270/435-480 nm (Peak A)and 330-350/420-480 nm (Peak C). High tyrosine-like intensity was observed in diatom red tide dispersion area, and tryptophan-like fluorescence was also found which was lower. High FIB/FIS showed that diatom red tide produced much tyrosine-like matter during dispersion. Peaks S, A and C had positive correlation with one another, and their distributions were similar, which decreased with distance increasing away from the shore. Good negative correlations between peaks S, A and C and salinity suggested that Jiangsu-Zhejiang coastal water was the same source of them. Correlations between fluorescence peak intensity and chlorophyll-a were not remarkable enough to clear the relationship between fluorescence and living algal matter. It was supposed that the living algal matter contributed little to the fluorescence intensity of algal dispersion seawater.

Variations in the endogenous fluorescence of rabbit corneas after mechanical property alterations

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Touchette, Genna; Zhu, Hong; Kochevar, Irene E.; Franco, Walfre

2017-09-01

Keratoconus is an eye disease in which the cornea progressively deforms due to loss of cornea mechanical rigidity, and thus causes deterioration of visual acuity. Techniques to characterize the mechanical characteristics of the cornea are important to better monitor changes and response to treatments. To investigate the feasibility of using the endogenous fluorescence of cornea for monitoring alterations of its mechanical rigidity, linear tensiometry was used to quantitate stiffness and Young's modulus (YM) after treatments that increase cornea stiffness (collagen photocross-linking) or decrease stiffness (enzymatic digestion). The endogenous ultraviolet fluorescence of cornea was also measured before and after these treatments. The fluorescence excitation/emission spectral ranges were 280 to 430/390 to 520 nm, respectively. A correlation analysis was carried out to identify fluorescence excitation/emission pairs whose intensity changes correlated with the stiffness. A positive correlation was found between variations in fluorescence intensity of the 415-/485-nm excitation/emission pair and YM of photocross-linked corneas. After treatment of corneas with pepsin, the YM decreased as the fluorescence intensity at 290-/390-nm wavelengths decreased. For weakening of corneas with collagenase, only qualitative changes in the fluorescence spectrum were observed. Changes in the concentration of native or newly created fluorescent molecular species contain information that may be directly or indirectly related to the mechanical structure of the cornea.

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

PubMed Central

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

2013-01-01

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

Highly sensitive detection of cancer cells using femtosecond dual-wavelength near-IR two-photon imaging.

PubMed

Starkey, Jean R; Makarov, Nikolay S; Drobizhev, Mikhail; Rebane, Aleksander

2012-07-01

We describe novel imaging protocols that allow detection of small cancer cell colonies deep inside tissue phantoms with high sensitivity and specificity. We compare fluorescence excited in Styryl-9M molecules by femtosecond pulses at near IR wavelengths, where Styryl-9M shows the largest dependence of the two-photon absorption (2PA) cross section on the local environment. We show that by calculating the normalized ratio of the two-photon excited fluorescence (2PEF) intensity at 1200 nm and 1100 nm excitation wavelengths we can achieve high sensitivity and specificity for determining the location of cancer cells surrounded by normal cells. The 2PEF results showed a positive correlation with the levels of MDR1 proteins expressed by the cells, and, for high MDR1 expressors, as few as ten cancer cells could be detected. Similar high sensitivity is also demonstrated for tumor colonies induced in mouse external ears. This technique could be useful in early cancer detection, and, perhaps, also in monitoring dormant cancer deposits.

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.

Two-Photon Absorption Properties of Gold Fluorescent Protein: A Combined Molecular Dynamics and Quantum Chemistry Study.

PubMed

Şimşek, Yusuf; Brown, Alex

2018-06-07

Molecular dynamics (MD) simulations were carried out to obtain the conformational changes of the chromophore in the gold fluorescent protein (PDB ID: 1OXF ). To obtain two-photon absorption (TPA) cross-sections, time dependent density functional theory (TD-DFT) computations were performed for chromophore geometries sampled along the trajectory. The TD-DFT computations used the CAM-B3LYP functional and 6-31+G(d) basis set. Results showed that two dihedral angles change remarkably over the simulation time. TPA cross-sections were found to average 13.82 GM for the excitation to S 1 computed from the equilibrium geometries; however, extending the structures with a water molecule and GLU residue, which make H bonds with the chromophore molecule, increased excitation energies and TPA cross-sections significantly. Besides the effects of the surrounding residues and the dihedrals on the spectroscopic properties, some bond lengths affected the excitation energies and the TPA cross-sections significantly (up to ±25-30%), while the effects of the bond angles were smaller (±5%). Overall the present results provide insight into the effects of the conformational flexibility on TPA (with gold fluorescent protein as a specific example) and suggest that further experimental measurements of TPA for the gold fluorescent protein should be undertaken.

Fluorescent biological aerosol particles (FBAPs) measured with the Waveband Integrated Bioaerosol Sensor WIBS-4: laboratory tests combined with a one year field study

NASA Astrophysics Data System (ADS)

Toprak, E.; Schnaiter, M.

2012-07-01

In this paper bioaerosol measurements conducted with the Waveband Integrated Bioaerosol Sensor mark 4 (WIBS-4) are presented. The measurements comprise aerosol chamber characterization experiments and a one-year ambient measurement period at a semi-rural site in South Western Germany. This study aims to investigate the sensitivity of WIBS-4 to biological and non-biological aerosols, performance of WIBS-4 for discrimination of several types of aerosols, and the detection and identification of biological particles in the ambient aerosol. Several types of biological and non-biological aerosol samples including spores, bacteria, pollen, mineral dust, ammonium sulphate, combustion soot, and fluorescent polystyrene spheres were analysed by WIBS-4 in the laboratory. The results confirm the sensitivity of the Ultra Violet Light Induced Fluorescence (UV-LIF) method to biological fluorophores and show the good discrimination capabilities of the two wavelengths excitation/two wavebands detection method applied in WIBS-4. However, a weak cross-sensitivity to non-biological fluorescent interferers remains and is discussed in this paper. All the laboratory studies have been undertaken in order to prepare WIBS-4 for ambient aerosol measurements. According to the one year ambient aerosol study, number concentration of fluorescent biological aerosol particles (FBAP) show strong seasonal and diurnal variability. The highest number concentration of FBAP was measured during the summer term and it decreases towards the winter period when colder and drier conditions are prevailing. Diurnal FBAP concentrations start to increase after sunset and reach maximum values during the late night and early morning hours. On the other hand the total aerosol number concentration was always higher during day time than during night time and a sharp decrease after sunset was observed. There was no correlation observed between the FBAP concentration and the meteorological parameters temperature, precipitation, wind direction and wind speed. However a clear correlation was identified between the FBAP number concentration and the relative humidity. Humidity controlled release mechanisms of some fungal spore species are discussed as a possible explanation.

The detection of T-Nos, a genetic element present in GMOs, by cross-priming isothermal amplification with real-time fluorescence.

PubMed

Zhang, Fang; Wang, Liu; Fan, Kai; Wu, Jian; Ying, Yibin

2014-05-01

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06 × 10(3) copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.

PubMed

Blackwell, Daniel J; Zak, Taylor J; Robia, Seth L

2016-09-20

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Optically-Enhanced, Near-IR, Silver Cluster Emission Altered by Single Base Changes in the DNA Template

PubMed Central

Fan, Chaoyang; Story, Sandra P.; Sengupta, Bidisha; Sartin, Matthew; Hsiang, Jung-Cheng; Perry, Joseph W.

2011-01-01

Few-atom silver clusters harbored by DNA are promising fluorophores due to their high molecular brightness along with their long- and short-term photostability. Furthermore, their emission rate can be enhanced when co-illuminated with low-energy light that optically depopulates the fluorescence-limiting dark state. The photophysical basis for this effect is evaluated for two near infrared-emitting clusters. Clusters emitting at ~800 nm form with C3AC3AC3TC3A and C3AC3AC3GC3A and both exhibit a trap state with λmax ~ 840 nm and an absorption cross section of 5–6 × 10−16 cm2/molec that can be optically depopulated. Transient absorption spectra, complemented by fluorescence correlation spectroscopy studies, show that the dark state has an inherent lifetime of 3–4 μs and that absorption from this state is accompanied by photoinduced crossover back to the emissive manifold of states with an action cross section of ~2 × 10−18 cm2/molec. Relative to C3AC3AC3TC3A, C3AC3AC3GC3A produces a longer-lived trap state and permits more facile passage back to the emissive manifold. With the C3AC3AC3AC3G template, a spectrally distinct cluster forms having emission at ~900 nm and its trap state has a ~four-fold shorter lifetime. These studies of optically-gated fluorescence bolster the critical role of the nucleobases on both the formation and excited state dynamics of these highly emissive metallic clusters. PMID:21568292

Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay.

PubMed

Tanigawa, Chihiro; Fujii, Yoshito; Miura, Masashi; Nzou, Samson Muuo; Mwangi, Anne Wanjiru; Nagi, Sachiyo; Hamano, Shinjiro; Njenga, Sammy M; Mbanefo, Evaristus Chibunna; Hirayama, Kenji; Mwau, Matilu; Kaneko, Satoshi

2015-01-01

Both Schistosoma mansoni and Schistosoma haematobium cause schistosomiasis in sub-Saharan Africa. We assessed the diagnostic value of selected Schistosoma antigens for the development of a multiplex serological immunoassay for sero-epidemiological surveillance. Diagnostic ability of recombinant antigens from S. mansoni and S. haematobium was assessed by Luminex multiplex immunoassay using plasma from school children in two areas of Kenya, endemic for different species of schistosomiasis. S. mansoni serine protease inhibitor (SERPIN) and Sm-RP26 showed significantly higher reactivity to patient plasma as compared to the control group. Sm-Filamin, Sm-GAPDH, Sm-GST, Sm-LAP1, Sm-LAP2, Sm-Sm31, Sm-Sm32 and Sm-Tropomyosin did not show difference in reactivity between S. mansoni infected and uninfected pupils. Sm-RP26 was cross-reactive to plasma from S. haematobium patients, whereas Sm-SERPIN was species-specific. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. ROC analysis for Sm-RP26, Sm-SERPIN and Sh-SERPIN showed AUC values of 0.833, 0.888 and 0.947, respectively. Using Spearman's rank correlation coefficient analysis, we also found significant positive correlation between the number of excreted eggs and median fluorescence intensity (MFI) from the multiplex immunoassays for Sm-SERPIN (ρ = 0.430, p-value = 0.003) and Sh-SERPIN (ρ = 0.433, p-value = 0.006). Sm-SERPIN is a promising species-specific diagnostic antigen. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. SERPINs showed correlation with the number of excreted eggs. These indicate prospects for inclusion of SERPINs in the multiplex serological immunoassay system.

Raman chemical imaging of explosive-contaminated fingerprints.

PubMed

Emmons, E D; Tripathi, A; Guicheteau, J A; Christesen, S D; Fountain, A W

2009-11-01

Raman chemical imaging (RCI) has been used to detect and identify explosives in contaminated fingerprints. Bright-field imaging is used to identify regions of interest within a fingerprint, which can then be examined to determine their chemical composition using RCI and fluorescence imaging. Results are presented where explosives in contaminated fingerprints are identified and their spatial distributions are obtained. Identification of explosives is obtained using Pearson's cosine cross-correlation technique using the characteristic region (500-1850 cm(-1)) of the spectrum. This study shows the ability to identify explosives nondestructively so that the fingerprint remains intact for further biometric analysis. Prospects for forensic examination of contaminated fingerprints are discussed.

FLUORESCENT-SERIOLOGICAL INVESTIGATIONS OF A PATHOGENIC FUNGUS (SPOROTRICHUM SCHENCKII),

DTIC Science & Technology

coloration of numerous other species of fungus no cross reactions with Sporotrichum schenkii were found. The use of this fluorescent coloring method for the diagnosis of Sporotrichosis is suggested. (Author)

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

NASA Astrophysics Data System (ADS)

Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

2017-09-01

Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

Differences in fluorescence profiles from breast cancer tissues due to changes in relative tryptophan content via energy transfer: tryptophan content correlates with histologic grade and tumor size but not with lymph node metastases

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Sordillo, Peter P.; Budansky, Yury; Pu, Yang; Alfano, Robert R.

2014-12-01

The correlation between histologic grade, an increasingly important measure of prognosis for patients with breast cancer, and tryptophan levels from tissues of 15 breast carcinoma patients was investigated. Changes in the relative content of key native organic biomolecule tryptophan were seen from the fluorescence spectra of cancerous and paired normal tissues with excitation wavelengths of 280 and 300 nm. Due to a large spectral overlap and matching excitation-emission spectra, fluorescence resonance energy transfer from tryptophan-donor to reduced nicotinamide adenine dinucleotides-acceptor was noted. We used the ratios of fluorescence intensities at their spectral emission peaks, or spectral fingerprint peaks, at 340, 440, and 460 nm. Higher ratios correlated strongly with high histologic grade, while lower-grade tumors had low ratios. Large tumor size also correlated with high ratios, while the number of lymph node metastases, a major factor in staging, was not correlated with tryptophan levels. High histologic grade correlates strongly with increased content of tryptophan in breast cancer tissues and suggests that measurement of tryptophan content may be useful as a part of the evaluation of these patients.

Distinguishing autofluorescence of normal, benign, and cancerous breast tissues through wavelet domain correlation studies.

PubMed

Gharekhan, Anita H; Arora, Siddharth; Oza, Ashok N; Sureshkumar, Mundan B; Pradhan, Asima; Panigrahi, Prasanta K

2011-08-01

Using the multiresolution ability of wavelets and effectiveness of singular value decomposition (SVD) to identify statistically robust parameters, we find a number of local and global features, capturing spectral correlations in the co- and cross-polarized channels, at different scales (of human breast tissues). The copolarized component, being sensitive to intrinsic fluorescence, shows different behavior for normal, benign, and cancerous tissues, in the emission domain of known fluorophores, whereas the perpendicular component, being more prone to the diffusive effect of scattering, points out differences in the Kernel-Smoother density estimate employed to the principal components, between malignant, normal, and benign tissues. The eigenvectors, corresponding to the dominant eigenvalues of the correlation matrix in SVD, also exhibit significant differences between the three tissue types, which clearly reflects the differences in the spectral correlation behavior. Interestingly, the most significant distinguishing feature manifests in the perpendicular component, corresponding to porphyrin emission range in the cancerous tissue. The fact that perpendicular component is strongly influenced by depolarization, and porphyrin emissions in cancerous tissue has been found to be strongly depolarized, may be the possible cause of the above observation.

Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

PubMed Central

Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R.; Blasi, Juan

2014-01-01

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

PubMed

Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R; Blasi, Juan

2014-01-01

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Determination of K shell absorption jump factors and jump ratios of 3d transition metals by measuring K shell fluorescence parameters.

PubMed

Kaçal, Mustafa Recep; Han, İbrahim; Akman, Ferdi

2015-01-01

Energy dispersive X-ray fluorescence technique (EDXRF) has been employed for measuring K-shell absorption jump factors and jump ratios for Ti, Cr, Fe, Co, Ni and Cu elements. The jump factors and jump ratios for these elements were determined by measuring K shell fluorescence parameters such as the Kα X-ray production cross-sections, K shell fluorescence yields, Kβ-to-Kα X-rays intensity ratios, total atomic absorption cross sections and mass attenuation coefficients. The measurements were performed using a Cd-109 radioactive point source and an Si(Li) detector in direct excitation and transmission experimental geometry. The measured values for jump factors and jump ratios were compared with theoretically calculated and the ones available in the literature. Copyright © 2014 Elsevier Ltd. All rights reserved.

Controlling Plasmon-Enhanced Fluorescence via Intersystem Crossing in Photoswitchable Molecules.

PubMed

Wang, Mingsong; Hartmann, Gregory; Wu, Zilong; Scarabelli, Leonardo; Rajeeva, Bharath Bangalore; Jarrett, Jeremy W; Perillo, Evan P; Dunn, Andrew K; Liz-Marzán, Luis M; Hwang, Gyeong S; Zheng, Yuebing

2017-10-01

By harnessing photoswitchable intersystem crossing (ISC) in spiropyran (SP) molecules, active control of plasmon-enhanced fluorescence in the hybrid systems of SP molecules and plasmonic nanostructures is achieved. Specifically, SP-derived merocyanine (MC) molecules formed by photochemical ring-opening reaction display efficient ISC due to their zwitterionic character. In contrast, ISC in quinoidal MC molecules formed by thermal ring-opening reaction is negligible. The high ISC rate can improve fluorescence quantum yield of the plasmon-modified spontaneous emission, only when the plasmonic electromagnetic field enhancement is sufficiently high. Along this line, extensive photomodulation of fluorescence is demonstrated by switching the ISC in MC molecules at Au nanoparticle aggregates, where strongly enhanced plasmonic hot spots exist. The ISC-mediated plasmon-enhanced fluorescence represents a new approach toward controlling the spontaneous emission of fluorophores near plasmonic nanostructures, which expands the applications of active molecular plasmonics in information processing, biosensing, and bioimaging. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Far wing depolarization of light - Generalized absorption profiles. [in laser fluorescence spectroscopy of Sr vapor

NASA Technical Reports Server (NTRS)

Thomann, P.; Burnett, K.; Cooper, J.

1981-01-01

An absorption (and/or emission) event which takes place during a strong collision is called a 'correlated event'. It is discussed how correlated events affect the far red wing depolarization of fluorescence. Attention is given to an atomic vapor which is irradiated by linearly polarized light of a frequency on the red side of the resonance line. Two limiting cases are considered, corresponding to excitation in the impact region and in the quasi-static wing. In the quasi-static wing, absorption of a photon followed by fluorescence (rather than Rayleigh scattering), occurs mostly during a collision. Correlated events dominate the scattering process. Expressions derived for the polarization of the fluorescent light are applied to far red wing depolarization. It is found that the polarization of the fluorescent light does not go to zero in the far wing, but depends crucially on the detailed nature of the anisotropy in the long-range part of the interatomic potential.

Effect of negative mechanical stress on the orientation of myosin cross-bridges in muscle fibers.

PubMed Central

Burghardt, T P; Ajtai, K

1989-01-01

The effect of positive and negative stress on myosin cross-bridge orientation in glycerinated muscle fibers was investigated by using fluorescence polarization spectroscopy of the emission from the covalent label tetramethyl-rhodamine-5-(and -6)-iodoacetamide (IATR) specifically modifying sulfhydryl one (SH1) on the myosin heavy chain. Positive tension was applied by stretching the fiber in rigor. Negative tension was applied in two steps by using a protocol introduced by Goldman et al. [Goldman, Y. E., McCray, J. A. & Vallette, D. P. (1988) J. Physiol. (London) 398, 75P]: relaxing a fiber at resting length and stretching it until the relaxed tension is appreciable and then placing the fiber in rigor and releasing the tension onto the rigor cross-bridges. We found, as have others, that positive tension has no effect on the fluorescence polarization spectrum from the SH1-bound probe, indicating that the cross-bridge does not rotate under these conditions. Negative tension, however, causes a change in the fluorescence polarization spectrum that indicates a probe rotation. The changes in the polarization spectrum from negative stress are partially reversed by the subsequent application of positive stress. It appears that negative tension strains the cross-bridge, or the cross-bridge domain containing SH1, and causes it to rotate. Images PMID:2526336

Plant stress detection by remote measurement of fluorescence

USGS Publications Warehouse

McFarlane, J. C.; Watson, Robert D.; Theisen, Arnold F.; Jackson, R. D.; Ehrler, W. L.; Pinter, P. J.; Idso, S. B.; Reginato, R. J.

1980-01-01

Chlorophyll fluorescence of mature lemon trees was measured with a Fraunhofer line discriminator (FLD). An increase in fluorescence was correlated with plant water stress as measured by stomatal resistance and twig water potential.

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.

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

PubMed

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

2010-12-01

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

A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Fassel, T. A.; Schultz, E.; Greaser, M. L.; Cassens, R. G.

1996-01-01

A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

The Potential Applications of Real-Time Monitoring of Water Quality in a Large Shallow Lake (Lake Taihu, China) Using a Chromophoric Dissolved Organic Matter Fluorescence Sensor

PubMed Central

Niu, Cheng; Zhang, Yunlin; Zhou, Yongqiang; Shi, Kun; Liu, Xiaohan; Qin, Boqiang

2014-01-01

This study presents results from field surveys performed over various seasons in a large, eutrophic, shallow lake (Lake Taihu, China) using an in situ chromophoric dissolved organic matter (CDOM) fluorescence sensor as a surrogate for other water quality parameters. These measurements identified highly significant empirical relationships between CDOM concentration measured using the in situ fluorescence sensor and CDOM absorption, fluorescence, dissolved organic carbon (DOC), chemical oxygen demand (COD) and total phosphorus (TP) concentrations. CDOM concentration expressed in quinine sulfate equivalent units, was highly correlated with the CDOM absorption coefficient (r2 = 0.80, p < 0.001), fluorescence intensities (Ex./Em. 370/460 nm) (r2 = 0.91, p < 0.001), the fluorescence index (r2 = 0.88, p < 0.001) and the humification index (r2 = 0.78, p < 0.001), suggesting that CDOM concentration measured using the in situ fluorescence sensor could act as a substitute for the CDOM absorption coefficient and fluorescence measured in the laboratory. Similarly, CDOM concentration was highly correlated with DOC concentration (r2 = 0.68, p < 0.001), indicating that in situ CDOM fluorescence sensor measurements could be a proxy for DOC concentration. In addition, significant positive correlations were found between laboratory CDOM absorption coefficients and COD (r2 = 0.83, p < 0.001), TP (r2 = 0.82, p < 0.001) concentrations, suggesting a potential further application for the real-time monitoring of water quality using an in situ CDOM fluorescence sensor. PMID:24984060

The potential applications of real-time monitoring of water quality in a large shallow lake (Lake Taihu, China) using a chromophoric dissolved organic matter fluorescence sensor.

PubMed

Niu, Cheng; Zhang, Yunlin; Zhou, Yongqiang; Shi, Kun; Liu, Xiaohan; Qin, Boqiang

2014-06-30

This study presents results from field surveys performed over various seasons in a large, eutrophic, shallow lake (Lake Taihu, China) using an in situ chromophoric dissolved organic matter (CDOM) fluorescence sensor as a surrogate for other water quality parameters. These measurements identified highly significant empirical relationships between CDOM concentration measured using the in situ fluorescence sensor and CDOM absorption, fluorescence, dissolved organic carbon (DOC), chemical oxygen demand (COD) and total phosphorus (TP) concentrations. CDOM concentration expressed in quinine sulfate equivalent units, was highly correlated with the CDOM absorption coefficient (r(2) = 0.80, p < 0.001), fluorescence intensities (Ex./Em. 370/460 nm) (r(2) = 0.91, p < 0.001), the fluorescence index (r(2) = 0.88, p < 0.001) and the humification index (r(2) = 0.78, p < 0.001), suggesting that CDOM concentration measured using the in situ fluorescence sensor could act as a substitute for the CDOM absorption coefficient and fluorescence measured in the laboratory. Similarly, CDOM concentration was highly correlated with DOC concentration (r(2) = 0.68, p < 0.001), indicating that in situ CDOM fluorescence sensor measurements could be a proxy for DOC concentration. In addition, significant positive correlations were found between laboratory CDOM absorption coefficients and COD (r(2) = 0.83, p < 0.001), TP (r(2) = 0.82, p < 0.001) concentrations, suggesting a potential further application for the real-time monitoring of water quality using an in situ CDOM fluorescence sensor.

Developing LED UV fluorescence sensors for online monitoring DOM and predicting DBPs formation potential during water treatment.

PubMed

Li, Wen-Tao; Jin, Jing; Li, Qiang; Wu, Chen-Fei; Lu, Hai; Zhou, Qing; Li, Ai-Min

2016-04-15

Online monitoring dissolved organic matter (DOM) is urgent for water treatment management. In this study, high performance size exclusion chromatography with multi-UV absorbance and multi-emission fluorescence scans were applied to spectrally characterize samples from 16 drinking water sources across Yangzi River and Huai River Watersheds. The UV absorbance indices at 254 nm and 280 nm referred to the same DOM components and concentration, and the 280 nm UV light could excite both protein-like and humic-like fluorescence. Hence a novel UV fluorescence sensor was developed out using only one UV280 light-emitting diode (LED) as light source. For all samples, enhanced coagulation was mainly effective for large molecular weight biopolymers; while anion exchange further substantially removed humic substances. During chlorination tests, UVA280 and UVA254 showed similar correlations with yields of disinfection byproducts (DBPs); the humic-like fluorescence obtained from LED sensors correlated well with both trihalomethanes and haloacetic acids yields, while the correlation between protein-like fluorescence and trihalomethanes was relatively poor. Anion exchange exhibited more reduction of DBPs yields as well as UV absorbance and fluorescence signals than enhanced coagulation. The results suggest that the LED UV fluorescence sensors are very promising for online monitoring DOM and predicting DBPs formation potential during water treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fabrication and biological imaging of polyhedral oligomeric silsesquioxane cross-linked fluorescent polymeric nanoparticles with aggregation-induced emission feature

NASA Astrophysics Data System (ADS)

Mao, Liucheng; Liu, Meiying; Xu, Dazhuang; Wan, Qing; Huang, Qiang; Jiang, Ruming; Shi, Yingge; Deng, Fengjie; Zhang, Xiaoyong; Wei, Yen

2017-11-01

Aggregation-induced emission (AIE) dyes based fluorescent polymeric nanoparticles (FNPs) have been intensively explored for biomedical applications. However, many of these AIE-active FNPs are relied on the self-assembly of amphiphilic copolymers, which are not stable in diluted solution. Therefore, the introduction of cross-linkages into these micelles has demonstrated to be an efficient route to overcome this stability problem and endow ultra-low critical micelle concentrations (CMC) of these AIE-active FNPs. In this work, we reported the fabrication of cross-linked AIE-active FNPs through controllable reversible addition fragmentation chain transfer polymerization by using commercially available octavinyl-T8-silsesquioxane (8-vinyl POSS) as the cross-linkage for the first time. The resultant cross-linked amphiphilic copolymers (named as PEG-POSS-PhE) are prone to self-assemble into stable core-shell nanoparticles with well water dispersity, strong red fluorescence and low CMC (0.0069 mg mL-1) in aqueous solution. More importantly, PEG-POSS-PhE FNPs possess some other properties such as high water dispersity, uniform morphology and small size, excellent biocompatibility and cellular internalization, providing great potential of PEG-POSS-PhE FNPs for biological imaging application.

Experimental Verification of the Individual Energy Dependencies of the Partial L-Shell Photoionization Cross Sections of Pd and Mo

NASA Astrophysics Data System (ADS)

Hönicke, Philipp; Kolbe, Michael; Müller, Matthias; Mantler, Michael; Krämer, Markus; Beckhoff, Burkhard

2014-10-01

An experimental method for the verification of the individually different energy dependencies of L1-, L2-, and L3- subshell photoionization cross sections is described. The results obtained for Pd and Mo are well in line with theory regarding both energy dependency and absolute values, and confirm the theoretically calculated cross sections by Scofield from the early 1970 s and, partially, more recent data by Trzhaskovskaya, Nefedov, and Yarzhemsky. The data also demonstrate the questionability of quantitative x-ray spectroscopical results based on the widely used fixed jump ratio approximated cross sections with energy independent ratios. The experiments are carried out by employing the radiometrically calibrated instrumentation of the Physikalisch-Technische Bundesanstalt at the electron storage ring BESSY II in Berlin; the obtained fluorescent intensities are thereby calibrated at an absolute level in reference to the International System of Units. Experimentally determined fixed fluorescence line ratios for each subshell are used for a reliable deconvolution of overlapping fluorescence lines. The relevant fundamental parameters of Mo and Pd are also determined experimentally in order to calculate the subshell photoionization cross sections independently of any database.

Correlative cryogenic tomography of cells using light and soft x-rays

PubMed Central

Smith, Elizabeth A.; Cinquin, Bertrand P.; Do, Myan; McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

2013-01-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryo-preserved state. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited to correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. PMID:24355261

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.

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

PubMed

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

2017-02-01

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

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

PubMed

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

2017-03-02

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

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

PubMed Central

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

2017-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2011-07-01

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

Dynamics and Size of Cross-Linking-Induced Lipid Nanodomains in Model Membranes

PubMed Central

Štefl, Martin; Šachl, Radek; Humpolíčková, Jana; Cebecauer, Marek; Macháň, Radek; Kolářová, Marie; Johansson, Lennart B.-Å.; Hof, Martin

2012-01-01

Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ∼8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation. PMID:22824274

Plasma Membrane Sterol Distribution Resembles the Surface Topography of Living Cells

PubMed Central

2007-01-01

Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8–2.5 μm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1—a putative raft marker—did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane. PMID:17065557

Measurement of protein-like fluorescence in river and waste water using a handheld spectrophotometer.

PubMed

Baker, Andy; Ward, David; Lieten, Shakti H; Periera, Ryan; Simpson, Ellie C; Slater, Malcolm

2004-07-01

Protein-like fluorescence intensity in rivers increases with increasing anthropogenic DOM inputs from sewerage and farm wastes. Here, a portable luminescence spectrophotometer was used to investigate if this technology could be used to provide both field scientists with a rapid pollution monitoring tool and process control engineers with a portable waste water monitoring device, through the measurement of river and waste water tryptophan-like fluorescence from a range of rivers in NE England and from effluents from within two waste water treatment plants. The portable spectrophotometer determined that waste waters and sewerage effluents had the highest tryptophan-like fluorescence intensity, urban streams had an intermediate tryptophan-like fluorescence intensity, and the upstream river samples of good water quality the lowest tryptophan-like fluorescence intensity. Replicate samples demonstrated that fluorescence intensity is reproducible to +/- 20% for low fluorescence, 'clean' river water samples and +/- 5% for urban water and waste waters. Correlations between fluorescence measured by the portable spectrophotometer with a conventional bench machine were 0.91; (Spearman's rho, n = 143), demonstrating that the portable spectrophotometer does correlate with tryptophan-like fluorescence intensity measured using the bench spectrophotometer.

Photonic reagents for concentration measurement of flu-orescent proteins with overlapping spectra

NASA Astrophysics Data System (ADS)

Goun, Alexei; Bondar, Denys I.; Er, Ali O.; Quine, Zachary; Rabitz, Herschel A.

2016-05-01

By exploiting photonic reagents (i.e., coherent control by shaped laser pulses), we employ Optimal Dynamic Discrimination (ODD) as a novel means for quantitatively characterizing mixtures of fluorescent proteins with a large spectral overlap. To illustrate ODD, we simultaneously measured concentrations of in vitro mixtures of Enhanced Blue Fluorescent Protein (EBFP) and Enhanced Cyan Fluorescent Protein (ECFP). Building on this foundational study, the ultimate goal is to exploit the capabilities of ODD for parallel monitoring of genetic and protein circuits by suppressing the spectral cross-talk among multiple fluorescent reporters.

Differentiation of Cariogenic Streptococci by Fluorescent Antibody1

PubMed Central

Jablon, James M.; Zinner, Doran D.

1966-01-01

Jablon, J. M. (University of Miami, Miami, Fla.), and D. D. Zinner. Differentiation of cariogenic streptococci by fluorescent antibody. J. Bacteriol. 92:1590–1596. 1966.—Eight strains of streptococci were isolated from human carious lesions by the fluorescent-antibody (FA) technique. Seven of these strains produced experimental caries in hamsters or rats maintained on a high sucrose diet. The eighth strain was noncariogenic in animals but possessed some antigenic components in common with the cariogenic strains. On the basis of antigen-antibody reactions by microprecipitin and agar-gel diffusion patterns, the strains were divided into four groups; these groups differed with regard to their cariogenic activity in hamsters. Fluorescein-conjugated antisera, prepared against the human strains, showed some cross-reactions which interfered with the efficacy of the FA technique in differentiating between the related streptococcal groups. To eliminate these cross-reactions, a small amount of related-strain antisera was added to the fluorescein-conjugated antisera to the cariogenic strains. This technique is effective in blocking cross-reactions and should be tried wherever cross-reactions are encountered in the FA technique. Images PMID:5334765

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

PubMed

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

2014-01-01

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

Synchrotron radiation analysis of possible correlations between metal status in human cementum and periodontal disease

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

Martin, R.R.; Naftel, S.J.; Nelson, A.J.

2010-03-16

Periodontitis is a serious disease that affects up to 50% of an adult population. It is a chronic condition involving inflammation of the periodontal ligament and associated tissues leading to eventual tooth loss. Some evidence suggests that trace metals, especially zinc and copper, may be involved in the onset and severity of periodontitis. Thus we have used synchrotron X-ray fluorescence imaging on cross sections of diseased and healthy teeth using a microbeam to explore the distribution of trace metals in cementum and adhering plaque. The comparison between diseased and healthy teeth indicates that there are elevated levels of zinc, coppermore » and nickel in diseased teeth as opposed to healthy teeth. This preliminary correlation between elevated levels of trace metals in the cementum and plaque of diseased teeth suggests that metals may play a role in the progress of periodontitis.« less

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

PubMed

Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

2017-04-03

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A theoretical investigation of two typical two-photon pH fluorescent probes.

PubMed

Xu, Zhong; Ren, Ai-Min; Guo, Jing-Fu; Liu, Xiao-Ting; Huang, Shuang; Feng, Ji-Kang

2013-01-01

Intracellular pH plays an important role in many cellular events, such as cell growth, endocytosis, cell adhesion and so on. Some pH fluorescent probes have been reported, but most of them are one-photon fluorescent probes, studies about two-photon fluorescent probes are very rare. In this work, the geometrical structure, electronic structure and one-photon properties of a series of two-photon pH fluorescent probes have been theoretically studied by using density functional theory (DFT) method. Their two-photon absorption (TPA) properties are calculated using the method of ZINDO/sum-over-states method. Two types of two-photon pH fluorescent probes have been investigated by theoretical methods. The mechanisms of the Photoinduced Charge Transfer (PCT) probes and the Photoinduced Electron Transfer (PET) probes are verified specifically. Some designed strategies of good two-photon pH fluorescent probes are suggested on the basis of the investigated results of two mechanisms. For the PCT probes, substituting a stronger electron-donating group for the terminal methoxyl group is an advisable choice to increase the TPA cross section. For the PET probes, the TPA cross sections increase upon protonation. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Fluorescence correlation spectroscopy: the case of subdiffusion.

PubMed

Lubelski, Ariel; Klafter, Joseph

2009-03-18

The theory of fluorescence correlation spectroscopy is revisited here for the case of subdiffusing molecules. Subdiffusion is assumed to stem from a continuous-time random walk process with a fat-tailed distribution of waiting times and can therefore be formulated in terms of a fractional diffusion equation (FDE). The FDE plays the central role in developing the fluorescence correlation spectroscopy expressions, analogous to the role played by the simple diffusion equation for regular systems. Due to the nonstationary nature of the continuous-time random walk/FDE, some interesting properties emerge that are amenable to experimental verification and may help in discriminating among subdiffusion mechanisms. In particular, the current approach predicts 1), a strong dependence of correlation functions on the initial time (aging); 2), sensitivity of correlation functions to the averaging procedure, ensemble versus time averaging (ergodicity breaking); and 3), that the basic mean-squared displacement observable depends on how the mean is taken.

Protein recognition by a pattern-generating fluorescent molecular probe.

PubMed

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Protein recognition by a pattern-generating fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M.; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M.; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Determination of Cancer Cell-Based pH-Sensitive Fluorescent Carbon Nanoparticles of Cross-Linked Polydopamine by Fluorescence Sensing of Alkaline Phosphatase Activity on Coated Surfaces and Aqueous Solution.

PubMed

Kang, Eun Bi; Choi, Cheong A; Mazrad, Zihnil Adha Islamy; Kim, Sung Han; In, Insik; Park, Sung Young

2017-12-19

The tumor-specific sensitive fluorescence sensing of cellular alkaline phosphatase (ALP) activity on the basis of host-guest specific and pH sensitivity was conducted on coated surfaces and aqueous states. Cross-linked fluorescent nanoparticles (C-FNP) consisting of β-cyclodextrin (β-CD)/boronic acid (BA) and fluorescent hyaluronic acid [FNP(HA)] were conjugated to fluorescent polydopamine [FNP(pDA)]. To determine the quenching effect of this system, hydrolysis of 4-nitrophenyl phosphate (NPP) to 4-nitrophenol (NP) was performed in the cavity of β-CD in the presence of ALP activated photoinduced electron transfer (PET) between NP and C-FNP. At an ALP level of 30-1000 U/L, NP caused off-emission of C-FNP because of their specific host-guest recognition. Fluorescence can be recovered under pH shock due to cleavage of the diol bond between β-CD and BA, resulting in release of NP from the fluorescent system. Sensitivity of the assays was assessed by confocal imaging not only in aqueous states, but also for the first time on coated surfaces in MDAMB-231 and MDCK cells. This novel system demonstrated high sensitivity to ALP through generation of good electron donor/acceptor pair during the PET process. Therefore, this fluorescence sensor system can be used to enhance ALP monitoring and cancer diagnosis on both coated surfaces and in aqueous states in clinical settings.

Fluorescence-based characterization of genetically encoded peptides that fold in live cells: progress toward a generic hairpin scaffold

NASA Astrophysics Data System (ADS)

Cheng, Zihao; Campbell, Robert E.

2007-02-01

Binding proteins suitable for expression and high affinity molecular recognition in the cytoplasm or nucleus of live cells have numerous applications in the biological sciences. In an effort to add a new minimal motif to the growing repertoire of validated non-immunoglobulin binding proteins, we have undertaken the development of a generic protein scaffold based on a single β-hairpin that can fold efficiently in the cytoplasm. We have developed a method, based on the measurement of fluorescence resonance energy transfer (FRET) between a genetically fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), that allows the structural stability of recombinant β-hairpin peptides to be rapidly assessed both in vitro and in vivo. We have previously reported the validation of this method when applied to a 16mer tryptophan zipper β-hairpin. We now describe the use of this method to evaluate the potential of a designed 20mer β-hairpin peptide with a 3rd Trp/Trp cross-strand pair to function as a generic protein scaffold. Quantitative analysis of the FRET efficiency, resistance to proteolysis (assayed by loss of FRET), and circular dichroism spectra revealed that the 20mer peptide is significantly more tolerant of destabilizing mutations than the 16mer peptide. Furthermore, we experimentally demonstrate that the in vitro determined β-hairpin stabilities are well correlated with in vivo β-hairpin stabilities as determined by FRET measurements of colonies of live bacteria expressing the recombinant peptides flanked by CFP and YFP. Finally, we report on our progress to develop highly folded 24mer and 28mer β-hairpin peptides through the use of fluorescence-based library screening.

"Sizing" the oligomers of Azami Green fluorescent protein with FCS and antibunching

NASA Astrophysics Data System (ADS)

Temirov, Jamshid; Werner, James H.; Goodwin, Peter M.; Bradbury, Andrew R. M.

2012-02-01

Fluorescent proteins are invaluable molecules in fluorescence microscopy and spectroscopy. The size and brightness of fluorescent proteins often dictates the application they may be used for. While a monomeric protein may be the least perturbative structure for labeling a protein in a cell, often oligomers (dimers and tetramers) of fluorescent proteins can be more stable. However, from a quantitative microscopy standpoint, it is important to realize the photophysical properties of monomers do not necessarily multiply by their number when they form oligomers. In this work we studied oligomerization states of the Azami Green (AG) protein with fluorescence correlation spectroscopy (FCS) and photon antibunching or photon pair correlation spectroscopy (PPCS). FCS was used to measure the hydrodynamic size of the oligomers, whereas antibunching was used to count the number of fluorescent emitters in the oligomers. The results exhibited that the dimers of AG were single emitters and the tetramers were dual-emitters, indicative of dipole-dipole interactions and energy transfer between the monomeric units. We also used these methods to estimate the number of fluorescent proteins displayed on T7 phage molecules.

Photochemical synthesis and photophysical properties of coumarins bearing extended polyaromatic rings studied by emission and transient absorption measurements.

PubMed

Yamaji, Minoru; Hakoda, Yuma; Okamoto, Hideki; Tani, Fumito

2017-04-12

We prepared a variety of coumarin derivatives having expanded π-electron systems along the direction crossing the C 3 -C 4 bond of the coumarin skeleton via a photochemical cyclization process and investigated their photophysical features as a function of the number (n) of the added benzene rings based on emission and transient absorption measurements. Upon increasing n, the fluorescence quantum yields of the π-extended coumarins increased. Expanding the π-electron system on the C 3 -C 4 bond of the coumarin skeleton was found to be efficient for increasing the fluorescence ability more than that on the C 7 -C 8 bond. Introducing the methoxy group at the 7-position was also efficient for enhancing the fluorescence quantum yield and rate of the expanded coumarins. The non-radiative process from the fluorescence state was not substantially influenced by the expanded π-electron system. The competitive process with the fluorescence was found to be intersystem crossing to the triplet state based on the observations of the triplet-triplet absorption. The effects of the expanded π-electron systems on the fluorescence ability were investigated with the aid of TD-DFT calculations.

A series of fluorene-based two-photon absorbing molecules: synthesis, linear and nonlinear characterization, and bioimaging

PubMed Central

Andrade, Carolina D.; Yanez, Ciceron O.; Rodriguez, Luis; Belfield, Kevin D.

2010-01-01

The synthesis, structural, and photophysical characterization of a series of new fluorescent donor–acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluorescent quantum yields, and, significantly, high two-photon absorption cross sections, making them well suited for two-photon fluorescence microscopy (2PFM) imaging. Confocal and two-photon fluorescence microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selectivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence microscopy. PMID:20481596

Steady-state chlorophyll fluorescence (Fs) measurements as a tool to follow variations of net CO2 assimilation and stomatal conductance during water-stress in C3 plants.

PubMed

Flexas, Jaume; Escalona, José Mariano; Evain, Sebastian; Gulías, Javier; Moya, Ismaël; Osmond, Charles Barry; Medrano, Hipólito

2002-02-01

Water stress experiments were performed with grapevines (Vitis vinifera L.) and other C3 plants in the field, in potted plants in the laboratory, and with detached leaves. It was found that, in all cases, the ratio of steady state chlorophyll fluorescence (Fs) normalized to dark-adapted intrinsic fluorescence (Fo) inversely correlated with non-photochemical quenching (NPQ). Also, at high irradiance, the ratio Fs/Fo was positively correlated with CO2 assimilation in air, with electron transport rate calculated from fluorescence, and with stomatal conductance, but no clear correlation was observed with qP. The significance of these relationships is discussed. The ratio Fs/Fo, measured with a portable instrument (PAM-2000) or with a remote sensing FIPAM system, provides a good method for the early detection of water stress, and may become a useful guide to irrigation requirements.

Spatio-energetic cross talk in photon counting detectors: Detector model and correlated Poisson data generator.

PubMed

Taguchi, Katsuyuki; Polster, Christoph; Lee, Okkyun; Stierstorfer, Karl; Kappler, Steffen

2016-12-01

An x-ray photon interacts with photon counting detectors (PCDs) and generates an electron charge cloud or multiple clouds. The clouds (thus, the photon energy) may be split between two adjacent PCD pixels when the interaction occurs near pixel boundaries, producing a count at both of the pixels. This is called double-counting with charge sharing. (A photoelectric effect with K-shell fluorescence x-ray emission would result in double-counting as well). As a result, PCD data are spatially and energetically correlated, although the output of individual PCD pixels is Poisson distributed. Major problems include the lack of a detector noise model for the spatio-energetic cross talk and lack of a computationally efficient simulation tool for generating correlated Poisson data. A Monte Carlo (MC) simulation can accurately simulate these phenomena and produce noisy data; however, it is not computationally efficient. In this study, the authors developed a new detector model and implemented it in an efficient software simulator that uses a Poisson random number generator to produce correlated noisy integer counts. The detector model takes the following effects into account: (1) detection efficiency; (2) incomplete charge collection and ballistic effect; (3) interaction with PCDs via photoelectric effect (with or without K-shell fluorescence x-ray emission, which may escape from the PCDs or be reabsorbed); and (4) electronic noise. The correlation was modeled by using these two simplifying assumptions: energy conservation and mutual exclusiveness. The mutual exclusiveness is that no more than two pixels measure energy from one photon. The effect of model parameters has been studied and results were compared with MC simulations. The agreement, with respect to the spectrum, was evaluated using the reduced χ 2 statistics or a weighted sum of squared errors, χ red 2 (≥1), where χ red 2 =1 indicates a perfect fit. The model produced spectra with flat field irradiation that qualitatively agree with previous studies. The spectra generated with different model and geometry parameters allowed for understanding the effect of the parameters on the spectrum and the correlation of data. The agreement between the model and MC data was very strong. The mean spectra with 90 keV and 140 kVp agreed exceptionally well: χ red 2 values were 1.049 with 90 keV data and 1.007 with 140 kVp data. The degrees of cross talk (in terms of the relative increase from single pixel irradiation to flat field irradiation) were 22% with 90 keV and 19% with 140 kVp for MC simulations, while they were 21% and 17%, respectively, for the model. The covariance was in strong agreement qualitatively, although it was overestimated. The noisy data generation was very efficient, taking less than a CPU minute as opposed to CPU hours for MC simulators. The authors have developed a novel, computationally efficient PCD model that takes into account double-counting and resulting spatio-energetic correlation between PCD pixels. The MC simulation validated the accuracy.

Application of normal fluorescence and stability-indicating derivative synchronous fluorescence spectroscopy for the determination of gliquidone in presence of its fluorescent alkaline degradation product

NASA Astrophysics Data System (ADS)

El-ghobashy, Mohamed R.; Yehia, Ali M.; Helmy, Aya H.; Youssef, Nadia F.

2018-01-01

Simple, smart and sensitive normal fluorescence and stability-indicating derivative synchronous spectrofluorimetric methods have been developed and validated for the determination of gliquidone in the drug substance and drug product. Normal spectrofluorimetric method of gliquidone was established in methanol at λ excitation 225 nm and λ emission 400 nm in concentration range 0.2-3 μg/ml with LOD equal 0.028. The fluorescence quantum yield of gliquidone was calculated using quinine sulfate as a reference and found to be 0.542. Stability-indicating first and third derivative synchronous fluorescence spectroscopy were successfully utilized to overcome the overlapped spectra in normal fluorescence of gliquidone and its alkaline degradation product. Derivative synchronous methods are based on using the synchronous fluorescence of gliquidone and its degradation product in methanol at Δ λ50 nm. Peak amplitude in the first derivative of synchronous fluorescence spectra was measured at 309 nm where degradation product showed zero-crossing without interference. The peak amplitudes in the third derivative of synchronous fluorescence spectra, peak to trough were measured at 316,329 nm where degradation product showed zero-crossing. The different experimental parameters affecting the normal and synchronous fluorescence intensity of gliquidone were studied and optimized. Moreover, the cited methods have been validated as per ICH guidelines. The peak amplitude-concentration plots of the derivative synchronous fluorescence were linear over the concentration range 0.05-2 μg/ml for gliquidone. Limits of detection were 0.020 and 0.022 in first and third derivative synchronous spectra, respectively. The adopted methods were successfully applied to commercial tablets and the results demonstrated that the derivative synchronous fluorescence spectroscopy is a powerful stability-indicating method, suitable for routine use with a short analysis time. Statistical comparison between the results obtained by normal fluorescence and derivative synchronous methods and the official one using student's t-test and F-ratio showed no significant difference regarding accuracy and precision.

k-Space Image Correlation Spectroscopy: A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics

PubMed Central

Kolin, David L.; Ronis, David; Wiseman, Paul W.

2006-01-01

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or “blinking”. Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of α5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection. PMID:16861272

Spectroscopic techniques to study the immune response in human saliva

NASA Astrophysics Data System (ADS)

Nepomnyashchaya, E.; Savchenko, E.; Velichko, E.; Bogomaz, T.; Aksenov, E.

2018-01-01

Studies of the immune response dynamics by means of spectroscopic techniques, i.e., laser correlation spectroscopy and fluorescence spectroscopy, are described. The laser correlation spectroscopy is aimed at measuring sizes of particles in biological fluids. The fluorescence spectroscopy allows studying of the conformational and other structural changings in immune complex. We have developed a new scheme of a laser correlation spectrometer and an original signal processing algorithm. We have suggested a new fluorescence detection scheme based on a prism and an integrating pin diode. The developed system based on the spectroscopic techniques allows studies of complex process in human saliva and opens some prospects for an individual treatment of immune diseases.

Alexandrite as a high-temperature pressure calibrant, and implications for the ruby-fluorescence scale

NASA Technical Reports Server (NTRS)

Jahren, A. H.; Kruger, M. B.; Jeanloz, Raymond

1992-01-01

The wavelength shifts of the R1 and R2 fluorescence lines of alexandrite (BeAl2O4:Cr(+3)) have been experimentally calibrated against the ruby-fluorescence scale as a function of both hydrostatic and nonhydrostatic pressures between 0 and 50 GPa, and simultaneously as a function of temperatures between 290 and 550 K. It is found that the pressure-temperature cross derivative of the fluorescence wavelength shifts are negligible for both ruby and alexandrite.

Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

NASA Astrophysics Data System (ADS)

Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

2015-05-01

We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

Viscosity-dependent diffusion of fluorescent particles using fluorescence correlation spectroscopy.

PubMed

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

2014-11-01

Fluorescent particles show the variety characteristics by the interaction with other particles and solvent. In order to investigate the relationship between the dynamic properties of fluorescent particles and solvent viscosity, particle diffusion in various solvents was evaluated using a fluorescence correlation spectroscopy. Upon analyzing the correlation functions of AF-647, Q-dot, and beads with different viscosity values, the diffusion time of all particles was observed to increase with increasing solvent viscosity, and the ratio of diffusion time to solvent viscosity, τ D /η, showed a linear dependence on particle size. The particle diffusion coefficients calculated from the diffusion time decreased with increasing solvent viscosity. Further, the hydrodynamic radii of AF-647, Q-dot, and beads were 0.98 ± 0.1 nm, 64.8 ± 3.23 nm, and 89.8 ± 4.91 nm, respectively, revealing a linear dependence on τ D /η, which suggests that the hydrodynamic radius of a particle strongly depends on both the physical size of the particle and solvent viscosity.

Validation of photosynthetic-fluorescence parameters as biomarkers for isoproturon toxic effect on alga Scenedesmus obliquus.

PubMed

Dewez, David; Didur, Olivier; Vincent-Héroux, Jonathan; Popovic, Radovan

2008-01-01

Photosynthetic-fluorescence parameters were investigated to be used as valid biomarkers of toxicity when alga Scenedesmus obliquus was exposed to isoproturon [3-(4-isopropylphenyl)-1,1-dimethylurea] effect. Chlorophyll fluorescence induction of algal cells treated with isoproturon showed inactivation of photosystem II (PSII) reaction centers and strong inhibition of PSII electron transport. A linear correlation was found (R2>or=0.861) between the change of cells density affected by isoproturon and the change of effective PSII quantum yield (PhiM'), photochemical quenching (qP) and relative photochemical quenching (qP(rel)) values. The cells density was also linearly dependent (R2=0.838) on the relative unquenched fluorescence parameter (UQF(rel)). Non-linear correlation was found (R2=0.937) only between cells density and the energy transfer efficiency from absorbed light to PSII reaction center (ABS/RC). The order of sensitivity determined by the EC-50% was: UQF(rel)>PhiM'>qP>qP(rel)>ABS/RC. Correlations between cells density and those photosynthetic-fluorescence parameters provide supporting evidence to use them as biomarkers of toxicity for environmental pollutants.

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

NASA Astrophysics Data System (ADS)

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

2006-10-01

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

[Flag leaf photosynthetic characteristics, change in chlorophyll fluorescence parameters, and their relationships with yield of winter wheat sowed in spring].

PubMed

Xu, Lan; Gao, Zhi-qang; An, Wei; Li, Yan-liang; Jiao, Xiong-fei; Wang, Chuang-yun

2016-01-01

With five good winter wheat cultivars selected from the middle and lower reaches of Yangtze River and Southwest China as test materials, a field experiment in Xinding basin area of Shanxi Province was conducted to study the photosynthetic characteristics, chlorophyll content, and chlorophyll fluorescence parameters of flag leaf at different sowing dates, as well as the correlations between these indices and yield for two years (2013-2014). The results showed that the difference in most fluorescence parameters except chlorophyll content among cultivars was significant. The correlations between these fluorescence parameters and yield were significant. The variation coefficient of chlorophyll (Chl) content was low (0.12-0.17), and that of performance index based on absorption (PIabs) was high (0.32-0.39), with the partial correlation coefficients of them with grain yield from 2013 to 2014 ranged in 0.70-0.81. Under the early sowing condition, the grain yield positively correlated with PIabs at flowering and filling stages and chlorophyll content at grain filling stage, but negatively correlated with the relative variable fluorescence at I point (Vi) at grain filling stage. About 81.1%-82.8% of grain yield were determined by the variations of PIabs, Chl, and Vi. Wheat cultivars had various performances in the treatments with different sowing dates and a consistent trend was observed in the two experimental years. Among these 5 cultivars, Yangmai 13 was suitable for early sowing, with the flag leaf photosynthetic rate (Pn), Chl, most fluorescence parame-ters, and grain yield showed obviously high levels. In conclusion, under early sowing condition chlorophyll content at grain filling stages, PIabs at flowering and filling stages, and Pn were important indices for selecting wheat cultivars with high photosynthetic efficiency.

Correlative cryogenic tomography of cells using light and soft x-rays.

PubMed

Smith, Elizabeth A; Cinquin, Bertrand P; Do, Myan; McDermott, Gerry; Le Gros, Mark A; Larabell, Carolyn A

2014-08-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited for correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. © 2013 Elsevier B.V. All rights reserved.

Entangled-photon coincidence fluorescence imaging

PubMed Central

Scarcelli, Giuliano; Yun, Seok H.

2009-01-01

We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as “detectors” breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations. PMID:18825257

UV-Vis Ratiometric Resonance Synchronous Spectroscopy for Determination of Nanoparticle and Molecular Optical Cross Sections.

PubMed

Nettles, Charles B; Zhou, Yadong; Zou, Shengli; Zhang, Dongmao

2016-03-01

Demonstrated herein is a UV-vis Ratiometric Resonance Synchronous Spectroscopic (R2S2, pronounced as "R-two-S-two" for simplicity) technique where the R2S2 spectrum is obtained by dividing the resonance synchronous spectrum of a NP-containing solution by the solvent resonance synchronous spectrum. Combined with conventional UV-vis measurements, this R2S2 method enables experimental quantification of the absolute optical cross sections for a wide range of molecular and nanoparticle (NP) materials that range optically from pure photon absorbers or scatterers to simultaneous photon absorbers and scatterers, simultaneous photon absorbers and emitters, and all the way to simultaneous photon absorbers, scatterers, and emitters in the UV-vis wavelength region. Example applications of this R2S2 method were demonstrated for quantifying the Rayleigh scattering cross sections of solvents including water and toluene, absorption and resonance light scattering cross sections for plasmonic gold nanoparticles, and absorption, scattering, and on-resonance fluorescence cross sections for semiconductor quantum dots (Qdots). On-resonance fluorescence quantum yields were quantified for the model molecular fluorophore Eosin Y and fluorescent Qdots CdSe and CdSe/ZnS. The insights and methodology presented in this work should be of broad significance in physical and biological science research that involves photon/matter interactions.

Photonic reagents for concentration measurement of flu-orescent proteins with overlapping spectra

PubMed Central

Goun, Alexei; Bondar, Denys I.; Er, Ali O.; Quine, Zachary; Rabitz, Herschel A.

2016-01-01

By exploiting photonic reagents (i.e., coherent control by shaped laser pulses), we employ Optimal Dynamic Discrimination (ODD) as a novel means for quantitatively characterizing mixtures of fluorescent proteins with a large spectral overlap. To illustrate ODD, we simultaneously measured concentrations of in vitro mixtures of Enhanced Blue Fluorescent Protein (EBFP) and Enhanced Cyan Fluorescent Protein (ECFP). Building on this foundational study, the ultimate goal is to exploit the capabilities of ODD for parallel monitoring of genetic and protein circuits by suppressing the spectral cross-talk among multiple fluorescent reporters. PMID:27181496

Identification of algae which interfere with the detection of Giardia cysts and Cryptosporidium oocysts and a method for alleviating this interference.

PubMed Central

Rodgers, M R; Flanigan, D J; Jakubowski, W

1995-01-01

Fifty-four algal species were tested for cross-reaction in the American Society for Testing and Materials Giardia/Cryptosporidium indirect immunofluorescence assay, and 24 showed some degree of fluorescence. Two species, Navicula minima and Synechococcus elongatus, exhibited a bright apple green fluorescence. The addition of goat serum to the assay mixture blocked the fluorescence of most nontarget organisms tested and also decreased the background fluorescence. Goat serum did not interfere with the fluorescence of Giardia cysts or Cryptosporidium oocysts or the identification of cyst and oocyst internal structures. PMID:7487013

Novel xenon calibration scheme for two-photon absorption laser induced fluorescence of hydrogen

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

Elliott, Drew; Scime, Earl; Short, Zachary, E-mail: zdshort@mix.wvu.edu

Two photon absorption laser induced fluorescence (TALIF) measurements of neutral hydrogen and its isotopes are typically calibrated by performing TALIF measurements on krypton with the same diagnostic system and using the known ratio of the absorption cross sections [K. Niemi et al., J. Phys. D 34, 2330 (2001)]. Here we present the measurements of a new calibration method based on a ground state xenon scheme for which the fluorescent emission wavelength is nearly identical to that of hydrogen, thereby eliminating chromatic effects in the collection optics and simplifying detector calibration. We determine that the ratio of the TALIF cross sectionsmore » of xenon and hydrogen is 0.024 ± 0.001.« less

Optical and Scintillation Properties of Polydimethyl-Diphenylsiloxane Based Organic Scintillators

NASA Astrophysics Data System (ADS)

Quaranta, Alberto; Carturan, Sara Maria; Marchi, Tommaso; Kravchuk, Vladimir L.; Gramegna, Fabiana; Maggioni, Gianluigi; Degerlier, Meltem

2010-04-01

Polysiloxane based scintillators with high light yield have been synthesized. The polymer consists in cross-linked polydimethyl-co-diphenylsiloxane with different molar percentages of phenyl units. 2,5-diphenyl oxazole (PPO) and 2,5-bis(5-ter-butyl-2-benzoxazolyl)thiophene (BBOT) have been dispersed in the polymer as dopants. The energy transfer and scintillation capabilities have been investigated, for two different amounts of phenyl groups in the polymer network and for different concentrations of dye molecules, by means of fluorescence spectroscopy, ion beam induced luminescence (IBIL) and scintillation yield measurements with ¿ particles from an 241Am source. The luminescence features and the scintillation yields have been correlated to the composition of the scintillators.

Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation

PubMed Central

Berezin, Mikhail; Zhan, Chun; Lee, Hyeran; Joo, Chulmin; Akers, Walter; Yazdanfar, Siavash; Achilefu, Samuel

2011-01-01

Two-photon (2P) optical properties of cyanine dyes were evaluated using a 2P fluorescence spectrophotometer with 1.55 μm excitation. We report the 2P characteristics of common NIR polymethine dyes, including their 2P action cross-sections and the 2P excited fluorescence lifetime. One of the dyes, DTTC showed the highest 2P action cross-section (~103 ± 19 GM) and relatively high 2P excited fluorescence lifetime and can be used as a scaffold for the synthesis of 2P molecular imaging probes. The 2P action cross-section of DTTC and the lifetime were also highly sensitive to the solvent polarity, providing other additional parameters for its use in optical imaging and the mechanism for probing environmental factors Overall, this study demonstrated the quantitative measurement of 2P properties of NIR dyes and established the foundation for designing molecular probes for 2P imaging applications in the NIR region. PMID:21866928

Dynamic cross-correlations between entangled biofilaments as they diffuse

PubMed Central

Tsang, Boyce; Dell, Zachary E.; Jiang, Lingxiang; Schweizer, Kenneth S.; Granick, Steve

2017-01-01

Entanglement in polymer and biological physics involves a state in which linear interthreaded macromolecules in isotropic liquids diffuse in a spatially anisotropic manner beyond a characteristic mesoscopic time and length scale (tube diameter). The physical reason is that linear macromolecules become transiently localized in directions transverse to their backbone but diffuse with relative ease parallel to it. Within the resulting broad spectrum of relaxation times there is an extended period before the longest relaxation time when filaments occupy a time-averaged cylindrical space of near-constant density. Here we show its implication with experiments based on fluorescence tracking of dilutely labeled macromolecules. The entangled pairs of aqueous F-actin biofilaments diffuse with separation-dependent dynamic cross-correlations that exceed those expected from continuum hydrodynamics up to strikingly large spatial distances of ≈15 µm, which is more than 104 times the size of the solvent water molecules in which they are dissolved, and is more than 50 times the dynamic tube diameter, but is almost equal to the filament length. Modeling this entangled system as a collection of rigid rods, we present a statistical mechanical theory that predicts these long-range dynamic correlations as an emergent consequence of an effective long-range interpolymer repulsion due to the de Gennes correlation hole, which is a combined consequence of chain connectivity and uncrossability. The key physical assumption needed to make theory and experiment agree is that solutions of entangled biofilaments localized in tubes that are effectively dynamically incompressible over the relevant intermediate time and length scales. PMID:28283664

Pancreatic tissue assessment using fluorescence and reflectance spectroscopy

NASA Astrophysics Data System (ADS)

Chandra, Malavika; Heidt, David; Simeone, Diane; McKenna, Barbara; Scheiman, James; Mycek, Mary-Ann

2007-07-01

The ability of multi-modal optical spectroscopy to detect signals from pancreatic tissue was demonstrated by studying human pancreatic cancer xenografts in mice and freshly excised human pancreatic tumor tissue. Measured optical spectra and fluorescence decays were correlated with tissue morphological and biochemical properties. The measured spectral features and decay times correlated well with expected pathological differences in normal, pancreatitis and adenocarcinoma tissue states. The observed differences between the fluorescence and reflectance properties of normal, pancreatitis and adenocarcinoma tissue indicate a possible application of multi-modal optical spectroscopy to differentiating between the three tissue classifications.

Silole-Based Red Fluorescent Organic Dots for Bright Two-Photon Fluorescence In vitro Cell and In vivo Blood Vessel Imaging.

PubMed

Chen, Bin; Feng, Guangxue; He, Bairong; Goh, Chiching; Xu, Shidang; Ramos-Ortiz, Gabriel; Aparicio-Ixta, Laura; Zhou, Jian; Ng, Laiguan; Zhao, Zujin; Liu, Bin; Tang, Ben Zhong

2016-02-10

Robust luminescent dyes with efficient two-photon fluorescence are highly desirable for biological imaging applications, but those suitable for organic dots fabrication are still rare because of aggregation-caused quenching. In this work, a red fluorescent silole, 2,5-bis[5-(dimesitylboranyl)thiophen-2-yl]-1-methyl-1,3,4-triphenylsilole ((MesB)2 DTTPS), is synthesized and characterized. (MesB)2 DTTPS exhibits enhanced fluorescence efficiency in nanoaggregates, indicative of aggregation-enhanced emission (AEE). The organic dots fabricated by encapsulating (MesB)2 DTTPS within lipid-PEG show red fluorescence peaking at 598 nm and a high fluorescence quantum yield of 32%. Upon excitation at 820 nm, the dots show a large two-photon absorption cross section of 3.43 × 10(5) GM, which yields a two-photon action cross section of 1.09 × 10(5) GM. These (MesB)2 DTTPS dots show good biocompatibility and are successfully applied to one-photon and two-photon fluorescence imaging of MCF-7 cells and two-photon in vivo visualization of the blood vascular of mouse muscle in a high-contrast and noninvasive manner. Moreover, the 3D blood vasculature located at the mouse ear skin with a depth of over 100 μm can also be visualized clearly, providing the spatiotemporal information about the whole blood vascular network. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purely Organic Thermally Activated Delayed Fluorescence Materials for Organic Light-Emitting Diodes.

PubMed

Wong, Michael Y; Zysman-Colman, Eli

2017-06-01

The design of thermally activated delayed fluorescence (TADF) materials both as emitters and as hosts is an exploding area of research. The replacement of phosphorescent metal complexes with inexpensive organic compounds in electroluminescent (EL) devices that demonstrate comparable performance metrics is paradigm shifting, as these new materials offer the possibility of developing low-cost lighting and displays. Here, a comprehensive review of TADF materials is presented, with a focus on linking their optoelectronic behavior with the performance of the organic light-emitting diode (OLED) and related EL devices. TADF emitters are cross-compared within specific color ranges, with a focus on blue, green-yellow, orange-red, and white OLEDs. Organic small-molecule, dendrimer, polymer, and exciplex emitters are all discussed within this review, as is their use as host materials. Correlations are provided between the structure of the TADF materials and their optoelectronic properties. The success of TADF materials has ushered in the next generation of OLEDs. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus.

PubMed

Yang, Yang; Qin, Xiaodong; Wang, Guangxiang; Zhang, Yuen; Shang, Youjun; Zhang, Zhidong

2015-12-02

Orf virus (ORFV) is the causative agent of Orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. A isothermal molecular diagnostic approach, termed recombinase polymerase amplification (RPA), is considered as an novel and rapid alternative techonology to PCR assay. In the present study, a novel fluorescent probe based on RPA assay (ORFV exo RPA assay) was developed. The developed ORFV exo RPA assay was capable of as low as 100 copies of ORFV DNA /reaction and was highly specific, with no cross-reaction with closely related viruses (capripox virus, foot-and-mouth disease virus or peste des petits ruminants virus). Further assessment with clinical samples showed that the developed ORFV exo RPA assay has good correlation with qPCR assays for detection of ORFV. These results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.

Red fluorescent biofilm: the thick, the old, and the cariogenic

PubMed Central

Volgenant, Catherine M.C.; Hoogenkamp, Michel A.; Buijs, Mark J.; Zaura, Egija; ten Cate, Jacob (Bob) M.; van der Veen, Monique H.

2016-01-01

Background Some dental plaque fluoresces red. The factors involved in this fluorescence are yet unknown. Objective The aim of this study was to assess systematically the effect of age, thickness, and cariogenicity on the extent of red fluorescence produced by in vitro microcosm biofilms. Design The effects of biofilm age and thickness on red fluorescence were tested in a constant depth film fermentor (CDFF) by growing biofilms of variable thicknesses that received a constant supply of defined mucin medium (DMM) and eight pulses of sucrose/day. The influence of cariogenicity on red fluorescence was tested by growing biofilm on dentin disks receiving DMM, supplemented with three or eight pulses of sucrose/day. The biofilms were analyzed at different time points after inoculation, up to 24 days. Emission spectra were measured using a fluorescence spectrophotometer (λexc405 nm) and the biofilms were photographed with a fluorescence camera. The composition of the biofilms was assessed using 454-pyrosequecing of the 16S rDNA gene. Results From day 7 onward, the biofilms emitted increasing intensities of red fluorescence as evidenced by the combined red fluorescence peaks. The red fluorescence intensity correlated with biofilm thickness but not in a linear way. Biofilm fluorescence also correlated with the imposed cariogenicity, evidenced by the induced dentin mineral loss. Increasing the biofilm age or increasing the sucrose pulsing frequency led to a shift in the microbial composition. These shifts in composition were accompanied by an increase in red fluorescence. Conclusions The current study shows that a thicker, older, or more cariogenic biofilm results in a higher intensity of red fluorescence. PMID:27060056

Plasmonically amplified bioassay - Total internal reflection fluorescence vs. epifluorescence geometry.

PubMed

Hageneder, Simone; Bauch, Martin; Dostalek, Jakub

2016-08-15

This paper investigates plasmonic amplification in two commonly used optical configurations for fluorescence readout of bioassays - epifluorescence (EPF) and total internal reflection fluorescence (TIRF). The plasmonic amplification in the EPF configuration was implemented by using crossed gold diffraction grating and Kretschmann geometry of attenuated total reflection method (ATR) was employed in the TIRF configuration. Identical assay, surface architecture for analyte capture, and optics for the excitation, collection and detection of emitted fluorescence light intensity were used in both TIRF and EPF configurations. Simulations predict that the crossed gold diffraction grating (EPF) can amplify the fluorescence signal by a factor of 10(2) by the combination of surface plasmon-enhanced excitation and directional surface plasmon-coupled emission in the red part of spectrum. This factor is about order of magnitude higher than that predicted for the Kretschmann geometry (TIRF) which only took advantage of the surface plasmon-enhanced excitation. When applied for the readout of sandwich interleukin 6 (IL-6) immunoassay, the plasmonically amplified EPF geometry designed for Alexa Fluor 647 labels offered 4-times higher fluorescence signal intensity compared to TIRF. Interestingly, both geometries allowed reaching the same detection limit of 0.4pM despite of the difference in the fluorescence signal enhancement. This is attributed to inherently lower background of fluorescence signal for TIRF geometry compared to that for EPF which compensates for the weaker fluorescence signal enhancement. The analysis of the inflammation biomarker IL-6 in serum at medically relevant concentrations and the utilization of plasmonic amplification for the fluorescence measurement of kinetics of surface affinity reactions are demonstrated for both EPF and TIRF readout. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Size-dependent fluorescence of bioaerosols: Mathematical model using fluorescing and absorbing molecules in bacteria

DOE PAGES

Hill, Steven C.; Williamson, Chatt C.; Doughty, David C.; ...

2015-02-02

This paper uses a mathematical model of fluorescent biological particles composed of bacteria and/or proteins (mostly as in Hill et al., 2013 [23]) to investigate the size-dependence of the total fluorescence emitted in all directions. The model applies to particles which have negligible reabsorption of fluorescence within the particle. The specific particles modeled here are composed of ovalbumin and of a generic Bacillus. The particles need not be spherical, and in some cases need not be homogeneous. However, the results calculated in this paper are for spherical homogeneous particles. Light absorbing and fluorescing molecules included in the model are aminomore » acids, nucleic acids, and several coenzymes. Here the excitation wavelength is 266 nm. The emission range, 300 to 370 nm, encompasses the fluorescence of tryptophan. The fluorescence cross section (C F) is calculated and compared with one set of published measured values. We investigate power law (Ad y) approximations to C F, where d is diameter, and A and y are parameters adjusted to fit the data, and examine how y varies with d and composition, including the fraction as water. The particle's fluorescence efficiency (Q F=C F/geometric-cross-section) can be written for homogeneous particles as Q absR F, where Q abs is the absorption efficiency, and R F, the fraction of the absorbed light emitted as fluorescence, is independent of size and shape. When Q F is plotted vs. m id or mi(m r-1)d, where m=m r+im i is the complex refractive index, the plots for different fractions of water in the particle tend to overlap.« less

Visualization of three pathways for macromolecule transport across cultured endothelium and their modification by flow.

PubMed

Ghim, Mean; Alpresa, Paola; Yang, Sung-Wook; Braakman, Sietse T; Gray, Stephen G; Sherwin, Spencer J; van Reeuwijk, Maarten; Weinberg, Peter D

2017-11-01

Transport of macromolecules across vascular endothelium and its modification by fluid mechanical forces are important for normal tissue function and in the development of atherosclerosis. However, the routes by which macromolecules cross endothelium, the hemodynamic stresses that maintain endothelial physiology or trigger disease, and the dependence of transendothelial transport on hemodynamic stresses are controversial. We visualized pathways for macromolecule transport and determined the effect on these pathways of different types of flow. Endothelial monolayers were cultured under static conditions or on an orbital shaker producing different flow profiles in different parts of the wells. Fluorescent tracers that bound to the substrate after crossing the endothelium were used to identify transport pathways. Maps of tracer distribution were compared with numerical simulations of flow to determine effects of different shear stress metrics on permeability. Albumin-sized tracers dominantly crossed the cultured endothelium via junctions between neighboring cells, high-density lipoprotein-sized tracers crossed at tricellular junctions, and low-density lipoprotein-sized tracers crossed through cells. Cells aligned close to the angle that minimized shear stresses across their long axis. The rate of paracellular transport under flow correlated with the magnitude of these minimized transverse stresses, whereas transport across cells was uniformly reduced by all types of flow. These results contradict the long-standing two-pore theory of solute transport across microvessel walls and the consensus view that endothelial cells align with the mean shear vector. They suggest that endothelial cells minimize transverse shear, supporting its postulated proatherogenic role. Preliminary data show that similar tracer techniques are practicable in vivo. NEW & NOTEWORTHY Solutes of increasing size crossed cultured endothelium through intercellular junctions, through tricellular junctions, or transcellularly. Cells aligned to minimize the shear stress acting across their long axis. Paracellular transport correlated with the level of this minimized shear, but transcellular transport was reduced uniformly by flow regardless of the shear profile. Copyright © 2017 the American Physiological Society.

A correlative study of Papanicolaou smear, fluorescent antibody, and culture for the diagnosis of Chlamydia trachomatis.

PubMed

Spence, M R; Barbacci, M; Kappus, E; Quinn, T

1986-11-01

A prospective study of 300 patients undergoing therapeutic termination of pregnancy was conducted. A Papanicolaou smear was obtained and a clinical evaluation of the cervix was made. Specimens from the cervix were examined by both direct fluorescent antibody and culture techniques for the presence of Chlamydia trachomatis. The presence of inflammation on Papanicolaou smear could be correlated with C trachomatis isolation. Papanicolaou smear findings consistent with C trachomatis lacked both sensitivity and specificity when compared with direct fluorescent antibody and/or culture techniques. A correlation was found between the clinical diagnosis of cervicitis and C trachomatis. This interrelationship was absent when the component findings of cervicitis (ectopy, friability, and purulent mucus) were examined independently.

Palus Somni - Anomalies in the correlation of Al/Si X-ray fluorescence intensity ratios and broad-spectrum visible albedos. [lunar surface mineralogy

NASA Technical Reports Server (NTRS)

Clark, P. E.; Andre, C. G.; Adler, I.; Weidner, J.; Podwysocki, M.

1976-01-01

The positive correlation between Al/Si X-ray fluorescence intensity ratios determined during the Apollo 15 lunar mission and a broad-spectrum visible albedo of the moon is quantitatively established. Linear regression analysis performed on 246 1 degree geographic cells of X-ray fluorescence intensity and visible albedo data points produced a statistically significant correlation coefficient of .78. Three distinct distributions of data were identified as (1) within one standard deviation of the regression line, (2) greater than one standard deviation below the line, and (3) greater than one standard deviation above the line. The latter two distributions of data were found to occupy distinct geographic areas in the Palus Somni region.

Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo

NASA Astrophysics Data System (ADS)

Zettergren, Eric; Swamy, Tushar; Runnels, Judith; Lin, Charles P.; Niedre, Mark

2012-07-01

Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a ‘diffuse fluorescence flow cytometer’ (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument.

Fluorescence Behavior and Dural Infiltration of Meningioma Analyzed by 5-Aminolevulinic Acid-Based Fluorescence: Operating Microscope Versus Mini-Spectrometer.

PubMed

Knipps, Johannes; Beseoglu, Kerim; Kamp, Marcel; Fischer, Igor; Felsberg, Joerg; Neumann, Lisa M; Steiger, Hans-Jakob; Cornelius, Jan F

2017-12-01

To compare fluorescence intensity of tumor specimens, as measured by a fluorescence-guided surgery microscope and a spectrometer, to evaluate tumor infiltration of dura mater around meningiomas with help of these 2 different 5-aminolevulinic acid (5-ALA)-based fluorescence tools, and to correlate fluorescence intensity with histopathologic data. In a clinical series, meningiomas were resected by 5-ALA fluorescence-guided surgery. Fluorescence intensity was semiquantitatively rated by the surgeon at predefined points. Biopsies were harvested and fluorescence intensity measured by a spectrometer and histopathologically analyzed. Sampling was realized at the level of the dura in a centrifugal direction. A total of 104 biopsies (n = 13 tumors) were analyzed. Specificity and sensitivity of the microscope were 0.96 and 0.53 and of the spectrometer 0.95 and 0.93, respectively. Fluorescence intensity as measured by the spectrometer was correlated to histologically confirmed tumor burden. In a centrifugal direction, tumor burden and fluorescence intensity continuously decreased (along the dural tail). Below a threshold value of 639 arbitrary units no tumor was histologically detectable. At the level of the dura the spectrometer was highly sensitive for detection of meningioma cells. The surgical microscope showed false negative results and missed residual tumor cells in more than one half of the cases. The complementary use of both fluorescence tools may improve resection quality. Copyright © 2017 Elsevier Inc. All rights reserved.

A New Methodology of Spatial Cross-Correlation Analysis

PubMed Central

Chen, Yanguang

2015-01-01

Spatial correlation modeling comprises both spatial autocorrelation and spatial cross-correlation processes. The spatial autocorrelation theory has been well-developed. It is necessary to advance the method of spatial cross-correlation analysis to supplement the autocorrelation analysis. This paper presents a set of models and analytical procedures for spatial cross-correlation analysis. By analogy with Moran’s index newly expressed in a spatial quadratic form, a theoretical framework is derived for geographical cross-correlation modeling. First, two sets of spatial cross-correlation coefficients are defined, including a global spatial cross-correlation coefficient and local spatial cross-correlation coefficients. Second, a pair of scatterplots of spatial cross-correlation is proposed, and the plots can be used to visually reveal the causality behind spatial systems. Based on the global cross-correlation coefficient, Pearson’s correlation coefficient can be decomposed into two parts: direct correlation (partial correlation) and indirect correlation (spatial cross-correlation). As an example, the methodology is applied to the relationships between China’s urbanization and economic development to illustrate how to model spatial cross-correlation phenomena. This study is an introduction to developing the theory of spatial cross-correlation, and future geographical spatial analysis might benefit from these models and indexes. PMID:25993120

A new methodology of spatial cross-correlation analysis.

PubMed

Chen, Yanguang

2015-01-01

Spatial correlation modeling comprises both spatial autocorrelation and spatial cross-correlation processes. The spatial autocorrelation theory has been well-developed. It is necessary to advance the method of spatial cross-correlation analysis to supplement the autocorrelation analysis. This paper presents a set of models and analytical procedures for spatial cross-correlation analysis. By analogy with Moran's index newly expressed in a spatial quadratic form, a theoretical framework is derived for geographical cross-correlation modeling. First, two sets of spatial cross-correlation coefficients are defined, including a global spatial cross-correlation coefficient and local spatial cross-correlation coefficients. Second, a pair of scatterplots of spatial cross-correlation is proposed, and the plots can be used to visually reveal the causality behind spatial systems. Based on the global cross-correlation coefficient, Pearson's correlation coefficient can be decomposed into two parts: direct correlation (partial correlation) and indirect correlation (spatial cross-correlation). As an example, the methodology is applied to the relationships between China's urbanization and economic development to illustrate how to model spatial cross-correlation phenomena. This study is an introduction to developing the theory of spatial cross-correlation, and future geographical spatial analysis might benefit from these models and indexes.

Versatile microfluidic total internal reflection (TIR)-based devices: application to microbeads velocity measurement and single molecule detection with upright and inverted microscope.

PubMed

Le, Nam Cao Hoai; Yokokawa, Ryuji; Dao, Dzung Viet; Nguyen, Thien Duy; Wells, John C; Sugiyama, Susumu

2009-01-21

A poly(dimethylsiloxane) (PDMS) chip for Total Internal Reflection (TIR)-based imaging and detection has been developed using Si bulk micromachining and PDMS casting. In this paper, we report the applications of the chip on both inverted and upright fluorescent microscopes and confirm that two types of sample delivery platforms, PDMS microchannel and glass microchannel, can be easily integrated depending on the magnification of an objective lens needed to visualize a sample. Although any device configuration can be achievable, here we performed two experiments to demonstrate the versatility of the microfluidic TIR-based devices. The first experiment was velocity measurement of Nile red microbeads with nominal diameter of 500 nm in a pressure-driven flow. The time-sequenced fluorescent images of microbeads, illuminated by an evanescent field, were cross-correlated by a Particle Image Velocimetry (PIV) program to obtain near-wall velocity field of the microbeads at various flow rates from 500 nl/min to 3000 nl/min. We then evaluated the capabilities of the device for Single Molecule Detection (SMD) of fluorescently labeled DNA molecules from 30 bp to 48.5 kbp and confirm that DNA molecules as short as 1105 bp were detectable. Our versatile, integrated device could provide low-cost and fast accessibility to Total Internal Reflection Fluorescent Microscopy (TIRFM) on both conventional upright and inverted microscopes. It could also be a useful component in a Micro-Total Analysis System (micro-TAS) to analyze nanoparticles or biomolecules near-wall transport or motion.

Monitoring of nucleophosmin oligomerization in live cells.

PubMed

Holoubek, Ales; Heřman, Petr; Sýkora, Jan; Brodská, Barbora; Humpolíčková, Jana; Kráčmarová, Markéta; Gášková, Dana; Hof, Martin; Kuželová, Kateřina

2018-06-14

Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells. © 2018 IOP Publishing Ltd.

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.

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

NASA Astrophysics Data System (ADS)

Li, Wenyan; Houston, Kevin D.; Houston, Jessica P.

2017-01-01

Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

Correlative Fluorescence and Electron Microscopy

PubMed Central

Schirra, Randall T.; Zhang, Peijun

2014-01-01

Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associate with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology have led to rapid improvement in the protocols and have ushered in a new generation of instruments to reach the next level of correlation – integration. PMID:25271959

Real-time measurements of airborne biologic particles using fluorescent particle counter to evaluate microbial contamination: results of a comparative study in an operating theater.

PubMed

Dai, Chunyang; Zhang, Yan; Ma, Xiaoling; Yin, Meiling; Zheng, Haiyang; Gu, Xuejun; Xie, Shaoqing; Jia, Hengmin; Zhang, Liang; Zhang, Weijun

2015-01-01

Airborne bacterial contamination poses a risk for surgical site infection, and routine surveillance of airborne bacteria is important. Traditional methods for detecting airborne bacteria are time consuming and strenuous. Measurement of biologic particle concentrations using a fluorescent particle counter is a novel method for evaluating air quality. The current study was to determine whether the number of biologic particles detected by the fluorescent particle counter can be used to indicate airborne bacterial counts in operating rooms. The study was performed in an operating theater at a university hospital in Hefei, China. The number of airborne biologic particles every minute was quantified using a fluorescent particle counter. Microbiologic air sampling was performed every 30 minutes using an Andersen air sampler (Pusong Electronic Instruments, Changzhou, China). Correlations between the 2 different methods were analyzed by Pearson correlation coefficients. A significant correlation was observed between biologic particle and bacterial counts (Pearson correlation coefficient = 0.76), and the counting results from 2 methods both increased substantially between operations, corresponding with human movements in the operating room. Fluorescent particle counters show potential as important tools for monitoring bacterial contamination in operating theatres. Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

PubMed

Tuijtel, Maarten W; Mulder, Aat A; Posthuma, Clara C; van der Hoeven, Barbara; Koster, Abraham J; Bárcena, Montserrat; Faas, Frank G A; Sharp, Thomas H

2017-09-05

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

LASER BIOLOGY AND MEDICINE: Application of laser fluorimetry for determining the influence of a single amino-acid substitution on the individual photophysical parameters of a fluorescent form of a fluorescent protein mRFP1

NASA Astrophysics Data System (ADS)

Banishev, A. A.; Vrzheshch, E. P.; Shirshin, E. A.

2009-03-01

Individual photophysical parameters of the chromophore of a fluorescent protein mRFP1 and its two mutants (amino-acid substitution at position 66 - mRFP1/ Q66C and mRFP1/Q66S proteins) are determined. For this purpose, apart from conventional methods of fluorimetry and spectrophotometry, nonlinear laser fluorimetry is used. It is shown that the individual extinction coefficients of the chromophore of proteins correlate (correlation coefficient above 0.9) with the volume of the substituted amino-acid residue at position 66 (similar to the positions of the absorption, fluorescence excitation and emission maxima).

A new serotyping method for Klebsiella species: development of the technique.

PubMed Central

Riser, E; Noone, P; Poulton, T A

1976-01-01

A new serotyping method for Klebsiella species using indirect immunofluorescence is described. Nonspecific fluorescence has been minimized by carrying out the capsular antigen-antibody reaction at pH 9.0. Commercial antisera have been tested with the 72 antigenic types of Klebsiella, and appropriate dilutions of each pool and specific antisera have been proposed for use in routine typing. Dilutions were chosen to allow strong fluorescence with each type and its specific antiserum and minimal fluorescence with cross reacting antisera. Where the pool antisera gave a weak reaction for one or more of the component types, it is recommended that the specific antisera for these types be added to the pool dilution. The few remaining cross reactions, with the pool and specific antisera in test dilution, are listed in a table. The unique cross reacting patterns of particular types have been found to be useful in identification. Typing Klebsiella by the fluorescent antibody technique is easy to perform and interpret; the results are reproducible, and it is less expensive than the existing capsular swelling method as it is more sensitive and requires less concentrated antisera. This new method of typing should facilitate detailed epidemiological studies of the mode of transmission of Klebsiella species in hospitals and thus allow more effective infection control measures to be instituted. Images PMID:777042

Highly efficient phosphorescent, TADF, and fluorescent OLEDs (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kim, Jang-Joo; Kim, Kwon-Hyeon; Moon, Chang-Ki; Shin, Hyun

2016-09-01

High efficiency OLEDs based on phosphorescent, thermally activated delayed fluorescent (TADF) and fluorescent emitters will be presented. We will show that EQEs over 60% is achievable if OLEDs are fabricated using organic semiconductors with the refractive indices of 1.5 and fully horizontal emitting dipoles without any extra light extracting structure. We will also show that reverse intersystem crossing RISC rate plays an important role to reduce the efficiency roll-off in efficient TADF and fluorescent OLEDs and a couple to methods will be presented to increase the RISC rate in the devices.

Nanoscale volume confinement and fluorescence enhancement with double nanohole aperture

PubMed Central

Regmi, Raju; Al Balushi, Ahmed A.; Rigneault, Hervé; Gordon, Reuven; Wenger, Jérôme

2015-01-01

Diffraction ultimately limits the fluorescence collected from a single molecule, and sets an upper limit to the maximum concentration to isolate a single molecule in the detection volume. To overcome these limitations, we introduce here the use of a double nanohole structure with 25 nm gap, and report enhanced detection of single fluorescent molecules in concentrated solutions exceeding 20 micromolar. The nanometer gap concentrates the light into an apex volume down to 70 zeptoliter (10−21 L), 7000-fold below the diffraction-limited confocal volume. Using fluorescence correlation spectroscopy and time-correlated photon counting, we measure fluorescence enhancement up to 100-fold, together with local density of optical states (LDOS) enhancement of 30-fold. The distinctive features of double nanoholes combining high local field enhancement, efficient background screening and relative nanofabrication simplicity offer new strategies for real time investigation of biochemical events with single molecule resolution at high concentrations. PMID:26511149

Excited-state absorption and fluorescence dynamics of Er3+:KY3F10

NASA Astrophysics Data System (ADS)

Labbé, C.; Doualan, J. L.; Moncorgé, R.; Braud, A.; Camy, P.

2018-05-01

We report here on a complete investigation of the excited-state absorption and fluorescence dynamics of Er3+ doped KY3F10 single crystals versus dopant concentrations and optical excitation conditions. Radiative and effective (including non-radiative relaxations) emission lifetimes and branching ratios are determined from a Judd-Ofelt analysis of the absorption spectra and via specific fluorescence experiments using wavelength selective laser excitations. Excited-state absorption and emission spectra are registered within seven spectral domains, i.e. 560 nm, 650 nm, 710 nm, 810 nm, 970 nm, 1550 nm and 2750 nm. A maximum gain cross-section of 0.93 × 10-21 cm2 is determined at the potential laser wavelength of 2.801 μm for a population ratio of 0.48. Saturation of fluorescence intensities and variations of population ratios versus pumping rates are registered and confronted with a rate equation model to derive the rates of the most important up-conversion and cross-relaxation energy transfers occurring at high dopant concentrations.

Multiphoton-Excited Fluorescence of Silicon-Vacancy Color Centers in Diamond

NASA Astrophysics Data System (ADS)

Higbie, J. M.; Perreault, J. D.; Acosta, V. M.; Belthangady, C.; Lebel, P.; Kim, M. H.; Nguyen, K.; Demas, V.; Bajaj, V.; Santori, C.

2017-05-01

Silicon-vacancy color centers in nanodiamonds are promising as fluorescent labels for biological applications, with a narrow, nonbleaching emission line at 738 nm. Two-photon excitation of this fluorescence offers the possibility of low-background detection at significant tissue depth with high three-dimensional spatial resolution. We measure the two-photon fluorescence cross section of a negatively charged silicon vacancy (Si -V- ) in ion-implanted bulk diamond to be 0.74 (19 )×10-50 cm4 s /photon at an excitation wavelength of 1040 nm. Compared to the diamond nitrogen-vacancy center, the expected detection threshold of a two-photon excited Si -V center is more than an order of magnitude lower, largely due to its much narrower linewidth. We also present measurements of two- and three-photon excitation spectra, finding an increase in the two-photon cross section with decreasing wavelength, and we discuss the physical interpretation of the spectra in the context of existing models of the Si -V energy-level structure.

Confocal Microscopy for the Histological Fluorescence Pattern of a Recurrent Atypical Meningioma: Case Report

PubMed Central

Whitson, Wesley J.; Valdes, Pablo A.; Harris, Brent T.; Paulsen, Keith D.; Roberts, David W.

2013-01-01

Background and Importance Fluorescence-guided resection with 5-aminolevulinic acid (5-ALA), which has shown promising results in the resection of malignant gliomas, has been used for meningioma resection in an attempt to more clearly delineate the tumor margin. However, no article has investigated the fluorescence pattern of meningiomas on a histological level. Understanding the microscopic pattern of fluorescence could help assess the precision and utility of using 5-ALA for these tumors. We present the case of a recurrent atypical meningioma operated on with 5-ALA fluorescence-guided resection for delineation of tumor tissue from surrounding uninvolved dura. Clinical Presentation A 53-year-old woman presented with recurrent atypical meningioma of the falx. Prior treatment included surgical resection 6 years earlier with subsequent fractionated radiation therapy and radiosurgery for tumor progression. The patient was given 5-ALA 20 mg/kg body weight dissolved in 100 mL water 3 hours before induction of anesthesia. Intraoperative fluorescence was coregistered with preoperative imaging. Neuropathological analysis of the resected falx with confocal microscopy enabled correlation of fluorescence with the extent of tumor on a histological level. Conclusion Fluorescence guidance allowed clear intraoperative delineation of tumor tissue from adjacent, uninvolved dura. On a microscopic level, there was a very close correlation of fluorescence with tumor, but some tumor cells did not fluoresce. PMID:21389893

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.

Discovering cell types in flow cytometry data with random matrix theory

NASA Astrophysics Data System (ADS)

Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

Flow cytometry is a widely used experimental technique in immunology research. During the experiments, peripheral blood mononuclear cells (PBMC) from a single patient, labeled with multiple fluorescent stains that bind to different proteins, are illuminated by a laser. The intensity of each stain on a single cell is recorded and reflects the amount of protein expressed by that cell. The data analysis focuses on identifying specific cell types related to a disease. Different cell types can be identified by the type and amount of protein they express. To date, this has most often been done manually by labelling a protein as expressed or not while ignoring the amount of expression. Using a cross correlation matrix of stain intensities, which contains both information on the proteins expressed and their amount, has been largely ignored by researchers as it suffers from measurement noise. Here we present an algorithm to identify cell types in flow cytometry data which uses random matrix theory (RMT) to reduce noise in a cross correlation matrix. We demonstrate our method using a published flow cytometry data set. Compared with previous analysis techniques, we were able to rediscover relevant cell types in an automatic way. Department of Physics, University of Maryland, College Park, MD 20742.

Application of Optical Diagnosis to Aged Low-Voltage Cable Insulation in Nuclear Plants

NASA Astrophysics Data System (ADS)

Katagiri, Junichi; Takezawa, Yoshitaka; Shouji, Hiroshi

We have developed a novel non-destructive optical diagnosis technique for low-voltage cable insulations used in nuclear power plants. The key features of this diagnosis are the use of two wavelengths to measure the change in reflective absorbance (ΔAR), the use of polarized light to measure crystallinity and the use of element volatilizing to measure fluorescence. Chemical kinetics is used to predict the lifetimes of the cable insulations. When cable insulations darken and harden by time degradation, the ΔAR and depolarization parameters increase. This means that the cross-linking density in the cable insulations increases due to deterioration reactions. When the cross-linking density of insulation increases, its elasticity, corresponding to the material's life, increases. Similarly, as the crystallinity increases due to the change in the high-order structure of the insulating resin caused by irradiation, its elongation property decreases. The elongation property of insulation is one of the most important parameters that can be used to evaluate material lifetimes, because it relates to elasticity. The ΔAR correlated with the elongation property, and the correlation coefficient of an accelerated experiment using model pieces was over 0.9. Thus, we concluded that this optical diagnosis should be applied to evaluate the degradation of cable insulations used in nuclear power plants.

Measurements of K shell absorption jump factors and jump ratios using EDXRF technique

NASA Astrophysics Data System (ADS)

Kacal, Mustafa Recep; Han, İbrahim; Akman, Ferdi

2015-04-01

In the present work, the K-shell absorption jump factors and jump ratios for 30 elements between Ti ( Z = 22) and Er ( Z = 68) were measured by energy dispersive X-ray fluorescence (EDXRF) technique. The jump factors and jump ratios for these elements were determined by measuring the K shell fluorescence parameters such as the Kα X-ray production cross-sections, K shell fluorescence yields, Kβ-to- Kα X-rays intensity ratios, total atomic absorption cross sections and mass attenuation coefficients. The measurements were performed using an Am-241 radioactive point source and a Si (Li) detector in direct excitation and transmission experimental geometry. The results for jump factors and jump ratios were compared with theoretically calculated and the ones available in the literature.

Low-temperature collisional quenching of NO A{sup 2}Σ{sup +}(v′ = 0) by NO(X{sup 2}Π) and O{sub 2} between 34 and 109 K

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

Sánchez-González, R.; Eveland, W. D.; West, N. A.

2014-08-21

We present measurements of collisional fluorescence quenching cross sections of NO(A{sup 2}Σ{sup +}, v′ = 0) by NO(X{sup 2}Π) and O{sub 2} between 34 and 109 K using a pulsed converging-diverging nozzle gas expansion, extending the temperature range of previous measurements. The thermally averaged fluorescence quenching cross sections for both species show a monotonic increase as temperature decreases in this temperature range, consistent with earlier observations. These new measurements, however, allow discrimination between predictions obtained by extrapolating fits of previous data using different functional forms that show discrepancies exceeding 120% for NO and 160% for O{sub 2} at 34 K.more » The measured self-quenching cross section is 52.9 Å{sup 2} near 112 K and increases to 64.1 Å{sup 2} at 35 K, whereas the O{sub 2} fluorescence quenching cross section is 42.9 Å{sup 2} at 109 K and increases to 58.3 Å{sup 2} at 34 K. Global fits of the quenching cross section temperature dependence show that, when including our current measurements, the low temperature behavior of the quenching cross sections for NO and O{sub 2} is better described by a parameterization that accounts for the long-range interactions leading to the collisional deactivation via an inverse power law model.« less

Boronlectin/Polyelectrolyte Ensembles as Artificial Tongue: Design, Construction, and Application for Discriminative Sensing of Complex Glycoconjugates from Panax ginseng.

PubMed

Zhang, Xiao-Tai; Wang, Shu; Xing, Guo-Wen

2017-02-01

Ginsenoside is a large family of triterpenoid saponins from Panax ginseng, which possesses various important biological functions. Due to the very similar structures of these complex glycoconjugates, it is crucial to develop a powerful analytic method to identify ginsenosides qualitatively or quantitatively. We herein report an eight-channel fluorescent sensor array as artificial tongue to achieve the discriminative sensing of ginsenosides. The fluorescent cross-responsive array was constructed by four boronlectins bearing flexible boronic acid moieties (FBAs) with multiple reactive sites and two linear poly(phenylene-ethynylene) (PPEs). An "on-off-on" response pattern was afforded on the basis of superquenching of fluorescent indicator PPEs and an analyte-induced allosteric indicator displacement (AID) process. Most importantly, it was found that the canonical distribution of ginsenoside data points analyzed by linear discriminant analysis (LDA) was highly correlated with the inherent molecular structures of the analytes, and the absence of overlaps among the five point groups reflected the effectiveness of the sensor array in the discrimination process. Almost all of the unknown ginsenoside samples at different concentrations were correctly identified on the basis of the established mathematical model. Our current work provided a general and constructive method to improve the quality assessment and control of ginseng and its extracts, which are useful and helpful for further discriminating other complex glycoconjugate families.

Fluorescence lifetime imaging of skin cancer

NASA Astrophysics Data System (ADS)

Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

2011-03-01

Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

PubMed

Lajevardipour, Alireza; Chon, James W M; Chattopadhyay, Amitabha; Clayton, Andrew H A

2016-11-22

Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C 6 -NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

Fluorescence spectroscopy for endogenous porphyrins in human facial skin

NASA Astrophysics Data System (ADS)

Seo, I.; Tseng, S. H.; Cula, G. O.; Bargo, P. R.; Kollias, N.

2009-02-01

The activity of certain bacteria in skin is known to correlate to the presence of porphyrins. In particular the presence of coproporphyrin produced by P.acnes inside plugged pores has been correlated to acne vulgaris. Another porphyrin encountered in skin is protoporphyrin IX, which is produced by the body in the pathway for production of heme. In the present work, a fluorescence spectroscopy system was developed to measure the characteristic spectrum and quantify the two types of porphyrins commonly present in human facial skin. The system is comprised of a Xe lamp both for fluorescence excitation and broadband light source for diffuse reflectance measurements. A computer-controlled filter wheel enables acquisition of sequential spectra, first excited by blue light at 405 nm then followed by the broadband light source, at the same location. The diffuse reflectance spectrum was used to correct the fluorescence spectrum due to the presence of skin chromophores, such as blood and melanin. The resulting fluorescence spectra were employed for the quantification of porphyrin concentration in a population of healthy subjects. The results show great variability on the concentration of these porphyrins and further studies are being conducted to correlate them with skin conditions such as inflammation and acne vulgaris.

Fluorescence guided surgery and tracer-dose, fact or fiction?

PubMed

KleinJan, Gijs H; Bunschoten, Anton; van den Berg, Nynke S; Olmos, Renato A Valdès; Klop, W Martin C; Horenblas, Simon; van der Poel, Henk G; Wester, Hans-Jürgen; van Leeuwen, Fijs W B

2016-09-01

Fluorescence guidance is an upcoming methodology to improve surgical accuracy. Challenging herein is the identification of the minimum dose at which the tracer can be detected with a clinical-grade fluorescence camera. Using a hybrid tracer such as indocyanine green (ICG)-(99m)Tc-nanocolloid, it has become possible to determine the accumulation of tracer and correlate this to intraoperative fluorescence-based identification rates. In the current study, we determined the lower detection limit of tracer at which intraoperative fluorescence guidance was still feasible. Size exclusion chromatography (SEC) provided a laboratory set-up to analyze the chemical content and to simulate the migratory behavior of ICG-nanocolloid in tissue. Tracer accumulation and intraoperative fluorescence detection findings were derived from a retrospective analysis of 20 head-and-neck melanoma patients, 40 penile and 20 prostate cancer patients scheduled for sentinel node (SN) biopsy using ICG-(99m)Tc-nanocolloid. In these patients, following tracer injection, single photon emission computed tomography fused with computed tomography (SPECT/CT) was used to identify the SN(s). The percentage injected dose (% ID), the amount of ICG (in nmol), and the concentration of ICG in the SNs (in μM) was assessed for SNs detected on SPECT/CT and correlated with the intraoperative fluorescence imaging findings. SEC determined that in the hybrid tracer formulation, 41 % (standard deviation: 12 %) of ICG was present in nanocolloid-bound form. In the SNs detected using fluorescence guidance a median of 0.88 % ID was present, compared to a median of 0.25 % ID in the non-fluorescent SNs (p-value < 0.001). The % ID values could be correlated to the amount ICG in a SN (range: 0.003-10.8 nmol) and the concentration of ICG in a SN (range: 0.006-64.6 μM). The ability to provide intraoperative fluorescence guidance is dependent on the amount and concentration of the fluorescent dye accumulated in the lesion(s) of interest. Our findings indicate that intraoperative fluorescence detection with ICG is possible above a μM concentration.

Multispectral fluorescence imaging for detection of bovine feces on Romaine lettuce and baby spinach leaves

USDA-ARS?s Scientific Manuscript database

Hyperspectral fluorescence imaging with ultraviolet-A excitation was used to evaluate the feasibility of two-waveband fluorescence algorithms for the detection of bovine fecal contaminants on the abaxial and adaxial surfaces of Romaine lettuce and baby spinach leaves. Correlation analysis was used t...

Integrated fluorescence correlation spectroscopy device for point-of-care clinical applications

PubMed Central

Olson, Eben; Torres, Richard; Levene, Michael J.

2013-01-01

We describe an optical system which reduces the cost and complexity of fluorescence correlation spectroscopy (FCS), intended to increase the suitability of the technique for clinical use. Integration of the focusing optics and sample chamber into a plastic component produces a design which is simple to align and operate. We validate the system by measurements on fluorescent dye, and compare the results to a commercial instrument. In addition, we demonstrate its application to measurements of concentration and multimerization of the clinically relevant protein von Willebrand factor (vWF) in human plasma. PMID:23847733

Temperature dependence of Ti:Sapphire fluorescence spectra for the design of cryogenic cooled Ti:Sapphire CPA laser.

PubMed

Burton, Harry; Debardelaben, Christopher; Amir, Wafa; Planchon, Thomas A

2017-03-20

The fluorescence spectra of titanium doped sapphire (Ti:Sapphire) crystals were measured for temperature ranging from 300K to 77K. The resulting gain cross-section line shapes were calculated and used in a three-dimensional amplification model to illustrate the importance of the precise knowledge of these fluorescence spectra for the design of cryogenic cooled Ti:Sapphire based chirped-pulse laser amplifiers.

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

Hill, Steven C.; Williamson, Chatt C.; Doughty, David C.

This paper uses a mathematical model of fluorescent biological particles composed of bacteria and/or proteins (mostly as in Hill et al., 2013 [23]) to investigate the size-dependence of the total fluorescence emitted in all directions. The model applies to particles which have negligible reabsorption of fluorescence within the particle. The specific particles modeled here are composed of ovalbumin and of a generic Bacillus. The particles need not be spherical, and in some cases need not be homogeneous. However, the results calculated in this paper are for spherical homogeneous particles. Light absorbing and fluorescing molecules included in the model are aminomore » acids, nucleic acids, and several coenzymes. Here the excitation wavelength is 266 nm. The emission range, 300 to 370 nm, encompasses the fluorescence of tryptophan. The fluorescence cross section (C F) is calculated and compared with one set of published measured values. We investigate power law (Ad y) approximations to C F, where d is diameter, and A and y are parameters adjusted to fit the data, and examine how y varies with d and composition, including the fraction as water. The particle's fluorescence efficiency (Q F=C F/geometric-cross-section) can be written for homogeneous particles as Q absR F, where Q abs is the absorption efficiency, and R F, the fraction of the absorbed light emitted as fluorescence, is independent of size and shape. When Q F is plotted vs. m id or mi(m r-1)d, where m=m r+im i is the complex refractive index, the plots for different fractions of water in the particle tend to overlap.« less

Novel insights into anoxic/aerobic(1)/aerobic(2) biological fluidized-bed system for coke wastewater treatment by fluorescence excitation-emission matrix spectra coupled with parallel factor analysis.

PubMed

Ou, Hua-Se; Wei, Chao-Hai; Mo, Ce-Hui; Wu, Hai-Zhen; Ren, Yuan; Feng, Chun-Hua

2014-10-01

Fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC) was applied to investigate the contaminant removal efficiency and fluorescent characteristic variations in a full scale coke wastewater (CWW) treatment plant with a novel anoxic/aerobic(1)/aerobic(2) (A/O(1)/O(2)) process, which combined with internal-loop fluidized-bed reactor. Routine monitoring results indicated that primary contaminants in CWW, such as phenols and free cyanide, were removed efficiently in A/O(1)/O(2) process (removal efficiency reached 99% and 95%, respectively). Three-dimensional excitation-emission matrix fluorescence spectroscopy and PARAFAC identified three fluorescent components, including two humic-like fluorescence components (C1 and C3) and one protein-like component (C2). Principal component analysis revealed that C1 and C2 correlated with COD (correlation coefficient (r)=0.782, p<0.01 and r=0.921, p<0.01), respectively) and phenols (r=0.796, p<0.01 and r=0.914, p<0.01, respectively), suggesting that C1 and C2 might be associated with the predominating aromatic contaminants in CWW. C3 correlated with mixed liquor suspended solids (r=0.863, p<0.01) in fluidized-bed reactors, suggesting that it might represent the biological dissolved organic matter. In A/O(1)/O(2) process, the fluorescence intensities of C1 and C2 consecutively decreased, indicating the degradation of aromatic contaminants. Correspondingly, the fluorescence intensity of C3 increased in aerobic(1) stage, suggesting an increase of biological dissolved organic matter. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of resin infiltration using quantitative light-induced fluorescence technology.

PubMed

Min, Ji-Hyun; Inaba, Daisuke; Kim, Baek-Il

2016-09-01

To determine whether quantitative light-induced fluorescence (QLF) technology can be used to classify the colour of teeth specimens before and after resin infiltration (RI) treatment, and calculate the correlation between the ΔF value and colour difference (ΔE) in fluorescence images of the specimens obtained using a QLF-digital (QLF-D) device. Sixty sound bovine permanent teeth specimens were immersed in demineralized solution. Two exposed windows were formed in each specimen, and RI treatment was applied to one of them. The ΔE values were obtained for the differences between a sound tooth surface (SS), an early dental caries surface (ECS) and an ECS treated with RI (RS) in white-light and fluorescence images obtained using QLF-D, respectively. The ΔF value was obtained from fluorescence images using dedicated software for QLF-D. The mean differences between the ΔE values obtained from the white-light and fluorescence images were analyzed by paired t-test. Pearson correlation analysis and Bland-Altman plots were applied to the differences between the ΔF value for ECS (ΔFSS-ECS) and the ΔE value between SS and ECS (ΔESS-ECS), and between the ΔF value for RS (ΔFSS-RS) and the ΔE value between SS and RS (ΔESS-RS) in fluorescence images. The ΔE values obtained from fluorescence images were three times higher than the ΔE values obtained from white-light images (p<0.001). Significant correlations were confirmed between ΔESS-ECS and ΔFSS-ECS (r=-0.492, p<0.001) and between ΔESS-RS and ΔFSS-RS (r=-0.661, p<0.001). QLF technology can be used to confirm the presence of RI in teeth. Copyright © 2016 Elsevier B.V. All rights reserved.

Can time-dependent density functional theory predict intersystem crossing in organic chromophores? A case study on benzo(bis)-X-diazole based donor-acceptor-donor type molecules.

PubMed

Tam, Teck Lip Dexter; Lin, Ting Ting; Chua, Ming Hui

2017-06-21

Here we utilized new diagnostic tools in time-dependent density functional theory to explain the trend of intersystem crossing in benzo(bis)-X-diazole based donor-acceptor-donor type molecules. These molecules display a wide range of fluorescence quantum yields and triplet yields, making them excellent candidates for testing the validity of these diagnostic tools. We believe that these tools are cost-effective and can be applied to structurally similar organic chromophores to predict/explain the trends of intersystem crossing, and thus fluorescence quantum yields and triplet yields without the use of complex and expensive multireference configuration interaction or multireference pertubation theory methods.

Changing blue fluorescent protein to green fluorescent protein using chemical RNA editing as a novel strategy in genetic restoration.

PubMed

Vu, Luyen T; Nguyen, Thanh T K; Alam, Shafiul; Sakamoto, Takashi; Fujimoto, Kenzo; Suzuki, Hitoshi; Tsukahara, Toshifumi

2015-11-01

Using the transition from cytosine of BFP (blue fluorescent protein) gene to uridine of GFP (green fluorescent protein) gene at position 199 as a model, we successfully controlled photochemical RNA editing to effect site-directed deamination of cytidine (C) to uridine (U). Oligodeoxynucleotides (ODNs) containing 5'-carboxyvinyl-2'-deoxyuridine ((CV) U) were used for reversible photoligation, and single-stranded 100-nt BFP DNA and in vitro-transcribed full-length BFP mRNA were the targets. Photo-cross-linking with the responsive ODNs was performed using UV (366 nm) irradiation, which was followed by heat treatment, and the cross-linked nucleotide was cleaved through photosplitting (UV, 312 nm). The products were analyzed using restriction fragment length polymorphism (RFLP) and fluorescence measurements. Western blotting and fluorescence-analysis results revealed that in vitro-translated proteins were synthesized from mRNAs after site-directed RNA editing. We detected substantial amounts of the target-base-substituted fragment using RFLP and observed highly reproducible spectra of the transition-GFP signal using fluorescence spectroscopy, which indicated protein stability. ODNc restored approximately 10% of the C-to-U transition. Thus, we successfully used non-enzymatic site-directed deamination for genetic restoration in vitro. In the near future, in vivo studies that include cultured cells and model animals will be conducted to treat genetic disorders. © 2015 John Wiley & Sons A/S.

Applications of fluorescence spectroscopy to problems of food safety: detection of fecal contamination and of the presence of central nervous system tissue and diagnosis of neurological disease

NASA Astrophysics Data System (ADS)

Adhikary, Ramkrishna; Bose, Sayantan; Casey, Thomas A.; Gapsch, Al; Rasmussen, Mark A.; Petrich, Jacob W.

2010-02-01

Applications of fluorescence spectroscopy that enable the real-time or rapid detection of fecal contamination on beef carcasses and the presence of central nervous system tissue in meat products are discussed. The former is achieved by employing spectroscopic signatures of chlorophyll metabolites; the latter, by exploiting the characteristic structure and intensity of lipofuscin in central nervous system tissue. The success of these techniques has led us to investigate the possibility of diagnosing scrapie in sheep by obtaining fluorescence spectra of the retina. Crucial to this diagnosis is the ability to obtain baseline correlations of lipofuscin fluorescence with age. A murine model was employed as a proof of principle of this correlation.

Sample-averaged biexciton quantum yield measured by solution-phase photon correlation.

PubMed

Beyler, Andrew P; Bischof, Thomas S; Cui, Jian; Coropceanu, Igor; Harris, Daniel K; Bawendi, Moungi G

2014-12-10

The brightness of nanoscale optical materials such as semiconductor nanocrystals is currently limited in high excitation flux applications by inefficient multiexciton fluorescence. We have devised a solution-phase photon correlation measurement that can conveniently and reliably measure the average biexciton-to-exciton quantum yield ratio of an entire sample without user selection bias. This technique can be used to investigate the multiexciton recombination dynamics of a broad scope of synthetically underdeveloped materials, including those with low exciton quantum yields and poor fluorescence stability. Here, we have applied this method to measure weak biexciton fluorescence in samples of visible-emitting InP/ZnS and InAs/ZnS core/shell nanocrystals, and to demonstrate that a rapid CdS shell growth procedure can markedly increase the biexciton fluorescence of CdSe nanocrystals.

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

PubMed Central

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

2013-01-01

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

Analyzing the performance of fluorescence parameters in the monitoring of leaf nitrogen content of paddy rice

PubMed Central

Yang, Jian; Gong, Wei; Shi, Shuo; Du, Lin; Sun, Jia; Song, Shalei; Chen, Biwu; Zhang, Zhenbing

2016-01-01

Leaf nitrogen content (LNC) is a significant factor which can be utilized to monitor the status of paddy rice and it requires a reliable approach for fast and precise quantification. This investigation aims to quantitatively analyze the correlation between fluorescence parameters and LNC based on laser-induced fluorescence (LIF) technology. The fluorescence parameters exhibited a consistent positive linear correlation with LNC in different growing years (2014 and 2015) and different rice cultivars. The R2 of the models varied from 0.6978 to 0.9045. Support vector machine (SVM) was then utilized to verify the feasibility of the fluorescence parameters for monitoring LNC. Comparison of the fluorescence parameters indicated that F740 is the most sensitive (the R2 of linear regression analysis of the between predicted and measured values changed from 0.8475 to 0.9226, and REs ranged from 3.52% to 4.83%) to the changes in LNC among all fluorescence parameters. Experimental results demonstrated that fluorescence parameters based on LIF technology combined with SVM is a potential method for realizing real-time, non-destructive monitoring of paddy rice LNC, which can provide guidance for the decision-making of farmers in their N fertilization strategies. PMID:27350029

Ethidium bromide as a marker of mtDNA replication in living cells

NASA Astrophysics Data System (ADS)

Villa, Anna Maria; Fusi, Paola; Pastori, Valentina; Amicarelli, Giulia; Pozzi, Chiara; Adlerstein, Daniel; Doglia, Silvia Maria

2012-04-01

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells.

Detection of organic residues on poultry processing equipment surfaces by LED-induced fluorescence imaging

USDA-ARS?s Scientific Manuscript database

Organic residues on equipment surfaces in poultry processing plants can generate cross- contamination and increase the risk of unsafe food for consumers. This research was aimed to investigate the potential of LED-induced fluorescence imaging technique for rapid inspection of stainless steel proces...

Use of a portable fluorescence imaging device to facilitate cleaning of deli slicers

USDA-ARS?s Scientific Manuscript database

Cross-contamination is a recognized food safety concern for deli departments, and deli slicers remain a particular concern. Our laboratory previously demonstrated that deli commodity residues can be detected using fluorescence imaging, and that the efficacy of cleaning and sanitation in produce proc...

Development of an Infrared Fluorescent Gas Analyzer.

ERIC Educational Resources Information Center

McClatchie, E. A.

A prototype model low level carbon monoxide analyzer was developed using fluorescent cell and negative chopping techniques to achieve a device superior to state of art NDIR (Nondispersive infrared) analyzers in stability and cross-sensitivity to other gaseous species. It is clear that this type of analyzer has that capacity. The prototype…

Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

NASA Astrophysics Data System (ADS)

Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.

2014-08-01

Primary biological aerosol particles (PBAPs) can contribute significantly to the coarse particle burden in many environments. PBAPs can thus influence climate and precipitation systems as cloud nuclei and can spread disease to humans, animals, and plants. Measurement data and techniques for PBAPs in natural environments at high time- and size resolution are, however, sparse, and so large uncertainties remain in the role that biological particles play in the Earth system. In this study two commercial real-time fluorescence particle sensors and a Sporewatch single-stage particle impactor were operated continuously from 2 August to 2 September 2010 at a rural sampling location in Killarney National Park in southwestern Ireland. A cascade impactor was operated periodically to collect size-resolved particles during exemplary periods. Here we report the first ambient comparison of a waveband integrated bioaerosol sensor (WIBS-4) with a ultraviolet aerodynamic particle sizer (UV-APS) and also compare these real-time fluorescence techniques with results of fluorescence and optical microscopy of impacted samples. Both real-time instruments showed qualitatively similar behavior, with increased fluorescent bioparticle concentrations at night, when relative humidity was highest and temperature was lowest. The fluorescent particle number from the FL3 channel of the WIBS-4 and from the UV-APS were strongly correlated and dominated by a 3 μm mode in the particle size distribution. The WIBS FL2 channel exhibited particle modes at approx. 1 and 3 μm, and each was correlated with the concentration of fungal spores commonly observed in air samples collected at the site (ascospores, basidiospores, Ganoderma spp.). The WIBS FL1 channel exhibited variable multimodal distributions turning into a broad featureless single mode after averaging, and exhibited poor correlation with fungal spore concentrations, which may be due to the detection of bacterial and non-biological fluorescent particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are relatively insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse-mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAPs observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.

Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

NASA Astrophysics Data System (ADS)

Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.

2014-02-01

Primary biological aerosol particles (PBAP) can contribute significantly to the coarse particle burden in many environments, may thus influence climate and precipitation systems as cloud nuclei, and can spread disease to humans, animals, and plants. Measurements of PBAP in natural environments taken at high time- and size- resolution are, however, sparse and so large uncertainties remain in the role that biological particles play in the Earth system. In this study two commercial real-time fluorescence particle sensors and a Sporewatch single-stage particle impactor were operated continuously from 2 August to 2 September 2010 at a rural sampling location in Killarney National Park in south western Ireland. A cascade impactor was operated periodically to collect size-resolved particles during exemplary periods. Here we report the first ambient comparison of the waveband integrated bioaerosol sensor (WIBS-4) with the ultraviolet aerodynamic particle sizer (UV-APS) and also compare these real-time fluorescence techniques with results of fluorescence and optical microscopy of impacted samples. Both real-time instruments showed qualitatively similar behaviour, with increased fluorescent bioparticle concentrations at night when relative humidity was highest and temperature was lowest. The fluorescent particle number from the FL3 channel of the WIBS-4 and from the UV-APS were strongly correlated and dominated by a 3 μm mode in the particle size distribution. The WIBS FL2 channel exhibited particle modes at approx. 1 and 3 μm, and each were correlated with the concentration of fungal spores commonly observed in air samples collected at the site (ascospores, basidiospores, Ganoderma spp.). The WIBS FL1 channel exhibited variable multi-modal distributions turning into a broad featureless single mode after averaging and exhibited poor correlation with fungal spore concentrations, which may be due to the detection of bacterial and non-biological fluorescent particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAP observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.

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.

[Retrospective, descriptive, observational study of treatment of multiple actinic keratoses with topical methyl aminolevulinate and red light: results in clinical practice and correlation with fluorescence imaging].

PubMed

Fernández-Guarino, M; Harto, A; Sánchez-Ronco, M; Pérez-García, B; Marquet, A; Jaén, P

2008-12-01

Actinic keratosis (AK) is one of the most common skin diseases seen in clinical practice. In the last 5 years, several studies assessing the efficacy of photodynamic therapy in the treatment of multiple AKs have been published. We aimed to assess the clinical outcomes of photodynamic therapy in patients with multiple AKs and the correlation of those outcomes with fluorescence imaging. In this retrospective, descriptive, observational study of 57 patients treated in our hospital with photodynamic therapy for multiple AKs, we recorded age, sex, and lesion site (face, scalp, and dorsum of the hands). All patients were treated in the same way: methyl aminolevulinic acid (Metvix) was applied for 3 hours and the skin then irradiated with red light at 630 nm, 37 J/cm(2), for 7.5 minutes (Aktilite). The response, remission duration, tolerance, number of sessions, and fluorescence images were recorded by site. The chi(2) test was used to assess between-site differences and the correlation between fluorescence imaging and clinical response. The greatest improvements were obtained for facial lesions; these required fewer sessions and remission lasted longer than lesions at other sites. The treatment was best tolerated on the dorsum of the hands. The fluorescence area and the reduction in intensity on applying treatment were found to be strongly and significantly correlated with the extent of clinical response. Overall, the outcomes of treatment of multiple AKs with photodynamic therapy are better for the face than for the scalp and dorsum of the hands. Fluorescence imaging may be an effective tool for predicting response to treatment.

Correlation between Wavelength Dispersive X-ray Fluorescence (WDXRF) analysis of hardened concrete for chlorides vs. Atomic Absorption (AA) analysis in accordance with AASHTO T- 260; sampling and testing for chloride ion in concrete and concrete raw mater

DOT National Transportation Integrated Search

2014-04-01

A correlation between Wavelength Dispersive X-ray Fluorescence(WDXRF) analysis of Hardened : Concrete for Chlorides and Atomic Absorption (AA) analysis (current method AASHTO T-260, procedure B) has been : found and a new method of analysis has been ...

Processes Affecting Variability of Fluorescence Signals from Benthic Targets in Shallow Waters

DTIC Science & Technology

1997-09-30

processes in the Department of Chemistry at Brookhaven National Laboratory. The model organisms used are primarily cultured zooxanthellae obtained from...and closed (Fm) photosystem II reaction centers in the zooxanthellae isolated from the fire coral, Montipora. The short lifetime curve corresponds...individual zooxanthellae strains, is highly correlated Figure 2. The correlation between the average fluorescence lifetimes, calculated from a four

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

Improved reference standards for femtosecond three-photon excitation of fluorescence in the wavelength range 950 - 1750 nm

NASA Astrophysics Data System (ADS)

Rebane, Aleksander; Mikhaylov, Alexander

2018-02-01

Fluorescence excited by instantaneous three-photon absorption (3PA) in organic fluorophores is gaining importance as a versatile modality for deep-tissue microscopy and imaging. However, due to technical difficulty of quantifying the higher-order nonlinear absorption cross-section, reliable 3PA cross section values, σ3PA, covering a broad spectral range have been so far not available. This lack of experimental data hinders us from gaining quantitative understanding of relevant structure-property relationships as well as impedes progress towards developing 3-photon fluorophores optimized for various applications. We report on measurement of the absolute 3PA cross section spectra in the 950 - 1750 nm range in a series of common organic fluorophores in various solvents: (a) Rhodamine 6G in deuterated methanol, (b) Coumarin 153 in DMSO and toluene, (c) Prodan in DMSO and toluene, (d) Fluorescein in pH11 buffer, (e) AF455 in toluene, (f) BDPAS in deuterated methylene chloride. In these experiments, we employ femtosecond wavelength-tunable optical parametric amplifier to excite fluorescence signal that has cubic dependence on the incident photon flux. Absolute values of σ3PA are determined using two complementary methods: (i) calibrating the fluorescence signal relative to one-photon (linear) excitation combined with accurate measurement of the pulse temporal- and spatial profile to determine the excitation photon flux and (ii) calibration of the cubic fluorescence signal relative to quadratic florescence excited in fluorophores with known two-photon absorption cross section. Depending on the method utilized, the peak σ3PA values have estimated accuracy 50% and vary in the range, σ3PA = 10-81 - 10-79 cm6 s2 photon-2 , depending on the system studied, with AF455 showing the most enhanced 3PA efficiency. The 3PA spectral shapes have estimated accuracy of 20% and show some unexpected deviations from corresponding one-photon spectral profiles.

Correlation and classification of single kernel fluorescence hyperspectral data with aflatoxin concentration in corn kernels inoculated with Aspergillus flavus spores.

PubMed

Yao, H; Hruska, Z; Kincaid, R; Brown, R; Cleveland, T; Bhatnagar, D

2010-05-01

The objective of this study was to examine the relationship between fluorescence emissions of corn kernels inoculated with Aspergillus flavus and aflatoxin contamination levels within the kernels. Aflatoxin contamination in corn has been a long-standing problem plaguing the grain industry with potentially devastating consequences to corn growers. In this study, aflatoxin-contaminated corn kernels were produced through artificial inoculation of corn ears in the field with toxigenic A. flavus spores. The kernel fluorescence emission data were taken with a fluorescence hyperspectral imaging system when corn kernels were excited with ultraviolet light. Raw fluorescence image data were preprocessed and regions of interest in each image were created for all kernels. The regions of interest were used to extract spectral signatures and statistical information. The aflatoxin contamination level of single corn kernels was then chemically measured using affinity column chromatography. A fluorescence peak shift phenomenon was noted among different groups of kernels with different aflatoxin contamination levels. The fluorescence peak shift was found to move more toward the longer wavelength in the blue region for the highly contaminated kernels and toward the shorter wavelengths for the clean kernels. Highly contaminated kernels were also found to have a lower fluorescence peak magnitude compared with the less contaminated kernels. It was also noted that a general negative correlation exists between measured aflatoxin and the fluorescence image bands in the blue and green regions. The correlation coefficients of determination, r(2), was 0.72 for the multiple linear regression model. The multivariate analysis of variance found that the fluorescence means of four aflatoxin groups, <1, 1-20, 20-100, and >or=100 ng g(-1) (parts per billion), were significantly different from each other at the 0.01 level of alpha. Classification accuracy under a two-class schema ranged from 0.84 to 0.91 when a threshold of either 20 or 100 ng g(-1) was used. Overall, the results indicate that fluorescence hyperspectral imaging may be applicable in estimating aflatoxin content in individual corn kernels.

Investigating the intersystem crossing rate and triplet quantum yield of Protoporphyrin IX by means of pulse train fluorescence technique

NASA Astrophysics Data System (ADS)

Gotardo, Fernando; Cocca, Leandro H. Z.; Acunha, Thiago V.; Longoni, Ana; Toldo, Josene; Gonçalves, Paulo F. B.; Iglesias, Bernardo A.; De Boni, Leonardo

2017-04-01

Photophysical investigations of PPIX were described in order to determine the triplet conversion efficiency. Time resolved fluorescence and pulse train fluorescence were employed to characterize the main mechanism responsible for deactivation of the first singlet excited state (excited singlet and triplet states). Single pulse and Z-Scan analysis were employed to measure the singlet excited state absorption cross-sections. Theoretical calculations were performed in order to get some properties of PPIX in ground state, first singlet and triplet excited state. A TD-DFT result shows a great possibility of ISC associated to out-of-plane distortions in porphyrinic ring. Furthermore, the B and Q bands in the calculated spectrum are assigned to the four frontier molecular orbitals as proposed by Gouterman for free-based porphyrins.

Estimating the Biodegradability of Treated Sewage Samples Using Synchronous Fluorescence Spectra

PubMed Central

Lai, Tien M.; Shin, Jae-Ki; Hur, Jin

2011-01-01

Synchronous fluorescence spectra (SFS) and the first derivative spectra of the influent versus the effluent wastewater samples were compared and the use of fluorescence indices is suggested as a means to estimate the biodegradability of the effluent wastewater. Three distinct peaks were identified from the SFS of the effluent wastewater samples. Protein-like fluorescence (PLF) was reduced, whereas fulvic and/or humic-like fluorescence (HLF) were enhanced, suggesting that the two fluorescence characteristics may represent biodegradable and refractory components, respectively. Five fluorescence indices were selected for the biodegradability estimation based on the spectral features changing from the influent to the effluent. Among the selected indices, the relative distribution of PLF to the total fluorescence area of SFS (Index II) exhibited the highest correlation coefficient with total organic carbon (TOC)-based biodegradability, which was even higher than those obtained with the traditional oxygen demand-based parameters. A multiple regression analysis using Index II and the area ratio of PLF to HLF (Index III) demonstrated the enhancement of the correlations from 0.558 to 0.711 for TOC-based biodegradability. The multiple regression equation finally obtained was 0.148 × Index II − 4.964 × Index III − 0.001 and 0.046 × Index II − 1.128 × Index III + 0.026. The fluorescence indices proposed here are expected to be utilized for successful development of real-time monitoring using a simple fluorescence sensing device for the biodegradability of treated sewage. PMID:22164023

Estimating the biodegradability of treated sewage samples using synchronous fluorescence spectra.

PubMed

Lai, Tien M; Shin, Jae-Ki; Hur, Jin

2011-01-01

Synchronous fluorescence spectra (SFS) and the first derivative spectra of the influent versus the effluent wastewater samples were compared and the use of fluorescence indices is suggested as a means to estimate the biodegradability of the effluent wastewater. Three distinct peaks were identified from the SFS of the effluent wastewater samples. Protein-like fluorescence (PLF) was reduced, whereas fulvic and/or humic-like fluorescence (HLF) were enhanced, suggesting that the two fluorescence characteristics may represent biodegradable and refractory components, respectively. Five fluorescence indices were selected for the biodegradability estimation based on the spectral features changing from the influent to the effluent. Among the selected indices, the relative distribution of PLF to the total fluorescence area of SFS (Index II) exhibited the highest correlation coefficient with total organic carbon (TOC)-based biodegradability, which was even higher than those obtained with the traditional oxygen demand-based parameters. A multiple regression analysis using Index II and the area ratio of PLF to HLF (Index III) demonstrated the enhancement of the correlations from 0.558 to 0.711 for TOC-based biodegradability. The multiple regression equation finally obtained was 0.148 × Index II - 4.964 × Index III - 0.001 and 0.046 × Index II - 1.128 × Index III + 0.026. The fluorescence indices proposed here are expected to be utilized for successful development of real-time monitoring using a simple fluorescence sensing device for the biodegradability of treated sewage.

Efficient triplet harvesting by fluorescent molecules through exciplexes for high efficiency organic light-emitting diodes

NASA Astrophysics Data System (ADS)

Park, Young-Seo; Kim, Kwon-Hyeon; Kim, Jang-Joo

2013-04-01

Efficient triplet harvesting from exciplexes by reverse intersystem crossing (RISC) is reported using a fluorescent molecular system composed of the 4,4',4″-tris(N-carbazolyl)-triphenylamine and bis-4,6-(3,5-di-3-pyridylphenyl)-2-methylpyrimidine. The exciplex forming material system shows the efficient delayed fluorescence emission. As a result, almost 100% PL efficiency at 35 K and 10% external quantum efficiency at 195 K are achieved from the exciplex. The delayed fluorescence of the exciplex clearly demonstrates that a significant proportion of the triplet exciplexes is harvested through the RISC.

Cross-correlations between Renminbi and four major currencies in the Renminbi currency basket

NASA Astrophysics Data System (ADS)

Wang, Gang-Jin; Xie, Chi

2013-03-01

We investigate the cross-correlations between Renminbi (CNY) and four major currencies (USD, EUR, JPY, and KRW) in the Renminbi currency basket, i.e., the cross-correlations of CNY-USD, CNY-EUR, CNY-JPY, and CNY-KRW. Qualitatively, using a statistical test in analogy to the Ljung-Box test, we find that cross-correlations significantly exist in CNY-USD, CNY-EUR, CNY-JPY, and CNY-KRW. Quantitatively, employing the detrended cross-correlation analysis (DCCA) method, we find that the cross-correlations of CNY-USD, CNY-EUR, CNY-JPY, and CNY-KRW are weakly persistent. We use the DCCA cross-correlation coefficient ρ to quantify the level of cross-correlations and find the currency weight in the Renminbi currency basket is arranged in the order of USD>EUR>JPY >KRW. Using the method of rolling windows, which can capture the time-varying cross-correlation scaling exponents, we find that: (i) CNY and USD are positively cross-correlated over time, but the cross-correlations of CNY-USD are anti-persistent during the US sub-prime crisis and the European debt crisis. (ii) The cross-correlation scaling exponents of CNY-EUR have the cyclical fluctuation with a nearly two-year cycle. (iii) CNY-JPY has long-term negative cross-correlations, during the European debt crisis, but CNY and KRW are positively cross-correlated.

Diiodobodipy-styrylbodipy Dyads: Preparation and Study of the Intersystem Crossing and Fluorescence Resonance Energy Transfer.

PubMed

Wang, Zhijia; Xie, Yun; Xu, Kejing; Zhao, Jianzhang; Glusac, Ksenija D

2015-07-02

2,6-Diiodobodipy-styrylbodipy dyads were prepared to study the competing intersystem crossing (ISC) and the fluorescence-resonance-energy-transfer (FRET), and its effect on the photophysical property of the dyads. In the dyads, 2,6-diiodobodipy moiety was used as singlet energy donor and the spin converter for triplet state formation, whereas the styrylbodipy was used as singlet and triplet energy acceptors, thus the competition between the ISC and FRET processes is established. The photophysical properties were studied with steady-state UV-vis absorption and fluorescence spectroscopy, electrochemical characterization, and femto/nanosecond time-resolved transient absorption spectroscopies. FRET was confirmed with steady state fluorescence quenching and fluorescence excitation spectra and ultrafast transient absorption spectroscopy (kFRET = 5.0 × 10(10) s(-1)). The singlet oxygen quantum yield (ΦΔ = 0.19) of the dyad was reduced as compared with that of the reference spin converter (2,6-diiodobodipy, ΦΔ = 0.85), thus the ISC was substantially inhibited by FRET. Photoinduced intramolecular electron transfer (ET) was studied by electrochemical data and fluorescence quenching. Intermolecular triplet energy transfer was studied with nanosecond transient absorption spectroscopy as an efficient (ΦTTET = 92%) and fast process (kTTET = 5.2 × 10(4) s(-1)). These results are useful for designing organic triplet photosensitizers and for the study of the photophysical properties.

Fluorescence of bioaerosols: mathematical model including primary fluorescing and absorbing molecules in bacteria.

PubMed

Hill, Steven C; Pan, Yong-Le; Williamson, Chatt; Santarpia, Joshua L; Hill, Hanna H

2013-09-23

This paper describes a mathematical model of fluorescent biological particles composed of bacteria, viruses, or proteins. The fluorescent and/or light absorbing molecules included in the model are amino acids (tryptophan, etc.); nucleic acids (DNA, RNA, etc.); coenzymes (nicotinamide adenine dinucleotides, flavins, and vitamins B₆ and K and variants of these); and dipicolinates. The concentrations, absorptivities, and fluorescence quantum yields are estimated from the literature, often with large uncertainties. The bioparticles in the model are spherical and homogeneous. Calculated fluorescence cross sections for particles excited at 266, 280, and 355 nm are compared with measured values from the literature for several bacteria, bacterial spores and albumins. The calculated 266- and 280-nm excited fluorescence is within a factor of 3.2 of the measurements for the vegetative cells and proteins, but overestimates the fluorescence of spores by a factor of 10 or more. This is the first reported modeling of the fluorescence of bioaerosols in which the primary fluorophores and absorbing molecules are included.

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

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.

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

PubMed Central

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

2014-01-01

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

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

DOE PAGES

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

2014-10-31

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

Detection of proximal caries using quantitative light-induced fluorescence-digital and laser fluorescence: a comparative study.

PubMed

Yoon, Hyung-In; Yoo, Min-Jeong; Park, Eun-Jin

2017-12-01

The purpose of this study was to evaluate the in vitro validity of quantitative light-induced fluorescence-digital (QLF-D) and laser fluorescence (DIAGNOdent) for assessing proximal caries in extracted premolars, using digital radiography as reference method. A total of 102 extracted premolars with similar lengths and shapes were used. A single operator conducted all the examinations using three different detection methods (bitewing radiography, QLF-D, and DIAGNOdent). The bitewing x-ray scale, QLF-D fluorescence loss (ΔF), and DIAGNOdent peak readings were compared and statistically analyzed. Each method showed an excellent reliability. The correlation coefficient between bitewing radiography and QLF-D, DIAGNOdent were -0.644 and 0.448, respectively, while the value between QLF-D and DIAGNOdent was -0.382. The kappa statistics for bitewing radiography and QLF-D had a higher diagnosis consensus than those for bitewing radiography and DIAGNOdent. The QLF-D was moderately to highly accurate (AUC = 0.753 - 0.908), while DIAGNOdent was moderately to less accurate (AUC = 0.622 - 0.784). All detection methods showed statistically significant correlation and high correlation between the bitewing radiography and QLF-D. QLF-D was found to be a valid and reliable alternative diagnostic method to digital bitewing radiography for in vitro detection of proximal caries.

In situ fluorescence spectroscopy correlates ionomer degradation to reactive oxygen species generation in an operating fuel cell.

PubMed

Prabhakaran, Venkateshkumar; Arges, Christopher G; Ramani, Vijay

2013-11-21

The rate of generation of reactive oxygen species (ROS) within the polymer electrolyte membrane (PEM) of an operating proton exchange member fuel cell (PEMFC) was monitored using in situ fluorescence spectroscopy. A modified barrier layer was introduced between the PEM and the electrocatalyst layer to eliminate metal-dye interactions and fluorescence resonance energy transfer (FRET) effects during measurements. Standard fuel cell operating parameters (temperature, relative humidity, and electrode potential) were systematically varied to evaluate their influence on the rate of ROS generation during PEMFC operation. Independently, the macroscopic rate of PEM degradation was measured by monitoring the fluoride ion emission rate (FER) in the effluent stream at each operating condition. The ROS generation reaction rate constant (estimated from the in situ fluorescence experiments) correlated perfectly with the measured FER across all conditions, demonstrating unequivocally for the first time that a direct correlation exists between in situ ROS generation and PEM macroscopic degradation. The activation energy for ROS generation within the PEM was estimated to be 12.5 kJ mol(-1).

Analysis of laser-induced-fluorescence carbon monoxide measurements in turbulent nonpremixed flames

NASA Astrophysics Data System (ADS)

Mokhov, A. V.; Levinsky, H. B.; van der Meij, C. E.; Jacobs, R. A. A. M.

1995-10-01

The influence of fluctuating concentrations and temperature on the laser-induced-fluorescence (LIF) measurement of CO in turbulent flames is described, under conditions in which the fluorescence and the temperature are measured independently. The analysis shows that correlations between CO concentration and temperature can bias the averaged mole fraction extracted from LIF measurements. The magnitude of the bias can exceed the order of the average CO mole fraction. Further, LIF measurements of CO concentrations in a turbulent, nonpremixed, natural gas flame are described. The averaged CO mole fractions are derived from the fluorescence measurements by the use of flame temperatures independently measured by coherent anti-Stokes Raman spectroscopy. Analysis of the fluctuations in measured temperature and fluorescence indicates that temperature and CO concentrations in flame regions with intensive mixing are indeed correlated. In the flame regions where burnout of CO has ceased, the LIF measurements of the CO mole fraction correspond to the probe measurements in exhaust.

Ex-vivo UV autofluorescence imaging and fluorescence spectroscopy of atherosclerotic pathology in human aorta

NASA Astrophysics Data System (ADS)

Lewis, William; Williams, Maura; Franco, Walfre

2017-02-01

The aim of our study was to identify fluorescence excitation-emission pairs correlated with atherosclerotic pathology in ex-vivo human aorta. Wide-field images of atherosclerotic human aorta were captured using UV and visible excitation and emission wavelength pairs of several known fluorophores to investigate correspondence with gross pathologic features. Fluorescence spectroscopy and histology were performed on 21 aortic samples. A matrix of Pearson correlation coefficients were determined for the relationship between relevant histologic features and the intensity of emission for 427 wavelength pairs. A multiple linear regression analysis indicated that elastin (370/460 nm) and tryptophan (290/340 nm) fluorescence predicted 58% of the variance in intima thickness (R-squared = 0.588, F(2,18) = 12.8, p=.0003), and 48% of the variance in media thickness (R-squared = 0.483, F(2,18) = 8.42, p=.002), suggesting that endogenous fluorescence intensity at these wavelengths can be utilized for improved pathologic characterization of atherosclerotic plaques.

Remote temperature measurements in femto-liter volumes using dual-focus-Fluorescence Correlation Spectroscopy.

PubMed

Müller, Claus B; Weiss, Kerstin; Loman, Anastasia; Enderlein, Jörg; Richtering, Walter

2009-05-07

Remote temperature measurements in microfluidic devices with micrometer spatial resolution are important for many applications in biology, biochemistry and chemistry. The most popular methods use the temperature-dependent fluorescence lifetime of Rhodamine B, or the temperature-dependent size of thermosensitive materials such as microgel particles. Here, we use the recently developed method of dual-focus fluorescence correlation spectroscopy (2fFCS) for measuring the absolute diffusion coefficient of small fluorescent molecules at nanomolar concentrations and show how these data can be used for remote temperature measurements on a micrometer scale. We perform comparative temperature measurements using all three methods and show that the accuracy of 2fFCS is comparable or even better than that achievable with Rhodamine B fluorescence lifetime measurements. The temperature dependent microgel swelling leads to an enhanced accuracy within a narrow temperature range around the volume phase transition temperature, but requires the availability of specific microgels, whereas 2fFCS is applicable under very general conditions.

Network inference from functional experimental data (Conference Presentation)

NASA Astrophysics Data System (ADS)

Desrosiers, Patrick; Labrecque, Simon; Tremblay, Maxime; Bélanger, Mathieu; De Dorlodot, Bertrand; Côté, Daniel C.

2016-03-01

Functional connectivity maps of neuronal networks are critical tools to understand how neurons form circuits, how information is encoded and processed by neurons, how memory is shaped, and how these basic processes are altered under pathological conditions. Current light microscopy allows to observe calcium or electrical activity of thousands of neurons simultaneously, yet assessing comprehensive connectivity maps directly from such data remains a non-trivial analytical task. There exist simple statistical methods, such as cross-correlation and Granger causality, but they only detect linear interactions between neurons. Other more involved inference methods inspired by information theory, such as mutual information and transfer entropy, identify more accurately connections between neurons but also require more computational resources. We carried out a comparative study of common connectivity inference methods. The relative accuracy and computational cost of each method was determined via simulated fluorescence traces generated with realistic computational models of interacting neurons in networks of different topologies (clustered or non-clustered) and sizes (10-1000 neurons). To bridge the computational and experimental works, we observed the intracellular calcium activity of live hippocampal neuronal cultures infected with the fluorescent calcium marker GCaMP6f. The spontaneous activity of the networks, consisting of 50-100 neurons per field of view, was recorded from 20 to 50 Hz on a microscope controlled by a homemade software. We implemented all connectivity inference methods in the software, which rapidly loads calcium fluorescence movies, segments the images, extracts the fluorescence traces, and assesses the functional connections (with strengths and directions) between each pair of neurons. We used this software to assess, in real time, the functional connectivity from real calcium imaging data in basal conditions, under plasticity protocols, and epileptic conditions.

Sample-Averaged Biexciton Quantum Yield Measured by Solution-Phase Photon Correlation

DOE PAGES

Beyler, Andrew P.; Bischof, Thomas S.; Cui, Jian; ...

2014-11-19

The brightness of nanoscale optical materials such as semiconductor nanocrystals is currently limited in high excitation flux applications by inefficient multiexciton fluorescence. We have devised a solution-phase photon correlation measurement that can conveniently and reliably measure the average biexciton-to-exciton quantum yield ratio of an entire sample without user selection bias. This technique can be used to investigate the multiexciton recombination dynamics of a broad scope of synthetically underdeveloped materials, including those with low exciton quantum yields and poor fluorescence stability. Here in this study, we have applied this method to measure weak biexciton fluorescence in samples of visible-emitting InP/ZnS andmore » InAs/ZnS core/shell nanocrystals, and to demonstrate that a rapid CdS shell growth procedure can markedly increase the biexciton fluorescence of CdSe nanocrystals.« less

Monitoring of an antigen manufacturing process.

PubMed

Zavatti, Vanessa; Budman, Hector; Legge, Raymond; Tamer, Melih

2016-06-01

Fluorescence spectroscopy in combination with multivariate statistical methods was employed as a tool for monitoring the manufacturing process of pertactin (PRN), one of the virulence factors of Bordetella pertussis utilized in whopping cough vaccines. Fluorophores such as amino acids and co-enzymes were detected throughout the process. The fluorescence data collected at different stages of the fermentation and purification process were treated employing principal component analysis (PCA). Through PCA, it was feasible to identify sources of variability in PRN production. Then, partial least square (PLS) was employed to correlate the fluorescence spectra obtained from pure PRN samples and the final protein content measured by a Kjeldahl test from these samples. In view that a statistically significant correlation was found between fluorescence and PRN levels, this approach could be further used as a method to predict the final protein content.

Sample-Averaged Biexciton Quantum Yield Measured by Solution-Phase Photon Correlation

PubMed Central

Beyler, Andrew P.; Bischof, Thomas S.; Cui, Jian; Coropceanu, Igor; Harris, Daniel K.; Bawendi, Moungi G.

2015-01-01

The brightness of nanoscale optical materials such as semiconductor nanocrystals is currently limited in high excitation flux applications by inefficient multiexciton fluorescence. We have devised a solution-phase photon correlation measurement that can conveniently and reliably measure the average biexciton-to-exciton quantum yield ratio of an entire sample without user selection bias. This technique can be used to investigate the multiexciton recombination dynamics of a broad scope of synthetically underdeveloped materials, including those with low exciton quantum yields and poor fluorescence stability. Here, we have applied this method to measure weak biexciton fluorescence in samples of visible-emitting InP/ZnS and InAs/ZnS core/shell nanocrystals, and to demonstrate that a rapid CdS shell growth procedure can markedly increase the biexciton fluorescence of CdSe nanocrystals. PMID:25409496

Laser-induced fluorescence ratios of Cajanus cajan L. under the stress of cadmium and its correlation with pigment content and pigment ratios.

PubMed

Maurya, Renu; Gopal, R

2008-04-01

Laser-induced fluorescence spectra were used to characterize the effect of cadmium on the pigment status of the leaves of Cajanus cajan L. Laser-induced fluorescence spectra of untreated as well as cadmium treated (0.01 mM, 0.10 mM, and 1.00 mM) Cajanus cajan L. were recorded using the 355 nm line of a Nd:YAG laser as the excitation source and a monochromator with an intensified charge-coupled device as a detector in the region 400-800 nm. The fluorescence intensity ratios (FIR) of control as well as treated Cajanus cajan L. have been calculated by evaluating curve fitted parameters using a Gaussian spectral function. In addition, some growth parameters, such as photosynthetic pigment content, were also measured. The 355 nm line of the laser-light-excited leaves not only showed a fluorescence emission in the red spectral region (650-800 nm), but also in the blue-green region (400-570 nm). The chlorophyll FIR F690/F740 strongly correlated with the photosynthetic pigment content (total chlorophyll and carotenoids) and its ratio. Consequently, a correlation was also seen between the ratio of the blue-green fluorescence F470/F540 and the photosynthetic pigment content. The results indicated that the plants treated with 0.01 mM of cadmium exhibited better growth, while higher concentrations of cadmium were hazardous for Cajanus cajan L.

Cross-correlations between crude oil and agricultural commodity markets

NASA Astrophysics Data System (ADS)

Liu, Li

2014-02-01

In this paper, we investigate cross-correlations between crude oil and agricultural commodity markets. Based on a popular statistical test proposed by Podobnik et al. (2009), we find that the linear return cross-correlations are significant at larger lag lengths and the volatility cross-correlations are highly significant at all of the lag lengths under consideration. Using a detrended cross-correlation analysis (DCCA), we find that the return cross-correlations are persistent for corn and soybean and anti-persistent for oat and soybean. The volatility cross-correlations are strongly persistent. Using a nonlinear cross-correlation measure, our results show that cross-correlations are relatively weak but they are significant for smaller time scales. For larger time scales, the cross-correlations are not significant. The reason may be that information transmission from crude oil market to agriculture markets can complete within a certain period of time. Finally, based on multifractal extension of DCCA, we find that the cross-correlations are multifractal and high oil prices partly contribute to food crisis during the period of 2006-mid-2008.

Rapid detection of malachite green in fish based on CdTe quantum dots coated with molecularly imprinted silica.

PubMed

Wu, Le; Lin, Zheng-Zhong; Zhong, Hui-Ping; Peng, Ai-Hong; Chen, Xiao-Mei; Huang, Zhi-Yong

2017-08-15

A sensitive fluorescence sensor for the detection of malachite green (MG) was fabricated by grafting molecularly imprinted polymers (MIPs) onto the surface of CdTe quantum dots (QDs). The MIP-coated QDs were synthesized via a reverse microemulsion method using (3-aminopropyl)triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) as functional monomer and cross-linker, respectively. The optimum molar ratio of MG, functional monomer and cross-linker was 1:3:10. The MIP-coated QDs exhibited uniform spheres with diameter around 49nm and excellent fluorescence emission at λ ex 370nm. A linear relationship with two segments between the relative fluorescence intensities and the MG concentrations ranging from 0.08 to 20μmol·L -1 could be obtained with a detection limit of 12μg·kg -1 . The fluorescent probe was successfully applied to the determination of MG in fish samples with the spiked recoveries ranging from 94.3% to 109.5% which were in accordance with those of the measurement by HPLC-UV. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of CDOM sources and their links with water quality in the lakes of Northeast China using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Zhao, Ying; Song, Kaishan; Wen, Zhidan; Fang, Chong; Shang, Yingxin; Lv, Lili

2017-07-01

The spatial distributions of the fluorescence intensities Fmax for chromophoric dissolved organic matter (CDOM) components, the fluorescence indices (FI370 and FI310) and their correlations with water quality of 19 lakes in the Songhua River Basin (SHRB) across semiarid regions of Northeast China were examined with the data collected in September 2012 and 2015. The 19 lakes were divided into two groups according to EC (threshold value = 800 μS cm-1): fresh water (N = 13) and brackish water lakes (N = 6). The fluorescent characteristics of CDOM in the 19 lakes were investigated using excitation-emission matrix fluorescence spectroscopy (EEM) coupled with parallel factor (PARAFAC) and multivariate analysis. Two humic-like components (C1 and C3), one tryptophan-like component (C2), and one tyrosine-like component (C4) were identified by PARAFAC. The component C4 was not included in subsequent analyses due to the strong scatter in some colloidal water samples from brackish water lakes. The correlations between Fmax for the three EEM-PARAFAC extracted CDOM components C1-C3, the fluorescence indices (FI370 and FI310) and the water quality parameters (i.e., TN, TP, Chl-a, pH, EC, turbidity (Turb) and dissolved organic carbon (DOC)) were determined by redundancy analysis (RDA). The results of RDA analysis showed that spatial variation in land cover, pollution sources, and salinity/EC gradients in water quality affected Fmax for the fluorescent components C1-C3 and the fluorescence indices (FI370 and FI310). Further examination indicated that the CDOM fluorescent components and the fluorescence indices (FI370 and FI310) did not significantly differ (t-test, p > 0.05) in fresh water (N = 13) and brackish water lakes (N = 6). There was a difference in the distribution of the average Fmax for the CDOM fluorescent components between C1 to C3 from agricultural sources and urban wastewater sources in hypereutrophic brackish water lakes. The Fmax for humic-like components C1 and C3 spatially varied with land cover among the 19 lakes. Our results indicated that the spatial distributions of Fmax for CDOM fluorescent components and their correlations with water quality can be evaluated by EEM-PARAFAC and multivariate analysis among the 19 lakes across semiarid regions of Northeast China, which has potential implication for lakes with similar genesis.

Ligand Assisted Stabilization of Fluorescence Nanoparticles; an Insight on the Fluorescence Characteristics, Dispersion Stability and DNA Loading Efficiency of Nanoparticles.

PubMed

Rhouati, Amina; Hayat, Akhtar; Mishra, Rupesh K; Bueno, Diana; Shahid, Shakir Ahmad; Muñoz, Roberto; Marty, Jean Louis

2016-07-01

This work reports on the ligand assisted stabilization of Fluospheres® carboxylate modified nanoparticles (FCMNPs), and subsequently investigation on the DNA loading capacity and fluorescence response of the modified particles. The designed fluorescence bioconjugate was characterized with enhanced fluorescence characteristics, good stability and large surface area with high DNA loading efficiency. For comparison purpose, bovine serum albumin (BSA) and polyethylene glycol (PEG) with three different length strands were used as cross linkers to modify the particles, and their DNA loading capacity and fluorescence characteristics were investigated. By comparing the performance of the particles, we found that the most improved fluorescence characteristics, enhanced DNA loading and high dispersion stability were obtained, when employing PEG of long spacer arm length. The designed fluorescence bioconjugate was observed to maintain all its characteristics under varying pH over an extended period of time. These types of bioconjugates are in great demand for fluorescence imaging and in vivo fluorescence biomedical application, especially when most of the as synthesized fluorescence particles cannot withstand to varying in vivo physiological conditions with decreases in fluorescence response and DNA loading efficiency.

Simultaneous one-dimensional fluorescence lifetime measurements of OH and CO in premixed flames

NASA Astrophysics Data System (ADS)

Jonsson, Malin; Ehn, Andreas; Christensen, Moah; Aldén, Marcus; Bood, Joakim

2014-04-01

A method for simultaneous measurements of fluorescence lifetimes of two species along a line is described. The experimental setup is based on picosecond laser pulses from two tunable optical parametric generator/optical parametric amplifier systems together with a streak camera. With an appropriate optical time delay between the two laser pulses, whose wavelengths are tuned to excite two different species, laser-induced fluorescence can be both detected temporally and spatially resolved by the streak camera. Hence, our method enables one-dimensional imaging of fluorescence lifetimes of two species in the same streak camera recording. The concept is demonstrated for fluorescence lifetime measurements of CO and OH in a laminar methane/air flame on a Bunsen-type burner. Measurements were taken in flames with four different equivalence ratios, namely ϕ = 0.9, 1.0, 1.15, and 1.25. The measured one-dimensional lifetime profiles generally agree well with lifetimes calculated from quenching cross sections found in the literature and quencher concentrations predicted by the GRI 3.0 mechanism. For OH, there is a systematic deviation of approximately 30 % between calculated and measured lifetimes. It is found that this is mainly due to the adiabatic assumption regarding the flame and uncertainty in H2O quenching cross section. This emphasizes the strength of measuring the quenching rates rather than relying on models. The measurement concept might be useful for single-shot measurements of fluorescence lifetimes of several species pairs of vital importance in combustion processes, hence allowing fluorescence signals to be corrected for quenching and ultimately yield quantitative concentration profiles.

Protein amyloids develop an intrinsic fluorescence signature during aggregation†

PubMed Central

Chan, Fiona T. S.; Kaminski Schierle, Gabriele S.; Kumita, Janet R.; Bertoncini, Carlos W.; Dobson, Christopher M.; Kaminski, Clemens F.

2017-01-01

We report observations of an intrinsic fluorescence in the visible range, which develops during the aggregation of a range of polypeptides, including the disease-related human peptides amyloid-β(1–40) and (1–42), lysozyme and tau. Characteristic fluorescence properties such as the emission lifetime and spectra were determined experimentally. This intrinsic fluorescence is independent of the presence of aromatic side-chain residues within the polypeptide structure. Rather, it appears to result from electronic levels that become available when the polypeptide chain folds into a cross-β sheet scaffold similar to what has been reported to take place in crystals. We use these findings to quantify protein aggregation in vitro by fluorescence imaging in a label-free manner. PMID:23420088

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

NASA Astrophysics Data System (ADS)

Just Sørensen, Thomas; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

2013-06-01

Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

Simultaneous AFM and fluorescence imaging: A method for aligning an AFM-tip with an excitation beam using a 2D galvanometer

NASA Astrophysics Data System (ADS)

Moores, A. N.; Cadby, A. J.

2018-02-01

Correlative fluorescence and atomic force microscopy (AFM) imaging is a highly attractive technique for use in biological imaging, enabling force and mechanical measurements of particular structures whose locations are known due to the specificity of fluorescence imaging. The ability to perform these two measurements simultaneously (rather than consecutively with post-processing correlation) is highly valuable because it would allow the mechanical properties of a structure to be tracked over time as changes in the sample occur. We present an instrument which allows simultaneous AFM and fluorescence imaging by aligning an incident fluorescence excitation beam with an AFM-tip. Alignment was performed by calibrating a 2D galvanometer present in the excitation beam path and using it to reposition the incident beam. Two programs were developed (one manual and one automated) which correlate sample features between the AFM and fluorescence images, calculating the distance required to translate the incident beam towards the AFM-tip. Using this method, we were able to obtain beam-tip alignment (and therefore field-of-view alignment) from an offset of >15 μm to within one micron in two iterations of the program. With the program running alongside data acquisition for real-time feedback between AFM and optical images, this offset was maintained over a time period of several hours. Not only does this eliminate the need to image large areas with both techniques to ensure that fields-of-view overlap, but it also raises the possibility of using this instrument for tip-enhanced fluorescence applications, a technique in which super-resolution images have previously been achieved.

Advances in Fluorescence Sensing Systems for the Remote Assessment of Nitrogen Supply in Field Corn

NASA Technical Reports Server (NTRS)

Corp, L. A.; Chappelle, E. W.; McMurtrey, J. E.; Daughtry, C. S. T.; Kim, M. S.

2000-01-01

The studies described herein were conducted to better define changes in fluorescence properties of leaves from field grown corn (Zea mays L.) as they relate to varying levels of nitrogen (N) fertilization. This research was directed toward: 1) providing a remote non-destructive sensing technique to aid in the determination of optimal rates of N fertilization in corn crops and, 2) defining parameters for further development of fluorescence instrumentation to be operated remotely at field canopy levels. Fluorescence imaging bands centered in the blue (450 nm), green (525 nm), red (680 nm), and far-red (740 nm) and ratios of these bands were compared with the following plant parameters: rates of photosynthesis, N:C ratio, pigment concentrations, and grain yields. Both the fluorescence and physiological measures exhibited similar curvilinear responses to N fertilization level while significant linear correlations were obtained among fluorescence bands and band ratios to certain physiological measures of plant productivity. The red / blue, red / green, far-red / blue, far-red /green fluorescence ratios are well suited for remote observation and provided high correlations to grain yield, LAI, N:C, and chlorophyll contents. The results from this investigation indicate that fluorescence technology could aid in the determination of N fertilization requirements for corn. This discussion will also address design concepts and preliminary field trials of a mobile field-based Laser Induced Fluorescence Imaging System (LIFIS) capable of simultaneously acquiring images of four fluorescence emission bands from areas of plant canopies equaling 1 sq m and greater without interference of ambient solar radiation.

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.

Transgenic nude mice ubiquitously expressing fluorescent proteins for color-coded imaging of the tumor microenvironment.

PubMed

Hoffman, Robert M

2014-01-01

We have developed a transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the β-actin promoter drives GFP expression in essentially all tissues. In the adult mice, many organs brightly expressed GFP, including the spleen, heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum as well as the circulatory system. The liver expressed GFP at a lesser level. The red fluorescent protein (RFP) transgenic nude mouse was obtained by crossing non-transgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives RFP (DsRed2) expression in essentially all tissues. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, liver, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. The cyan fluorescent protein (CFP) nude mouse was developed by crossing nontransgenic nude mice with the transgenic CK/ECFP mouse in which the β-actin promoter drives expression of CFP in almost all tissues. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescence signals of all internal organs, which vary in intensity. The GFP, RFP, and CFP nude mice when transplanted with cancer cells of another color are powerful models for color-coded imaging of the tumor microenvironment (TME) at the cellular level.

Real-time detection of airborne fluorescent bioparticles in Antarctica

NASA Astrophysics Data System (ADS)

Crawford, Ian; Gallagher, Martin W.; Bower, Keith N.; Choularton, Thomas W.; Flynn, Michael J.; Ruske, Simon; Listowski, Constantino; Brough, Neil; Lachlan-Cope, Thomas; Fleming, Zoë L.; Foot, Virginia E.; Stanley, Warren R.

2017-12-01

We demonstrate, for the first time, continuous real-time observations of airborne bio-fluorescent aerosols recorded at the British Antarctic Survey's Halley VI Research Station, located on the Brunt Ice Shelf close to the Weddell Sea coast (lat 75°34'59'' S, long 26°10'0'' W) during Antarctic summer, 2015. As part of the NERC MAC (Microphysics of Antarctic Clouds) aircraft aerosol cloud interaction project, observations with a real-time ultraviolet-light-induced fluorescence (UV-LIF) spectrometer were conducted to quantify airborne biological containing particle concentrations along with dust particles as a function of wind speed and direction over a 3-week period. Significant, intermittent enhancements of both non- and bio-fluorescent particles were observed to varying degrees in very specific wind directions and during strong wind events. Analysis of the particle UV-induced emission spectra, particle sizes and shapes recorded during these events suggest the majority of particles were likely a subset of dust with weak fluorescence emission responses. A minor fraction, however, were likely primary biological particles that were very strongly fluorescent, with a subset identified as likely being pollen based on comparison with laboratory data obtained using the same instrument. A strong correlation of bio-fluorescent particles with wind speed was observed in some, but not all, periods. Interestingly, the fraction of fluorescent particles to total particle concentration also increased significantly with wind speed during these events. The enhancement in concentrations of these particles could be interpreted as due to resuspension from the local ice surface but more likely due to emissions from distal sources within Antarctica as well as intercontinental transport. Likely distal sources identified by back trajectory analyses and dispersion modelling were the coastal ice margin zones in Halley Bay consisting of bird colonies with likely associated high bacterial activity together with contributions from exposed ice margin bacterial colonies but also long-range transport from the southern coasts of Argentina and Chile. Dispersion modelling also demonstrated emissions from shipping lanes, and therefore marine anthropogenic sources cannot be ruled out. Average total concentrations of total fluorescent aerosols were found to be 1.9 ± 2.6 L-1 over a 3-week period crossing over from November into December, but peak concentrations during intermittent enhancement events could be up to several tens per litre. While this short pilot study is not intended to be generally representative of Antarctic aerosol, it demonstrates the usefulness of the UV-LIF measurement technique for quantification of airborne bioaerosol concentrations and to understand their dispersion. The potential importance for microbial colonisation of Antarctica is highlighted.

[Derivative synchronous fluorimetry for determination of synthetic food dyes in food].

PubMed

Xie, Zhi-Hai; Wang, Ling-Yan; Liu, Yu; Cai, Qing; Wang, Hai-Li; Yan, Hong-Tao

2014-05-01

The first order derivative synchronous fluoremetry was proposed for simultaneous determination of sunset yellow and ponceau 4R The effect of different experimental conditions, such as different pH for character of fluorescence spectra and the choosing of the optimal wavelength difference were studied. It was showed that the zero-crossing points were at 313. 6 nm for ponceau 4R and at 302. 8 nm for sunset yellow in first order derivative synchronous fluorescence spectra. Therefore, 313. 6 and 302. 8 nm were selected for the determination of sunset yellow and ponceau 4R when delta lambda= 130 nm. This method could minimize interference without preseparation. The linear ranges of sunset yellow and ponceau 4R were from 0. 1 to 2. 0 mg L-1 and from 0. 1 to 4. 0 mg L-1 with correlation coefficient 0. 996 6 and 0. 999 2, the detection limits were 0. 041 and 0. 019 mg L-1 , RS-Ds were 4. 6% and 4. 8% (n=6), respectively. The recoveries varied from 91. 0% to 110%. The proposed method was successfully applied in simultaneous determination of sunset yellow and Ponceau 4R in food.

Differential serological testing by simultaneous indirect immunofluorescent antibody test in canine leishmaniosis and ehrlichiosis.

PubMed

Guillén Llera, J L; López García, M L; Martín Reinoso, E; De Vivar González, R

2002-11-11

A mixed indirect fluorescence antibody test (IFAT), based on cultured promastigotes Leishmania infantum and formol-inactivated suspension of cells infected with the bacteria Ehrlichia canis, was applied to make a differential diagnosis between canine ehrlichiosis and leishmaniosis. A titre greater than 80 was considered positive for antibodies to E. canis and suggestive of antibodies to L. infantum. Positive sera were titrated subsequently by serial dilutions to confirm antibodies positive to Leishmania and establishing the antibody titre of both pathogens. Fluorescence was absent with negative control sera and background staining was minimal. No serological cross-reactions between positive sera for L. infantum or E. canis were detected. Results obtained by mixed IFAT did not differ when the same serum IFAT standard was compared. The test showed equivalent sensitivity (100%). The specifities were 100% for L. infantum and 98.5% for E. canis. The equivalence in sensitivity was confirmed by calculating the correlation coefficient between IFAT standards and mixed IFAT (r>or=0.99 for both pathogens). The results of our investigations demonstrated that mixed IFAT is a specific means of establishing serological differential diagnosis of canine leishmaniosis and ehrlichiosis.

In vitro particle image velocity measurements in a model root canal: flow around a polymer rotary finishing file.

PubMed

Koch, Jon D; Smith, Nicholas A; Garces, Daniel; Gao, Luyang; Olsen, F Kris

2014-03-01

Root canal irrigation is vital to thorough debridement and disinfection, but the mechanisms that contribute to its effectiveness are complex and uncertain. Traditionally, studies in this area have relied on before-and-after static comparisons to assess effectiveness, but new in situ tools are being developed to provide real-time assessments of irrigation. The aim in this work was to measure a cross section of the velocity field in the fluid flow around a polymer rotary finishing file in a model root canal. Fluorescent microparticles were seeded into an optically accessible acrylic root canal model. A polymer rotary finishing file was activated in a static position. After laser excitation, fluorescence from the microparticles was imaged onto a frame-transfer camera. Two consecutive images were cross-correlated to provide a measurement of a projected, 2-dimensional velocity field. The method reveals that fluid velocities can be much higher than the velocity of the file because of the shape of the file. Furthermore, these high velocities are in the axial direction of the canal rather than only in the direct of motion of the file. Particle image velocimetry indicates that fluid velocities induced by the rotating file can be much larger than the speed of the file. Particle image velocimetry can provide qualitative insight and quantitative measurements that may be useful for validating computational fluid dynamic models and connecting clinical observations to physical explanations in dental research. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Dibenzopyrrolo[1,2-a][1,8]naphthyridines: Synthesis and Structural Modification of Fluorescent L-Shaped Heteroarenes.

PubMed

Tateno, Kotaro; Ogawa, Rie; Sakamoto, Ryota; Tsuchiya, Mizuho; Kutsumura, Noriki; Otani, Takashi; Ono, Kosuke; Kawai, Hidetoshi; Saito, Takao

2018-01-19

The L-shaped, π-extended pentacycle dibenzopyrrolo[1,2-a][1,8]naphthyridine and its derivatives were synthesized using two methods: fully intramolecular [2 + 2 + 2] cycloaddition and oxidative aromatization using substituted carbodiimide and modification of an electron-rich indole ring of an L-shaped skeleton via electrophilic reaction and cross-coupling. These L-shaped compounds emitted fluorescence in high quantum yield. The position of substituents affected the fluorescence color through two different mechanisms, π-conjugation and skeletal distortion, which caused the substituted L-shaped compounds to emit fluorescence in a variety of colors and to exhibit solvato-fluorochromism.

Bulky Counterions: Enhancing the Two-Photon Excited Fluorescence of Gold Nanoclusters.

PubMed

Bertorelle, Franck; Moulin, Christophe; Soleilhac, Antonin; Comby-Zerbino, Clothilde; Dugourd, Philippe; Russier-Antoine, Isabelle; Brevet, Pierre-François; Antoine, Rodolphe

2018-01-19

Increasing fluorescence quantum yields of ligand-protected gold nanoclusters has attracted wide research interest. The strategy consisting in using bulky counterions has been found to dramatically enhance the fluorescence. In this Communication, we push forward this concept to the nonlinear optical regime. We show that by an appropriate choice of bulky counterions and of solvent, a 30-fold increase in two-photon excited fluorescence (TPEF) signal at ≈600 nm for gold nanoclusters can be obtained. This would correspond to a TPEF cross-section in the range of 0.1 to 1 GM. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cross-correlations between crude oil and exchange markets for selected oil rich economies

NASA Astrophysics Data System (ADS)

Li, Jianfeng; Lu, Xinsheng; Zhou, Ying

2016-07-01

Using multifractal detrended cross-correlation analysis (MF-DCCA), this paper studies the cross-correlation behavior between crude oil market and five selected exchange rate markets. The dataset covers the period of January 1,1996-December 31,2014, and contains 4,633 observations for each of the series, including daily closing prices of crude oil, Australian Dollars, Canadian Dollars, Mexican Pesos, Russian Rubles, and South African Rand. Our empirical results obtained from cross-correlation statistic and cross-correlation coefficient have confirmed the existence of cross-correlations, and the MF-DCCA results have demonstrated a strong multifractality between cross-correlated crude oil market and exchange rate markets in both short term and long term. Using rolling window analysis, we have also found the persistent cross-correlations between the exchange rates and crude oil returns, and the cross-correlation scaling exponents exhibit volatility during some time periods due to its sensitivity to sudden events.

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.

Weighted partial least squares based on the error and variance of the recovery rate in calibration set.

PubMed

Yu, Shaohui; Xiao, Xue; Ding, Hong; Xu, Ge; Li, Haixia; Liu, Jing

2017-08-05

The quantitative analysis is very difficult for the emission-excitation fluorescence spectroscopy of multi-component mixtures whose fluorescence peaks are serious overlapping. As an effective method for the quantitative analysis, partial least squares can extract the latent variables from both the independent variables and the dependent variables, so it can model for multiple correlations between variables. However, there are some factors that usually affect the prediction results of partial least squares, such as the noise, the distribution and amount of the samples in calibration set etc. This work focuses on the problems in the calibration set that are mentioned above. Firstly, the outliers in the calibration set are removed by leave-one-out cross-validation. Then, according to two different prediction requirements, the EWPLS method and the VWPLS method are proposed. The independent variables and dependent variables are weighted in the EWPLS method by the maximum error of the recovery rate and weighted in the VWPLS method by the maximum variance of the recovery rate. Three organic matters with serious overlapping excitation-emission fluorescence spectroscopy are selected for the experiments. The step adjustment parameter, the iteration number and the sample amount in the calibration set are discussed. The results show the EWPLS method and the VWPLS method are superior to the PLS method especially for the case of small samples in the calibration set. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiple Velocity Profile Measurements in Hypersonic Flows Using Sequentially-Imaged Fluorescence Tagging

NASA Technical Reports Server (NTRS)

Bathel, Brett F.; Danehy, Paul M.; Inman, Jennifer A.; Jones, Stephen B.; Ivey,Christopher b.; Goyne, Christopher P.

2010-01-01

Nitric-oxide planar laser-induced fluorescence (NO PLIF) was used to perform velocity measurements in hypersonic flows by generating multiple tagged lines which fluoresce as they convect downstream. For each laser pulse, a single interline, progressive scan intensified CCD (charge-coupled device) camera was used to obtain two sequential images of the NO molecules that had been tagged by the laser. The CCD configuration allowed for sub-microsecond acquisition of both images, resulting in sub-microsecond temporal resolution as well as sub-mm spatial resolution (0.5-mm horizontal, 0.7-mm vertical). Determination of axial velocity was made by application of a cross-correlation analysis of the horizontal shift of individual tagged lines. A numerical study of measured velocity error due to a uniform and linearly-varying collisional rate distribution was performed. Quantification of systematic errors, the contribution of gating/exposure duration errors, and the influence of collision rate on temporal uncertainty were made. Quantification of the spatial uncertainty depended upon the signal-to-noise ratio of the acquired profiles. This velocity measurement technique has been demonstrated for two hypersonic flow experiments: (1) a reaction control system (RCS) jet on an Orion Crew Exploration Vehicle (CEV) wind tunnel model and (2) a 10-degree half-angle wedge containing a 2-mm tall, 4-mm wide cylindrical boundary layer trip. The experiments were performed at the NASA Langley Research Center's 31-Inch Mach 10 Air Tunnel.

Microsecond protein dynamics observed at the single-molecule level

NASA Astrophysics Data System (ADS)

Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

2015-07-01

How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.

Microsecond protein dynamics observed at the single-molecule level

PubMed Central

Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

2015-01-01

How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767

Highly stable lipid-encapsulation of fluorescent nanodiamonds for bioimaging applications.

PubMed

Sotoma, Shingo; Hsieh, Feng-Jen; Chen, Yen-Wei; Tsai, Pei-Chang; Chang, Huan-Cheng

2018-01-23

Highly stable lipid-encapsulated fluorescent nanodiamonds (FNDs) are produced by photo-crosslinking of diacetylene-containing lipids physically attached to the FND surface. Not only is this coating method simple and fast, but also it gives the FND-lipid hybrids favorable properties for bioapplications. The hybrids are useful as fluorescent biolabels as well as fiducial markers for correlative light and electron microscopy.

Nanodiamonds as multi-purpose labels for microscopy.

PubMed

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

Fluorescence Guided PDT for Optimization of Skin Cancer Treatment

NASA Astrophysics Data System (ADS)

Blanco, Kate; Moriyama, Lilian; Inada, Natalia; Kurachi, Cristina; Salvio, Ana; Leite, Everson; Menezes, Priscila; Bagnato, Vanderlei

2015-04-01

The photodynamic therapy (PDT) is an alternative technique that can be indicated for superficial basal cell carcinoma (sBCC), Bowen’s disease and actinic keratosis with high efficiency. The objective of this study is to present the importance of fluorescence imaging for PDT guidance and monitoring in real time. Confirming that the lesion is well prepared and the photosensitizer shows a homogenous distribution, the outcome after few PDT sessions will be positive and the recurrence should be lower. Our proposition in this study is use the widefield fluorescence imaging to evaluate the PDT protocol in situ and in real time for each lesion. This evaluation procedure is performed in two steps: first with the monitoring of the production of protoporphyrin IX (PpIX) induced by methyl aminolevulinate (MAL), an derivative of 5-aminolevulinic acid (ALA) and second with the detection of PpIX photobleaching after illumination. The fluorescence images provide information correlated with distinct clinical features and with the treatment outcome. Eight BCC lesions are presented and discussed in this study. Different fluorescence patterns of PpIX production and photobleaching could be correlated with the treatment response. The presented results show the potential of using widefield fluorescence imaging as a guidance tool to customized PDT.

Study on fluorescence of Maillard reaction compounds in breakfast cereals.

PubMed

Delgado-Andrade, Cristina; Rufián-Henares, José A; Morales, Francisco J

2006-09-01

During the advanced stage of the Maillard reaction (MR) in food processing and cooking, Amadori rearrangement products undergo dehydration and fission and fluorescent substances are formed. Free and total (free + linked to the protein backbone) fluorescence (FIC) due to Maillard compounds in 60 commercial breakfast cereals was evaluated. Pronase was used for efficient release of linked fluorescent Maillard compounds from the protein backbone. Results were correlated with some heat-induced markers of the extent of the MR or sugar caramelisation during cereal processing, such as hydroxymethylfurfural, furfural, glucosilisomaltol and furosine. The effect of sample composition (dietary-fibre added, protein, etc.) on levels of FIC, expressed as fluorescence intensity (FI) per milligram of sample, is discussed. FIC is significantly correlated to the protein content of the sample and fluorescent Maillard compounds are mainly linked to the protein backbone. The ratio of total-FIC to free-FIC was 10.4-fold for corn-based, wheat-based and multicereal-based breakfast cereals but significantly higher in rice-based samples. Addition of dietary fibre or honey increased the FIC values. Data support the usefulness of FIC measurement as an unspecific heat-induced marker in breakfast cereals.

Multiscale Detrended Cross-Correlation Analysis of STOCK Markets

NASA Astrophysics Data System (ADS)

Yin, Yi; Shang, Pengjian

2014-06-01

In this paper, we employ the detrended cross-correlation analysis (DCCA) to investigate the cross-correlations between different stock markets. We report the results of cross-correlated behaviors in US, Chinese and European stock markets in period 1997-2012 by using DCCA method. The DCCA shows the cross-correlated behaviors of intra-regional and inter-regional stock markets in the short and long term which display the similarities and differences of cross-correlated behaviors simply and roughly and the persistence of cross-correlated behaviors of fluctuations. Then, because of the limitation and inapplicability of DCCA method, we propose multiscale detrended cross-correlation analysis (MSDCCA) method to avoid "a priori" selecting the ranges of scales over which two coefficients of the classical DCCA method are identified, and employ MSDCCA to reanalyze these cross-correlations to exhibit some important details such as the existence and position of minimum, maximum and bimodal distribution which are lost if the scale structure is described by two coefficients only and essential differences and similarities in the scale structures of cross-correlation of intra-regional and inter-regional markets. More statistical characteristics of cross-correlation obtained by MSDCCA method help us to understand how two different stock markets influence each other and to analyze the influence from thus two inter-regional markets on the cross-correlation in detail, thus we get a richer and more detailed knowledge of the complex evolutions of dynamics of the cross-correlations between stock markets. The application of MSDCCA is important to promote our understanding of the internal mechanisms and structures of financial markets and helps to forecast the stock indices based on our current results demonstrated the cross-correlations between stock indices. We also discuss the MSDCCA methods of secant rolling window with different sizes and, lastly, provide some relevant implications and issue.

Multifractal detrended cross-correlation analysis in the MENA area

NASA Astrophysics Data System (ADS)

El Alaoui, Marwane; Benbachir, Saâd

2013-12-01

In this paper, we investigated multifractal cross-correlations qualitatively and quantitatively using a cross-correlation test and the Multifractal detrended cross-correlation analysis method (MF-DCCA) for markets in the MENA area. We used cross-correlation coefficients to measure the level of this correlation. The analysis concerns four stock market indices of Morocco, Tunisia, Egypt and Jordan. The countries chosen are signatory of the Agadir agreement concerning the establishment of a free trade area comprising Arab Mediterranean countries. We computed the bivariate generalized Hurst exponent, Rényi exponent and spectrum of singularity for each pair of indices to measure quantitatively the cross-correlations. By analyzing the results, we found the existence of multifractal cross-correlations between all of these markets. We compared the spectrum width of these indices; we also found which pair of indices has a strong multifractal cross-correlation.

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

PubMed

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

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Laboratory studies of in vivo fluorescence of phytoplankton

NASA Technical Reports Server (NTRS)

Brown, C. A., Jr.; Farmer, F. H.; Jarrett, O., Jr.; Staton, W. L.

1978-01-01

A lidar system is developed that uses four selected excitation wavelengths to induce chlorophyll 'a' fluorescence which is indicative of both the concentration and diversity of phytoplankton. The operating principles of the system and the results of measurements of phytoplankton fluorescence in a controlled laboratory environment are presented. A comparative study of results from lidar fluorosensor laboratory tank tests using representative species of phytoplankton in single and multispecies cultures from each of four color groups reveals that (1) there is good correlation between the fluorescence of chlorophyll 'a' remotely simulated and detected by the lidar system and in-situ measurements using four similar excitation wavelengths in a flow-through fluorometer; (2) good correlation exists between the total chlorophyll 'a' calculated from lidar-fluorosensor data and measurements obtained by the Strickland-Parsons method; and (3) the lidar fluorosensor can provide an index of population diversity.

Tracking Silent Hypersensitivity Reactions to Asparaginase during Leukemia Therapy Using Single-Chip Indirect Plasmonic and Fluorescence Immunosensing.

PubMed

Charbonneau, David M; Breault-Turcot, Julien; Sinnett, Daniel; Krajinovic, Maja; Leclerc, Jean-Marie; Masson, Jean-François; Pelletier, Joelle N

2017-12-22

Microbial asparaginase is an essential component of chemotherapy for the treatment of childhood acute lymphoblastic leukemia (cALL). Silent hypersensitivity reactions to this microbial enzyme need to be monitored accurately during treatment to avoid adverse effects of the drug and its silent inactivation. Here, we present a dual-response anti-asparaginase sensor that combines indirect SPR and fluorescence on a single chip to perform ELISA-type immunosensing, and correlate measurements with classical ELISA. Analysis of serum samples from children undergoing cALL therapy revealed a clear correlation between single-chip indirect SPR/fluorescence immunosensing and ELISA used in clinical settings (R 2 > 0.9). We also report that the portable SPR/fluorescence system had a better sensitivity than classical ELISA to detect antibodies in clinical samples with low antigenicity. This work demonstrates the reliability of dual sensing for monitoring clinically relevant antibody titers in clinical serum samples.

Fluorescence spectroscopy for assessment of liver transplantation grafts concerning graft viability and patient survival

NASA Astrophysics Data System (ADS)

Vollet Filho, José D.; da Silveira, Marina R.; Castro-e-Silva, Orlando; Bagnato, Vanderlei S.; Kurachi, Cristina

2015-06-01

Evaluating transplantation grafts at harvest is essential for its success. Laser-induced fluorescence spectroscopy (LIFS) can help monitoring changes in metabolic/structural conditions of tissue during transplantation. The aim of the present study is to correlate LIFSobtained spectra of human hepatic grafts during liver transplantation with post-operative patients' mortality rate and biochemical parameters, establishing a method to exclude nonviable grafts before implantation. Orthotopic liver transplantation, piggyback technique was performed in 15 patients. LIFS was performed under 408nm excitation. Collection was performed immediately after opening donor's abdominal cavity, after cold perfusion, end of back-table period, and 5 min and 1 h after warm perfusion at recipient. Fluorescence information was compared to lactate, creatinine, bilirubin and INR levels and to survival status. LIFS was sensitive to liver changes during transplantation stages. Study-in-progress; initial results indicate correlation between fluorescence and life/death status of patients.

Detrended cross-correlation analysis on RMB exchange rate and Hang Seng China Enterprises Index

NASA Astrophysics Data System (ADS)

Ruan, Qingsong; Yang, Bingchan; Ma, Guofeng

2017-02-01

In this paper, we investigate the cross-correlations between the Hang Seng China Enterprises Index and RMB exchange markets on the basis of a cross-correlation statistic test and multifractal detrended cross-correlation analysis (MF-DCCA). MF-DCCA has, at best, serious limitations for most of the signals describing complex natural processes and often indicates multifractal cross-correlations when there are none. In order to prevent these false multifractal cross-correlations, we apply MFCCA to verify the cross-correlations. Qualitatively, we find that the return series of the Hang Seng China Enterprises Index and RMB exchange markets were, overall, significantly cross-correlated based on the statistical analysis. Quantitatively, we find that the cross-correlations between the stock index and RMB exchange markets were strongly multifractal, and the multifractal degree of the onshore RMB exchange markets was somewhat larger than the offshore RMB exchange markets. Moreover, we use the absolute return series to investigate and confirm the fact of multifractality. The results from the rolling windows show that the short-term cross-correlations between volatility series remain high.

1.083 μm laser operation in Nd,Mg:LiTaO3 crystal

NASA Astrophysics Data System (ADS)

Hu, P. C.; Hang, Y.; Li, R.; Gong, J.; Yin, J. G.; Zhao, C. C.; He, X. M.; Yu, T.; Zhang, L. H.; Chen, W. B.; Zhu, Y. Y.

2011-10-01

Nd,Mg:LiTaO3 single crystal with high optical quality was grown by Czochralski technique. Absorption and fluorescence spectra were investigated. The peak absorption cross section at 806.5 nm and peak emission cross section at 1091 nm are 6.81×10-20 and 3.28×10-20 cm2, respectively. The fluorescence lifetime was measured to be 129 μs. With a laser-diode as the pump source, a maximum 375 mW continuous-wave laser output at 1083 nm has been obtained with a slope efficiency of 7.2% with respect to the pump power.

Photoabsorption and photodissociation of molecules important in the interstellar medium

NASA Technical Reports Server (NTRS)

Lee, Long C.; Suto, Masako

1991-01-01

The photoabsorption, photodissociation, and fluorescence cross sections of interstellar molecules are measured at 90 to 250 nm. These quantitative optical data are needed for the understanding of the formation and destruction processes of molecules under the intense interstellar UV radiation field. Research covering the following topics is presented: (1) fluorescences from photoexcitation of CH4, CH3OH, and CH3SH; (2) NO gamma emission from photoexcitation of NO; (3) photoexcitation cross sections of aromatic molecules; (4) IR emission from UV excitation of HONO2; (5) IR emission from UV excitation of benzene and methyl-derivitives; and (6) IR emission from UV excitation of polycyclic aromatic hydrocarbon molecules.

[Pediatric pneumonia, pleural effusion, and pericarditis following cat scratch disease and serological cross-reactions among Bartonella henselae and Rickettsia japonica determined by indirect fluorescence antibodies].

PubMed

Takeda, Nobue; Ishiwada, Naruhiko; Fukasawa, Chie; Furuya, Yumiko; Tsuneoka, Hidehiro; Tsukahara, Masato; Kohno, Yoichi

2007-03-01

Cat scratch disease is associated with a variety of systemic manifestations. We report a pediatric case associated with pneumonia, pleural effusion, and pericarditis. A 3-year-old boy developed prolonged fever unresponsive to antibiotic treatment, including azithromycin and minocycline. Although the fever resolved with corticosteroid treatment, Bartonella henselae IgG titer was positive in indirect fluorescence antibodies, as was Rickettsia japonica IgG titer. Both titers were significantly reduced by serum absorption with B. henselae antigens, and we observed a serological cross-reaction between B. henselae and R. japonica.

An explanation of the very low fluorescence and phosphorescence in pyridine: a CASSCF/CASMP2 study

NASA Astrophysics Data System (ADS)

Varras, Panayiotis C.; Gritzapis, Panagiotis S.; Fylaktakidou, Konstantina C.

2018-01-01

In this work, we applied the multiconfigurational complete active space self-consistent field method and the multiconfigurational second-order perturbation theory CASMP2 to study the fundamental excited states of pyridine and its possible photophysical and photochemical transformations. Our calculations, which are in agreement with the experimental results corresponding to excitations around the 0-0 transition, showed that the very low experimentally observed fluorescence of pyridine is due to the presence of two almost isoenergetic crossings, one of triple character, S1/T1/S0 and the other of S1/S0 character. Both crossings are below the minimum of S1(nπ*) and have a common transition state (S1(TS)) with a very low energy barrier (1.85 kcal/mol or 0.08 eV at the CASMP2 level of theory) separating them. A third triple crossing of the type S1/T1/S0 lying lower with respect to the other two elucidates the observed T1→S0 radiationless transition. This explains not only pyridine's very low fluorescence and phosphorescence but also its almost negligible photochemistry, showing that photophysics is the prevalent process in this molecule.

Multifractal detrended cross-correlations between the CSI 300 index futures and the spot markets based on high-frequency data

NASA Astrophysics Data System (ADS)

Cao, Guangxi; Han, Yan; Cui, Weijun; Guo, Yu

2014-11-01

The cross-correlation between the China Securities Index 300 (CSI 300) index futures and the spot markets based on high-frequency data is discussed in this paper. We empirically analyze the cross-correlation by using the multifractal detrended cross-correlation analysis (MF-DCCA), and investigate further the characteristics of asymmetry, frequency difference, and transmission direction of the cross-correlation. The results indicate that the cross-correlation between the two markets is significant and multifractal. Meanwhile, weak asymmetries exist in the cross-correlation, and higher data frequency results in a lower multifractality degree of the cross-correlation. The causal relationship between the two markets is bidirectional, but the CSI 300 index futures market has greater impact on the spot market.

Synthesis of yellow and red fluorescent 1,3a,6a-triazapentalenes and the theoretical investigation of their optical properties† †Electronic supplementary information (ESI) available: the experimental details for the synthesis of the triazapentalenes and the fluorescent cell staining, the absorption and fluorescence spectra, and the 1H and 13C NMR spectra. Also given are the molecular orbitals, the natural charges, the dipole moments, and the Cartesian coordinates of the triazapentalenes (1a, 1b, 1g, 1e, and 1f). See DOI: 10.1039/c4sc02780a Click here for additional data file.

PubMed Central

Osawa, Ayumi; Mera, Akane; Tano, Fumi; Chuman, Yoshiro; Sakuda, Eri; Taketsugu, Tetsuya; Sakaguchi, Kazuyasu; Kitamura, Noboru

2015-01-01

To expand the originally developed fluorescent 1,3a,6a-triazapentalenes as fluorescent labelling reagents, the fluorescence wavelength of the 1,3a,6a-triazapentalenes was extended to the red color region. Based on the noteworthy correlation of the fluorescence wavelength with the inductive effect of the 2-substituent, electron-deficient 2-(2-cyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene and 2-(2,6-dicyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene were synthesized. The former exhibited yellow fluorescence and the latter exhibited red fluorescence, and both compounds exhibited large Stokes shifts, and the 1,3a,6a-triazapentalene system enabled the same fluorescent chromophore to cover the entire region of visible wavelengths. The potential applications of the 1,3a,6a-triazapentalenes as fluorescent probes in the fields of the life sciences were investigated, and the 1,3a,6a-triazapentalene system was clearly proven to be useful as a fluorescent reagent for live cell imaging. Quantum chemical calculations were performed to investigate the optical properties of the 1,3a,6a-triazapentalenes. These calculations revealed that the excitation involves a significant charge-transfer from the 1,3a,6a-triazapentalene skeleton to the 2-substituent. The calculated absorption and fluorescence wavelengths showed a good correlation with the experimental ones, and thus the system could enable the theoretical design of substituents with the desired optical properties. PMID:29560196

Long-term correlations and cross-correlations in IBovespa and constituent companies

NASA Astrophysics Data System (ADS)

de Lima, Neílson F.; Fernandes, Leonardo H. S.; Jale, Jader S.; de Mattos Neto, Paulo S. G.; Stošić, Tatijana; Stošić, Borko; Ferreira, Tiago A. E.

2018-02-01

We study auto-correlations and cross-correlations of IBovespa index and its constituent companies. We use Detrended Fluctuation Analysis (DFA) to quantify auto-correlations and Detrended Cross-Correlation Analysis (DCCA) to quantify cross-correlations in absolute returns of daily closing prices of IBovespa and the individual companies. We find persistent long-term correlations and cross-correlations which are weaker than those found for USA market. Our results indicate that market indices of developing markets exhibit weaker coupling with its constituents than for mature developed markets.

Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy.

PubMed

Gröner, Nadine; Capoulade, Jérémie; Cremer, Christoph; Wachsmuth, Malte

2010-09-27

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

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

Gallagher-Jones, Marcus; Dias, Carlos Sato Baraldi; Pryor, Alan

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here in this paper, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features inmore » the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe 3O 4 core encased by a 25-nm-thick fluorescent silica (SiO 2) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles.« less

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

DOE PAGES

Gallagher-Jones, Marcus; Dias, Carlos Sato Baraldi; Pryor, Alan; ...

2017-07-06

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here in this paper, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features inmore » the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe 3O 4 core encased by a 25-nm-thick fluorescent silica (SiO 2) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles.« less

Fracture Toughness, Mechanical Property, And Chemical Characterization Of A Critical Modification To The NASA SLS Solid Booster Internal Material System

NASA Technical Reports Server (NTRS)

Pancoast, Justin; Garrett, William; Moe, Gulia

2015-01-01

A modified propellant-liner-insulation (PLI) bondline in the Space Launch System (SLS) solid rocket booster required characterization for flight certification. The chemical changes to the PLI bondline and the required additional processing have been correlated to mechanical responses of the materials across the bondline. Mechanical properties testing and analyses included fracture toughness, tensile, and shear tests. Chemical properties testing and analyses included Fourier transform infrared (FTIR) spectroscopy, cross-link density, high-performance liquid chromatography (HPLC), gas chromatography (GC), gel permeation chromatography (GPC), and wave dispersion X-ray fluorescence (WDXRF). The testing identified the presence of the expected new materials and found the functional bondline performance of the new PLI system was not significantly changed from the old system.

Price-volume multifractal analysis of the Moroccan stock market

NASA Astrophysics Data System (ADS)

El Alaoui, Marwane

2017-11-01

In this paper, we analyzed price-volume multifractal cross-correlations of Moroccan Stock Exchange. We chose the period from January 1st 2000 to January 20th 2017 to investigate the multifractal behavior of price change and volume change series. Then, we used multifractal detrended cross-correlations analysis method (MF-DCCA) and multifractal detrended fluctuation analysis (MF-DFA) to analyze the series. We computed bivariate generalized Hurst exponent, Rényi exponent and spectrum of singularity for each pair of indices to measure quantitatively cross-correlations. Furthermore, we used detrended cross-correlations coefficient (DCCA) and cross-correlation test (Q(m)) to analyze cross-correlation quantitatively and qualitatively. By analyzing results, we found existence of price-volume multifractal cross-correlations. The spectrum width has a strong multifractal cross-correlation. We remarked that volume change series is anti-persistent when we analyzed the generalized Hurst exponent for all moments q. The cross-correlation test showed the presence of a significant cross-correlation. However, DCCA coefficient had a small positive value, which means that the level of correlation is not very significant. Finally, we analyzed sources of multifractality and their degree of contribution in the series.

Density measurement in air with saturable absorbing seed gas

NASA Technical Reports Server (NTRS)

Baganoff, D.

1982-01-01

Approaches which have the potential to make density measurements in a compressible flow, where one or more laser beams are used as probes, were investigated. Saturation in sulfur hexafluoride iodine and a crossed beam technique where one beam acts as a saturating beam and the other is at low intensity and acts as a probe beam are considered. It is shown that a balance between an increase in fluorescence intensity with increasing pressure from line broadening and the normal decrease in intensity with increasing pressure from quenching can be used to develop a linear relation between fluorescence intensity and number density and lead to a new density measurement scheme. The method is used to obtain a density image of the cross section of an iodine seeded underexpanded supersonic jet of nitrogen, by illuminating the cross section by a sheet of laser light.

Application of portable online LED UV fluorescence sensor to predict the degradation of dissolved organic matter and trace organic contaminants during ozonation.

PubMed

Li, Wen-Tao; Majewsky, Marius; Abbt-Braun, Gudrun; Horn, Harald; Jin, Jing; Li, Qiang; Zhou, Qing; Li, Ai-Min

2016-09-15

This work aims to correlate signals of LED UV/fluorescence sensor with the degradation of dissolved organic matter (DOM) and trace-level organic contaminants (TOrCs) during ozonation process. Six sets of bench-scale ozonation kinetic experiments incorporated with three different water matrices and 14 TOrCs of different reactivity (group I ∼ V) were conducted. Calibrated by tryptophan and humic substances standards and verified by the lab benchtop spectroscopy, the newly developed portable/online LED sensor, which measures the UV280 absorbance, protein-like and humic-like fluorescence simultaneously, was feasible to monitor chromophores and fluorophores with good sensitivity and accuracy. The liquid chromatography with organic carbon detector combined with 2D synchronous correlation analysis further demonstrated how the DOM components of large molecular weight were transformed into small moieties as a function of the decrease of humic-like fluorescence. For TOrCs, their removal rates were well correlated with the decrease of the LED UV/fluorescence signals, and their elimination patterns were mainly determined by their reactivity with O3 and hydroxyl radicals. At approximately 50% reduction of humic-like fluorescence almost complete oxidation of TOrCs of group I and II was reached, a similar removal percentage (25-75%) of TOrCs of group III and IV, and a poor removal percentage (<25%) of group V. This study might contribute to the smart control of advanced oxidation processes for the water and wastewater treatment in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantitative analysis of tear film fluorescence and discomfort during tear film instability and thinning.

PubMed

Begley, Carolyn; Simpson, Trefford; Liu, Haixia; Salvo, Eliza; Wu, Ziwei; Bradley, Arthur; Situ, Ping

2013-04-12

The purpose of this study was to test the association between tear film fluorescence changes during tear break-up (TBU) or thinning and the concurrent ocular sensory response. Sixteen subjects kept one eye open as long as possible (MBI), indicated their discomfort level continuously, and rated ocular sensations of irritation, stinging, burning, pricking, and cooling using visual analog scales (VAS). Fluorescence of the tear film was quantified by a pixel-based analysis of the median pixel intensity (PI), TBU, and percentage of dark pixels (DarkPix) over time. A cutoff of 5% TBU was used to divide subjects into either break-up (BU) or minimal break-up (BUmin) groups. Tear film fluorescence decreased (median PI) and the percentage of TBU and DarkPix increased in all trials, with the rate significantly greater in the BU than the BUmin group (Mann-Whitney U test, P < 0.05). The rate of increasing discomfort during trials was highly correlated with the rate of decrease in median PI and developing TBU (Spearman's, r ≥ 0.70). Significant correlations were found between corneal fluorescence, MBI, and sensory measures. Concentration quenching of fluorescein dye with tear film thinning best explains decreasing tear film fluorescence during trials. This was highly correlated with increasing ocular discomfort, suggesting that both tear film thinning and TBU stimulate underlying corneal nerves, although TBU produced more rapid stimulation. Slow increases in tear film hyperosmolarity may cause the gradual increase in discomfort during slow tear film thinning, whereas the sharp increases in discomfort during TBU suggest a more complex stimulus.

Fluorescence fingerprinting properties for exploring water-soluble organic compounds in PM2.5 in an industrial city of northwest China

NASA Astrophysics Data System (ADS)

Qin, Juanjuan; Zhang, Leiming; Zhou, Xueming; Duan, Jingchun; Mu, Situ; Xiao, Kang; Hu, Jingnan; Tan, Jihua

2018-07-01

Excitation-emission matrix (EEM) spectra were used to analyze the fluorescence properties of water-soluble organic compounds (WSOC) in PM2.5 during winter and summer seasons in Lanzhou city, northwest China. Protein-like substances, humic-like substances and microbial by-products were found to be the main fluorophoric organic matter. Humification index (HIX), biological index (BIX) and fluorescence index (FI) were 1.2 ± 0.1, 1.4 ± 0.1 and 1.7 ± 0.2 in winter and 2.0 ± 0.3, 1.3 ± 0.1 and 1.2 ± 0.4 in summer, respectively, indicating higher aromaticity of WSOC in summer and terrestrial biogenic sources of WSOC in both seasons. Strong correlations were found between regional average fluorescence intensities (RAFI) at the fluorescence regions of II-V and water-soluble inorganic ions (K+, Cl-, NO3- and SO42-) in winter with the Spearman correlation coefficients ρ being larger than 0.7 and mostly around 0.9, suggesting significant contributions of bioaerosols, coal combustion and vehicle exhaust to PM2.5. Moderate correlations were found between RAFI and K+, Cl-, and NO3- in summer, indicating the more important roles of biogenic and vehicle exhaust than coal combustion sources in summer. High temperature in winter was conducive to and in summer prohibitive to polycondensation of WSOC, indicating the existence of an optimum temperature for such a process to occur. High relative humidity was unfavorable to the formation of fluorescent WSOC.

Laser Induced Optical Pumping Measurements of Cross Sections for Fine and Hyperfine Structure Transitions in Sodium Induced by Collisions with Helium Argon Atoms

NASA Technical Reports Server (NTRS)

Dobson, Chris C.; Sung, C. C.

1998-01-01

Optical pumping of the ground states of sodium can radically alter the shape of the laser induced fluorescence excitation spectrum, complicating measurements of temperature, pressure, etc., which are based on these spectra. Modeling of the fluorescence using rate equations for the eight hyperfine states of the sodium D manifolds can be used to quantify the contribution to the ground state pumping of transitions among the hyperfine excited states induced by collisions with buffer gas atoms. This model is used here to determine, from the shape of experimental spectra, cross sections for (Delta)F transitions of the P(sub 3/2) state induced by collisions with helium and argon atoms, for a range of values assumed for the P(sub 1/2), (Delta)F cross sections. The hyperfine cross sections measured using this method, which is thought to be novel, are compared with cross sections for transitions involving polarized magnetic substates, m(sub F), measured previously using polarization sensitive absorption. Also, fine structure transition ((Delta)J) cross sections were measured in the pumped vapor, giving agreement with previous measurements made in the absence of pumping.

Laser-Induced Optical Pumping Measurements of Cross Section for Fine- and Hyperfine-Structure Transitions in Sodium Induced by Collisions with Helium and Argon Atoms

NASA Technical Reports Server (NTRS)

Dobson, Chris C.; Sung, C. C.

1999-01-01

Optical pumping of the ground states of sodium can radically alter the shape of the laser-induced fluorescence excitation spectrum, complicating measurements of temperature, pressure, etc., which are based on these spectra. Modeling of the fluorescence using rate equations for the eight hyperfine states of the sodium D manifolds can be used to quantify the contribution to the ground state pumping of transitions among the hyperfine excited states induced by collisions with buffer gas atoms. This model is used here to determine, from the shape of experimental spectra, cross sections lor DELTA.F transitions of the P(sub 3/2) state induced by collisions with helium and argon atoms, for a range of values assumed for the P(sub 1/2), DELTA.F cross sections. The hyperfine cross sections measured using this method, which to our knowledge is novel, are compared with cross sections for transitions involving polarized magnetic substates m(sub F) measured previously using polarization sensitive absorption. Also, fine-structure transition cross sections were measured in the pumped vapor, giving agreement with previous measurements made in the absence of pumping.

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.

Establishing and validating the fluorescent amyloid ligand h-FTAA (heptamer formyl thiophene acetic acid) to identify transthyretin amyloid deposits in carpal tunnel syndrome.

PubMed

Hahn, Katharina; Nilsson, K Peter R; Hammarström, Per; Urban, Peter; Meliss, Rolf Rüdiger; Behrens, Hans-Michael; Krüger, Sandra; Röcken, Christoph

2017-06-01

Transthyretin-derived (ATTR) amyloidosis is a frequent finding in carpal tunnel syndrome. We tested the following hypotheses: the novel fluorescent amyloid ligand heptameric formic thiophene acetic acid (h-FTAA) has a superior sensitivity for the detection of amyloid compared with Congo red-staining; Amyloid load correlates with patient gender and/or patient age. We retrieved 208 resection specimens obtained from 184 patients with ATTR amyloid in the carpal tunnel. Serial sections were stained with Congo red, h-FTAA and an antibody directed against transthyretin (TTR). Stained sections were digitalized and forwarded to computational analyses. The amount of amyloid was correlated with patient demographics. Amyloid stained intensely with h-FTAA and an anti-TTR-antibody. Congo red-staining combined with fluorescence microscopy was significantly less sensitive than h-FTAA-fluorescence and TTR-immunostaining: the highest percentage area was found in TTR-immunostained sections, followed by h-FTAA and Congo red. The Pearson correlation coefficient was .8 (Congo red vs. h-FTAA) and .9 (TTR vs. h-FTAA). Amyloid load correlated with patient gender, anatomical site and patient age. h-FTAA is a highly sensitive method to detect even small amounts of ATTR amyloid in the carpal tunnel. The staining protocol is easy and h-FTAA may be a much more sensitive procedure to detect amyloid at an earlier stage.

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

PubMed

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

2017-02-01

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

Antibodies induced by vaccination with purified chick embryo cell culture vaccine (PCECV) cross-neutralize non-classical bat lyssavirus strains.

PubMed

Malerczyk, Claudius; Selhorst, Thomas; Tordo, Noël; Moore, Susan; Müller, Thomas

2009-08-27

Tissue-culture vaccines like purified chick embryo cell vaccine (PCECV) have been shown to provide protection against classical rabies virus (RABV) via pre-exposure or post-exposure prophylaxis. A cross-neutralization study was conducted using a panel of 100 human sera, to determine, to what extent after vaccination with PCECV protection exists against non-classical bat lyssavirus strains like European bat lyssavirus (EBLV) type 1 and 2 and Australian bat lyssavirus (ABLV). Virus neutralizing antibody (VNA) concentrations against the rabies virus variants CVS-11, ABLV, EBLV-1 and EBLV-2 were determined by using a modified rapid fluorescent focus inhibition test. For ABLV and EBLV-2, the comparison to CVS-11 revealed almost identical results (100% adequate VNA concentrations >or=0.5 IU/mL; correlation coefficient r(2)=0.69 and 0.77, respectively), while for EBLV-1 more scattering was observed (97% adequate VNA concentrations; r(2)=0.50). In conclusion, vaccination with PCECV produces adequate VNA concentrations against classical RABV as well as non-classical lyssavirus strains ABLV, EBLV-1, and EBLV-2.

Photochemical studies of a fluorescent chlorophyll catabolite--source of bright blue fluorescence in plant tissue and efficient sensitizer of singlet oxygen.

PubMed

Jockusch, Steffen; Turro, Nicholas J; Banala, Srinivas; Kräutler, Bernhard

2014-02-01

Fluorescent chlorophyll catabolites (FCCs) are fleeting intermediates of chlorophyll breakdown, which is seen as an enzyme controlled detoxification process of the chlorophylls in plants. However, some plants accumulate large amounts of persistent FCCs, such as in senescent leaves and in peels of yellow bananas. The photophysical properties of such a persistent FCC (Me-sFCC) were investigated in detail. FCCs absorb in the near UV spectral region and show blue fluorescence (max at 437 nm). The Me-sFCC fluorescence had a quantum yield of 0.21 (lifetime 1.6 ns). Photoexcited Me-sFCC intersystem crosses into the triplet state (quantum yield 0.6) and generates efficiently singlet oxygen (quantum yield 0.59). The efficient generation of singlet oxygen makes fluorescent chlorophyll catabolites phototoxic, but might also be useful as a (stress) signal and for defense of the plant tissue against infection by pathogens.

Fluorescence correlation spectroscopy: principles and applications.

PubMed

Bacia, Kirsten; Haustein, Elke; Schwille, Petra

2014-07-01

Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed. © 2014 Cold Spring Harbor Laboratory Press.

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy

PubMed Central

Brooker, Gary; Siegel, Nisan; Wang, Victor; Rosen, Joseph

2011-01-01

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging. PMID:21445140

Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

PubMed

Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

2016-01-05

A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results. Copyright © 2015 Elsevier B.V. All rights reserved.

Superiorities of time-correlated single-photon counting against standard fluorimetry in exploiting the potential of fluorochromized oligonucleotide probes for biomedical investigation

NASA Astrophysics Data System (ADS)

Lamperti, Marco; Nardo, Luca; Bondani, Maria

2015-05-01

Site-specific fluorescence-resonance-energy-transfer donor-acceptor dual-labelled oligonucleotide probes are widely used in state-of-art biotechnological applications. Such applications include their usage as primers in polymerase chain reaction. However, the steady-state fluorescence intensity signal emitted by these molecular tools strongly depends from the specificities of the probe conformation. For this reason, the information which can be reliably inferred by steady-state fluorimetry performed on such samples is forcedly confined to a semi-qualitative level. Namely, fluorescent emission is frequently used as ON/OFF indicator of the probe hybridization state, i.e. detection of fluorescence signals indicates either hybridization to or detachment from the template DNA of the probe. Nonetheless, a fully quantitative analysis of their fluorescence emission properties would disclose other exciting applications of dual-labelled probes in biosensing. Here we show how time-correlated single-photon counting can be applied to get rid of the technical limitations and interpretational ambiguities plaguing the intensity analysis, and to derive information on the template DNA reaching single-base.

Cross-correlations and influence in world gold markets

NASA Astrophysics Data System (ADS)

Lin, Min; Wang, Gang-Jin; Xie, Chi; Stanley, H. Eugene

2018-01-01

Using the detrended cross-correlation analysis (DCCA) coefficient and the detrended partial cross-correlation analysis (DPCCA) coefficient, we investigate cross-correlations and net cross-correlations among five major world gold markets (London, New York, Shanghai, Tokyo, and Mumbai) at different time scales. We propose multiscale influence measures for examining the influence of individual markets on other markets and on the entire system. We find (i) that the cross-correlations, net cross-correlations, and net influences among the five gold markets vary across time scales, (ii) that the cross-market correlation between London and New York at each time scale is intense and inherent, meaning that the influence of other gold markets on the London-New York market is negligible, (iii) that the remaining cross-market correlations (i.e., those other than London-New York) are greatly affected by other gold markets, and (iv) that the London gold market significantly affects the other four gold markets and dominates the world-wide gold market. Our multiscale findings give market participants and market regulators new information on cross-market linkages in the world-wide gold market.

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

Shcheslavskiy, V. I.; Institute of Biomedical Technologies, Nizhny Novgorod State Medical Academy, Minin and Pozharsky Square, 10/1, Nizhny Novgorod 603005; Neubauer, A.

We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.

A Passive Method for Detecting Vegetation Stress from Orbit: Chlorophyll Fluorescence Spectra from Fraunhofer Lines

NASA Technical Reports Server (NTRS)

Theisen, Arnold F.

2000-01-01

Solar-stimulated chlorophyll fluorescence measured with the Fraunhofer line depth method has correlated well with vegetation stress in previous studies. However, the instruments used in those studies were limited to a single solar absorption line (e.g. 656.3 nm), obviating the red/far-red ratio (R/FR) method. Optics and detector technology have reached the level whereby multiple, very narrow Fraunhofer lines are resolvable. Thirteen such lines span the visible spectrum in the red to far-red region where chlorophyll fluorescence occurs. Fluorescence intensities at the 13 Fraunhofer line wavelengths were used to model emission spectra. The source data were collected for summer and fall bean crops (Phaseolus vulgaris L.) subjected to various levels of nitrogen fertilization. The intensities were adjusted to account for Fraunhofer line depth and atmospheric transmittance. Multiple R/FR fluorescence ratios, calculated from the modeled fluorescence spectra, correlated strongly with leaf chlorophyll concentration and well with applied nitrogen. The ratio yielding the best correlation with chlorophyll utilized red fluorescence at the 694.5 nm Fraunhofer line and farred fluorescence at the 755.6 nm Fraunhofer line. Twenty R/FR ratios, each evaluated for the maximum differential between low and high (optimal) nitrogen treatments, ranked higher in some cases and lower in others, possibly related to the time of year the crops were grown and the stage of growth of the crops. Ratios with 728.9 nm and 738.9 nm in the denominator consistently ranked in the lowest and next lowest quartile, respectively. Ratios of the 656.3 nm Fraunhofer line and the 755.6 nm line consistently ranked highest for the summer crop. Ratios with 755.6 nm in the denominator ranked in the upper quartile for 10 out of 12 measurement dates. Differences in ratio ranking indicate that physiological conditions may be estimated using selected ratios of Fraunhofer lines within the context of R/FR analysis. A passive instrument designed to monitor R/FR chlorophyll fluorescence (i.e. vegetation stress) from orbit could be built today.

Relating Trp-Glu dipeptide fluorescence to molecular conformation: the role of the discrete Chi 1 and Chi 2 angles.

PubMed

Eisenberg, Azaria Solomon; Juszczak, Laura J

2013-07-05

Molecular dynamics (MD), coupled with fluorescence data for charged dipeptides of tryptophanyl glutamic acid (Trp-Glu), reveal a detailed picture of how specific conformation affects fluorescence. Fluorescence emission spectra and time-resolved emission measurements have been collected for all four charged species. MD simulations 20 to 30 ns in length have also been carried out for the Trp-Glu species, as simulation provides aqueous phase conformational data that can be correlated with the fluorescence data. The calculations show that each dipeptide species is characterized by a similar set of six, discrete Chi 1, Chi 2 dihedral angle pairs. The preferred Chi 1 angles--60°, 180°, and 300°--play the significant role in positioning the terminal amine relative to the indole ring. A Chi 1 angle of 60° results in the arching of the backbone over the indole ring and no interaction of the ring with the terminal amine. Chi 1 values of 180° and 300° result in an extension of the backbone away from the indole ring and a NH3 cation-π interaction with indole. This interaction is believed responsible for charge transfer quenching. Two fluorescence lifetimes and their corresponding amplitudes correlate with the Chi 1 angle probability distribution for all four charged Trp-Glu dipeptides. Fluorescence emission band maxima are also consistent with the proposed pattern of terminal amine cation quenching of fluorescence. Copyright © 2013 Wiley Periodicals, Inc.

Robust Smoothing: Smoothing Parameter Selection and Applications to Fluorescence Spectroscopy∂

PubMed Central

Lee, Jong Soo; Cox, Dennis D.

2009-01-01

Fluorescence spectroscopy has emerged in recent years as an effective way to detect cervical cancer. Investigation of the data preprocessing stage uncovered a need for a robust smoothing to extract the signal from the noise. Various robust smoothing methods for estimating fluorescence emission spectra are compared and data driven methods for the selection of smoothing parameter are suggested. The methods currently implemented in R for smoothing parameter selection proved to be unsatisfactory, and a computationally efficient procedure that approximates robust leave-one-out cross validation is presented. PMID:20729976

Assessment of β-zone peripapillary atrophy by optical coherence tomography and scanning laser ophthalmoscopy imaging in glaucoma patients

PubMed Central

Seidensticker, Florian; Reznicek, Lukas; Mann, Thomas; Hübert, Irene; Kampik, Anselm; Ulbig, Michael; Hirneiss, Christoph; Neubauer, Aljoscha S; Kernt, Marcus

2014-01-01

Purpose To assess β-zone peripapillary atrophy (β-PPA) using spectral domain optical coherence tomography (SD-OCT), scanning laser ophthalmoscopy (SLO), and fundus auto-fluorescence (FAF) imaging in patients with primary open-angle glaucoma with advanced glaucomatous visual field defects. Methods A consecutive, prospective series of 82 study eyes with primary open-angle glaucoma were included in this study. All study participants underwent a full ophthalmic examination followed by SD-OCT, wide-field SLO, and FAF imaging of the optic nerve head and the peripapillary region. Results Eighty-four glaucomatous eyes were included in our prospective study. Correlation analyses for horizontally and vertically obtained β-PPA for all three imaging modalities (color SLO, FAF, and SD-OCT) revealed highest correlations between FAF and color SLO (Pearson correlation coefficient: 0.904 [P<0.001] for horizontal β-PPA and 0.786 [P<0.001] for vertical β-PPA). Bland–Altman plotting revealed highest agreements between color SLO and FAF, with −2.1 pixels ±1.96 standard deviation (SD) for horizontal β-PPA, SD: 10.5 pixels and 2.4 pixels ±1.96 SD for vertical β-PPA. Conclusion β-PPA can be assessed using en-face SLO and cross-sectional SD-OCT imaging. Correlation analyses revealed highest correlations between color SLO and FAF imaging, while correlations between SLO and SD-OCT were weak. A more precise structural definition of β-PPA is needed. PMID:25061270

Cross-correlations between agricultural commodity futures markets in the US and China

NASA Astrophysics Data System (ADS)

Li, Zhihui; Lu, Xinsheng

2012-08-01

This paper examines the cross-correlation properties of agricultural futures markets between the US and China using a cross-correlation statistic test and multifractal detrended cross-correlation analysis (MF-DCCA). The results show that the cross-correlations between the two geographically distant markets for four pairs of important agricultural commodities futures are significantly multifractal. By introducing the concept of a “crossover”, we find that the multifractality of cross-correlations between the two markets is not long lasting. The cross-correlations in the short term are more strongly multifractal, but they are weakly so in the long term. Moreover, cross-correlations of small fluctuations are persistent and those of large fluctuations are anti-persistent in the short term while cross-correlations of all kinds of fluctuations for soy bean and soy meal futures are persistent and for corn and wheat futures are anti-persistent in the long term. We also find that cross-correlation exponents are less than the averaged generalized Hurst exponent when q<0 and more than the averaged generalized Hurst exponent when q>0 in the short term, while in the long term they are almost the same.

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.

Thermally activated delayed fluorescence organic dots for two-photon fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

He, Tingchao; Ren, Can; Li, Zhuohua; Xiao, Shuyu; Li, Junzi; Lin, Xiaodong; Ye, Chuanxiang; Zhang, Junmin; Guo, Lihong; Hu, Wenbo; Chen, Rui

2018-05-01

Autofluorescence is a major challenge in complex tissue imaging when molecules present in the biological tissue compete with the fluorophore. This issue may be resolved by designing organic molecules with long fluorescence lifetimes. The present work reports the two-photon absorption (TPA) properties of a thermally activated delayed fluorescence (TADF) molecule with carbazole as the electron donor and dicyanobenzene as the electron acceptor (i.e., 4CzIPN). The results indicate that 4CzIPN exhibits a moderate TPA cross-section (˜9 × 10-50 cm4 s photon-1), high fluorescence quantum yield, and a long fluorescence lifetime (˜1.47 μs). 4CzIPN was compactly encapsulated into an amphiphilic copolymer via nanoprecipitation to achieve water-soluble organic dots. Interestingly, 4CzIPN organic dots have been utilized in applications involving two-photon fluorescence lifetime imaging (FLIM). Our work aptly demonstrates that TADF molecules are promising candidates of nonlinear optical probes for developing next-generation multiphoton FLIM applications.

Contrasting characteristics of aqueous reactive species induced by cross-field and linear-field plasma jets

NASA Astrophysics Data System (ADS)

Xu, Han; Chen, Chen; Liu, Dingxin; Xu, Dehui; Liu, Zhijie; Wang, Xiaohua; Kong, Michael G.

2017-06-01

A comparative study on aqueous reactive species in deionized water treated by two types of plasma jets is presented. Classified by the direction of the electric field in the jet device, a linear-field jet and cross-field jet have been set up. Concentrations of several aqueous reactive species are measured quantitatively by chemical fluorescent assays and electron spin resonance spectrometer. Results show that these two-type plasma jets would generate approximately the same gaseous reactive species under the same discharge power, but the linear-field plasma jet is much more efficient at delivering those species to the remote deionized water. This leads to a much more aqueous short-lived species including OH and \\text{O}2- produced in water, which are mainly correlated to the solvation of gaseous short-lived species such as ions and electrons. Regarding the long-lived species of aqueous H2O2, the concentration grows faster when treated by the linear-field plasma jet in the initial stage, but after 10 min it is similar to that treated by the cross-field counterpart due to the vapor-liquid equilibrium. The aqueous peroxynitrite is also predicted to be produced as a result of the air inclusion in the feeding gas.

Fluorescence of fungi in superficial and deep fungal infections

PubMed Central

Elston, Dirk M

2001-01-01

Background Fluorescence of many fungi is noted when H&E stained sections are examined under a fluorescent microscope. In theory, this phenomenon could aid in the diagnosis of cutaneous and disseminated fungal infections without the delay associated with special stains. Seventy-six cases of superficial and deep fungal infections and 3 cases of protothecosis were studied to determine the clinical usefulness of this technique. Results In most cases, fluorescence was noted, but was not intense. Fluorescence of fungi did not correlate with the age of the specimen. In most cases, organisms in H&E stained sections were more easily identified with routine light microscopy than with fluorescent microscopy. Conclusion This report suggests that in H&E stained skin specimens, fluorescent microscopy is of little benefit in the identification of fungal organisms. PMID:11602016

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.

Biophysical Characterization of Copolymer-Protected Gene Vectors (COPROGs)

PubMed Central

Hönig, Daniel; DeRouchey, Jason; Jungmann, Ralf; Koch, Christian; Plank, Christian; Rädler, Joachim O.

2010-01-01

A copolymer-protected gene vector (COPROG) is a three component gene delivery system consisting of a preformed DNA and branched polyethylenimine (bPEI) complex subsequently modified by the addition of a copolymer (P6YE5C) incorporating both poly(ethylene glycol) (PEG) and anionic peptides. Using fluorescence correlation spectroscopy (FCS) and atomic force microscopy (AFM), we characterized and compared the self-assembly of bPEI/DNA particles and COPROG complexes. In low salt buffer, both bPEI/DNA and COPROG formulations form stable nanoparticles with hydrodynamic radii between 60–120 nm. COPROG particles, as compared to bPEI/DNA, show greatly improved particle stability to both physiological salt as well as low pH conditions. Binding stoichiometry of the three-component COPROG system was investigated by dual-color fluorescence cross-correlation spectroscopy (FCCS). It was found that a significant fraction of P6YE5C copolymer aggregates with excess bPEI forming bPEI/P6YE5C “ghost complexes” with no DNA inside. The ratio of ghost particles to COPROG complexes is about 4:1. In addition we find a large fraction of excess P6YE5C copolymer, which remains unbound in solution. We observe a 2–4 fold enhanced reporter gene expression with COPROG formulations at various equivalents as compared to bPEI-DNA alone. We believe that both complex stabilization as well as the capture of excess bPEI into ghost particles induced by the copolymer is responsible for the improvement in gene expression. PMID:20672861

Correlative FRET: new method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture

NASA Astrophysics Data System (ADS)

Shinogle-Decker, Heather; Martinez-Rivera, Noraida; O'Brien, John; Powell, Richard D.; Joshi, Vishwas N.; Connell, Samuel; Rosa-Molinar, Eduardo

2018-02-01

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence; in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies.

INFLUENCE OF ROOT COLONIZING BACTERIA ON THE DEFENSE RESPONSES OF BEAN

EPA Science Inventory

Colonization of plant roots by fluorescent pseudomonads has been correlated with disease suppression. ne mechanism may involve altered defense responses in the plant upon colonization. ltered defense responses were observed in bean (Phaseolus vulgaris) inoculated with fluorescent...

Efficacy of NGR peptide-modified PEGylated quantum dots for crossing the blood-brain barrier and targeted fluorescence imaging of glioma and tumor vasculature.

PubMed

Huang, Ning; Cheng, Si; Zhang, Xiang; Tian, Qi; Pi, Jiangli; Tang, Jun; Huang, Qing; Wang, Feng; Chen, Jin; Xie, Zongyi; Xu, Zhongye; Chen, Weifu; Zheng, Huzhi; Cheng, Yuan

2017-01-01

Delivery of imaging agents to brain glioma is challenging because the blood-brain barrier (BBB) functions as a physiological checkpoint guarding the central nervous system from circulating large molecules. Moreover, the ability of existing probes to target glioma has been insufficient and needs to be improved. In present study, PEG-based long circulation, CdSe/ZnS quantum dots (QDs)-based nanoscale and fluorescence, asparagines-glycine-arginine peptides (NGR)-based specific CD13 recognition were integrated to design and synthesize a novel nanoprobe by conjugating biotinylated NGR peptides to avidin-PEG-coated QDs. Our data showed that the NGR-PEG-QDs were nanoscale with less than 100 nm and were stable in various pH (4.0~8.0). These nanomaterials with non-toxic concentrations could cross the BBB and target CD13-overexpressing glioma and tumor vasculature in vitro and in vivo, contributing to fluorescence imaging of this brain malignancy. These achievements allowed groundbreaking technological advances in targeted fluorescence imaging for the diagnosis and surgical removal of glioma, facilitating potential transformation toward clinical nanomedicine. Copyright © 2016 Elsevier Inc. All rights reserved.

Direct detection of RDX vapor using a conjugated polymer network.

PubMed

Gopalakrishnan, Deepti; Dichtel, William R

2013-06-05

1,3,5-Trinitroperhydro-1,3,5-triazine (RDX) is a principal component of plastic explosives used in acts of terrorism and within improvised explosive devices, among others. Approaches to detect RDX compatible with remote, "stand-off" sampling that do not require preconcentration strategies, such as the swabs commonly employed in airports, will benefit military and civilian security. Such detection remains a significant challenge because RDX is 10(3) less volatile than 1,3,5-trinitrotoluene (TNT), corresponding to a parts-per-trillion vapor pressure under ambient conditions. Therefore, while fluorescence quenching of conjugated polymers is sufficiently sensitive to detect TNT vapors, RDX vapor detection is undemonstrated. Here we report a cross-linked phenylene vinylene polymer network whose fluorescence is quenched by trace amounts of RDX introduced from solution or the vapor phase. Fluorescence quenching is reduced, but remains significant, when partially degraded RDX is employed, suggesting that the polymer responds to RDX itself. The polymer network also responds to TNT and PETN similarly introduced from solution or the vapor phase. Pure solvents, volatile amines, and the outgassed vapors from lipstick or sunscreen do not quench polymer fluorescence. The established success of TNT sensors based on fluorescence quenching makes this a material of interest for real-world explosive sensors and will motivate further interest in cross-linked polymers and framework materials for sensing applications.

Dynamics of diamond nanoparticles in solution and cells.

PubMed

Neugart, Felix; Zappe, Andrea; Jelezko, Fedor; Tietz, C; Boudou, Jean Paul; Krueger, Anke; Wrachtrup, Jörg

2007-12-01

The fluorescence and motional dynamics of single diamond nanocrystals in buffer solution and in living cells is investigated. Stable hydrosols of nanodiamonds in buffer solutions are investigated by fluorescence correlation spectroscopy. Measurement of the effective hydrodynamic radius yields particles of 48 nm diameter, which is in excellent agreement with atomic force microscopy measurements made on the same particles. Fluorescence correlation spectroscopy measurements indicate that nanocrystals easily form aggregates when the buffer pH is changed. This tendency is reduced when the surface of the diamonds is covered with surfactants. Upon incubation, cells spontaneously take up nanocrystals that uniformly distribute in cells. Most of the particles get immobilized within a few minutes. The binding of streptavidin to biotinylated aggregates of 4 nm diameter nanodiamonds is demonstrated.

Trapping, Deformation, and Rotation of Giant Unilamellar Vesicles in Octode Dielectrophoretic Field Cages

PubMed Central

Korlach, J.; Reichle, C.; Müller, T.; Schnelle, T.; Webb, W. W.

2005-01-01

The behavior of freestanding lipid bilayer membranes under the influence of dielectric force potentials was studied by trapping, holding, and rotating individual giant unilamellar vesicles (GUVs) inside dielectrophoretic microfield cages. Using laser scanning confocal microscopy and three-dimensional image reconstructions of GUVs labeled with fluorescent membrane probes, field strength and frequency-dependent vesicle deformations were observed which are explained by calculations of the dielectric force potentials inside the cage. Dynamical membrane properties under the influence of the field cage were studied by fluorescence correlation spectroscopy, circumventing potential artifacts associated with measurements involving GUV immobilization on support surfaces. Lipid transport could be accelerated markedly by the applied fields, aided by hydrodynamic fluid streaming which was also studied by fluorescence correlation spectroscopy. PMID:15863477

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging.

PubMed

Schmidt, F N; Zimmermann, E A; Campbell, G M; Sroga, G E; Püschel, K; Amling, M; Tang, S Y; Vashishth, D; Busse, B

2017-04-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic cross-links, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692cm -1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p<0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high-performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. Copyright © 2017 Elsevier Inc. All rights reserved.

PyCorrFit-generic data evaluation for fluorescence correlation spectroscopy.

PubMed

Müller, Paul; Schwille, Petra; Weidemann, Thomas

2014-09-01

We present a graphical user interface (PyCorrFit) for the fitting of theoretical model functions to experimental data obtained by fluorescence correlation spectroscopy (FCS). The program supports many data file formats and features a set of tools specialized in FCS data evaluation. The Python source code is freely available for download from the PyCorrFit web page at http://pycorrfit.craban.de. We offer binaries for Ubuntu Linux, Mac OS X and Microsoft Windows. © The Author 2014. Published by Oxford University Press.

Two-Photon Fluorescence Correlation Spectroscopy

NASA Technical Reports Server (NTRS)

Zimmerli, Gregory A.; Fischer, David G.

2002-01-01

We will describe a two-photon microscope currently under development at the NASA Glenn Research Center. It is composed of a Coherent Mira 900 tunable, pulsed Titanium:Sapphire laser system, an Olympus Fluoview 300 confocal scanning head, and a Leica DM IRE inverted microscope. It will be used in conjunction with a technique known as fluorescence correlation spectroscopy (FCS) to study intracellular protein dynamics. We will briefly explain the advantages of the two-photon system over a conventional confocal microscope, and provide some preliminary experimental results.

Intensity and amplitude correlations in the fluorescence from atoms with interacting Rydberg states

NASA Astrophysics Data System (ADS)

Xu, Qing; Mølmer, Klaus

2015-09-01

We explore the fluorescence signals from a pair of atoms driven towards Rydberg states on a three-level ladder transition. The dipole-dipole interactions between Rydberg excited atoms significantly distort the dark state and electromagnetically induced transparency behavior observed with independent atoms and, thus, their steady-state light emission. We calculate and analyze the temporal correlations between intensities and amplitudes of the signals emitted by the atoms and explain their origin in the atomic Rydberg state interactions.

Mating compatibility in the parasitic protist Trypanosoma brucei.

PubMed

Peacock, Lori; Ferris, Vanessa; Bailey, Mick; Gibson, Wendy

2014-02-21

Genetic exchange has been described in several kinetoplastid parasites, but the most well-studied mating system is that of Trypanosoma brucei, the causative organism of African sleeping sickness. Sexual reproduction takes place in the salivary glands (SG) of the tsetse vector and involves meiosis and production of haploid gametes. Few genetic crosses have been carried out to date and consequently there is little information about the mating compatibility of different trypanosomes. In other single-celled eukaryotes, mating compatibility is typically determined by a system of two or more mating types (MT). Here we investigated the MT system in T. brucei. We analysed a large series of F1, F2 and back crosses by pairwise co-transmission of red and green fluorescent cloned cell lines through experimental tsetse flies. To analyse each cross, trypanosomes were cloned from fly SG containing a mixture of both parents, and genotyped by microsatellites and molecular karyotype. To investigate mating compatibility at the level of individual cells, we directly observed the behaviour of SG-derived gametes in intra- or interclonal mixtures of red and green fluorescent trypanosomes ex vivo. Hybrid progeny were found in all F1 and F2 crosses and most of the back crosses. The success of individual crosses was highly variable as judged by the number of hybrid clones produced, suggesting a range of mating compatibilities among F1 progeny. As well as hybrids, large numbers of recombinant genotypes resulting from intraclonal mating (selfers) were found in some crosses. In ex vivo mixtures, red and green fluorescent trypanosome gametes were observed to pair up and interact via their flagella in both inter- and intraclonal combinations. While yellow hybrid trypanosomes were frequently observed in interclonal mixtures, such evidence of cytoplasmic exchange was rare in the intraclonal mixtures. The outcomes of individual crosses, particularly back crosses, were variable in numbers of both hybrid and selfer clones produced, and do not readily fit a simple two MT model. From comparison of the behaviour of trypanosome gametes in inter- and intraclonal mixtures, we infer that mating compatibility is controlled at the level of gamete fusion.

Mating compatibility in the parasitic protist Trypanosoma brucei

PubMed Central

2014-01-01

Background Genetic exchange has been described in several kinetoplastid parasites, but the most well-studied mating system is that of Trypanosoma brucei, the causative organism of African sleeping sickness. Sexual reproduction takes place in the salivary glands (SG) of the tsetse vector and involves meiosis and production of haploid gametes. Few genetic crosses have been carried out to date and consequently there is little information about the mating compatibility of different trypanosomes. In other single-celled eukaryotes, mating compatibility is typically determined by a system of two or more mating types (MT). Here we investigated the MT system in T. brucei. Methods We analysed a large series of F1, F2 and back crosses by pairwise co-transmission of red and green fluorescent cloned cell lines through experimental tsetse flies. To analyse each cross, trypanosomes were cloned from fly SG containing a mixture of both parents, and genotyped by microsatellites and molecular karyotype. To investigate mating compatibility at the level of individual cells, we directly observed the behaviour of SG-derived gametes in intra- or interclonal mixtures of red and green fluorescent trypanosomes ex vivo. Results Hybrid progeny were found in all F1 and F2 crosses and most of the back crosses. The success of individual crosses was highly variable as judged by the number of hybrid clones produced, suggesting a range of mating compatibilities among F1 progeny. As well as hybrids, large numbers of recombinant genotypes resulting from intraclonal mating (selfers) were found in some crosses. In ex vivo mixtures, red and green fluorescent trypanosome gametes were observed to pair up and interact via their flagella in both inter- and intraclonal combinations. While yellow hybrid trypanosomes were frequently observed in interclonal mixtures, such evidence of cytoplasmic exchange was rare in the intraclonal mixtures. Conclusions The outcomes of individual crosses, particularly back crosses, were variable in numbers of both hybrid and selfer clones produced, and do not readily fit a simple two MT model. From comparison of the behaviour of trypanosome gametes in inter- and intraclonal mixtures, we infer that mating compatibility is controlled at the level of gamete fusion. PMID:24559099

Quantifying the range of cross-correlated fluctuations using a q- L dependent AHXA coefficient

NASA Astrophysics Data System (ADS)

Wang, Fang; Wang, Lin; Chen, Yuming

2018-03-01

Recently, based on analogous height cross-correlation analysis (AHXA), a cross-correlation coefficient ρ×(L) has been proposed to quantify the levels of cross-correlation on different temporal scales for bivariate series. A limitation of this coefficient is that it cannot capture the full information of cross-correlations on amplitude of fluctuations. In fact, it only detects the cross-correlation at a specific order fluctuation, which might neglect some important information inherited from other order fluctuations. To overcome this disadvantage, in this work, based on the scaling of the qth order covariance and time delay L, we define a two-parameter dependent cross-correlation coefficient ρq(L) to detect and quantify the range and level of cross-correlations. This new version of ρq(L) coefficient leads to the formation of a ρq(L) surface, which not only is able to quantify the level of cross-correlations, but also allows us to identify the range of fluctuation amplitudes that are correlated in two given signals. Applications to the classical ARFIMA models and the binomial multifractal series illustrate the feasibility of this new coefficient ρq(L) . In addition, a statistical test is proposed to quantify the existence of cross-correlations between two given series. Applying our method to the real life empirical data from the 1999-2000 California electricity market, we find that the California power crisis in 2000 destroys the cross-correlation between the price and the load series but does not affect the correlation of the load series during and before the crisis.

Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

PubMed Central

Olsen, Katja N.; Budde, Birgitte B.; Siegumfeldt, Henrik; Rechinger, K. Björn; Jakobsen, Mogens; Ingmer, Hanne

2002-01-01

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments. PMID:12147523

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

NASA Astrophysics Data System (ADS)

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

2015-11-01

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

Fluorescent single-digit detonation nanodiamond for biomedical applications

NASA Astrophysics Data System (ADS)

Nunn, Nicholas; d’Amora, Marta; Prabhakar, Neeraj; Panich, Alexander M.; Froumin, Natalya; Torelli, Marco D.; Vlasov, Igor; Reineck, Philipp; Gibson, Brant; Rosenholm, Jessica M.; Giordani, Silvia; Shenderova, Olga

2018-07-01

Detonation nanodiamonds (DNDs) have emerged as promising candidates for a variety of biomedical applications, thanks to different physicochemical and biological properties, such as small size and reactive surfaces. In this study, we propose carbon dot decorated single digit (4–5 nm diameter) primary particles of detonation nanodiamond as promising fluorescent probes. Due to their intrinsic fluorescence originating from tiny (1–2 atomic layer thickness) carbonaceous structures on their surfaces, they exhibit brightness suitable for in vitro imaging. Moreover, this material offers a unique, cost effective alternative to sub-10 nm nanodiamonds containing fluorescent nitrogen-vacancy color centers, which have not yet been produced at large scale. In this paper, carbon dot decorated nanodiamonds are characterized by several analytical techniques. In addition, the efficient cellular uptake and fluorescence of these particles are observed in vitro on MDA-MD-231 breast cancer cells by means of confocal imaging. Finally, the in vivo biocompatibility of carbon dot decorated nanodiamonds is demonstrated in zebrafish during the development. Our results indicate the potential of single-digit detonation nanodiamonds as biocompatible fluorescent probes. This unique material will find application in correlative light and electron microscopy, where small sized NDs can be attached to antibodies to act as a suitable dual marker for intracellular correlative microscopy of biomolecules.

Laser-Induced Fluorescence (LIF) from plant foliage

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Williams, D. L.

1986-01-01

The fluorescence spectra and fluorescence induction kinetics of green plants excited at 337 nm by a laser were studied. They correlate with plant type, as well as with changes in the physiology of the plant as the result of stress. The plant types studied include herbaceous dicots, monocots, hardwoods, conifers, and algae. These plant types could be identified on the basis of differences in either the number of fluorescent bands or the relative intensity of the bands. Differences in fluorescent spectra which could be related to vigor status are observed in conifers located in an area of high atmospheric deposition. Changes in the fluorescence spectra and induction kinetics are also seen in plants grown under conditions of nutrient deficiency and drought stress.

Laser-Induced Fluorescence (LIF) from plant foliage

NASA Technical Reports Server (NTRS)

Chappelle, Emmett W.; Williams, Darrel L.

1987-01-01

The fluorescence spectra and fluorescence induction kinetics of green plants excited at 337 nm by a laser were studied. They correlate with plant type, as well as with changes in the physiology of the plant as the result of stress. The plant types studied include herbaceous dicots, monocots, hardwoods, conifers, and algae. These plant types could be identified on the basis of differences in either the number of fluorescent bands or the relative intensity of the bands. Differences in fluorescent spectra which could be related to vigor status are observed in conifers located in an area of high atmospheric deposition. Changes in the fluorescence spectra and induction kinetics are also seen in plants grown under conditions of nutrient deficiency and drought stress.

The effects of common risk factors on stock returns: A detrended cross-correlation analysis

NASA Astrophysics Data System (ADS)

Ruan, Qingsong; Yang, Bingchan

2017-10-01

In this paper, we investigate the cross-correlations between Fama and French three factors and the return of American industries on the basis of cross-correlation statistic test and multifractal detrended cross-correlation analysis (MF-DCCA). Qualitatively, we find that the return series of Fama and French three factors and American industries were overall significantly cross-correlated based on the analysis of a statistic. Quantitatively, we find that the cross-correlations between three factors and the return of American industries were strongly multifractal, and applying MF-DCCA we also investigate the cross-correlation of industry returns and residuals. We find that there exists multifractality of industry returns and residuals. The result of correlation coefficients we can verify that there exist other factors which influence the industry returns except Fama three factors.

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...

Synthesis, prodigious two-photon absorption cross sections and electrochemical properties of a series of triphenylamine-based chromophores

NASA Astrophysics Data System (ADS)

Li, Rui; Li, Dandan; Fei, Wenwen; Tan, Jingyun; Li, Shengli; Zhou, Hongping; Zhang, Shengyi; Wu, Jieying; Tian, Yupeng

2014-06-01

A series of triphenylamine-based chromophores (L1-3) with donor-π-donor (D-π-D) model have been designed and synthesized via solid phase Wittig reaction. Their one/two-photon fluorescence and electrochemical properties have been investigated. The results show that L2 and L3 exhibited strong and wide-dispersed two-photon-excited fluorescence (TPEF) in different solvents. Chromophore L3 displays the strongest intensity two-photon absorption activity and large cross-sections (>3600 GM) in the range of 680-840 nm in THF, the largest δ up to 8899 GM in the near-IR range, and the measured maximum TPA cross-sections per molecular weight (δmax/MW) is 8.64 GM/g (L3) in THF. Significantly, it also exhibits good solubility in common organic solvents when the chromophore was modified by polyether units as peripheral groups.

Laser induced fluorescence technique for detecting organic matter in East China Sea

NASA Astrophysics Data System (ADS)

Chen, Peng; Wang, Tianyu; Pan, Delu; Huang, Haiqing

2017-10-01

A laser induced fluorescence (LIF) technique for fast diagnosing chromophoric dissolved organic matter (CDOM) in water is discussed. We have developed a new field-portable laser fluorometer for rapid fluorescence measurements. In addtion, the fluorescence spectral characteristics of fluorescent constituents (e.g., CDOM, chlorophyll-a) were analyzed with a spectral deconvolution method of bi-Gaussian peak function. In situ measurements by the LIF technique compared well with values measured by conventional spectrophotometer method in laboratory. A significant correlation (R2 = 0.93) was observed between fluorescence by the technique and absorption by laboratory spectrophotometer. Influence of temperature variation on LIF measurement was investigated in lab and a temperature coefficient was deduced for fluorescence correction. Distributions of CDOM fluorescence measured using this technique in the East China Sea coast were presented. The in situ result demonstrated the utility of the LIF technique for rapid detecting dissolved organic matter.

SEROLOGICAL TYPING OF STAPHYLOCOCCI BY MEANS OF FLUORESCENT ANTIBODIES I.

PubMed Central

Cohen, Jay O.; Oeding, Per

1962-01-01

Cohen, Jay O. (Communicable Disease Center, Atlanta, Ga.) and Per Oeding. Serological typing of staphylococci by means of fluorescent antibodies. I. Development of specific reagents for seven serological factors. J. Bacteriol. 84:735–741. 1962—Fluorescent antibody reagents for identifying seven antigenic factors of staphylococci have been prepared. The fluorescent staining reactions of these reagents were compared to the agglutination reactions with diagnostic cultures of coagulase-positive staphylococci. Correlation between the two serological tests was almost complete with factors a, b, i, and k. The c fluorescent antibody reagent had a somewhat broader spectrum of activity than the corresponding agglutination serum, whereas the m fluorescent antibody reagent stained fewer strains than were agglutinated in m serum. The fluorescent antibody reagent for h factor stained strains possessing h1 factor but not strains possessing only h2 factor. Fluorescent antibody reagents for specific staphylococcal factors did not stain strains of group A streptococci. PMID:14022057

Atomic carbon emission from photodissociation of CO2. [planetary atmospheric chemistry

NASA Technical Reports Server (NTRS)

Wu, C. Y. R.; Phillips, E.; Lee, L. C.; Judge, D. L.

1978-01-01

Atomic carbon fluorescence, C I 1561, 1657, and 1931 A, has been observed from photodissociation of CO2, and the production cross sections have been measured. A line emission source provided the primary photons at wavelengths from threshold to 420 A. The present results suggest that the excited carbon atoms are produced by total dissociation of CO2 into three atoms. The cross sections for producing the O I 1304-A fluorescence through photodissociation of CO2 are found to be less than 0.01 Mb in the wavelength region from 420 to 835 A. The present data have implications with respect to photochemical processes in the atmospheres of Mars and Venus.

Spectroscopy of high index contrast Yb:Ta2O5 waveguides for lasing applications

NASA Astrophysics Data System (ADS)

Aghajani, A.; Murugan, G. S.; Sessions, N. P.; Apostolopoulos, V.; Wilkinson, J. S.

2015-06-01

Ytterbium-doped waveguides are required for compact integrated lasers and Yb- doped Ta2O5 is a promising candidate material. The design, fabrication and spectroscopic characterisation of Yb:Ta2O5 rib waveguides are described. The peak absorption cross-section was measured to be 2.75×10-20 cm2 at 975 nm. The emission spectrum was found to have a fluorescence emission peak at a wavelength of 976 nm with a peak cross-section of 2.9×10-20 cm2 and a second broad fluorescence band spanning from 990 nm to 1090 nm. The excited- state life time was measured to be 260 μs.

Study on discrimination of oral cancer from normal using blood plasma based on fluorescence steady and excited state at excitation wavelength 280 nm

NASA Astrophysics Data System (ADS)

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

2016-03-01

Many research works based on fluorescence spectroscopy have proven its potential in the diagnosis of various diseases using the spectral signatures of the native key fluorophores such as tryptophan, tyrosine, collagen, NADH, FAD and porphyrin. These fluorophores distribution, concentration and their conformation may be changed depending upon the pathological and metabolic conditions of cells and tissues. In this study, we have made an attempt to characterize the blood plasma of normal subject and oral cancer patients by native fluorescence spectroscopy at 280 nm excitation. Further, the fluorescence data were analyzed by employing the multivariate statistical method - linear discriminant analyses (LDA) using leaves one out cross validation method. The results illustrate the potential of fluorescence spectroscopy technique in the diagnosis of oral cancer using blood plasma.

Photo-degradation behaviour of roseoflavin in some aqueous solutions

NASA Astrophysics Data System (ADS)

Tyagi, A.; Penzkofer, A.; Mathes, T.; Hegemann, P.

2010-03-01

An absorption and emission spectroscopic characterization of roseoflavin (8-dimethylamino-8-demethyl-riboflavin, RoF) in aqueous solutions was carried out. The studies were concentrated on roseoflavin in pH 8 phosphate buffer. Absorption cross-section spectra, fluorescence excitation spectra, fluorescence quantum distributions, fluorescence quantum yields and fluorescence lifetimes were determined. The fluorescence of RoF is quenched by photo-induced intra-molecular charge-transfer at room temperature. The photo-degradation of RoF in un-buffered water, in Tris-HCl buffer, and in phosphate buffer was studied. Phosphate buffer and to a smaller extent Tris buffer catalyse the RoF photo-degradation. Photo-excitation of the primary photoproduct, 8-methylamino-riboflavin (8-MNH-RF), enhanced the RoF degradation by triplet 8-MNH-RF - singlet RoF excitation transfer with subsequent triplet-state RoF degradation.

Near-IR Two-Photon Fluorescent Sensor for K(+) Imaging in Live Cells.

PubMed

Sui, Binglin; Yue, Xiling; Kim, Bosung; Belfield, Kevin D

2015-08-19

A new two-photon excited fluorescent K(+) sensor is reported. The sensor comprises three moieties, a highly selective K(+) chelator as the K(+) recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (>52-fold) in detecting K(+) over other physiological metal cations. Upon binding K(+), the sensor switches from nonfluorescent to highly fluorescent, emitting red to near-IR (NIR) fluorescence. The sensor exhibited a good two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K(+) sensing in living cells.

An optical fiber taper fluorescent probe for detection of nitro-explosives based on tetraphenylethylene with aggregation-induced emission

NASA Astrophysics Data System (ADS)

Liu, Fukun; Cui, Minxin; Ma, Jiajun; Zou, Gang; Zhang, Qijin

2017-07-01

In this work, we report a novel optical fiber taper fluorescent probe for detection of nitro-explosives. The probe was fabricated by an in-situ photo-plating through evanescent wave and transmitted light initiated thiol-ene ;click; reaction, from which a cross-linked fluorescence porous polymer film was covalently bonded on the surface of the fiber taper. The film exhibits well-organized porous structure due to the presence of polyhedral oligomeric vinylsilsesquioxane moieties, and simultaneously displays strong fluorescence from tetraphenylethylene with aggregation-induced emission property. These two characters make the probe show a remarkable sensitivity, anti-photo-bleaching and a repeatability in detection of TNT and DNT vapors by fluorescence quenching. In addition, the detection is not interfered in the presence of other volatile organic gases.

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

NASA Astrophysics Data System (ADS)

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

1992-04-01

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

Fluorescence detection of trace TNT by novel cross-linking electropolymerized films both in vapor and aqueous medium.

PubMed

Nie, Heran; Lv, Ying; Yao, Liang; Pan, Yuyu; Zhao, Yang; Li, Peng; Sun, Guannan; Ma, Yuguang; Zhang, Ming

2014-01-15

Electropolymerized (EP) films with high fluorescent efficiency are introduced to the detection of trace 2,4,6-trinitrotoluene (TNT). Three electroactive materials TCPC, OCPC and OCz have been synthesized and their EP films have been demonstrated to be sensitive to TNT. Among them, the TCPC EP films have displayed the highest sensitivity to TNT in both vapor and aqueous medium, even in the natural water. It is proposed that the good performances would be caused by the following two factors: first, the cross-linking network of EP films can generate the cavities which benefit the TNT penetration, and remarkably increase the contact area between the EP films and TNT; second, the frontier orbits distribution leads the fast photo-induced electron transfer (PET) from the TCPC EP films to TNT. Our results prove that these EP films are promising TNT sensing candidates and provide a new method to prepare fluorescent porous films. Copyright © 2013 Elsevier B.V. All rights reserved.

Guest concentration, bias current, and temperature-dependent sign inversion of magneto-electroluminescence in thermally activated delayed fluorescence devices

NASA Astrophysics Data System (ADS)

Deng, Junquan; Jia, Weiyao; Chen, Yingbing; Liu, Dongyu; Hu, Yeqian; Xiong, Zuhong

2017-03-01

Non-emissive triplet excited states in devices that undergo thermally activated delayed fluorescence (TADF) can be up-converted to singlet excited states via reverse intersystem crossing (RISC), which leads to an enhanced electroluminescence efficiency. Exciton-based fluorescence devices always exhibit a positive magneto-electroluminescence (MEL) because intersystem crossing (ISC) can be suppressed effectively by an external magnetic field. Conversely, TADF devices should exhibit a negative MEL because RISC is suppressed by the external magnetic field. Intriguingly, we observed a positive MEL in TADF devices. Moreover, the sign of the MEL was either positive or negative, and depended on experimental conditions, including doping concentration, current density and temperature. The MEL observed from our TADF devices demonstrated that ISC in the host material and RISC in the guest material coexisted. These competing processes were affected by the experimental conditions, which led to the sign change of the MEL. This work gives important insight into the energy transfer processes and the evolution of excited states in TADF devices.

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2015-09-11

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

Advanced glycation End-products (AGEs): an emerging concern for processed food industries.

PubMed

Sharma, Chetan; Kaur, Amarjeet; Thind, S S; Singh, Baljit; Raina, Shiveta

2015-12-01

The global food industry is expected to increase more than US $ 7 trillion by 2014. This rise in processed food sector shows that more and more people are diverging towards modern processed foods. As modern diets are largely heat processed, they are more prone to contain high levels of advanced glycation end products (AGEs). AGEs are a group of complex and heterogeneous compounds which are known as brown and fluorescent cross-linking substances such as pentosidine, non-fluorescent cross-linking products such as methylglyoxal-lysine dimers (MOLD), or non-fluorescent, non-cross linking adducts such as carboxymethyllysine (CML) and pyrraline (a pyrrole aldehyde). The chemistry of the AGEs formation, absorption and bioavailability and their patho-biochemistry particularly in relation to different complications like diabetes and ageing discussed. The concept of AGEs receptor - RAGE is mentioned. AGEs contribute to a variety of microvascular and macrovascular complications through the formation of cross-links between molecules in the basement membrane of the extracellular matrix and by engaging the receptor for advanced glycation end products (RAGE). Different methods of detection and quantification along with types of agents used for the treatment of AGEs are reviewed. Generally, ELISA or LC-MS methods are used for analysis of foods and body fluids, however lack of universally established method highlighted. The inhibitory effect of bioactive components on AGEs by trapping variety of chemical moieties discussed. The emerging evidence about the adverse effects of AGEs makes it necessary to investigate the different therapies to inhibit AGEs.

The Application of Fluorescence In Situ Hybridization in Different Ploidy Levels Cross-Breeding of Lily

PubMed Central

Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

2015-01-01

21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid ‘Freya’ had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true. PMID:26010356

The application of fluorescence in situ hybridization in different ploidy levels cross-breeding of lily.

PubMed

Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

2015-01-01

21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid 'Freya' had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true.

Tracking microbial contamination in retail environments using fluorescent powder--a retail delicatessen environment example.

PubMed

Sirsat, Sujata A; Kim, Kawon; Gibson, Kristen E; Crandall, Phillip G; Ricke, Steven C; Neal, Jack A

2014-03-05

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g. small farms), hospitals, nursing homes, cruise ships, and hotels.

Separating non-diffuse component from ambient seismic noise cross-correlation in southern California­­

NASA Astrophysics Data System (ADS)

Liu, X.; Beroza, G. C.; Nakata, N.

2017-12-01

Cross-correlation of fully diffuse wavefields provides Green's function between receivers, although the ambient noise field in the real world contains both diffuse and non-diffuse fields. The non-diffuse field potentially degrades the correlation functions. We attempt to blindly separate the diffuse and the non-diffuse components from cross-correlations of ambient seismic noise and analyze the potential bias caused by the non-diffuse components. We compute the 9-component noise cross-correlations for 17 stations in southern California. For the Rayleigh wave components, we assume that the cross-correlation of multiply scattered waves (diffuse component) is independent from the cross-correlation of ocean microseismic quasi-point source responses (non-diffuse component), and the cross-correlation function of ambient seismic data is the sum of both components. Thus we can blindly separate the non-diffuse component due to physical point sources and the more diffuse component due to cross-correlation of multiply scattered noise based on their statistical independence. We also perform beamforming over different frequency bands for the cross-correlations before and after the separation, and we find that the decomposed Rayleigh wave represents more coherent features among all Rayleigh wave polarization cross-correlation components. We show that after separating the non-diffuse component, the Frequency-Time Analysis results are less ambiguous. In addition, we estimate the bias in phase velocity on the raw cross-correlation data due to the non-diffuse component. We also apply this technique to a few borehole stations in Groningen, the Netherlands, to demonstrate its applicability in different instrument/geology settings.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

PyCCF: Python Cross Correlation Function for reverberation mapping studies

NASA Astrophysics Data System (ADS)

Sun, Mouyuan; Grier, C. J.; Peterson, B. M.

2018-05-01

PyCCF emulates a Fortran program written by B. Peterson for use with reverberation mapping. The code cross correlates two light curves that are unevenly sampled using linear interpolation and measures the peak and centroid of the cross-correlation function. In addition, it is possible to run Monto Carlo iterations using flux randomization and random subset selection (RSS) to produce cross-correlation centroid distributions to estimate the uncertainties in the cross correlation results.

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.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Detection of Subclinical Arthritis in Mice by a Thrombin Receptor-Derived Imaging Agent.

PubMed

Friedman, Beth; Whitney, Michael A; Savariar, Elamprakash N; Caneda, Christa; Steinbach, Paul; Xiong, Qing; Hingorani, Dina V; Crisp, Jessica; Adams, Stephen R; Kenner, Michael; Lippert, Csilla N; Nguyen, Quyen T; Guma, Monica; Tsien, Roger Y; Corr, Maripat

2018-01-01

Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPP NleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPP NleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. In chronic arthritis, RACPP NleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPP NleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPP NleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. RACPP NleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPP NleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression. © 2017, American College of Rheumatology.

Ultrastructural Mapping of the Zebrafish Gastrointestinal System as a Basis for Experimental Drug Studies

PubMed Central

Shami, Gerald J.; Morsch, Marco; Chung, Roger S.; Braet, Filip

2016-01-01

Research in the field of gastroenterology is increasingly focused on the use of alternative nonrodent model organisms to provide new experimental tools to study chronic diseases. The zebrafish is a particularly valuable experimental platform to explore organ and cell structure-function relationships under relevant biological and pathobiological settings. This is due to its optical transparency and its close-to-human genetic makeup. To-date, the structure-function properties of the GIS of the zebrafish are relatively unexplored and limited to histology and fluorescent microscopy. Occasionally those studies include EM of a given subcellular process but lack the required full histological picture. In this work, we employed a novel combined biomolecular imaging approach in order to cross-correlate 3D ultrastructure over different length scales (optical-, X-ray micro-CT, and high-resolution EM). Our correlated imaging studies and subsequent data modelling provide to our knowledge the first detailed 3D picture of the zebrafish larvae GIS. Our results provide unequivocally a limit of confidence for studying various digestive disorders and drug delivery pathways in the zebrafish. PMID:27340669

Ultrastructural Mapping of the Zebrafish Gastrointestinal System as a Basis for Experimental Drug Studies.

PubMed

Cheng, Delfine; Shami, Gerald J; Morsch, Marco; Chung, Roger S; Braet, Filip

2016-01-01

Research in the field of gastroenterology is increasingly focused on the use of alternative nonrodent model organisms to provide new experimental tools to study chronic diseases. The zebrafish is a particularly valuable experimental platform to explore organ and cell structure-function relationships under relevant biological and pathobiological settings. This is due to its optical transparency and its close-to-human genetic makeup. To-date, the structure-function properties of the GIS of the zebrafish are relatively unexplored and limited to histology and fluorescent microscopy. Occasionally those studies include EM of a given subcellular process but lack the required full histological picture. In this work, we employed a novel combined biomolecular imaging approach in order to cross-correlate 3D ultrastructure over different length scales (optical-, X-ray micro-CT, and high-resolution EM). Our correlated imaging studies and subsequent data modelling provide to our knowledge the first detailed 3D picture of the zebrafish larvae GIS. Our results provide unequivocally a limit of confidence for studying various digestive disorders and drug delivery pathways in the zebrafish.

Measurement of Turbulent Fluxes of Swirling Flow in a Scaled Up Multi Inlet Vortex Reactor

NASA Astrophysics Data System (ADS)

Olsen, Michael; Hitimana, Emmanual; Hill, James; Fox, Rodney

2017-11-01

The multi-inlet vortex reactor (MIVR) has been developed for use in the FlashNanoprecipitation (FNP) process. The MIVR has four identical square inlets connected to a central cylindrical mixing chamber with one common outlet creating a highly turbulent swirling flow dominated by a strong vortex in the center. Efficient FNP requires rapid mixing within the MIVR. To investigate the mixing, instantaneous velocity and concentration fields were acquired using simultaneous stereoscopic particle image velocimetry and planar laser-induced fluorescence. The simultaneous velocity and concentration data were used to determine turbulent fluxes and spatial cross-correlations of velocity and concentration fluctuations. The measurements were performed for four inlet flow Reynolds numbers (3250, 4875, 6500, and 8125) and at three measurement planes within the reactor. A correlation between turbulent fluxes and vortex strength was found. For all Reynolds numbers, turbulent fluxes are maximum in the vortex dominated central region of the reactor and decay away from the vortex. Increasing Reynolds number increased turbulent fluxes and subsequently enhanced mixing. The mixing performance was confirmed by determining coefficients of concentration variance within the reactor.

Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin*

PubMed Central

Courson, David S.; Rock, Ronald S.

2010-01-01

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and α-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. α-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

Multifractal detrended cross-correlation analysis on gold, crude oil and foreign exchange rate time series

NASA Astrophysics Data System (ADS)

Pal, Mayukha; Madhusudana Rao, P.; Manimaran, P.

2014-12-01

We apply the recently developed multifractal detrended cross-correlation analysis method to investigate the cross-correlation behavior and fractal nature between two non-stationary time series. We analyze the daily return price of gold, West Texas Intermediate and Brent crude oil, foreign exchange rate data, over a period of 18 years. The cross correlation has been measured from the Hurst scaling exponents and the singularity spectrum quantitatively. From the results, the existence of multifractal cross-correlation between all of these time series is found. We also found that the cross correlation between gold and oil prices possess uncorrelated behavior and the remaining bivariate time series possess persistent behavior. It was observed for five bivariate series that the cross-correlation exponents are less than the calculated average generalized Hurst exponents (GHE) for q<0 and greater than GHE when q>0 and for one bivariate series the cross-correlation exponent is greater than GHE for all q values.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

NASA Astrophysics Data System (ADS)

Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Gurung, Arun Bahadur; Bhattacharjee, Atanu; Aguan, Kripamoy; Mitra, Sivaprasad

2018-01-01

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Ecological changes in oral microcosm biofilm during maturation

NASA Astrophysics Data System (ADS)

Kim, Young-Seok; Kang, Si-Mook; Lee, Eun-Song; Lee, Ji Hyun; Kim, Bo-Ra; Kim, Baek-Il

2016-10-01

The aim of this study was to evaluate the ecological changes in the biofilm at different stages of maturation using 16S rDNA gene amplicon sequencing and to identify correlations between red/green (R/G) fluorescence ratio and ecological changes. An oral microcosm biofilm was initiated from the saliva of a single donor and grown anaerobically for up to 10 days in basal medium mucin. Quantitative light-induced fluorescence analysis was shown that the R/G ratio of the biofilm increased consistently, but the slope rapidly decreased after six days. The bacterial compositions of 10 species also consistently changed over time. However, there was no significant correlation between each bacteria and red fluorescence. The monitoring of the maturation process of oral microcosm biofilm over 10 days revealed that the R/G ratio and the bacterial composition within biofilm consistently changed. Therefore, the R/G fluorescence ratio of biofilm may be related with its ecological change rather than specific bacteria.

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.

Community detection for fluorescent lifetime microscopy image segmentation

NASA Astrophysics Data System (ADS)

Hu, Dandan; Sarder, Pinaki; Ronhovde, Peter; Achilefu, Samuel; Nussinov, Zohar

2014-03-01

Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes.1 In the current paper, we further explore this method in FLT images of ex vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or "solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1:25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.

Real-time visualization of oxidative stress in a floating macrophyte Lemna minor L. exposed to cadmium, copper, menadione, and AAPH.

PubMed

Razinger, Jaka; Drinovec, Luka; Zrimec, Alexis

2010-12-01

An ultra-sensitive digital imaging system was employed to visualize oxidative stress in intact L. minor plants exposed to Cd, Cu, menadione, AAPH, and ascorbate in real time. The increase of ROS production was assessed by measuring the rate of fluorescence intensity increases of the test medium supplemented with a fluorescing probe (dichlorofluorescein diacetate). The addition of 100 μM CdCl₂ or 100 μM CuSO₄ to the growth medium resulted in a significant increase of medium fluorescence. Additionally, CuSO₄ caused a significantly higher fluorescence intensity than CdCl₂ did. A strong positive correlation (R² = 0.99) between menadione concentration and fluorescence intensity was observed. The positive correlation between AAPH concentration and fluorescence intensity was not as strong as in the case of menadione (R² = 0.81). Menadione induced a stronger oxidative stress than similar concentration of AAPH. The addition of 100 μM ascorbate to L. minor treated with 50 μM menadione significantly reduced the fluorescence intensity increase. A linear trend of the fluorescence increase was observed in all treatments, indicating that chemical-induced oxidative stress is a gradual process and that the applied concentrations of the chemicals caused a constant increased production of ROS with different intensities, depending on the treatment. This is the combined result of a gradual diminishing of antioxidant reserves and accumulating oxidative damage. The observed rates of ROS production were slower than those in the studies using cell cultures. Copyright © 2009 Wiley Periodicals, Inc.

[Study on optical characteristics of chromophoric dissolved organic matter (CDOM) in rainwater by fluorescence excitation-emission matrix and absorbance spectroscopy].

PubMed

Cheng, Yuan-yue; Guo, Wei-dong; Long, Ai-min; Chen, Shao-yong

2010-09-01

The optical characteristics of chromophoric dissolved organic matter (CDOM) were determined in rain samples collected in Xiamen Island, during a rainy season in 2007, using fluorescence excitation-emission matrix spectroscopy associated with UV-Vis absorbance spectra. Results showed that the absorbance spectra of CDOM in rain samples decreased exponentially with wavelength. The absorbance coefficient at 300 nm [a(300)] ranged from 0.27 to 3.45 m(-1), which would be used as an index of CDOM abundance, and the mean value was 1.08 m(-1). The content of earlier stage of precipitation events was higher than that of later stage of precipitation events, which implied that anthropogenic sources or atmospheric pollution or air mass types were important contributors to CDOM levels in precipitation. EEMs spectra showed 4 types of fluorescence signals (2 humic-like fluorescence peaks and 2 protein-like fluorescence peaks) in rainwater samples, and there were significant positive correlations of peak A with C and peak B with S, showing their same sources or some relationship of the two humic-like substance and the two protein-like substance. The strong positive correlations of the two humic-like fluorescence peaks with a(300), suggested that the chromophores responsible for absorbance might be the same as fluorophores responsible for fluorescence. Results showed that the presence of highly absorbing and fluorescing CDOM in rainwater is of significant importance in atmospheric chemistry and might play a previously unrecognized role in the wavelength dependent spectral attenuation of solar radiation by atmospheric waters.

δ-aminolevulinic acid–induced protoporphyrin IX concentration correlates with histopathologic markers of malignancy in human gliomas: the need for quantitative fluorescence-guided resection to identify regions of increasing malignancy

PubMed Central

Valdés, Pablo A.; Kim, Anthony; Brantsch, Marco; Niu, Carolyn; Moses, Ziev B.; Tosteson, Tor D.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.; Harris, Brent T.

2011-01-01

Extent of resection is a major goal and prognostic factor in the treatment of gliomas. In this study we evaluate whether quantitative ex vivo tissue measurements of δ-aminolevulinic acid–induced protoporphyrin IX (PpIX) identify regions of increasing malignancy in low- and high-grade gliomas beyond the capabilities of current fluorescence imaging in patients undergoing fluorescence-guided resection (FGR). Surgical specimens were collected from 133 biopsies in 23 patients and processed for ex vivo neuropathological analysis: PpIX fluorimetry to measure PpIX concentrations (CPpIX) and Ki-67 immunohistochemistry to assess tissue proliferation. Samples displaying visible levels of fluorescence showed significantly higher levels of CPpIX and tissue proliferation. CPpIX was strongly correlated with histopathological score (nonparametric) and tissue proliferation (parametric), such that increasing levels of CPpIX were identified with regions of increasing malignancy. Furthermore, a large percentage of tumor-positive biopsy sites (∼40%) that were not visibly fluorescent under the operating microscope had levels of CPpIX greater than 0.1 µg/mL, which indicates that significant PpIX accumulation exists below the detection threshold of current fluorescence imaging. Although PpIX fluorescence is recognized as a visual biomarker for neurosurgical resection guidance, these data show that it is quantitatively related at the microscopic level to increasing malignancy in both low- and high-grade gliomas. This work suggests a need for improved PpIX fluorescence detection technologies to achieve better sensitivity and quantification of PpIX in tissue during surgery. PMID:21798847

Quantification of Material Fluorescence and Light Scattering Cross Sections Using Ratiometric Bandwidth-Varied Polarized Resonance Synchronous Spectroscopy.

PubMed

Xu, Joanna Xiuzhu; Hu, Juan; Zhang, Dongmao

2018-05-25

Presented herein is the ratiometric bandwidth-varied polarized resonance synchronous spectroscopy (BVPRS2) method for quantification of material optical activity spectra. These include the sample light absorption and scattering cross-section spectrum, the scattering depolarization spectrum, and the fluorescence emission cross-section and depolarization spectrum in the wavelength region where the sample both absorbs and emits. This ratiometric BVPRS2 spectroscopic method is a self-contained technique capable of quantitatively decoupling material fluorescence and light scattering signal contribution to its ratiometric BVPRS2 spectra through the linear curve-fitting of the ratiometric BVPRS2 signal as a function of the wavelength bandwidth used in the PRS2 measurements. Example applications of this new spectroscopic method are demonstrated with materials that can be approximated as pure scatterers, simultaneous photon absorbers/emitters, simultaneous photon absorbers/scatterers, and finally simultaneous photon absorbers/scatterers/emitters. Because the only instruments needed for this ratiometric BVPRS2 technique are the conventional UV-vis spectrophotometer and spectrofluorometer, this work should open doors for routine decomposition of material UV-vis extinction spectrum into its absorption and scattering component spectra. The methodology and insights provided in this work should be of broad significance to all chemical research that involves photon/matter interactions.

Silver nanoparticle-induced degranulation observed with quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yang, Wenzhong; Lee, Seungrag; Lee, Jiyong; Bae, Yoonsung; Kim, Dugyoung

2010-07-01

Monitoring a degranulation process in a live mast cell is a quite important issue in immunology and pharmacology. Because the size of a granule is normally much smaller than the resolution limit of an optical microscope system, there is no direct real-time live cell imaging technique for observing degranulation processes except for fluorescence imaging techniques. In this research, we propose optical quantitative phase microscopy (QPM) as a new observation tool to study degranulation processes in a live mast cell without any fluorescence labeling. We measure the cell volumes and the cross sectional profiles (x-z plane) of an RBL-2H3 cell and a HeLa cell, before and after they are exposed to calcium ionophore A23187 and silver nanoparticles (AgNPs). We verify that the volume and the cross sectional line profile of the RBL-2H3 cell were changed significantly when it was exposed to A23187. When 50 μg/mL of AgNP is used instead of A23187, the measurements of cell volume and cross sectional profiles indicate that RBL-2H3 cells also follow degranulation processes. Degranulation processes for these cells are verified by monitoring the increase of intracellular calcium ([Ca2+]i) and histamine with fluorescent methods.

Evidence of a glycemic threshold for the formation of pentosidine in diabetic dog lens but not in collagen.

PubMed

Nagaraj, R H; Kern, T S; Sell, D R; Fogarty, J; Engerman, R L; Monnier, V M

1996-05-01

The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross-links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 +/- 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 +/- 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging

PubMed Central

Schmidt, F.N.; Zimmermann, E.A.; Campbell, G.M.; Sroga, G.E.; Püschel, K.; Amling, M.; Tang, S. Y.; Vashishth, D.; Busse, B.

2017-01-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic crosslinks, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692 cm−1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p< 0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. PMID:28109917

Analyzing the Cross-Correlation Between Onshore and Offshore RMB Exchange Rates Based on Multifractal Detrended Cross-Correlation Analysis (MF-DCCA)

NASA Astrophysics Data System (ADS)

Xie, Chi; Zhou, Yingying; Wang, Gangjin; Yan, Xinguo

We use the multifractal detrended cross-correlation analysis (MF-DCCA) method to explore the multifractal behavior of the cross-correlation between exchange rates of onshore RMB (CNY) and offshore RMB (CNH) against US dollar (USD). The empirical data are daily prices of CNY/USD and CNH/USD from May 1, 2012 to February 29, 2016. The results demonstrate that: (i) the cross-correlation between CNY/USD and CNH/USD is persistent and its fluctuation is smaller when the order of fluctuation function is negative than that when the order is positive; (ii) the multifractal behavior of the cross-correlation between CNY/USD and CNH/USD is significant during the sample period; (iii) the dynamic Hurst exponents obtained by the rolling windows analysis show that the cross-correlation is stable when the global economic situation is good and volatile in bad situation; and (iv) the non-normal distribution of original data has a greater effect on the multifractality of the cross-correlation between CNY/USD and CNH/USD than the temporary correlation.

[Excitation transfer between high-lying states in K2 in collisions with ground state K and H2 molecules].

PubMed

Shen, Xiao-Yan; Liu, Jing; Dai, Kang; Shen, Yi-Fan

2010-02-01

Pure potassium vapor or K-H2 mixture was irradiated in a glass fluorescence cell with pulses of 710 nm radiation from an OPO laser, populating K2 (1lambda(g)) state by two-photon absorption. Cross sections for 1lambda(g)-3lambda(g) transfer in K2 were determined using methods of molecular fluorescence. During the experiments with pure K vapor, the cell temperature was varied between 553 and 603 K. The K number density was determined spectroscopically by the white-light absorption measurement in the blue wing of the self-broadened resonance D2 line. The resulting fluorescence included a direct component emitted in the decay of the optically excitation and a sensitized component arising from the collisionally populated state. The decay signal of time-resolved fluorescence from1lambda(g) -->1 1sigma(u)+ transition was monitored. It was seen that just after the laser pulse the fluorescence of the photoexcited level decreased exponentially. The effective lifetimes of the 1lambda(g) state can be resolved. The plot of reciprocal of effective lifetimes of the 1lambda(g) state against K densities yielded the slope that indicated the total cross section for deactivation and the intercept that provided the radiative lifetime of the state. The radiative lifetime (20 +/- 2) ns was obtained. The cross section for deactivation of the K2(1lambda(g)) molecules by collisions with K is (2.5 +/- 0.3) x 10(-14) cm2. The time-resolved intensities of the K23lambda(g) --> 1 3sigma(u)+ (484 nm) line were measured. The radiative lifetime (16.0 +/- 3.2) ns and the total cross section (2.5 +/- 0.6) x 10(-14) cm2 for deactivation of the K2 (3lambda(g)) state can also be determined through the analogous procedure. The time-integrated intensities of 1lambda(g) --> 1 1sigma(u)+ and 3lambda(g) --> 1 3sigma(u)+ transitions were measured. The cross section (1.1 +/- 0.3) x10(-14) cm2 was obtained for K2 (1lambda(g))+ K --> K2 (3lambda(g)) + K collisions. During the experiments with K-H2 mixture, the cell temperature was kept constant at 553 K. The H2 pressure was varied between 40 and 400 Pa. The effects of K2-K collisions could not be neglected. These effects were subtracted out using the results of the pure K experiments. The cross section (2.7 +/- 1.1) x 10(-15) cm2 was obtained for K2 (1lambda(g)) + H2 --> K2 (3lambda(g))+H2 collisions. The cross section is (6.8 +/- 2.7) x 10(-15) cm2 for K2 (3lambda(g)) + H2 --> states out of K2 (3lambda(g)) + H2 collisions.

Vanadium fine-structure K-shell electron impact ionization cross sections for fast-electron diagnostic in laser–solid experiments

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

Palmeri, P., E-mail: patrick.palmeri@umons.ac.be; Quinet, P., E-mail: pascal.quinet@umons.ac.be; IPNAS, Université de Liège, B-4000 Liège

2015-09-15

The K-shell electron impact ionization (EII) cross section, along with the K-shell fluorescence yield, is one of the key atomic parameters for fast-electron diagnostic in laser–solid experiments through the K-shell emission cross section. In addition, in a campaign dedicated to the modeling of the K lines of astrophysical interest (Palmeri et al. (2012)), the K-shell fluorescence yields for the K-vacancy fine-structure atomic levels of all the vanadium isonuclear ions have been calculated. In this study, the K-shell EII cross sections connecting the ground and the metastable levels of the parent vanadium ions to the daughter ions K-vacancy levels considered in Palmerimore » et al. (2012) have been determined. The relativistic distorted-wave (DW) approximation implemented in the FAC atomic code has been used for the incident electron kinetic energies up to 20 times the K-shell threshold energies. Moreover, the resulting DW cross sections have been extrapolated at higher energies using the asymptotic behavior of the modified relativistic binary encounter Bethe model (MRBEB) of Guerra et al. (2012) with the density-effect correction proposed by Davies et al. (2013)« less

L i ( i = 1-3) subshell X-ray production cross sections and fluorescence yields for some elements with 56 ⩽ Z ⩽ 68 at 22.6 keV

NASA Astrophysics Data System (ADS)

Chauhan, Yogeshwar; Tiwari, M. K.; Puri, Sanjiv

2008-01-01

The L k ( k = l, α, β 1,4, β 3,6, β 2,15,9,10,7, γ 1,5 and γ 2,3,4) X-ray production (XRP) cross sections have been measured for six elements with 56 ⩽ Z ⩽ 68 at 22.6 keV incident photon energy using the EDXRF spectrometer. The incident photon intensity, detector efficiency and geometrical factors have been determined from the K X-ray yields emitted from elemental targets with 22 ⩽ Z ⩽ 42 in the same geometrical setup and from knowledge of the K XRP cross sections. The L 1 and L 2 subshell fluorescence yields have been deduced from the present measured L k XRP cross sections using the relativistic Hartree-Fock-Slater (HFS) model based photoionization cross sections. The present deduced ω1 (exp) values have been found to be, on an average, higher by 15% and 20% than those based on the Dirac-Hartree-Slater (DHS) model and the semi-empirical values compiled by Krause, respectively, for elements with 60 ⩽ Z ⩽ 68.

M ξ, M αβ, M γ and M m X-ray production cross-sections for elements with 71⩽ z⩽92 at 5.96 keV photon energy

NASA Astrophysics Data System (ADS)

Sharma, Manju; Sharma, Veena; Kumar, Sanjeev; Puri, S.; Singh, Nirmal

2006-11-01

The M ξ, M αβ, M γ and M m X-ray production (XRP) cross-sections have been measured for the elements with 71⩽ Z⩽92 at 5.96 keV incident photon energy satisfying EM1< Einc< EL3, where EM1(L3) is the M 1(L 3) subshell binding energy. These XRP cross-sections have been calculated using photoionization cross-sections based on the relativistic Dirac-Hartree-Slater (RDHS) model with three sets of X-ray emission rates, fluorescence, Coster-Kronig and super Coster-Kronig yields based on (i) the non-relativistic Hartree-Slater (NRHS) potential model, (ii) the RDHS model and (iii) the relativistic Dirac-Fock (RDF) model. For the third set, the M i ( i=1-5) subshell fluorescence yields have been calculated using the RDF model-based X-ray emission rates and total widths reevaluated to incorporate the RDF model-based radiative widths. The measured cross-sections have been compared with the calculated values to check the applicability of the physical parameters based on different models.

Complexation of β-cyclodextrin with dual molecular probes bearing fluorescent and paramagnetic moieties linked by short polyether chains.

PubMed

Mocanu, S; Matei, I; Ionescu, S; Tecuceanu, V; Marinescu, G; Ionita, P; Culita, D; Leonties, A; Ionita, Gabriela

2017-10-18

Electron paramagnetic resonance (EPR) and fluorescence spectroscopies provide molecular-level insights on the interaction of paramagnetic and fluorescent species with the microenvironment. A series of dual molecular probes bearing fluorescent and paramagnetic moieties linked by flexible short polyether chains have been synthesized. These new molecular probes open the possibility to investigate various multi-component systems such as host-guest systems, polymeric micelles, gels and protein solutions by using EPR and fluorescence spectroscopies concertedly. The EPR and fluorescence spectra of these compounds show that the dependence of the rotational correlation time and fluorescence quantum yield on the chain length of the linker is not linear, due to the flexibility of the polyether linker. The quenching effect of the nitroxide moiety on the fluorescence intensity of the pyrene group varies with the linker length and flexibility. The interaction of these dual molecular probes with β-cyclodextrin, in solution and in polymeric gels, was evaluated and demonstrated by analysis of EPR and fluorescence spectra.

Protoporphyrin IX fluorescence as potential indicator of psoriasis severity and progression.

PubMed

Wang, Bo; Xu, Yu-Ting; Zhang, Li; Zheng, Jie; Sroka, Ronald; Wang, Hong-Wei; Wang, Xiu-Li

2017-09-01

In psoriatic lesions, fluorescence diagnosis with blue light can detect protoporphyrin IX accumulation, especially after topical 5-aminolaevulinic acid (ALA) application. However, variable fluorescence distributions, interpersonal variations and long incubation time limit its wide application in clinic. This study is aimed to identify a consistent and convenient method to facilitate diagnosis and evaluation of psoriatic lesions. 104 psoriatic lesions from 30 patients were evaluated. Single lesion PSI scoring and fluorescence by macrospectrofluorometry were recorded on each lesion before and after treatment with narrow-band UVB. Punctate red fluorescence, emitted mainly by protoporphyrin IX, is observed in some psoriatic lesions. According to psoriasis severity index, fluorescence-positive lesions are more severe than lesions without fluorescence. We found a significant positive correlation between psoriasis severity and fluorescence intensity from protoporphyrin IX. Protoporphyrin IX-induced red fluorescence can be used as a novel and convenient approach for psoriasis diagnosis and progression evaluation. Copyright © 2017. Published by Elsevier B.V.

M sub-shell X-ray fluorescence cross-section measurements for six elements in the range Z = 78-92 at tuned synchrotron photon energies 5, 7 and 9 keV.

NASA Astrophysics Data System (ADS)

Bansal, Himani; Tiwari, M. K.; Mittal, Raj

2018-01-01

M sub-shell X-ray fluorescence cross-sections of elements Pt, Au, Hg, Pb, Th and U have been measured with linearly polarized photon beams from Indus-II synchrotron source at Raja Ramanna Centre for Advanced Technology (RRCAT), India at tuned 5, 7 and 9 keV energies less than the L3 edge energy of elements. Measurements at present energies and elements are not available in literature. Therefore, measured cross-sections for Mξ, Mδ, Mα, Mβ, Mγ, Mm1 and Mm2 group of X-rays were compared with calculated theoretical values based upon Non Relativistic Hartree-Slater (NRHS) and relativistic Dirac-Fork (DF) and Dirac-Hartree-Slater (DHS) models. The measured cross-sections along with our earlier quoted measurements at 8 and 10 keV by Kaur et al. [Nucl. Instrum. Meth. B, 2014; 320: 37] are found in good agreement with DF and DHS values around 20% deviations and are highly deviated from NRHS values. Most of the spots of observed high deviations in measured and theoretical cross-sections are found to coincide with the presence of crisscrosses/sharp variations in contributing physical parameters photo-ionization cross-sections σMi's and Coster-Kronig yields fij's with Zs.

In vivo characterization of a bigenic fluorescent mouse model of Alzheimer's disease with neurodegeneration.

PubMed

Crowe, Sarah E; Ellis-Davies, Graham C R

2013-07-01

The loss of cognitive function in Alzheimer's disease (AD) patients is strongly correlated with the loss of neurons in various regions of the brain. We have created a new fluorescent bigenic mouse model of AD by crossing "H-line" yellow fluorescent protein (YFP) mice with the 5xFAD mouse model, which we call the 5XY mouse model. The 5xFAD mouse has been shown to have significant loss of L5 pyramidal neurons by 12 months of age. These neurons are transgenically labeled with YFP in the 5XY mouse, which enable longitudinal imaging of structural changes. In the 5XY mice, we observed an appearance of axonal dystrophies, with two distinct morphologies in the early stages of the disease progression. Simple swelling dystrophies are transient in nature and are not directly associated with amyloid plaques. Rosette dystrophies are more complex structures that remained stable throughout all imaging sessions, and always surrounded an amyloid plaque. Plaque growth was followed over 4 weeks, and significant growth was seen between weekly imaging sessions. In addition to axonal dystrophy appearance and plaque growth, we were able to follow spine stability in 4-month old 5XY mice, which revealed no significant loss of spines. 5XY mice also showed a striking shrinkage of the neocortex at older ages (12-14 months). The 5XY mouse model may be a valuable tool for studying specific events in the degeneration of the neocortex, and may suggest new avenues for therapeutic intervention. Copyright © 2013 Wiley Periodicals, Inc.

Heavy Atom Effect of Bromine Significantly Enhances Exciton Utilization of Delayed Fluorescence Luminogens.

PubMed

Gan, Shifeng; Hu, Shimin; Li, Xiang-Long; Zeng, Jiajie; Zhang, Dongdong; Huang, Tianyu; Luo, Wenwen; Zhao, Zujin; Duan, Lian; Su, Shi-Jian; Tang, Ben Zhong

2018-05-23

Raising triplet exciton utilization of pure organic luminescent materials is of significant importance for efficiency advancement of organic light-emitting diodes (OLEDs). Herein, by introducing bromine atom(s) onto a typical molecule (bis(carbazol-9-yl)-4,5-dicyanobenzene) with thermally activated delayed fluorescence, we demonstrate that the heavy atom effect of bromine can increase spin-orbit coupling and promote the reverse intersystem crossing, which endow the molecules with more distinct delayed fluorescence. In consequence, the triplet exciton utilization is improved greatly with the increase of bromine atoms, affording apparently advanced external quantum efficiencies of OLEDs. Utilizing the enhancement effect of bromine atoms on delayed fluorescence should be a simple and promising design concept for efficient organic luminogens with high exciton utilization.

Simultaneous two-dimensional laser-induced-fluorescence measurements of argon ions.

PubMed

Hansen, A K; Galante, Matthew; McCarren, Dustin; Sears, Stephanie; Scime, E E

2010-10-01

Recent laser upgrades on the Hot Helicon Experiment at West Virginia University have enabled multiplexed simultaneous measurements of the ion velocity distribution function at a single location, expanding our capabilities in laser-induced fluorescence diagnostics. The laser output is split into two beams, each modulated with an optical chopper and injected perpendicular and parallel to the magnetic field. Light from the crossing point of the beams is transported to a narrow-band photomultiplier tube filtered at the fluorescence wavelength and monitored by two lock-in amplifiers, each referenced to one of the two chopper frequencies.

Photon-Counting H33D Detector for Biological Fluorescence Imaging

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.

2010-01-01

We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:20151021

Accumulation of Maillard reaction products in skin collagen in diabetes and aging.

PubMed Central

Dyer, D G; Dunn, J A; Thorpe, S R; Bailie, K E; Lyons, T J; McCance, D R; Baynes, J W

1993-01-01

To investigate the contribution of glycation and oxidation reactions to the modification of insoluble collagen in aging and diabetes, Maillard reaction products were measured in skin collagen from 39 type 1 diabetic patients and 52 nondiabetic control subjects. Compounds studied included fructoselysine (FL), the initial glycation product, and the glycoxidation products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine, formed during later Maillard reactions. Collagen-linked fluorescence was also studied. In nondiabetic subjects, glycation of collagen (FL content) increased only 33% between 20 and 85 yr of age. In contrast, CML, pentosidine and fluorescence increased five-fold, correlating strongly with age. In diabetic patients, collagen FL was increased threefold compared with nondiabetic subjects, correlating strongly with glycated hemoglobin but not with age. Collagen CML, pentosidine and fluorescence were increased up to twofold in diabetic compared with control patients: this could be explained by the increase in glycation alone, without invoking increased oxidative stress. There were strong correlations among CML, pentosidine and fluorescence in both groups, providing evidence for age-dependent chemical modification of collagen via the Maillard reaction, and acceleration of this process in diabetes. These results support the description of diabetes as a disease characterized by accelerated chemical aging of long-lived tissue proteins. PMID:8514858

Oligomeric State of Lipocalin-1 (LCN1) by Multiangle Laser Light Scattering and Fluorescence Anisotropy Decay

PubMed Central

Gasymov, Oktay K.; Abduragimov, Adil R.; Merschak, Petra; Redl, Bernhard; Glasgow, Ben J.

2007-01-01

Multiangle laser light scattering and fluorescence anisotropy decay measurements clarified the oligomeric states of native and recombinant tear lipocalin (lipocalin-1, TL). Native TL is monomeric. Recombinant TL (5-68 μM) with or without the histidine tag shows less than 7% dimer formation that is not in equilibrium with the monomeric form. Fluorescence anisotropy decay showed a correlation time of 9-10 ns for TL (10 μM- 1mM). Hydrodynamic calculations based on the crystallographic structure of a monomeric TL mutant closely concur with the observed correlation time. The solution properties calculated with HYDROPRO and SOLPRO programs from the available crystallographic structure of a monomeric TL mutant concur closely with the observed fluorescence anisotropy decay. The resulting model shows that protein topology is the major determinant of rotational correlation time and accounts for deviation from the Stokes-Einstein relation. The data challenge previous gel filtration studies to show that native TL exists predominantly as a monomer in solution rather than as a dimer. Delipidation of TL results in a formation of a complex oligomeric state (up to 25%). These findings are important as the dynamic processes in the tear film are limited by diffusional, translational as well as rotational, properties of the protein. PMID:17869594

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

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

Ross, J.B.A.; Schwartz, G.P.; Laws, W.R.

1992-02-18

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

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

PubMed

Killingsworth, Murray C; Bobryshev, Yuri V

2016-08-07

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

PubMed Central

2013-01-01

Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules. PMID:23548159

Comparison of five techniques for the detection of Renibacterium salmoninarum in adult coho salmon.

USGS Publications Warehouse

Pascho, R.J.; Elliott, D.G.; Mallett, R.W.; Mulcahy, D.

1987-01-01

Samples of kidney, spleen, coelomic fluid, and blood from 56 sexually mature coho salmon Oncorhynchus kisutch were examined for infection by Renibacterium salmoninarum by five methods. The overall prevalence (all sample types combined) of R. salmoninarum in the fish was 100% by the enzyme-linked immunosorbent assay, 86% by the combined results of the direct fluorescent antibody and the direct filtration-fluorescent antibody techniques, 39% by culture, 11% by counterimmunoelectrophoresis, and 5% by agarose gel immunodiffusion. There was a significant positive correlation (P < 0.001) between the enzyme-linked immunosorbent assay absorbance levels and the counts by fluorescent antibody techniques for kidney, spleen, and coelomic fluid, and significant positive correlations (P < 0.001) in enzyme-linked immunosorbent assay absorbance levels for all four of the sample types.

Fluorescence Correlation Spectroscopy to Study Diffusion of Polymer Chains within Layered Hydrogen-Bonded Polymer Films

NASA Astrophysics Data System (ADS)

Pristinski, Denis; Kharlampieva, Evguenia; Sukhishvili, Svetlana

2002-03-01

Fluorescence Correlation Spectroscopy (FCS) has been used to probe molecular motions within polymer multilayers formed by hydrogen-bonding sequential self-assembly. Polyethylene glycol (PEG) molecules were end-labeled with the fluorescent tags, and self-assembled with polymethacrylic acid (PMAA) using layer-by-layer deposition. We have found that molecules included in the top adsorbed layer have significant mobility at the millisecond time scale, probably due to translational diffusion. However, their dynamics deviate from classical Brownian motion with a single diffusion time. Possible reasons for the deviation are discussed. We found that motions were significantly slowed with increasing depth within the PEG/PMAA multilayer. This phenomena occured in a narrow pH range around 4.0 in which intermolecular interactions were relatively weak.

Saturated fluorescence measurements of the hydroxyl radical in laminar high-pressure flames

NASA Technical Reports Server (NTRS)

Carter, Campbell D.; King, Galen B.; Laurendeau, Normand M.

1990-01-01

The efficacy of laser saturated fluorescence (LSF) for OH concentration measurements in high pressure flames was studied theoretically and experimentally. Using a numerical model describing the interaction of hydroxyl with nonuniform laser excitation, the effect of pressure on the validity of the balanced cross-rate model was studied along with the sensitivity of the depopulation of the laser-coupled levels to the ratio of rate coefficients describing: (1) electronic quenching to (sup 2) Sigma (+) (v double prime greater than 0), and (2) vibrational relaxation from v double prime greater than 0 to v double prime = 0. At sufficiently high pressures and near-saturated conditions, the total population of the laser-coupled levels reaches an asymptotic value, which is insensitive to the degree of saturation. When the ratio of electronic quenching to vibrational relaxation is small and the rate of coefficients for rotational transfer in the ground and excited electronic states are nearly the same, the balanced cross-rate model remains a good approximation for all pressures. When the above ratio is large, depopulation of the laser-coupled levels becomes significant at high pressures, and thus the balanced cross-rate model no longer holds. Under these conditions, however, knowledge of the depletion of the laser-coupled levels can be used to correct the model. A combustion facility for operation up to 20 atm was developed to allow LSF measurements of OH in high pressure flames. Using this facility, partial saturation in laminar high pressure (less than or equal to 12.3 atm) C2H6/O2/N2 flames was achieved. To evaluate the limits of the balanced cross-rate model, absorption and calibrated LSF measurements at 3.1 and 6.1 atm were compared. The fluorescence voltages were calibrated with absorption measurements in an atmospheric flame and corrected for their finite sensitivity to quenching with: (1) estimated quenching rate coefficients, and (2) an in situ measurement from a technique employing two fluorescence detection geometries.

NIST/ISAC standardization study: variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads.

PubMed

Hoffman, Robert A; Wang, Lili; Bigos, Martin; Nolan, John P

2012-09-01

Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue™), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned fluorescence standard beads. Copyright © 2012 International Society for Advancement of Cytometry.

Signal Digitizer and Cross-Correlation Application Specific Integrated Circuit

NASA Technical Reports Server (NTRS)

Baranauskas, Gytis (Inventor); Lim, Boon H. (Inventor); Baranauskas, Dalius (Inventor); Zelenin, Denis (Inventor); Kangaslahti, Pekka (Inventor); Tanner, Alan B. (Inventor)

2017-01-01

According to one embodiment, a cross-correlator comprises a plurality of analog front ends (AFEs), a cross-correlation circuit and a data serializer. Each of the AFEs comprises a variable gain amplifier (VGA) and a corresponding analog-to-digital converter (ADC) in which the VGA receives and modifies a unique analog signal associates with a measured analog radio frequency (RF) signal and the ADC produces digital data associated with the modified analog signal. Communicatively coupled to the AFEs, the cross-correlation circuit performs a cross-correlation operation on the digital data produced from different measured analog RF signals. The data serializer is communicatively coupled to the summing and cross-correlating matrix and continuously outputs a prescribed amount of the correlated digital data.

Two-photon microscopy and spectroscopy based on a compact confocal scanning head

NASA Astrophysics Data System (ADS)

Diaspro, Alberto; Chirico, Giberto; Federici, Federico; Cannone, Fabio; Beretta, Sabrina; Robello, Mauro; Olivini, Francesca; Ramoino, Paola

2001-07-01

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two- photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

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.

Development of Fluorescence Surrogates to Predict the Photochemical Transformation of Pharmaceuticals in Wastewater Effluents.

PubMed

Yan, Shuwen; Yao, Bo; Lian, Lushi; Lu, Xinchen; Snyder, Shane A; Li, Rui; Song, Weihua

2017-03-07

The photochemical transformation of pharmaceutical and personal care products (PPCPs) in wastewater effluents is an emerging concern for environmental scientists. In the current study, the photodegradation of 29 PPCPs was examined in effluents under simulated solar irradiation. Direct photodegradation, triplet state effluent organic matter ( 3 EfOM*)-mediated and hydroxyl radical (HO • )-mediated degradation are three major pathways in the removal process. With the photodegradation of trace levels of PPCPs, the excitation-emission matrix (EEM) fluorescence intensities of the effluents were also gradually reduced. Therefore, fluorescence peaks have been identified, for the first time, as appropriate surrogates to assess the photodegradation of PPCPs. The humic-like fluorescence peak is linked to direct photolysis-labile PPCPs, such as naproxen, ronidazole, diclofenac, ornidazole, tinidazole, chloramphenicol, flumequine, ciprofloxacin, methadone, and dimetridazole. The tyrosine-like EEM peak is associated with HO • /CO 3 •- -labile PPCPs, such as trimethoprim, ibuprofen, gemfibrozil, atenolol, carbamazepine, and cephalexin. The tryptophan-like peak is associated with 3 EfOM*-labile PPCPs, such as clenbuterol, metoprolol, venlafaxine, bisphenol A, propranolol, ractopamine, salbutamol, roxithromycin, clarithromycin, azithromycin, famotidine, terbutaline, and erythromycin. The reduction in EEM fluorescence correlates well with the removal of PPCPs, allowing a model to be constructed. The solar-driven removal of EEM fluorescence was applied to predict the attenuation of 11 PPCPs in five field samples. A close correlation between the predicted results and the experimental results suggests that fluorescence may be a suitable surrogate for monitoring the solar-driven photodegradation of PPCPs in effluents.

Use of fluorescence EEM to monitor the removal of emerging contaminants in full scale wastewater treatment plants.

PubMed

Sgroi, Massimiliano; Roccaro, Paolo; Korshin, Gregory V; Greco, Valentina; Sciuto, Sebastiano; Anumol, Tarun; Snyder, Shane A; Vagliasindi, Federico G A

2017-02-05

This study investigated the applicability of different techniques for fluorescence excitation/emission matrices data interpretations, including peak-picking method, fluorescence regional integration and PARAFAC modelling, to act as surrogates in predicting emerging trace organic compounds (ETOrCs) removal during conventional wastewater treatments that usually comprise primary and secondary treatments. Results showed that fluorescence indexes developed using alternative methodologies but indicative of a same dissolved organic matter component resulted in similar predictions of the removal of the target compounds. The peak index defined by the excitation/emission wavelength positions (λ ex/ λ em ) 225/290nm and related to aromatic proteins and tyrosine-like fluorescence was determined to be a particularly suitable surrogate for monitoring ETOrCs that had very high removal rates (average removal >70%) (i.e., triclosan, caffeine and ibuprofen). The peak index defined by λ ex/ λ em =245/440nm and the PARAFAC component with wavelength of the maxima λ ex/ λ em =245, 350/450, both identified as humic-like fluorescence, were found remarkably well correlated with ETOrCs such as atenolol, naproxen and gemfibrozil that were moderately removed (51-70% average removal). Finally, the PARAFAC component with wavelength of the maxima λ ex/ λ em =<240, 315/380 identified as microbial humic-like fluorescence was the only index correlated with the removal of the antibiotic trimethoprim (average removal 68%). Copyright © 2016 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

Yilmaz, Hasan

2016-03-01

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

Depth-resolved fluorescence of biological tissue

NASA Astrophysics Data System (ADS)

Wu, Yicong; Xi, Peng; Cheung, Tak-Hong; Yim, So Fan; Yu, Mei-Yung; Qu, Jianan Y.

2005-06-01

The depth-resolved autofluorescence ofrabbit oral tissue, normal and dysplastic human ectocervical tissue within l20μm depth were investigated utilizing a confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of oral and ectocervical tissue, strong keratin fluorescence with the spectral characteristics similar to collagen was observed. The fluorescence signal from epithelial tissue between the keratinizing layer and stroma can be well resolved. Furthermore, NADH and FADfluorescence measured from the underlying non-keratinizing epithelial layer were strongly correlated to the tissue pathology. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

Correlated FLIM and PLIM for cell metabolism

NASA Astrophysics Data System (ADS)

Rück, A.; Breymayer, J.; Kalinina, S.

2016-03-01

Correlated imaging of phosphorescence and fluorescence lifetime parameters of metabolic markers is a challenge for direct investigating mechanisms related to cell metabolism and oxygen tension. A large variety of clinical phenotypes is associated with mitochondrial defects accomplished with changes in cell metabolism. In many cases the hypoxic microenvironment of cancer cells shifts metabolism from oxidative phosphorylation (OXPHOS) to anaerobic or aerobic glycolysis, a process known as "Warburg" effect. Also during stem cell differentiation a switch in cell metabolism is observed. A defective mitochondrial function associated with hypoxia has been invoked in many complex disorders such as type 2 diabetes, Alzheimers disease, cardiac ischemia/reperfusion injury, tissue inflammation and cancer. Cellular responses to oxygen tension have been studied extensively, optical imaging techniques based on time correlated single photon counting (TCSPC) to detect the underlying metabolic mechanisms are therefore of prominent interest. They offer the possibility by inspecting fluorescence decay characteristics of intrinsic coenzymes to directly image metabolic pathways. Moreover oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescent probe. The combination of both fluorescence lifetime imaging (FLIM) of coenzymes like NADH and FAD and phosphorescence lifetime (PLIM) of phosphorescent dyes could provide valuable information about correlation of metabolic pathways and oxygen tension.

Facile fabrication of core cross-linked micelles by RAFT polymerization and enzyme-mediated reaction.

PubMed

Wu, Yukun; Lai, Quanyong; Lai, Shuqi; Wu, Jing; Wang, Wei; Yuan, Zhi

2014-06-01

Polymeric micelles formed in aqueous solution by assembly of amphiphilic block copolymers have been extensively investigated due to their great potential as drug carriers. However, the stability of polymeric assembly is still one of the major challenges in delivering drugs to tissues and cells. Here, we report a facile route to fabricate core cross-linked (CCL) micelles using an enzymatic polymerization as the cross-linking method. We present synthesis of poly(ethylene glycol)-block-poly(N-isopropyl acrylamide-co-N-(4-hydroxyphenethyl) acrylamide) diblock copolymer PEG-b-P(NIPAAm-co-NHPAAm) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The diblock copolymer was then self-assembled into non-cross-linked (NCL) micelles upon heating above the lower critical solution temperature (LCST), and subsequently cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) as enzyme and oxidant. The characterization of the diblock copolymer and micelles were studied by NMR, DLS, UV-vis, and fluorescence spectroscopy. The fluorescence study reveals that the cross-linking process endows the micelles with much lower critical micelle concentration (CMC). In addition, the drug release study shows that the CCL micelles have lower release amount of doxorubicin (DOX) than the NCL micelles due to the enhanced stability of the CCL micelles by core cross-linking process. Copyright © 2014 Elsevier B.V. All rights reserved.

Fluorescence spectroscopy of the retina from scrapie-infected mice

USDA-ARS?s Scientific Manuscript database

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

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

NASA Astrophysics Data System (ADS)

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-03-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

Direct Evidence of Intrinsic Blue Fluorescence from Oligomeric Interfaces of Human Serum Albumin.

PubMed

Bhattacharya, Arpan; Bhowmik, Soumitra; Singh, Amit K; Kodgire, Prashant; Das, Apurba K; Mukherjee, Tushar Kanti

2017-10-10

The molecular origin behind the concentration-dependent intrinsic blue fluorescence of human serum albumin (HSA) is not known yet. This unusual blue fluorescence is believed to be a characteristic feature of amyloid-like fibrils of protein/peptide and originates due to the delocalization of peptide bond electrons through the extended hydrogen bond networks of cross-β-sheet structure. Herein, by combining the results of spectroscopy, size exclusion chromatography, native gel electrophoresis, and confocal microscopy, we have shown that the intrinsic blue fluorescence of HSA exclusively originates from oligomeric interfaces devoid of any amyloid-like fibrillar structure. Our study suggests that this low energy fluorescence band is not due to any particular residue/sequence, but rather it is a common feature of self-assembled peptide bonds. The present findings of intrinsic blue fluorescence from oligomeric interfaces pave the way for future applications of this unique visual phenomenon for early stage detection of various protein aggregation related human diseases.

Porphyrin involvement in redshift fluorescence in dentin decay

NASA Astrophysics Data System (ADS)

Slimani, A.; Panayotov, I.; Levallois, B.; Cloitre, T.; Gergely, C.; Bec, N.; Larroque, C.; Tassery, H.; Cuisinier, F.

2014-05-01

The aim of this study was to evaluate the porphyrin involvement in the red fluorescence observed in dental caries with Soprolife® light-induced fluorescence camera in treatments mode (SOPRO, ACTEON Group, La Ciotat, France) and Vistacam® camera (DÜRR DENTAL AG, Bietigheim-Bissingen, Germany). The International Caries Detection and Assessment System (ICDAS) was used to rand the samples. Human teeth cross-sections, ranked from ICDAS score 0 to 6, were examined by epi-fluorescence microscopy and Confocal Raman microscopy. Comparable studies were done with Protoporphyrin IX, Porphyrin I and Pentosidine solutions. An RGB analysis of Soprolife® images was performed using ImageJ Software (1.46r, National Institutes of Health, USA). Fluorescence spectroscopy and MicroRaman spectroscopy revealed the presence of Protoporphyrin IX, in carious enamel, dentin and dental plaque. However, the presence of porphyrin I and pentosidine cannot be excluded. The results indicated that not only porphyrin were implicated in the red fluorescence, Advanced Glygation Endproducts (AGEs) of the Maillard reaction also contributed to this phenomenon.

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

PubMed Central

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-01-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy. PMID:28300146

Fluorescent photochromes of diarylethene series: synthesis and properties

NASA Astrophysics Data System (ADS)

Shirinian, Valerii Z.; Lonshakov, D. V.; Lvov, A. G.; Krayushkin, Mikhail M.

2013-06-01

The research data in the field of fluorescent photochromic di(het)arylethenes published over the last decade are summarized. The characteristics of these compounds significant for their application in the design of molecular optical memory systems and photocontrolled switches are considered. The main types of diarylethenes and methods for their synthesis are described, and the correlations between structure and spectral properties, in particular, fluorescence characteristics are analyzed. Considerable attention is given to the means for endowing diarylethenes with fluorescence properties as one of the most promising methods for data readout from molecular information carriers. The bibliography includes 203 references.

The nature of multiphoton fluorescence from red blood cells

NASA Astrophysics Data System (ADS)

Saytashev, Ilyas; Murphy, Michael; Osseiran, Sam; Spence, Dana M.; Evans, Conor L.; Dantus, Marcos

2016-03-01

We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC's, but longer more intense pulses lead to their destruction.

Structural and biophysical properties of h-FANCI ARM repeat protein.

PubMed

Siddiqui, Mohd Quadir; Choudhary, Rajan Kumar; Thapa, Pankaj; Kulkarni, Neha; Rajpurohit, Yogendra S; Misra, Hari S; Gadewal, Nikhil; Kumar, Satish; Hasan, Syed K; Varma, Ashok K

2017-11-01

Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.

Dynamic organization of myristoylated Src in the live cell plasma membrane

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

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Dynamic organization of myristoylated Src in the live cell plasma membrane

DOE PAGES

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.; ...

2016-01-15

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Dendrimer internalization and intracellular trafficking in living cells.

PubMed

Albertazzi, Lorenzo; Serresi, Michela; Albanese, Alberto; Beltram, Fabio

2010-06-07

The ability of dendrimers to cross cell membranes is of much interest for their application in drug and gene delivery. Recent studies demonstrate that dendrimers are capable to enter cells by endocytosis, but the intracellular pathway following their internalization remains controversial. In this study we use confocal fluorescence microscopy to elucidate the intracellular trafficking properties of PAMAM dendrimers with high spatial and temporal resolution in living HeLa cells. Macromolecules of different chemical functionality (neutral, cationic and lipidated), size (from G2 up to G6) and surface charge are investigated and their internalization properties correlated with the molecular structure. Toxicity and internalization data are discussed that allow the identification of dendrimers maximizing intracellular uptake with the minimum effect on cell viability. Time-lapse imaging and colocalization assays with fluorescent biomarkers for endocytic vesicles demonstrate that dendrimers are internalized by both clathrin-dependent endocytosis and macropinocytosis and are eventually delivered to the lysosomal compartment. Moreover we analyzed the uptake of dendrimers in additional cell lines of practical interest for therapeutic purposes. These measurements together with a direct comparison with TAT peptides demonstrate that PAMAM dendrimers possess similar properties to these widely used cell-penetrating peptides and thanks to their chemical tunability may represent a valid alternative for drug and gene delivery.

The use of dwell time cross-correlation functions to study single-ion channel gating kinetics.

PubMed Central

Ball, F G; Kerry, C J; Ramsey, R L; Sansom, M S; Usherwood, P N

1988-01-01

The derivation of cross-correlation functions from single-channel dwell (open and closed) times is described. Simulation of single-channel data for simple gating models, alongside theoretical treatment, is used to demonstrate the relationship of cross-correlation functions to underlying gating mechanisms. It is shown that time irreversibility of gating kinetics may be revealed in cross-correlation functions. Application of cross-correlation function analysis to data derived from the locust muscle glutamate receptor-channel provides evidence for multiple gateway states and time reversibility of gating. A model for the gating of this channel is used to show the effect of omission of brief channel events on cross-correlation functions. PMID:2462924

Laser-induced tissue fluorescence in radiofrequency tissue-fusion characterization.

PubMed

Su, Lei; Fonseca, Martina B; Arya, Shobhit; Kudo, Hiromi; Goldin, Robert; Hanna, George B; Elson, Daniel S

2014-01-01

Heat-induced tissue fusion is an important procedure in modern surgery and can greatly reduce trauma, complications, and mortality during minimally invasive surgical blood vessel anastomosis, but it may also have further benefits if applied to other tissue types such as small and large intestine anastomoses. We present a tissue-fusion characterization technology using laser-induced fluorescence spectroscopy, which provides further insight into tissue constituent variations at the molecular level. In particular, an increase of fluorescence intensity in 450- to 550-nm range for 375- and 405-nm excitation suggests that the collagen cross-linking in fused tissues increased. Our experimental and statistical analyses showed that, by using fluorescence spectral data, good fusion could be differentiated from other cases with an accuracy of more than 95%. This suggests that the fluorescence spectroscopy could be potentially used as a feedback control method in online tissue-fusion monitoring.

Absorption and emission spectroscopic characterisation of 8-amino-riboflavin

NASA Astrophysics Data System (ADS)

Tyagi, A.; Zirak, P.; Penzkofer, A.; Mathes, T.; Hegemann, P.; Mack, M.; Ghisla, S.

2009-10-01

The flavin dye 8-amino-8-demethyl- D-riboflavin (AF) in the solvents water, DMSO, methanol, and chloroform/DMSO was studied by absorption and fluorescence spectroscopy. The first absorption band is red-shifted compared to riboflavin, and blue-shifted compared to roseoflavin (8-dimethylamino-8-demethyl-D-riboflavin). The fluorescence quantum yield of AF in the studied solvents varies between 20% and 50%. The fluorescence lifetimes were found to be in the 2-5 ns range. AF is well soluble in DMSO, weakly soluble in water and methanol, and practically insoluble in chloroform. The limited solubility causes AF aggregation, which was seen in differences between measured absorption spectra and fluorescence excitation spectra. Light scattering in the dye absorption region is discussed and approximate absorption cross-section spectra are determined from the combined measurement of transmission and fluorescence excitation spectra. The photo-stability of AF was studied by prolonged light exposure. The photo-degradation routes of AF are discussed.

Robust Statistical Detection of Power-Law Cross-Correlation.

PubMed

Blythe, Duncan A J; Nikulin, Vadim V; Müller, Klaus-Robert

2016-06-02

We show that widely used approaches in statistical physics incorrectly indicate the existence of power-law cross-correlations between financial stock market fluctuations measured over several years and the neuronal activity of the human brain lasting for only a few minutes. While such cross-correlations are nonsensical, no current methodology allows them to be reliably discarded, leaving researchers at greater risk when the spurious nature of cross-correlations is not clear from the unrelated origin of the time series and rather requires careful statistical estimation. Here we propose a theory and method (PLCC-test) which allows us to rigorously and robustly test for power-law cross-correlations, correctly detecting genuine and discarding spurious cross-correlations, thus establishing meaningful relationships between processes in complex physical systems. Our method reveals for the first time the presence of power-law cross-correlations between amplitudes of the alpha and beta frequency ranges of the human electroencephalogram.

Robust Statistical Detection of Power-Law Cross-Correlation

PubMed Central

Blythe, Duncan A. J.; Nikulin, Vadim V.; Müller, Klaus-Robert

2016-01-01

We show that widely used approaches in statistical physics incorrectly indicate the existence of power-law cross-correlations between financial stock market fluctuations measured over several years and the neuronal activity of the human brain lasting for only a few minutes. While such cross-correlations are nonsensical, no current methodology allows them to be reliably discarded, leaving researchers at greater risk when the spurious nature of cross-correlations is not clear from the unrelated origin of the time series and rather requires careful statistical estimation. Here we propose a theory and method (PLCC-test) which allows us to rigorously and robustly test for power-law cross-correlations, correctly detecting genuine and discarding spurious cross-correlations, thus establishing meaningful relationships between processes in complex physical systems. Our method reveals for the first time the presence of power-law cross-correlations between amplitudes of the alpha and beta frequency ranges of the human electroencephalogram. PMID:27250630

Construction of a 'turn-on' fluorescent probe system for His-tagged proteins.

PubMed

Murata, Atsushi; Arai, Satoshi; Yoon, Su-In; Takabayashi, Masao; Ozaki, Miwako; Takeoka, Shinji

2010-12-01

Hexahistidine ((His)(6)) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)(6), and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni(2+), can bind (His)(6). The system is turned off when Dabcyl-(His)(6) is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)(6)-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)(6)-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His)(6)-protein. Copyright © 2010 Elsevier Ltd. All rights reserved.

Determination of biological activity from fluorescence-lifetime measurements in Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Rudek, F.; Baselt, T.; Lempe, B.; Taudt, C.; Hartmann, P.

2015-03-01

The importance of fluorescence lifetime measurement as an optical analysis tool is growing. Many applications already exist in order to determine the fluorescence lifetime, but the majority of these require the addition of fluorescence-active substances to enable measurements. Every usage of such foreign materials has an associated risk. This paper investigates the use of auto-fluorescing substances in Saccharomyces cerevisiae (Baker's yeast) as a risk free alternative to fluorescence-active substance enabled measurements. The experimental setup uses a nitrogen laser with a pulse length of 350 ps and a wavelength of 337 nm. The excited sample emits light due to fluorescence of NADH/NADPH and collagen. A fast photodiode collects the light at the output of an appropriate high-pass edge-filter at 400 nm. Fluorescence lifetimes can be determined from the decay of the measurement signals, which in turn characterizes the individual materials and their surrounding environment. Information about the quantity of the fluorescence active substances can also be measured based on the received signal intensity. The correlation between the fluorescence lifetime and the metabolic state of Saccharomyces cerevisiae was investigated and is presented here.

Cross-correlations between RMB exchange rate and international commodity markets

NASA Astrophysics Data System (ADS)

Lu, Xinsheng; Li, Jianfeng; Zhou, Ying; Qian, Yubo

2017-11-01

This paper employs multifractal detrended analysis (MF-DFA) and multifractal detrended cross-correlation analysis (MF-DCCA) to study cross-correlation behaviors between China's RMB exchange rate market and four international commodity markets, using a comprehensive set of data covering the period from 22 July 2005 to 15 March 2016. Our empirical results from MF-DFA indicate that the RMB exchange rate is the most inefficient among the 4 selected markets. The results from quantitative analysis have testified the existence of cross-correlations and the result from MF-DCCA have further confirmed a strong multifractal behavior between RMB exchange rate and international commodity markets. We also demonstrate that the recent financial crisis has significant impact on the cross-correlated behavior. Through the rolling window analysis, we find that the RMB exchange rates and international commodity prices are anti-persistent cross-correlated. The main sources of multifractality in the cross-correlations are long-range correlations between RMB exchange rate and the aggregate commodity, energy and metals index.

Elemental and Molecular Segregation in Oil Paintings due to Lead Soap Degradation.

PubMed

Chen-Wiegart, Yu-Chen Karen; Catalano, Jaclyn; Williams, Garth J; Murphy, Anna; Yao, Yao; Zumbulyadis, Nicholas; Centeno, Silvia A; Dybowski, Cecil; Thieme, Juergen

2017-09-14

The formation of Pb, Zn, and Cu carboxylates (soaps) has caused visible deterioration in hundreds of oil paintings dating from the 15th century to the present. Through transport phenomena not yet understood, free fatty acids in the oil binding medium migrate through the paint and react with pigments containing heavy metals to form soaps. To investigate the complex correlation among the elemental segregation, types of chemical compounds formed, and possible mechanisms of the reactions, a paint sample cross-section from a 15th century oil painting was examined by synchrotron X-ray techniques. X-ray fluorescence (XRF) microscopy, quantified with elemental correlation density distribution, showed Pb and Sn segregation in the soap-affected areas. X-ray absorption near edge structure (XANES) around the Pb-L3 absorption edge showed that Pb pigments and Pb soaps can be distinguished while micro-XANES gave further information on the chemical heterogeneity in the paint film. The advantages and limitations of these synchrotron-based techniques are discussed and compared to those of methods routinely used to analyze paint samples. The results presented set the stage for improving the information extracted from samples removed from works of art and for correlating observations in model paint samples to those in the naturally aged samples, to shed light onto the mechanism of soap formation.

The influence of the Tbeta level upon fluorescence and laser properties of aromatic compounds.

PubMed

Nijegorodov, N; Winkoun, D P; Nkoma, J S

2004-07-01

The fluorescence and laser properties of seven specially chosen aromatic compounds are studied at 293 degrees C. The quantum yield of fluorescence, gamma, decay times, tauf, of the deaerated and non-deaerated solutions are measured. The oscillator strength, fe, fluorescence rate constants, kf, natural lifetimes, tauT0, and intersystem crossing rate constants, kST, are calculated. Some laser parameters are calculated or measured experimentally. It is found that the position of the Tbeta level plays an important role in the fluorescence and laser properties of aromatic compounds. If the Tbeta level is situated below the Sp level, it decreases the quantum yield of fluorescence and the decay time and increases the threshold of laser action. If, due to some structural changes of a molecule, the Tbeta level is situated higher than the Sp level, then the quantum yield of fluorescence and the decay times are increasing and the threshold of laser action is decreasing. Such influence of the position of the Tbeta level upon fluorescence and laser properties of aromatic compound is explained by the fact that the Sp level mixes with the Tbeta level more readily than with other taupipi* levels.

Polarized fluorescence for skin cancer diagnostic with a multi-aperture camera

NASA Astrophysics Data System (ADS)

Kandimalla, Haripriya; Ramella-Roman, Jessica C.

2008-02-01

Polarized fluorescence has shown some promising results in assessment of skin cancer margins. Researchers have used tetracycline and cross polarization imaging for nonmelanoma skin cancer demarcation as well as investigating endogenous skin polarized fluorescence. In this paper we present a new instrument for polarized fluorescence imaging, able to calculate the full fluorescence Stokes vector in one snapshot. The core of our system is a multi-aperture camera constructed with a two by two lenslet array. Three of the lenses have polarizing elements in front of them, oriented at 0°, + 45°and 90° with respect to light source polarization. A flash lamp combined with a polarizer parallel to the source-camera-sample plane and a UV filter is used as an excitation source. A blue filter in front of the camera system is used to collect only the fluorescent emission of interest and filter out the incident light. In-vitro tests of endogenous and exogenous polarized fluorescence on collagen rich material like bovine tendon were performed and Stokes vector of polarized fluorescence calculated. The system has the advantage of eliminating moving artifacts with the collection of different polarization states and stoke vector in a single snap shot.