Quantitative calculations of fluorescence polarization and absorption anisotropy kinetics of double- and triple-chromophore complexes with energy transfer.

PubMed Central

Demidov, A A

1994-01-01

A new method is presented for calculation of the fluorescence depolarization and kinetics of absorption anisotropy for molecular complexes with a limited number of chromophores. The method considers absorption and emission of light by both chromophores, and also energy transfer between them, with regard to their mutual orientations. The chromophores in each individual complex are rigidly positioned. The complexes are randomly distributed and oriented in space, and there is no energy transfer between them. The new "practical" formula for absorption anisotropy and fluorescence depolarization kinetics, P(t) = [3B(t) - 1 + 2A(t)]/[3 + B(t) + 4A(t)], is derived both for double- and triple-chromophore complexes with delta-pulse excitation. The parameter B(t) is given by (a) B(t) = cos2(theta) for double-chromophore complexes, and (b) B(t) = q12(t)cos2(theta 12) + q13(t)-cos2(theta 13) + q23(t)cos2(theta 23) for triple-chromophore complexes, where q12(t) + q13(t) + q23(t) = 1. Here theta ij are the angles between the chromophore transition dipole moments in the individual molecular complex. The parameters qij(t) and A(t) are dependent on chromophore spectroscopic features and on the rates of energy transfer. PMID:7696461

Selective binding of choline by a phosphate-coordination-based triple helicate featuring an aromatic box.

PubMed

Jia, Chuandong; Zuo, Wei; Yang, Dong; Chen, Yanming; Cao, Liping; Custelcean, Radu; Hostaš, Jiří; Hobza, Pavel; Glaser, Robert; Wang, Yao-Yu; Yang, Xiao-Juan; Wu, Biao

2017-10-16

In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. This similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives, as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, L-carnitine, and glycine betaine effectively.The choline-binding protein ChoX exhibits a synergistic dual-site binding mode that allows it to discriminate choline over structural analogues. Here, the authors design a biomimetic triple anion helicate receptor whose selectivity for choline arises from a similar binding mechanism.

Experimental characterization of pairwise correlations from triple quantum correlated beams generated by cascaded four-wave mixing processes

NASA Astrophysics Data System (ADS)

Wang, Wei; Cao, Leiming; Lou, Yanbo; Du, Jinjian; Jing, Jietai

2018-01-01

We theoretically and experimentally characterize the performance of the pairwise correlations from triple quantum correlated beams based on the cascaded four-wave mixing (FWM) processes. The pairwise correlations between any two of the beams are theoretically calculated and experimentally measured. The experimental and theoretical results are in good agreement. We find that two of the three pairwise correlations can be in the quantum regime. The other pairwise correlation is always in the classical regime. In addition, we also measure the triple-beam correlation which is always in the quantum regime. Such unbalanced and controllable pairwise correlation structures may be taken as advantages in practical quantum communications, for example, hierarchical quantum secret sharing. Our results also open the way for the classification and application of quantum states generated from the cascaded FWM processes.

Corresponding states correlation for temperature dependent surface tension of normal saturated liquids

NASA Astrophysics Data System (ADS)

Yi, Huili; Tian, Jianxiang

2014-07-01

A new simple correlation based on the principle of corresponding state is proposed to estimate the temperature-dependent surface tension of normal saturated liquids. The correlation is a linear one and strongly stands for 41 saturated normal liquids. The new correlation requires only the triple point temperature, triple point surface tension and critical point temperature as input and is able to represent the experimental surface tension data for these 41 saturated normal liquids with a mean absolute average percent deviation of 1.26% in the temperature regions considered. For most substances, the temperature covers the range from the triple temperature to the one beyond the boiling temperature.

Improvements of low-detection-limit filter-free fluorescence sensor developed by charge accumulation operation

NASA Astrophysics Data System (ADS)

Tanaka, Kiyotsugu; Choi, Yong Joon; Moriwaki, Yu; Hizawa, Takeshi; Iwata, Tatsuya; Dasai, Fumihiro; Kimura, Yasuyuki; Takahashi, Kazuhiro; Sawada, Kazuaki

2017-04-01

We developed a low-detection-limit filter-free fluorescence sensor by a charge accumulation technique. For charge accumulation, a floating diffusion amplifier (FDA), which included a floating diffusion capacitor, a transfer gate, and a source follower circuit, was used. To integrate CMOS circuits with the filter-free fluorescence sensor, we adopted a triple-well process to isolate transistors from the sensor on a single chip. We detected 0.1 nW fluorescence under the illumination of excitation light by 1.5 ms accumulation, which was one order of magnitude greater than that of a previous current detection sensor.

Laser speckle strain and deformation sensor using linear array image cross-correlation method for specifically arranged triple-beam triple-camera configuration

NASA Technical Reports Server (NTRS)

Sarrafzadeh-Khoee, Adel K. (Inventor)

2000-01-01

The invention provides a method of triple-beam and triple-sensor in a laser speckle strain/deformation measurement system. The triple-beam/triple-camera configuration combined with sequential timing of laser beam shutters is capable of providing indications of surface strain and structure deformations. The strain and deformation quantities, the four variables of surface strain, in-plane displacement, out-of-plane displacement and tilt, are determined in closed form solutions.

Chelators Exhibiting Triple Fluorescence.

DTIC Science & Technology

1998-08-31

l-NN-dimethylamino-propane, forms an intramolecular 9 exciplex between the phenyl and amino end groups. The formation of an intramolecular exciplex 10...alkyl amino chains. e.g. 3-(4-cyanophenyl)-l-N.N- 21 dimethylaminopropane (CNP3NM, Fig. I b), can form intramolecular exciplexes which arise due to 2...for intramolecular exciplex formation in CNP3NM is indicated by 4 the strong. red-shifted fluorescence observed, and the complete absence of LE

New generalized corresponding states correlation for surface tension of normal saturated liquids

NASA Astrophysics Data System (ADS)

Yi, Huili; Tian, Jianxiang

2015-08-01

A new simple correlation based on the principle of corresponding state is proposed to estimate the temperature-dependent surface tension of normal saturated liquids. The new correlation contains three coefficients obtained by fitting 17,051 surface tension data of 38 saturated normal liquids. These 38 liquids contain refrigerants, hydrocarbons and some other inorganic liquids. The new correlation requires only the triple point temperature, triple point surface tension and critical point temperature as input and is able to well represent the experimental surface tension data for each of the 38 saturated normal liquids from the triple temperature up to the point near the critical point. The new correlation gives absolute average deviations (AAD) values below 3% for all of these 38 liquids with the only exception being octane with AAD=4.30%. Thus, the new correlation gives better overall results in comparison with other correlations for these 38 normal saturated liquids.

Confinement of carbon dots localizing to the ultrathin layered double hydroxides toward simultaneous triple-mode bioimaging and photothermal therapy.

PubMed

Weng, Yangziwan; Guan, Shanyue; Lu, Heng; Meng, Xiangmin; Kaassis, Abdessamad Y; Ren, Xiaoxue; Qu, Xiaozhong; Sun, Chenghua; Xie, Zheng; Zhou, Shuyun

2018-07-01

It is a great challenge to develop multifunctional nanocarriers for cancer diagnosis and therapy. Herein, versatile CDs/ICG-uLDHs nanovehicles for triple-modal fluorescence/photoacoustic/two-photon bioimaging and effective photothermal therapy were prepared via a facile self-assembly of red emission carbon dots (CDs), indocyanine green (ICG) with the ultrathin layered double hydroxides (uLDHs). Due to the J-aggregates of ICG constructed in the self-assembly process, CDs/ICG-uLDHs was able to stabilize the photothermal agent ICG and enhanced its photothermal efficiency. Furthermore, the unique confinement effect of uLDHs has extended the fluorescence lifetime of CDs in favor of bioimaging. Considering the excellent in vitro and in vivo phototherapeutics and multimodal imaging effects, this work provides a promising platform for the construction of multifunctional theranostic nanocarrier system for the cancer treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

A triple helical calcium-based coordination polymer with strong blue fluorescent emission

NASA Astrophysics Data System (ADS)

Yu, Liang-Cai; Chen, Zhen-Feng; Liang, Hong; Zhou, Chun-Shan; Li, Yan

2005-08-01

A hydrothermal reaction of 1,3-dicyanobenzene and Ca(OH)2 yielded a triple helical calcium-based coordination polymer of the formula, C20H25Ca2.50O18.50 (1). The 1,3-benzenecarboxylate anion, found in the final product was generated in situ during the synthesis by the hydrolysis of 1,3-dicyanobenzene. X-ray diffraction study shows that the complex 1 crystallizes in the monoclinic system, C2/c space group, a=15.5701(5), b=21.4445(7), c=17.1601(6) Å, β=111.7400(7)°, V=5322.1(3) Å3, Z=8, Dc=1.651 Mg/m3. The calcium atoms show differences in the coordination environments. Complex 1 emits strong blue fluorescent light (λem(max)=419 nm) when it is excited by UV light (λex(max)=316 nm) in the solid state at room temperature.

Selective binding of choline by a phosphate-coordination-based triple helicate featuring an aromatic box

DOE PAGES

Jia, Chuandong; Zuo, Wei; Yang, Dong; ...

2017-10-16

In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. Here, this similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives,more » as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, l-carnitine, and glycine betaine effectively.« less

Progressive accumulation of ubiquitin and disappearance of alpha-synuclein epitope in multiple system atrophy-associated glial cytoplasmic inclusions: triple fluorescence study combined with Gallyas-Braak method.

PubMed

Sakamoto, Masaki; Uchihara, Toshiki; Nakamura, Ayako; Mizutani, Toshio; Mizusawa, Hidehiro

2005-10-01

Alpha-synuclein (alphaS) and ubiquitin (Ub) are shared constituents of glial cytoplasmic inclusions (GCIs) and Lewy bodies (LBs), both composed of fibrillary structures. Staining profiles of GCIs were investigated with triple immunofluorescence involving immunostaining for alphaS and Ub, both amplified with catalyzed reporter deposition, and a fluorochrome, thiazin red (TR) that has an affinity to fibrillary structures. After observation for the triple-fluorescent images, the sections were subsequently stained with the Gallyas-Braak method. Sections of putamen, cerebellar white matter and motor cortex from patients suffering from multiple system atrophy (MSA) with varying duration of the disease (4-15 years) were quantified for these staining profiles of Gallyas-positive GCIs. Although most of GCIs were positive for Ub and variably positive for alphaS, they were consistently negative for TR. The result was opposite in LBs in Lewy body disease with variable affinity to TR, suggesting that the construction of GCIs is different from that of LBs. These four staining features (alphaS, Ub, TR and Gallyas) alone failed to exhibit apparent correlation with disease duration, lesion site or severity of degeneration as reported previously. The fraction of alphaS-negative and Ub-positive GCIs, however, linearly increased along the disease progression, while that of alphaS-positive and Ub-negative GCIs decreased in contrast. This reciprocal change suggests that alphaS immunoreactivity in GCIs is being replaced by Ub immunoreactivity during the disease progression, which resulted in the ultimate predominance of alphaS-negative and Ub-positive GCIs in the most advanced case. Interestingly, this predominance of alphaS-negative and Ub-positive GCIs was a feature of motor cortex, where degeneration usually remains mild in spite of robust appearance of Gallyas-positive GCIs. Another fraction, alphaS-positive and Ub-positive GCIs were frequent in cerebellar white matter, suggesting that GCI evolution is heterogeneous and dependent also on area examined. Progressive accumulation of Ub with concomitant disappearance of alphaS epitope and their colocalization, partly shared with LBs, may represent a process of GCI formation, possibly linked to an aspect of degeneration in MSA.

Perturbative triples correction for local pair natural orbital based explicitly correlated CCSD(F12*) using Laplace transformation techniques.

PubMed

Schmitz, Gunnar; Hättig, Christof

2016-12-21

We present an implementation of pair natural orbital coupled cluster singles and doubles with perturbative triples, PNO-CCSD(T), which avoids the quasi-canonical triples approximation (T0) where couplings due to off-diagonal Fock matrix elements are neglected. A numerical Laplace transformation of the canonical expression for the perturbative (T) triples correction is used to avoid an I/O and storage bottleneck for the triples amplitudes. Results for a test set of reaction energies show that only very few Laplace grid points are needed to obtain converged energy differences and that PNO-CCSD(T) is a more robust approximation than PNO-CCSD(T0) with a reduced mean absolute deviation from canonical CCSD(T) results. We combine the PNO-based (T) triples correction with the explicitly correlated PNO-CCSD(F12*) method and investigate the use of specialized F12-PNOs in the conventional triples correction. We find that no significant additional errors are introduced and that PNO-CCSD(F12*)(T) can be applied in a black box manner.

Explicitly correlated coupled-cluster theory using cusp conditions. II. Treatment of connected triple excitations.

PubMed

Köhn, Andreas

2010-11-07

The coupled-cluster singles and doubles method augmented with single Slater-type correlation factors (CCSD-F12) determined by the cusp conditions (also denoted as SP ansatz) yields results close to the basis set limit with only small overhead compared to conventional CCSD. Quantitative calculations on many-electron systems, however, require to include the effect of connected triple excitations at least. In this contribution, the recently proposed [A. Köhn, J. Chem. Phys. 130, 131101 (2009)] extended SP ansatz and its application to the noniterative triples correction CCSD(T) is reviewed. The approach allows to include explicit correlation into connected triple excitations without introducing additional unknown parameters. The explicit expressions are presented and analyzed, and possible simplifications to arrive at a computationally efficient scheme are suggested. Numerical tests based on an implementation obtained by an automated approach are presented. Using a partial wave expansion for the neon atom, we can show that the proposed ansatz indeed leads to the expected (L(max)+1)(-7) convergence of the noniterative triples correction, where L(max) is the maximum angular momentum in the orbital expansion. Further results are reported for a test set of 29 molecules, employing Peterson's F12-optimized basis sets. We find that the customary approach of using the conventional noniterative triples correction on top of a CCSD-F12 calculation leads to significant basis set errors. This, however, is not always directly visible for total CCSD(T) energies due to fortuitous error compensation. The new approach offers a thoroughly explicitly correlated CCSD(T)-F12 method with improved basis set convergence of the triples contributions to both total and relative energies.

Normal range of hepatic fat fraction on dual- and triple-echo fat quantification MR in children.

PubMed

Shin, Hyun Joo; Kim, Hyun Gi; Kim, Myung-Joon; Koh, Hong; Kim, Ha Yan; Roh, Yun Ho; Lee, Mi-Jung

2015-01-01

To evaluate hepatic fat fraction on dual- and triple-echo gradient-recalled echo MRI sequences in healthy children. We retrospectively reviewed the records of children in a medical check-up clinic from May 2012 to November 2013. We excluded children with abnormal laboratory findings or those who were overweight. Hepatic fat fraction was measured on dual- and triple-echo sequences using 3T MRI. We compared fat fractions using the Wilcoxon signed rank test and the Bland-Altman 95% limits of agreement. The correlation between fat fractions and clinical and laboratory findings was evaluated using Spearman's correlation test, and the cut-off values of fat fractions for diagnosing fatty liver were obtained from reference intervals. In 54 children (M:F = 26:28; 5-15 years; mean 9 years), the dual fat fraction (0.1-8.0%; median 1.6%) was not different from the triple fat fraction (0.4-6.5%; median 2.7%) (p = 0.010). The dual- and triple-echo fat fractions showed good agreement using a Bland-Altman plot (-0.6 ± 2.8%). Eight children (14.8%) on dual-echo sequences and six (11.1%) on triple-echo sequences had greater than 5% fat fraction. From these children, six out of eight children on dual-echo sequences and four out of six children on triple-echo sequences had a 5-6% hepatic fat fraction. When using a cut-off value of a 6% fat fraction derived from a reference interval, only 3.7% of children were diagnosed with fatty liver. There was no significant correlation between clinical and laboratory findings with dual and triple-echo fat fractions. Dual fat fraction was not different from triple fat fraction. We suggest a cut-off value of a 6% fat fraction is more appropriate for diagnosing fatty liver on both dual- and triple-echo sequences in children.

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.

Experimental characterization of quantum correlated triple beams generated by cascaded four-wave mixing processes

NASA Astrophysics Data System (ADS)

Qin, Zhongzhong; Cao, Leiming; Jing, Jietai

2015-05-01

Quantum correlations and entanglement shared among multiple modes are fundamental ingredients of most continuous-variable quantum technologies. Recently, a method used to generate multiple quantum correlated beams using cascaded four-wave mixing (FWM) processes was theoretically proposed and experimentally realized by our group [Z. Qin et al., Phys. Rev. Lett. 113, 023602 (2014)]. Our study of triple-beam quantum correlation paves the way to showing the tripartite entanglement in our system. Our system also promises to find applications in quantum information and precision measurement such as the controlled quantum communications, the generation of multiple quantum correlated images, and the realization of a multiport nonlinear interferometer. For its applications, the degree of quantum correlation is a crucial figure of merit. In this letter, we experimentally study how various parameters, such as the cell temperatures, one-photon, and two-photon detunings, influence the degree of quantum correlation between the triple beams generated from the cascaded two-FWM configuration.

Experimental characterization of quantum correlated triple beams generated by cascaded four-wave mixing processes

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

Qin, Zhongzhong; Cao, Leiming; Jing, Jietai, E-mail: jtjing@phy.ecnu.edu.cn

2015-05-25

Quantum correlations and entanglement shared among multiple modes are fundamental ingredients of most continuous-variable quantum technologies. Recently, a method used to generate multiple quantum correlated beams using cascaded four-wave mixing (FWM) processes was theoretically proposed and experimentally realized by our group [Z. Qin et al., Phys. Rev. Lett. 113, 023602 (2014)]. Our study of triple-beam quantum correlation paves the way to showing the tripartite entanglement in our system. Our system also promises to find applications in quantum information and precision measurement such as the controlled quantum communications, the generation of multiple quantum correlated images, and the realization of a multiportmore » nonlinear interferometer. For its applications, the degree of quantum correlation is a crucial figure of merit. In this letter, we experimentally study how various parameters, such as the cell temperatures, one-photon, and two-photon detunings, influence the degree of quantum correlation between the triple beams generated from the cascaded two-FWM configuration.« less

Modeling the Blood-Brain Barrier in a 3D triple co-culture microfluidic system.

PubMed

Adriani, G; Ma, D; Pavesi, A; Goh, E L K; Kamm, R D

2015-01-01

The need for a blood-brain barrier (BBB) model that accurately mimics the physiological characteristics of the in-vivo situation is well-recognized by researchers in academia and industry. However, there is currently no in-vitro model allowing studies of neuronal growth and/or function influenced by factors from the blood that cross through the BBB. Therefore, we established a 3D triple co-culture microfluidic system using human umbilical vein endothelial cells (HUVEC) together with primary rat astrocytes and neurons. Immunostaining confirmed the successful triple co-culture system consisting of an intact BBB with tight intercellular junctions in the endothelial monolayer. The BBB selective permeability was determined by a fluorescent-based assay using dextrans of different molecular weights. Finally, neuron functionality was demonstrated by calcium imaging.

Flies and humans share a motion estimation strategy that exploits natural scene statistics

PubMed Central

Clark, Damon A.; Fitzgerald, James E.; Ales, Justin M.; Gohl, Daryl M.; Silies, Marion A.; Norcia, Anthony M.; Clandinin, Thomas R.

2014-01-01

Sighted animals extract motion information from visual scenes by processing spatiotemporal patterns of light falling on the retina. The dominant models for motion estimation exploit intensity correlations only between pairs of points in space and time. Moving natural scenes, however, contain more complex correlations. Here we show that fly and human visual systems encode the combined direction and contrast polarity of moving edges using triple correlations that enhance motion estimation in natural environments. Both species extract triple correlations with neural substrates tuned for light or dark edges, and sensitivity to specific triple correlations is retained even as light and dark edge motion signals are combined. Thus, both species separately process light and dark image contrasts to capture motion signatures that can improve estimation accuracy. This striking convergence argues that statistical structures in natural scenes have profoundly affected visual processing, driving a common computational strategy over 500 million years of evolution. PMID:24390225

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

PubMed Central

Warner, Katherine Deigan; Chen, Michael C.; Song, Wenjiao; Strack, Rita L.; Thorn, Andrea; Jaffrey, Samie R.; Ferré-D’Amaré, Adrian R.

2014-01-01

Green fluorescent protein (GFP) and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro evolved RNA mimic of GFP, which as genetically encoded fusions, makes possible live-cell, real-time imaging of biological RNAs, without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we have solved its co-crystal structure bound to its cognate exogenous chromophore, revealing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex, and an unpaired guanine. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs, and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure has guided the design of a miniaturized 'Baby Spinach', and provides the foundation for structure-driven design and tuning of fluorescent RNAs. PMID:25026079

Methods of separation of variables in turbulence theory

NASA Technical Reports Server (NTRS)

Tsuge, S.

1978-01-01

Two schemes of closing turbulent moment equations are proposed both of which make double correlation equations separated into single-point equations. The first is based on neglected triple correlation, leading to an equation differing from small perturbed gasdynamic equations where the separation constant appears as the frequency. Grid-produced turbulence is described in this light as time-independent, cylindrically-isotropic turbulence. Application to wall turbulence guided by a new asymptotic method for the Orr-Sommerfeld equation reveals a neutrally stable mode of essentially three dimensional nature. The second closure scheme is based on an assumption of identity of the separated variables through which triple and quadruple correlations are formed. The resulting equation adds, to its equivalent of the first scheme, an integral of nonlinear convolution in the frequency describing a role due to triple correlation of direct energy-cascading.

Evaluation of the charge transfer efficiency of organic thin-film photovoltaic devices fabricated using a photoprecursor approach.

PubMed

Masuo, Sadahiro; Sato, Wataru; Yamaguchi, Yuji; Suzuki, Mitsuharu; Nakayama, Ken-ichi; Yamada, Hiroko

2015-05-01

Recently, a unique 'photoprecursor approach' was reported as a new option to fabricate a p-i-n triple-layer organic photovoltaic device (OPV) through solution processes. By fabricating the p-i-n architecture using two kinds of photoprecursors and a [6,6]-phenyl C71 butyric acid methyl ester (PC71BM) as the donor and the acceptor, the p-i-n OPVs afforded a higher photovoltaic efficiency than the corresponding p-n devices and i-devices, while the photovoltaic efficiency of p-i-n OPVs depended on the photoprecursors. In this work, the charge transfer efficiency of the i-devices composed of the photoprecursors and PC71BM was investigated using high-sensitivity fluorescence microspectroscopy combined with a time-correlated single photon counting technique to elucidate the photovoltaic efficiency depending on the photoprecursors and the effects of the p-i-n architecture. The spatially resolved fluorescence images and fluorescence lifetime measurements clearly indicated that the compatibility of the photoprecursors with PC71BM influences the charge transfer and the photovoltaic efficiencies. Although the charge transfer efficiency of the i-device was quite high, the photovoltaic efficiency of the i-device was much lower than that of the p-i-n device. These results imply that the carrier generation and carrier transportation efficiencies can be increased by fabricating the p-i-n architecture.

Reference Correlation for the Viscosity of Ammonia from the Triple Point to 725 K and up to 50 MPa

NASA Astrophysics Data System (ADS)

Monogenidou, S. A.; Assael, M. J.; Huber, M. L.

2018-06-01

This paper presents a new wide-ranging correlation for the viscosity of ammonia based on critically evaluated experimental data. The correlation is designed to be used with a recently developed equation of state, and it is valid from the triple point to 725 K at pressures up to 50 MPa. The estimated uncertainty varies depending on the temperature and pressure, from 0.6% to 5%. The correlation behaves in a physically reasonable manner when extrapolated to 100 MPa; however, care should be taken when using the correlation outside of the validated range.

A trident dithienylethene-perylenemonoimide dyad with super fluorescence switching speed and ratio

NASA Astrophysics Data System (ADS)

Li, Chong; Yan, Hui; Zhao, Ling-Xi; Zhang, Guo-Feng; Hu, Zhe; Huang, Zhen-Li; Zhu, Ming-Qiang

2014-12-01

Photoswitchable fluorescent diarylethenes are promising in molecular optical memory and photonic devices. However, the performance of current diarylethenes is far from satisfactory because of the scarcity of high-speed switching capability and large fluorescence on-off ratio. Here we report a trident perylenemonoimide dyad modified by triple dithienylethenes whose photochromic fluorescence quenching ratio at the photostationary state exceeds 10,000 and the fluorescence quenching efficiency is close to 100% within seconds of ultraviolet irradiation. The highly sensitive fluorescence on/off switching of the trident dyad enables recyclable fluorescence patterning and all-optical transistors. The prototype optical device based on the trident dyad enables the optical switching of incident light and conversion from incident light wavelength to transmitted light wavelength, which is all-optically controlled, reversible and wavelength-convertible. In addition, the trident dyad-staining block copolymer vesicles are observed via optical nanoimaging with a sub-100 nm resolution, portending a potential prospect of the dithienylethene dyad in super-resolution imaging.

A trident dithienylethene-perylenemonoimide dyad with super fluorescence switching speed and ratio.

PubMed

Li, Chong; Yan, Hui; Zhao, Ling-Xi; Zhang, Guo-Feng; Hu, Zhe; Huang, Zhen-Li; Zhu, Ming-Qiang

2014-12-12

Photoswitchable fluorescent diarylethenes are promising in molecular optical memory and photonic devices. However, the performance of current diarylethenes is far from satisfactory because of the scarcity of high-speed switching capability and large fluorescence on-off ratio. Here we report a trident perylenemonoimide dyad modified by triple dithienylethenes whose photochromic fluorescence quenching ratio at the photostationary state exceeds 10,000 and the fluorescence quenching efficiency is close to 100% within seconds of ultraviolet irradiation. The highly sensitive fluorescence on/off switching of the trident dyad enables recyclable fluorescence patterning and all-optical transistors. The prototype optical device based on the trident dyad enables the optical switching of incident light and conversion from incident light wavelength to transmitted light wavelength, which is all-optically controlled, reversible and wavelength-convertible. In addition, the trident dyad-staining block copolymer vesicles are observed via optical nanoimaging with a sub-100 nm resolution, portending a potential prospect of the dithienylethene dyad in super-resolution imaging.

Deficiency of Ferritin Heavy-Chain Nuclear Import in Triple A Syndrome Implies Nuclear Oxidative Damage as the Primary Disease Mechanism

PubMed Central

Storr, Helen L.; Kind, Barbara; Parfitt, David A.; Chapple, J. Paul; Lorenz, M.; Koehler, Katrin; Huebner, Angela; Clark, Adrian J. L.

2009-01-01

Triple A syndrome is a rare autosomal recessive disorder characterized by ACTH-resistant adrenal failure, alacrima, achalasia, and progressive neurological manifestations. The majority of cases are associated with mutations in the AAAS gene, which encodes a novel, 60-kDa WD-repeat nuclear pore protein, alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN) of unknown function. Our aim was to elucidate the functional role of ALADIN by determining its interacting protein partners using the bacterial two-hybrid (B2-H) technique. Nonidentical cDNA fragments were identified from both a HeLa S-3 cell and human cerebellar cDNA library that encoded the full-length ferritin heavy chain protein (FTH1). This interaction was confirmed by both co-immunoprecipitation and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. Immunoblotting showed that fibroblasts from triple A patients (with known AAAS mutations) lack nuclear FTH1, suggesting that the nuclear translocation of FTH1 is defective. Cells transfected with FTH1 and visualized by confocal microscopy had very little nuclear FTH1, but when cotransfected with AAAS, FTH1 is readily visible in the nuclei. Therefore, FTH1 nuclear translocation is enhanced when ALADIN is coexpressed in these cells. In addition to its well known iron storage role, FTH1 has been shown to protect the nucleus from oxidative damage. Apoptosis of neuronal cells induced by hydrogen peroxide was significantly reduced by transfection of AAAS or by FTH1 or maximally by both genes together. Taken together, this work offers a plausible mechanism for the progressive clinical features of triple A syndrome. PMID:19855093

Improved sensitivity of ochratoxin A analysis in coffee using high-performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS).

PubMed

Kokina, Aija; Pugajeva, Iveta; Bartkevics, Vadims

2016-01-01

A novel and sensitive method utilising high-performance liquid chromatography coupled to triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS) was developed in order to analyse the content of ochratoxin A (OTA) in coffee samples. The introduction of the triple-stage MS scanning mode (MS(3)) has been shown to increase greatly sensitivity and selectivity by eliminating the high chromatographic baseline caused by interference of complex coffee matrices. The analysis included the sample preparation procedure involving extraction of OTA using a methanol-water mixture and clean-up by immunoaffinity columns and detection using the MS(3) scanning mode of LC-QqQLIT-MS/MS. The proposed method offered a good linear correlation (r(2) > 0.998), excellent precision (RSD < 2.9%) and recovery (94%). The limit of quantification (LOQ) for coffee beans and espresso beverages was 0.010 and 0.003 µg kg(-1), respectively. The developed procedure was compared with traditional methods employing liquid chromatography coupled to fluorescent and tandem quadrupole detectors in conjunction with QuEChERS and solid-phase extraction. The proposed method was successfully applied to the determination of OTA in 15 samples of coffee beans and in 15 samples of espresso coffee beverages obtained from the Latvian market. OTA was found in 10 samples of coffee beans and in two samples of espresso in the ranges of 0.018-1.80 µg kg(-1) and 0.020-0.440 µg l(-1), respectively. No samples exceeded the maximum permitted level of OTA in the European Union (5.0 µg kg(-1)).

A new triple system DNA-Nanosilver-Berberine for cancer therapy

NASA Astrophysics Data System (ADS)

Grebinyk, Anna; Yashchuk, Valeriy; Bashmakova, Nataliya; Gryn, Dmytro; Hagemann, Tobias; Naumenko, Antonina; Kutsevol, Nataliya; Dandekar, Thomas; Frohme, Marcus

2018-03-01

The isoquinoline quaternary alkaloid Berberine possesses a variety of pharmacological properties that suggests its promising application for an anticancer delivery system design utilizing its ability to intercalate DNA. In the current work, we have investigated the effects of Berberine on the human T cell leukemia cell line in vitro. Fluorescent microscopy of leukemic cells revealed Berberine nuclear localization. The results showed that Berberine inhibited leukemic cell growth in a time- and dose-dependent manner, that was associated with reactive oxygen species production intensification and caspase 3/7 activity increase with followed apoptosis induction. Berberine was used as a toxic and phototoxic agent for triple system synthesis along with DNA as a carrier and nanosilver as a plasmonic accelerator of Berberine electronic transitions and high energy emission absorbent centers. The proposed method allows to obtain the complex of DNA with Berberine molecules and silver nanoparticles. The optical properties of free components as well as their various combinations, including the final triple system DNA-Nanosilver-Berberine, were investigated. Obtained results support the possibility to use the triple system DNA-Nanosilver-Berberine as an alternative therapeutic agent for cancer treatment.

Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

PubMed

Stawikowski, Maciej J; Stawikowska, Roma; Fields, Gregg B

2015-05-19

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

Development of base pressure similarity parameters for application to space shuttle launch vehicle power-on aerodynamic testing

NASA Technical Reports Server (NTRS)

Sulyma, P. R.; Penny, M. M.

1978-01-01

A base pressure data correlation study was conducted to define exhaust plume similarity parameters for use in Space Shuttle power-on launch vehicle aerodynamic test programs. Data correlations were performed for single bodies having, respectively, single and triple nozzle configurations and for a triple body configuration with single nozzles on each of the outside bodies. Base pressure similarity parameters were found to differ for the single nozzle and triple nozzle configurations. However, the correlation parameter for each was found to be a strong function of the nozzle exit momentum. Results of the data base evaluation are presented indicating an assessment of all data points. Analytical/experimental data comparisons were made for nozzle calibrations and correction factors derived, where indicated for use in nozzle exit plane data calculations.

Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range.

PubMed

Milles, Sigrid; Koehler, Christine; Gambin, Yann; Deniz, Ashok A; Lemke, Edward A

2012-10-01

Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.

Intramolecular Three-Colour Single Pair FRET of Intrinsically Disordered Proteins with Increased Dynamic Range

PubMed Central

Milles, Sigrid; Koehler, Christine; Gambin, Yann

2012-01-01

Single molecule observation of fluorescence resonance energy transfer can be used to provide insights into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins. PMID:22739670

Black phosphorus nanosheets for rapid microRNA detection.

PubMed

Zhou, Jie; Li, Zhongjun; Ying, Ming; Liu, Maixian; Wang, Xiaomei; Wang, Xianyou; Cao, Liwei; Zhang, Han; Xu, Gaixia

2018-03-15

Herein, for the first time, a sensitive sensing platform for rapid detection of microRNA was developed by employing black phosphorus nanosheets as the fluorescence quenching material. The biosensor displayed a good linear response to microRNA ranging from 10 nM to 1000 nM. Moreover, the biosensor could distinguish triple nucleotide polymorphism.

A non-destructive method for characterizing phenotypes and growth of a Bacillus subtilis biofilm using fluorescence microscopy

NASA Astrophysics Data System (ADS)

Koehler, Stephan; Wang, Xiaoling; Wilking, James; Weitz, Dave

2015-11-01

We develop an imaging technique for characterizing growth of biofilms using a triple fluorescent labeled strain for the three main phenotypes of a Bacillus subtilis biofilm on an agar substrate. We find that the biofilm does not flow across the substrate and thus growth is due to colonization at the periphery and thickening of the interior regions. We obtain local height and its composition of the three main phenotypes, which are motile, matrix-producing and sporulating, as well as the non-fluorescent material, which can be spores, dormant or dead cells or extracellular matrix. This technique is suitable for the study of biofilm growth and inhibition for different conditions such as biocides or bioremediation.

Remote imaging laser-induced breakdown spectroscopy and laser-induced fluorescence spectroscopy using nanosecond pulses from a mobile lidar system.

PubMed

Grönlund, Rasmus; Lundqvist, Mats; Svanberg, Sune

2006-08-01

A mobile lidar system was used in remote imaging laser-induced breakdown spectroscopy (LIBS) and laser-induced fluorescence (LIF) experiments. Also, computer-controlled remote ablation of a chosen area was demonstrated, relevant to cleaning of cultural heritage items. Nanosecond frequency-tripled Nd:YAG laser pulses at 355 nm were employed in experiments with a stand-off distance of 60 meters using pulse energies of up to 170 mJ. By coaxial transmission and common folding of the transmission and reception optical paths using a large computer-controlled mirror, full elemental imaging capability was achieved on composite targets. Different spectral identification algorithms were compared in producing thematic data based on plasma or fluorescence light.

Towards a Rational Model for the Triple Velocity Correlations of Turbulence

NASA Technical Reports Server (NTRS)

Younis, B. A.; Gatski, T. B.; Speziale, C. G.

1999-01-01

This paper presents a rational approach to modelling the triple velocity correlations that appear in the transport equations for the Reynolds stresses. All existing models of these correlations have largely been formulated on phenomenological grounds and are defective in one important aspect: they all neglect to allow for the dependence of these correlations on the local gradients of mean velocity. The mathematical necessity for this dependence will be demonstrated in the paper. The present contribution lies in the novel use of Group Representation Theory to determine the most general tensorial form of these correlations in terms of all the second- and third-order tensor quantities that appear in the exact equations that govern their evolution. The requisite representation did not exist in the literature and therefore had to be developed specifically for this purpose by Professor G. F. Smith. The outcome of this work is a mathematical framework for the construction of algebraic, explicit, and rational models for the triple velocity correlations that are theoretically consistent and include all the correct dependencies. Previous models are reviewed, and all are shown to be an incomplete subset of this new representation, even to lowest order.

Characterization of fluorescent-dissolved organic matter and identification of specific fluorophores in textile effluents.

PubMed

Li, Wentao; Xu, Zixiao; Wu, Qian; Li, Yan; Shuang, Chendong; Li, Aimin

2015-03-01

This study focused on the characterization of fluorescent-dissolved organic matter and identification of specific fluorophores in textile effluents. Samples from different textile wastewater treatment plants were characterized by high-performance liquid chromatography and size exclusion chromatography as well as fluorescence excitation-emission matrix spectra. Despite the highly heterogeneous textile effluents, the fluorescent components and their physicochemical properties were found relatively invariable, which is beneficial for the combination of biological and physicochemical treatment processes. The humic-like substance with triple-excitation peaks (excitation (Ex) 250, 310, 365/emission (Em) 460 nm) presented as the specific fluorescence indicator in textile effluents. It was also the major contributor to UV absorbance at 254 nm and resulted in the brown color of biologically treated textile effluents. By spectral comparison, the specific fluorophore in textile effluents could be attributed to the intermediate structure of azo dyes 1-amino-2-naphthol, which was transferred into the special humic-like substances during biological treatment.

Use of spectroscopic techniques for uranium(VI)/montmorillonite interaction modeling.

PubMed

Kowal-Fouchard, A; Drot, R; Simoni, E; Ehrhardt, J J

2004-03-01

To experimentally identify both clay sorption sites and sorption equilibria and to understand the retention mechanisms at a molecular level, we have characterized the structure of hexavalent uranium surface complexes resulting from the interaction between the uranyl ions and the surface retention groups of a montmorillonite clay. We have performed laser-induced fluorescence spectroscopy (LIFS) and X-ray photoelectron spectroscopy (XPS) on uranyl ion loaded montmorillonite. These structural results were then compared to those obtained from the study of uranyl ions sorbed onto an alumina and also from U(VI) sorbed on an amorphous silica. This experimental approach allowed for a clear determination of the reactive surface sites of montmorillonite for U(VI) sorption. The lifetime values and the U4f XPS spectra of uranium(VI) sorbed on montmorillonite have shown that this ion is sorbed on both exchange and edge sites. The comparison of U(VI)/clay and U(VI)/oxide systems has determined that the interaction between uranyl ions and montmorillonite edge sites occurs via both [triple bond]AlOH and [triple bond]SiOH surface groups and involves three distinct surface complexes. The surface complexation modeling of the U(VI)/montmorillonite sorption edges was determined using the constant capacitance model and the above experimental constraints. The following equilibria were found to account for the uranyl sorption mechanisms onto montmorillonite for metal concentrations ranged from 10(-6) to 10(-3) M and two ionic strengths (0.1 and 0.5 M): 2[triple bond]XNa + UO2(2+) <==> ([triple bond]X)2UO2 + 2Na+, log K0(exch) = 3.0; [triple bond]Al(OH)2 + UO2(2+) <==> [triple bond]Al(OH)2UO2(2+), log K0(Al) = 14.9; [triple bond]Si(OH)2 + UO2(2+) <==> [triple bond]SiO2UO2 + 2H+, log K0(Si1) = -3.8; and [triple bond]Si(OH)2 + 3UO2(2+) + 5H2O <==> [triple bond]SiO2(UO2)3(OH)5- + 7H+, log K0(Si2) = -20.0.

Genetic events in the progression of adenoid cystic carcinoma of the breast to high-grade triple-negative breast cancer.

PubMed

Fusco, Nicola; Geyer, Felipe C; De Filippo, Maria R; Martelotto, Luciano G; Ng, Charlotte K Y; Piscuoglio, Salvatore; Guerini-Rocco, Elena; Schultheis, Anne M; Fuhrmann, Laetitia; Wang, Lu; Jungbluth, Achim A; Burke, Kathleen A; Lim, Raymond S; Vincent-Salomon, Anne; Bamba, Masamichi; Moritani, Suzuko; Badve, Sunil S; Ichihara, Shu; Ellis, Ian O; Reis-Filho, Jorge S; Weigelt, Britta

2016-11-01

Adenoid cystic carcinoma of the breast is a rare histological type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Although the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intratumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by the MYB-NFIB fusion gene and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-negative breast cancer of no special type may involve the selection of neoplastic clones and/or the acquisition of additional genetic alterations.

Genetic events in the progression of adenoid cystic carcinoma of the breast to high-grade triple-negative breast cancer

PubMed Central

Fusco, Nicola; Geyer, Felipe C; De Filippo, Maria R; Martelotto, Luciano G; Ng, Charlotte K Y; Piscuoglio, Salvatore; Guerini-Rocco, Elena; Schultheis, Anne M; Fuhrmann, Laetitia; Wang, Lu; Jungbluth, Achim A; Burke, Kathleen A; Lim, Raymond S; Vincent-Salomon, Anne; Bamba, Masamichi; Moritani, Suzuko; Badve, Sunil S; Ichihara, Shu; Ellis, Ian O; Reis-Filho, Jorge S; Weigelt, Britta

2016-01-01

Adenoid cystic carcinoma of the breast is a rare histologic type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Whilst the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intra-tumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by MYB-NFIB fusion gene, and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-negative breast cancer of no special type may involve the selection of neoplastic clones and/ or the acquisition of additional genetic alterations. PMID:27491809

Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation

NASA Astrophysics Data System (ADS)

Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George

2012-03-01

With traditional 2-color Förster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFPNWASP/ Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1- F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.

Quantum-Dot-Based Theranostic Micelles Conjugated with an Anti-EGFR Nanobody for Triple-Negative Breast Cancer Therapy.

PubMed

Wang, Yuyuan; Wang, Yidan; Chen, Guojun; Li, Yitong; Xu, Wei; Gong, Shaoqin

2017-09-13

A quantum-dot (QD)-based micelle conjugated with an anti-epidermal growth factor receptor (EGFR) nanobody (Nb) and loaded with an anticancer drug, aminoflavone (AF), has been engineered for EGFR-overexpressing cancer theranostics. The near-infrared (NIR) fluorescence of the indium phosphate core/zinc sulfide shell QDs (InP/ZnS QDs) allowed for in vivo nanoparticle biodistribution studies. The anti-EGFR nanobody 7D12 conjugation improved the cellular uptake and cytotoxicity of the QD-based micelles in EGFR-overexpressing MDA-MB-468 triple-negative breast cancer (TNBC) cells. In comparison with the AF-encapsulated nontargeted (i.e., without Nb conjugation) micelles, the AF-encapsulated Nb-conjugated (i.e., targeted) micelles accumulated in tumors at higher concentrations, leading to more effective tumor regression in an orthotopic triple-negative breast cancer xenograft mouse model. Furthermore, there was no systemic toxicity observed with the treatments. Thus, this QD-based Nb-conjugated micelle may serve as an effective theranostic nanoplatform for EGFR-overexpressing cancers such as TNBCs.

Solvent induced fluorescence enhancement of graphene oxide studied by ultrafast spectroscopy

NASA Astrophysics Data System (ADS)

Zhao, Litao; Chen, Jinquan; He, Xiaoxiao; Yu, Xiantong; Yan, Shujun; Zhang, Sanjun; Pan, Haifeng; Xu, Jianhua

2018-05-01

Femtosecond transient absorption (TA) spectroscopy combined with picosecond time resolved fluorescence (TRF) were used to reveal the fluorescence kinetics of graphene oxide (GO) in water, ethanol and water-ethanol mixtures. Size-independent fluorescence of GO were observed in water, and pH-dependent fluorescence spectra could be fitted well by a triple emission relaxation with peaks around 440 nm, 500 nm, and 590 nm respectively. The results indicate that polycyclic aromatic hydrocarbons (PAHs) linked by oxygen-containing functional groups dominate GO's fluorescence emission. GO's fluorescence quantum yield was measured to be 2.8% in ethanol but 1.2% in water. The three decay components fluorescence decay, as well as the transient absorption dynamics with an offset, confirmed this solvent induced fluorescence enhancement. GO's Raman spectral signals showed that GO in ethanol has a smaller average size of PAHs than that of GO in water. Therefore, besides other enhancement effects reported in literatures, we proposed that solvents could also change the size of PAHs, resulting in a photoluminescence enhancement. Our experimental data demonstrates that GO's quantum yield could be up to 2.8% in water and 8.4% in ethanol and this observation may help ones to improve GO's photoluminescence efficiency as well as its applications in solution.

A label-free fluorescent direct detection of live Salmonella typhimurium using cascade triple trigger sequences-regenerated strand displacement amplification and hairpin template-generated-scaffolded silver nanoclusters.

PubMed

Zhang, Peng; Liu, Hui; Li, Xiaocheng; Ma, Suzhen; Men, Shuai; Wei, Heng; Cui, Jingjing; Wang, Hongning

2017-01-15

The harm of Salmonella typhimurium (S. typhimurium) to public health mainly by the consumption of contaminated agricultural products or water stresses an urgent need for rapid detection methods to help control the spread of S. typhimurium. In this work, an intelligently designed sensor system took creative advantage of triple trigger sequences-regenerated strand displacement amplification and self-protective hairpin template-generated-scaffolded silver nanoclusters (AgNCs) for the first time. In the presence of live S. typhimurium, single-stranded trigger sequences were released from aptamer-trigger sequences complex, initiating a branch migration to open the hairpin template I containing complementary scaffolds of AgNCs. Then the first strand displacement amplification was induced to produce numerous scaffolds of AgNCs and reporter strands which initiated a branch migration to open the hairpin template II containing complementary scaffolds of AgNCs. Then the second strand displacement amplification was induced to generate numerous scaffolds of AgNCs and trigger sequences which initiated the third branch migration and strand displacement amplification to produce numerous scaffolds of AgNCs and reporter strands in succession. Cyclically, the reproduction of the trigger sequences and cascade successive production of scaffolds were achieved successfully, forming highly fluorescent AgNCs, thus providing significantly enhanced fluorescent signals to achieve ultrasensitive detection of live S. typhimurium down to 50 CFU/mL with a linear range from 10 2 to 10 7 CFU/mL. It is the first report on a fluorescent biosensor for detecting viable S. typhimurium directly, which can distinguish from heat denatured S. typhimurium. And it develops a new strategy to generate the DNA-scaffolds for forming AgNCs. Copyright © 2016 Elsevier B.V. All rights reserved.

Corresponding state-based correlations for the temperature-dependent surface tension of saturated hydrocarbons

NASA Astrophysics Data System (ADS)

Tian, Jianxiang; Zhang, Cuihua; Zhang, Laibin; Zheng, Mengmeng; Liu, Shuzhen

2017-10-01

Based on the recent progresses on the corresponding state-based correlations for the temperature-dependent surface tension of saturated fluids [I. Cachadiña, A. Mulero and J. X. Tian, Fluid Phase Equilibr. 442 (2017) 68; J. X. Tian, M. M. Zheng, H. L. Yi, L. B. Zhang and S. Z. Liu, Mod. Phys. Lett. B 31 (2017) 1750110], we proposed a new correlation for saturated hydrocarbons. This correlation includes three fluid-independent parameters and inquires the critical temperature, the triple-point temperature and the surface tension at the triple-point temperature as inputs for each hydrocarbon. Results show that this correlation can reproduce NIST data with absolute average deviation (AAD) less than 1% for 10 out of 19 hydrocarbons and AAD less than 5% for 17 out of 19 hydrocarbons, clearly better than other correlations.

Orbit Alignment in Triple Stars

NASA Astrophysics Data System (ADS)

Tokovinin, Andrei

2017-08-01

The statistics of the angle Φ between orbital angular momenta in hierarchical triple systems with known inner visual or astrometric orbits are studied. A correlation between apparent revolution directions proves the partial orbit alignment known from earlier works. The alignment is strong in triples with outer projected separation less than ˜50 au, where the average Φ is about 20^\\circ . In contrast, outer orbits wider than 1000 au are not aligned with the inner orbits. It is established that the orbit alignment decreases with the increasing mass of the primary component. The average eccentricity of inner orbits in well-aligned triples is smaller than in randomly aligned ones. These findings highlight the role of dissipative interactions with gas in defining the orbital architecture of low-mass triple systems. On the other hand, chaotic dynamics apparently played a role in shaping more massive hierarchies. The analysis of projected configurations and triples with known inner and outer orbits indicates that the distribution of Φ is likely bimodal, where 80% of triples have {{Φ }}< 70^\\circ and the remaining ones are randomly aligned.

An improved triple collocation algorithm for decomposing autocorrelated and white soil moisture retrieval errors

USDA-ARS?s Scientific Manuscript database

If not properly account for, auto-correlated errors in observations can lead to inaccurate results in soil moisture data analysis and reanalysis. Here, we propose a more generalized form of the triple collocation algorithm (GTC) capable of decomposing the total error variance of remotely-sensed surf...

Highly active antiretroviral therapy including protease inhibitors does not confer a unique CD4 cell benefit. The AVANTI and INCAS Study Groups.

PubMed

2000-07-07

To determine if triple combination therapy, particularly including HIV protease inhibitors (PI), confers an unique immunological benefit that is independent of reductions of plasma viral load (pVL). The correlation between changes from baseline in CD4 cell count and pVL was examined at all time points up to 52 weeks in three randomized clinical trials (AVANTI-2, AVANTI-3 and INCAS) that compared dual nucleoside therapy with triple combination therapy. Individual pVL and CD4 cell counts changes from baseline were entered into multivariate linear regression models for patients receiving double therapy and for those receiving triple therapy including a PI and/or a non-nucleoside reverse transcriptase inhibitor (NNRTI), and the null hypothesis was tested. After 52 weeks of therapy, the relationship between changes from baseline CD4 cell count and pVL was independent of whether patients were assigned double or triple therapy (P = 0.23 and 0.69 for intercept and slope, respectively), or whether patients were assigned triple therapy including a PI or triple therapy including an NNRTI (P = 0.92 and 0.95, respectively). Less than 5% of patients ever had 'discordant' increases in both CD4 cell count and pVL compared with baseline, and this proportion was unrelated to the class of therapy used. 'Discordant' decreases from baseline in both parameters were observed in up to 35% of individuals. The correlation between pVL and CD4 cell count changes from baseline improved over time on therapy, regardless of the therapeutic regimen involved. The data provide no evidence for a CD4 cell count benefit of highly active antiretroviral therapy (HAART) unique to triple therapy or PI-containing regimens.

Triple Junctions, Boninites, and a New Microplate in the Western Pacific

NASA Astrophysics Data System (ADS)

Flores, J. A.; Casey, J.

2017-12-01

A new microplate has been discovered while trying to correlate melting processes in subduction zones that are forming boninites along the southern Mariana Plate. The westward boundary between the Mariana plate and the Philippine Sea plate is along a well-defined back-arc spreading center. The southern extension of this spreading center to the intersection with the Mariana Trench does not have a recognized morphological boundary. Previous work has hypothesized that subduction beneath a spreading center provides conditions required for boninite petrogenesis. Therefore, the exact location of the trench-trench-ridge triple junction needs to be found and correlated with known boninite locations. The triple junction was found using fault plane solutions to constrain the southern boundary of the two plates as it transects across the forearc. Normal faults suggest the triple junction to be at approximately 11.9N 144.1W; slip direction of reverse faults associated with the subducting plate are dominantly north-south west of this junction and northwest-southeast on the east side. While locating the southern boundary, the nucleation of a new spreading center that creates a ridge-ridge-ridge triple junction was found. The main spreading center trends mostly north-south until about 12.5N 143W, where two other spreading centers meet. The western spreading zone trends mostly east-west and seems to be in its infancy whereas there is another spreading center trending northwest-southeast. It is this last spreading center that forms the trench-ridge-trench triple junction. Discovery of these triple junctions isolates a piece of lithosphere that we interpret to be a new microplate that we name the Challenger Microplate.

Multimodal Spectral Imaging of Cells Using a Transmission Diffraction Grating on a Light Microscope

PubMed Central

Isailovic, Dragan; Xu, Yang; Copus, Tyler; Saraswat, Suraj; Nauli, Surya M.

2011-01-01

A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield or DIC spectral microscopy, respectively. The presented spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens. PMID:21639978

4-amino-1H-benzo[g]quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides.

PubMed

Godde, F; Toulmé, J J; Moreau, S

2000-08-01

We developed a new fluorescent analog of cytosine, the 4-amino-1H-benzo[g]quinazoline-2-one, which constitute a probe sensitive to pH. The 2'-O-Me ribonucleoside derivative of this heterocycle was synthesized and exhibited a fluorescence emission centered at 456 nm, characterized by four major excitation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of Phi = 0.62 at pH 7.1. The fluorescence emission maximum shifted from 456 to 492 nm when pH was decreased from 7.1 to 2.1. The pK(a) (4) was close to that of cytosine (4.17). When introduced in triplex forming oligonucleotides this new nucleoside can be used to reveal the protonation state of triplets in triple-stranded structures. Complex formation was detected by a significant quenching of fluorescence emission (approximately 88%) and the N-3 protonation of the quinazoline ring by a shift of the emission maximum from 485 to 465 nm. Using this probe we unambiguously showed that triplex formation of the pyrimidine motif does not require the protonation of all 4-amino-2-one pyrimidine rings.

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.

Long Term Non-Invasive Imaging of Embryonic Stem Cells Using Reporter Genes

PubMed Central

Sun, Ning; Lee, Andrew; Wu, Joseph C.

2013-01-01

Development of non-invasive and accurate methods to track cell fate following delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. Here we describe a protocol for the in vivo monitoring of stem cell survival, proliferation, and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes using lentiviral transduction. We used fluorescence activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography imaging to track them in vivo, and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny. PMID:19617890

Double Photoionization of helium atom using Screening Potential Approach

NASA Astrophysics Data System (ADS)

Saha, Haripada

2014-05-01

The triple differential cross section for double Photoionization of helium atom will be investigated using our recently extended MCHF method. It is well known that electron correlation effects in both the initial and the final states are very important. To incorporate these effects we will use the multi-configuration Hartree-Fock method to account for electron correlation in the initial state. The electron correlation in the final state will be taken into account using the angle-dependent screening potential approximation. The triple differential cross section (TDCS) will be calculated for 20 eV photon energy, which has experimental results. Our results will be compared with available experimental and the theoretical observations.

Experimental triple-slit interference in a strongly driven V-type artificial atom

NASA Astrophysics Data System (ADS)

Dada, Adetunmise C.; Santana, Ted S.; Koutroumanis, Antonios; Ma, Yong; Park, Suk-In; Song, Jindong; Gerardot, Brian D.

2017-08-01

Rabi oscillations of a two-level atom appear as a quantum interference effect between the amplitudes associated with atomic superpositions, in analogy with the classic double-slit experiment which manifests a sinusoidal interference pattern. By extension, through direct detection of time-resolved resonance fluorescence from a quantum-dot neutral exciton driven in the Rabi regime, we experimentally demonstrate triple-slit-type quantum interference via quantum erasure in a V-type three-level artificial atom. This result is of fundamental interest in the experimental studies of the properties of V-type three-level systems and may pave the way for further insight into their coherence properties as well as applications for quantum information schemes. It also suggests quantum dots as candidates for multipath-interference experiments for probing foundational concepts in quantum physics.

Characterization of triple layers

NASA Astrophysics Data System (ADS)

Otero, Toribio F.; Cortes, M. Teresa

2001-07-01

We reported the characterization of a macroscopic electrochemomechanical actuator like triple layer (3x1 cm) formed by polypyrrole)/double- sided, non-conducting and flexible tape/ polypyrrole that works in liquid electrolytes under electrochemical control. This triple layer has characteristics of artificial muscle. The influence of variables that control the volume change in the polymer as electrolyte concentration, or temperature has been studied. Variations of time, energy and charge under different conditions are described. We have found that the triple layer acts, at the same time, as sensor and actuator. Therefore, physical magnitudes like the electrolyte concentration or the temperature in the cell can be obtained from electrical energy consumed by a muscle. We have evaluated the influence of variables as area of the triple layer or the trailing weight, which don't participate in the electrochemical reaction. We propose an explication to the results, which show a correlation between the trailed mass and the consumed charge required to move a constant angle those masses by the triple layer. When different surface areas of the triple layer has been evaluated we found that the consumed electrical charge is proportional to area (the mass) of the triple layer. The triple layer can make macroscopical movements in short times, their position is absolutely controlled with the electrical charge, and it has capacity to lift masses. These characteristics allow their use in the design of tools. So, we present a macroscopic tool constituted by two triple layers, which allows catch and translate objects in liquid medium (nipper).

Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers.

PubMed

Sherwood, Carly A; Eastham, Ashley; Lee, Lik Wee; Risler, Jenni; Vitek, Olga; Martin, Daniel B

2009-09-01

Multiple reaction monitoring mass spectrometry (MRM-MS) is a technique for high-sensitivity targeted analysis. In proteomics, MRM-MS can be used to monitor and quantify a peptide based on the production of expected fragment peaks from the selected peptide precursor ion. The choice of which fragment ions to monitor in order to achieve maximum sensitivity in MRM-MS can potentially be guided by existing MS/MS spectra. However, because the majority of discovery experiments are performed on ion trap platforms, there is concern in the field regarding the generalizability of these spectra to MRM-MS on a triple quadrupole instrument. In light of this concern, many operators perform an optimization step to determine the most intense fragments for a target peptide on a triple quadrupole mass spectrometer. We have addressed this issue by targeting, on a triple quadrupole, the top six y-ion peaks from ion trap-derived consensus library spectra for 258 doubly charged peptides from three different sample sets and quantifying the observed elution curves. This analysis revealed a strong correlation between the y-ion peak rank order and relative intensity across platforms. This suggests that y-type ions obtained from ion trap-based library spectra are well-suited for generating MRM-MS assays for triple quadrupoles and that optimization is not required for each target peptide.

Strong correlation in incremental full configuration interaction

NASA Astrophysics Data System (ADS)

Zimmerman, Paul M.

2017-06-01

Incremental Full Configuration Interaction (iFCI) reaches high accuracy electronic energies via a many-body expansion of the correlation energy. In this work, the Perfect Pairing (PP) ansatz replaces the Hartree-Fock reference of the original iFCI method. This substitution captures a large amount of correlation at zero-order, which allows iFCI to recover the remaining correlation energy with low-order increments. The resulting approach, PP-iFCI, is size consistent, size extensive, and systematically improvable with increasing order of incremental expansion. Tests on multiple single bond, multiple double bond, and triple bond dissociations of main group polyatomics using double and triple zeta basis sets demonstrate the power of the method for handling strong correlation. The smooth dissociation profiles that result from PP-iFCI show that FCI-quality ground state computations are now within reach for systems with up to about 10 heavy atoms.

Multispectroscopic and Theoretical Exploration of the Comparative Binding Aspects of Bioflavonoid Fisetin with Triple- and Double-Helical Forms of RNA.

PubMed

Bhuiya, Sutanwi; Haque, Lucy; Goswami, Rapti; Das, Suman

2017-12-14

The interactions of RNA triplex (U.A*U) and duplex (A.U) with naturally occurring flavonoid fisetin (FTN) have been examined at pH 7.0 using various spectroscopic, viscometric, and theoretical studies. Experimental observations showed that the ligand binds with both double- and triple-helical forms of RNA, although the binding affinity is greater for the triplex structure (5.94 × 10 6 M -1 ) compared to that for the duplex counterpart (1.0 × 10 5 M -1 ). Thermal melting experiments revealed that the Hoogsteen base-paired third strand of triplex was stabilized to a greater extent (∼14 °C) compared with the Watson-Crick base-paired second strand (∼4 °C) in the presence of FTN. From fluorimetric study, we observed that U.A*U and A.U primarily bind to the photoproduced tautomer of FTN in the excited state. Steady-state and time-resolved anisotropy measurements illustrate considerable modulations of the spectroscopic properties of the tautomeric FTN within the RNA environment. Viscometric, fluorescence quenching, and thermal melting studies all together support the mode of binding to be intercalation. Theoretical study explains the experimental absorption and emission (dual fluorescence) behavior of FTN along with the excited-state intramolecular proton transfer process.

Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience.

PubMed

Kim, Sally A; Schwille, Petra

2003-10-01

Based on time-averaging fluctuation analysis of small fluorescent molecular ensembles in equilibrium, fluorescence correlation spectroscopy has recently been applied to investigate processes in the intracellular milieu. The exquisite sensitivity of fluorescence correlation spectroscopy provides access to a multitude of measurement parameters (rates of diffusion, local concentration, states of aggregation and molecular interactions) in real time with fast temporal and high spatial resolution. The introduction of dual-color cross-correlation, imaging, two-photon excitation, and coincidence analysis coupled with fluorescence correlation spectroscopy has expanded the utility of the technique to encompass a wide range of promising applications in living cells that may provide unprecedented insight into understanding the molecular mechanisms of intracellular neurobiological processes.

Comparison of the quadratic configuration interaction and coupled cluster approaches to electron correlation including the effect of triple excitations

NASA Technical Reports Server (NTRS)

Taylor, Peter R.; Lee, Timothy J.; Rendell, Alistair P.

1990-01-01

The recently proposed quadratic configuration interaction (QCI) method is compared with the more rigorous coupled cluster (CC) approach for a variety of chemical systems. Some of these systems are well represented by a single-determinant reference function and others are not. The finite order singles and doubles correlation energy, the perturbational triples correlation energy, and a recently devised diagnostic for estimating the importance of multireference effects are considered. The spectroscopic constants of CuH, the equilibrium structure of cis-(NO)2 and the binding energies of Be3, Be4, Mg3, and Mg4 were calculated using both approaches. The diagnostic for estimating multireference character clearly demonstrates that the QCI method becomes less satisfactory than the CC approach as non-dynamical correlation becomes more important, in agreement with a perturbational analysis of the two methods and the numerical estimates of the triple excitation energies they yield. The results for CuH show that the differences between the two methods become more apparent as the chemical systems under investigation becomes more multireference in nature and the QCI results consequently become less reliable. Nonetheless, when the system of interest is dominated by a single reference determinant both QCI and CC give very similar results.

High Expression of CCR7 Predicts Lymph Node Metastasis and Good Prognosis in Triple Negative Breast Cancer.

PubMed

Li, Xuelu; Sun, Siwen; Li, Ning; Gao, Jiyue; Yu, Jing; Zhao, Jinbo; Li, Man; Zhao, Zuowei

2017-01-01

Previous preclinical and clinical studies have reported a positive correlation between the expression of the C-C chemokine receptor 7 (CCR7) and the incidence of lymph node metastasis in breast cancer. However, the prognostic relevance of CCR7 expression in breast cancer remains contradictory till now. The aim of this study is to assess the correlation of the CCR7 expression with other clinicopathological features and prognosis in breast cancer. The CCR7 gene amplification and mRNA expression levels from approximately 3,000 patients were retrieved from human breast cancer databases and analyzed. Furthermore, a total of 188 primary triple negative breast cancer patients were enrolled in this study (diagnosed since January 2009 to January 2013 from the Second Hospital of Dalian Medical University). The protein levels of CCR7 were examined by immunohistochemistry using paraffin-embedded tumor tissues. The analysis of gene amplification and mRNA levels showed the expression of CCR7 in breast cancer correlated with better prognosis. When we compared the CCR7 expressions in different subtypes, the basal-like group showed the highest expression of CCR7 and exhibited a better prognosis. Consistently, Kaplan-Meier analysis of 188 triple negative breast cancer patients showed that the prognosis of patients with positive CCR7 expression was significantly better than those with negative expression (HR=0.642, p=0.0275). Additionally, we also observed a positive correlation between lymph node metastasis and the CCR7 expression (p=0.0096). Our results indicated that elevated CCR7 expression as a marker for increased lymph node metastasis, in addition to serve as an independent prognostic indicator for better overall survival in triple negative breast cancer patients. © 2017 The Author(s). Published by S. Karger AG, Basel.

Combinatorial Effects of Lapatinib and Rapamycin in Triple-Negative Breast Cancer Cells

PubMed Central

Liu, Tongrui; Yacoub, Rami; Taliaferro-Smith, LaTonia D.; Sun, Shi-Yong; Graham, Tisheeka R.; Dolan, Ryan; Lobo, Christine; Tighiouart, Mourad; Yang, Lily; Adams, Amy; O'Regan, Ruth M.

2016-01-01

Triple-negative breast cancers, which lack estrogen receptor, progesterone receptor, and HER2/neu overexpression, account for approximately 15% of breast cancers, but occur more commonly in African Americans. The poor survival outcomes seen with triple-negative breast cancers patients are, in part, due to a lack of therapeutic targets. Epidermal growth factor receptor (EGFR) is overexpressed in 50% of triple-negative breast cancers, but EGFR inhibitors have not been effective in patients with metastatic breast cancers. However, mTOR inhibition has been shown to reverse resistance to EGFR inhibitors. We examined the combination effects of mTOR inhibition with EGFR inhibition in triple-negative breast cancer in vitro and in vivo. The combination of EGFR inhibition by using lapatinib and mTOR inhibition with rapamycin resulted in significantly greater cytotoxicity than the single agents alone and these effects were synergistic in vitro. The combination of rapamycin and lapatinib significantly decreased growth of triple-negative breast cancers in vivo compared with either agent alone. EGFR inhibition abrogated the expression of rapamycin-induced activated Akt in triple-negative breast cancer cells in vitro. The combination of EGFR and mTOR inhibition resulted in increased apoptosis in some, but not all, triple-negative cell lines, and these apoptotic effects correlated with a decrease in activated eukaryotic translation initiation factor (eIF4E). These results suggest that mTOR inhibitors could sensitize a subset of triple-negative breast cancers to EGFR inhibitors. Given the paucity of effective targeted agents in triple-negative breast cancers, these results warrant further evaluation. PMID:21690228

Fluorescent Protein Aided Insights on Plastids and their Extensions: A Critical Appraisal

PubMed Central

Delfosse, Kathleen; Wozny, Michael R.; Jaipargas, Erica-Ashley; Barton, Kiah A.; Anderson, Cole; Mathur, Jaideep

2016-01-01

Multi-colored fluorescent proteins targeted to plastids have provided new insights on the dynamic behavior of these organelles and their interactions with other cytoplasmic components and compartments. Sub-plastidic components such as thylakoids, stroma, the inner and outer membranes of the plastid envelope, nucleoids, plastoglobuli, and starch grains have been efficiently highlighted in living plant cells. In addition, stroma filled membrane extensions called stromules have drawn attention to the dynamic nature of the plastid and its interactions with the rest of the cell. Use of dual and triple fluorescent protein combinations has begun to reveal plastid interactions with mitochondria, the nucleus, the endoplasmic reticulum and F-actin and suggests integral roles of plastids in retrograde signaling, cell to cell communication as well as plant-pathogen interactions. While the rapid advances and insights achieved through fluorescent protein based research on plastids are commendable it is necessary to endorse meaningful observations but subject others to closer scrutiny. Here, in order to develop a better and more comprehensive understanding of plastids and their extensions we provide a critical appraisal of recent information that has been acquired using targeted fluorescent protein probes. PMID:26834765

On the Melting Curve of Sulfur Hexafluoride

NASA Astrophysics Data System (ADS)

Harvey, Allan H.

2017-12-01

A previous correlation for the melting curve of sulfur hexafluoride (SF6) is inconsistent with the thermodynamic slope at the triple point derived from the Clapeyron equation. It is shown that this is probably due to the previous authors combining an accurate measurement of the triple point with melting-curve data that were distorted by impurities. A new equation is proposed that is consistent with the Clapeyron slope.

H(C)P and H(P)C triple-resonance experiments at natural abundance employing long-range couplings.

PubMed

Malon, Michal; Koshino, Hiroyuki

2007-09-01

Modified two-dimensional (2D) triple-resonance H(C)P and H(P)C experiments based on INEPT/HMQC and double-INEPT schemes are applied to the study of organophosphorus compounds at natural abundances. The implementation of effective (1)H--(13)C gradient selection, additional purging pulsed field gradients, spinlock pulses, and improved phase cycling is demonstrated to allow weak correlation signals based on long-range couplings to be readily observed. Through the combination of two heteronuclear long-range coupling constants, (n)J(CH) and (n)J(PC) in H(C)P experiments or (n)J(PH) and (n)J(PC) in H(P)C experiments, protons can be correlated to a second heteronucleus through 4-7 chemical bonds. These experiments thus overcome the inherit limitations of classical (1)H-X HMBC experiments, which require a nonzero value of the heteronuclear coupling constant (n)J(XH). Ultra-broadband inversion composite pulses are successfully employed in the H(P)C INEPT/HMQC and H(P)C double-INEPT pulse sequences to increase the utility of the experiments and the quality of obtained spectra. This work extends and completes a set of 2D phase-sensitive triple-resonance experiments applicable at natural abundances, and also offers insight into the methodology of triple-resonance experiments and the application of pulsed field gradients. A one-dimensional triple-resonance experiment employing carbon detection is suggested for accurate determination of small (n)J(PC).

On the universal behavior of some thermodynamic properties along the whole liquid-vapor coexistence curve

NASA Astrophysics Data System (ADS)

Román, F. L.; White, J. A.; Velasco, S.; Mulero, A.

2005-09-01

When thermodynamic properties of a pure substance are transformed to reduced form by using both critical- and triple-point values, the corresponding experimental data along the whole liquid-vapor coexistence curve can be correlated with a very simple analytical expression that interpolates between the behavior near the triple and the critical points. The leading terms of this expression contain only two parameters: the critical exponent and the slope at the triple point. For a given thermodynamic property, the critical exponent has a universal character but the slope at the triple point can vary significantly from one substance to another. However, for certain thermodynamic properties including the difference of coexisting densities, the enthalpy of vaporization, and the surface tension of the saturated liquid, one finds that the slope at the triple point also has a nearly universal value for a wide class of fluids. These thermodynamic properties thus show a corresponding apparently universal behavior along the whole coexistence curve.

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.

In Vitro Studies of Primary Explosive Blast Loading on Neurons

DTIC Science & Technology

2015-09-01

blast but was significantly higher for the triple blast. Membrane permeability was also evaluated by calcein dye . Calcein is normally a membrane...impermeable dye ; however, upon damage to the plasma membrane, leakage of the dye into the cytosol can occur, causing an increase in the fluorescence of the...intensities were significantly higher for the injured cells compared with the control and sham. However, the difference in dye uptake between the singly and

The Triple Roles of Glutathione for a DNA-Cleaving DNAzyme and Development of a Fluorescent Glutathione/Cu2+-Dependent DNAzyme Sensor for Detection of Cu2+ in Drinking Water.

PubMed

Wang, Shijin; Liu, Chengcheng; Li, Guiying; Sheng, Yongjie; Sun, Yanhong; Rui, Hongyue; Zhang, Jin; Xu, Jiacui; Jiang, Dazhi

2017-03-24

Pistol-like DNAzyme (PLDz) is an oxidative DNA-cleaving catalytic DNA with ascorbic acid as cofactor. Herein, glutathione was induced into the reaction system to maintain reduced ascorbic acid levels for higher efficient cleavage. However, data indicated that glutathione played triple roles in PLDz-catalyzed reactions. Glutathione alone had no effect on PLDz, and showed inhibitory effect on ascorbic acid-induced PLDz catalysis, but exhibited stimulating effect on Cu 2+ -promoted self-cleavage of PLDz. Further analysis of the effect of glutathione/Cu 2+ on PLDz indicated that H 2 O 2 played a key role in PLDz catalysis. Finally, we developed a fluorescent Cu 2+ sensor (PL-Cu 1.0) based on the relationship between glutathione/Cu 2+ and catalytic activity of PLDz. The fluorescent intensity showed a linear response toward the logarithm concentration of Cu 2+ over the range from 80 nM to 30 μM, with a detection limit of 21.1 nM. PL-Cu 1.0 provided only detection of Cu 2+ over other divalent metal ions. Ca 2+ and Mg 2+ could not interfere with Cu 2+ detection even at a 1000-fold concentration. We further applied PL-Cu 1.0 for Cu 2+ detection in tap and bottled water. Water stored in copper taps overnight had relatively high Cu 2+ concentrations, with a maximum 22.3 μM. Trace Cu 2+ (52.2 nM) in deep spring was detected among the tested bottled water. Therefore, PL-Cu 1.0 is feasible to detect Cu 2+ in drinking water, with a practical application.

Blood-brain barrier-penetrating amphiphilic polymer nanoparticles deliver docetaxel for the treatment of brain metastases of triple negative breast cancer.

PubMed

He, Chunsheng; Cai, Ping; Li, Jason; Zhang, Tian; Lin, Lucy; Abbasi, Azhar Z; Henderson, Jeffrey T; Rauth, Andrew Michael; Wu, Xiao Yu

2017-01-28

Brain metastasis is a fatal disease with limited treatment options and very short survival. Although systemic chemotherapy has some effect on peripheral metastases of breast cancer, it is ineffective in treating brain metastasis due largely to the blood-brain barrier (BBB). Here we developed a BBB-penetrating amphiphilic polymer-lipid nanoparticle (NP) system that efficiently delivered anti-mitotic drug docetaxel (DTX) for the treatment of brain metastasis of triple negative breast cancer (TNBC). We evaluated the biodistribution, brain accumulation, pharmacokinetics and efficacy of DTX-NP in a mouse model of brain metastasis of TNBC. Confocal fluorescence microscopy revealed extravasation of dye-loaded NPs from intact brain microvessels in healthy mice. DTX-NP also extravasated from brain microvessels and accumulated in micrometastasis lesions in the brain. Intravenously injected DTX-NPs increased the blood circulation time of DTX by 5.5-fold and the AUC 0-24h in tumor-bearing brain by 5-fold compared to the clinically used DTX formulation Taxotere® . The kinetics of NPs in the brain, determined by ex vivo fluorescence imaging, showed synchronization with DTX kinetics in the brain measured by LC-MS/MS. This result confirmed successful delivery of DTX by the NPs into the brain and suggested that ex vivo fluorescence imaging of NP could be an effective and quick means for probing drug disposition in the brain. Treatment with the DTX-NP formulation delayed tumor growth by 11-fold and prolonged median survival of tumor-bearing mice by 94% compared to an equivalent dose of Taxotere®, without inducing histological changes in the major organs. Copyright © 2016 Elsevier B.V. All rights reserved.

Correlations for the Viscosity of 2,3,3,3-Tetrafluoroprop-1-ene (R1234yf) and trans-1,3,3,3-Tetrafluoropropene (R1234ze(E))†

PubMed Central

Huber, Marcia L.; Assael, Marc J.

2016-01-01

Due to concerns about global warming, there is interest in 2,3,3,3-Tetrafluoroprop-1-ene (R1234yf) and trans-1,3,3,3-Tetrafluoropropene (R1234ze(E)) as potential replacements for refrigerants with high global warming potential (GWP). In this manuscript we survey available data and provide viscosity correlations that cover the entire fluid range including vapor, liquid, and supercritical regions. The correlation for R1234yf is valid from the triple point (220 K) to 410 K at pressures up to 30 MPa, and the correlation for R1234ze(E) is valid from the triple point (169 K) to 420 K at pressures up to 100 MPa. The estimated uncertainty for both correlations at a 95 % confidence level is 2 % for the liquid phase over the temperature range 243 K to 363 K at pressures to 30 MPa, and 3 % for the gas phase at atmospheric pressure. PMID:27840461

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.

Migration of grain boundaries and triple junctions in high-purity aluminum during annealing after slight cold rolling

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

Yin, Wenhong; School of Mechanical Engineering, Shandong University of Technology, Zibo 255049; Wang, Weiguo, E-mail: wang_weiguo@vip.163.com

Grain orientations and grain boundary migrations near triple junctions in a high purity aluminum were analyzed by electron back scattered diffraction. The results indicate that there are good correlations between the Schmid factors or Taylor factors and the misorientation values of point to original point in grains near the triple junctions in a slightly deformed sample. Grains with higher Schmid factors or lower Taylor factors typically correspond to higher misorientation values near the triple junctions. In a subsequent annealing at 400 °C, both grain boundaries and triple junctions migrate, but the former leave ghost lines. During such migration, a grainmore » boundary grows from the grain with lower Schmid factor (higher Taylor factor) into the grain with higher Schmid factor (lower Taylor factor). Usually, the amount of migration of a grain boundary is considerably greater than that of a triple junction, and the grain boundary becomes more curved after migration. These observations indicate that the triple junctions have drag effects on grain boundary migration. - Highlights: • Polycrystalline aluminum with fine grains about 30 μm were used. • Off-line in situ EBSD was used to identify TJs before and after annealing. • Grains with higher SFs have higher misorientation values near TJs after deformation. • Grain boundaries grow from hard grains into soft grains during annealing. • Triple junctions have drag effects on grain boundaries migration.« less

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.

Polarization spectroscopy of the sodium dimer utilizing a triple-resonance technique in the presence of argon

NASA Astrophysics Data System (ADS)

Arndt, Phillip; Horton, Timothy; McFarland, Jacob; Bayram, Burcin; Miami University Spectroscopy Team

2015-05-01

The collisional dynamics of molecular sodium in the 61Σg electronic state is under investigation using a triple resonance technique in the presence of argon. A continuous wave ring dye laser is used to populate specific rovibrational levels of the A1Σu electronic state. A pump-probe technique is then employed where the pump laser populates the 61Σg state, and the probe laser dumps the population to the B1Σu state. From this level, fluorescence is detected as the system decays to the X1Σg state. We measure the polarization of this signal in the presence of various argon pressures. We will present our current work as well as the processes involved in the experiment. Financial support from the National Science Foundation (Grant No. NSF-PHY-1309571) is gratefully acknowledged.

Fabrication of triple-labeled polyelectrolyte microcapsules for localized ratiometric pH sensing.

PubMed

Song, Xiaoxue; Li, Huanbin; Tong, Weijun; Gao, Changyou

2014-02-15

Encapsulation of pH sensitive fluorophores as reporting molecules provides a powerful approach to visualize the transportation of multilayer capsules. In this study, two pH sensitive dyes (fluorescein and oregon green) and one pH insensitive dye (rhodamine B) were simultaneously labeled on the microcapsules to fabricate ratiometric pH sensors. The fluorescence of the triple-labeled microcapsule sensors was robust and nearly independent of other intracellular species. With a dynamic pH measurement range of 3.3-6.5, the microcapsules can report their localized pH at a real time. Cell culture experiments showed that the microcapsules could be internalized by RAW 246.7 cells naturally and finally accumulated in acidic organelles with a pH value of 5.08 ± 0.59 (mean ± s.d.; n=162). Copyright © 2013 Elsevier Inc. All rights reserved.

Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy

NASA Astrophysics Data System (ADS)

Su, Yu-Cheng; Burnouf, Pierre-Alain; Chuang, Kuo-Hsiang; Chen, Bing-Mae; Cheng, Tian-Lu; Roffler, Steve R.

2017-06-01

Triple-negative breast cancer (TNBC) lacks effective treatment options due to the absence of traditional therapeutic targets. The epidermal growth factor receptor (EGFR) has emerged as a promising target for TNBC therapy because it is overexpressed in about 50% of TNBC patients. Here we describe a PEG engager that simultaneously binds polyethylene glycol and EGFR to deliver PEGylated nanomedicines to EGFR+ TNBC. The PEG engager displays conditional internalization by remaining on the surface of TNBC cells until contact with PEGylated nanocarriers triggers rapid engulfment of nanocargos. PEG engager enhances the anti-proliferative activity of PEG-liposomal doxorubicin to EGFR+ TNBC cells by up to 100-fold with potency dependent on EGFR expression levels. The PEG engager significantly increases retention of fluorescent PEG probes and enhances the antitumour activity of PEGylated liposomal doxorubicin in human TNBC xenografts. PEG engagers with specificity for EGFR are promising for improved treatment of EGFR+ TNBC patients.

On universality of scaling law describing roughness of triple line.

PubMed

Bormashenko, Edward; Musin, Albina; Whyman, Gene; Barkay, Zahava; Zinigrad, Michael

2015-01-01

The fine structure of the three-phase (triple) line was studied for different liquids, various topographies of micro-rough substrates and various wetting regimes. Wetting of porous and pillar-based micro-scaled polymer surfaces was investigated. The triple line was visualized with the environmental scanning electron microscope and scanning electron microscope for the "frozen" triple lines. The value of the roughness exponent ζ for water (ice)/rough polymer systems was located within 0.55-0.63. For epoxy glue/rough polymer systems somewhat lower values of the exponent, 0.42 < ζ < 0.54, were established. The obtained values of ζ were close for the Cassie and Wenzel wetting regimes, different liquids, and different substrates' topographies. Thus, the above values of the exponent are to a great extent universal. The switch of the exponent, when the roughness size approaches to the correlation length of the defects, is also universal.

Interaction of thionine with triple-, double-, and single-stranded RNAs.

PubMed

Lozano, Héctor J; García, Begoña; Busto, Natalia; Leal, José M

2013-01-10

The interaction of thionine with triple, double, and single RNA helices has been fully characterized by thermodynamic and kinetic methods. The nature of the interaction of thionine with the synthetic polynucleotides poly(rU), poly(rA)·poly(rU), and poly(rA)·2poly(rU) has been studied at pH = 7.0 and 25 °C by UV absorbance, fluorescence, circular dichroism spectroscopy, viscometry, differential scanning calorimetry, and T-jump kinetic measurements. The results show that at I = 0.1 M thionine binds to a single poly(rU) strand, destabilizes the poly(rA)·2poly(rU) triplex by external binding, and intercalates into poly(rA)·poly(rU) with similar affinity to the thionine/DNA intercalated complex (Paul, P.; Kumar, G. S. J. Fluoresc. 2012, 22, 71-80). On the other hand, the differential scanning calorimetry measurements performed with thionine display a point in which the heat capacity remains unaltered, revealing the equilibrium of isothermal denaturation: thionine/poly(rA)·2poly(rU) + thionine ⇌ thionine/poly(rA)·poly(rU) + thionine/poly(rU), an outcome supported by the other techniques used. The denaturation equilibrium constant, K(D) (25 °C) = 522 M(-1), was evaluated from the affinity with the single, duplex, and triplex RNA.

MYC/BCL2/BCL6 triple hit lymphoma: a study of 40 patients with a comparison to MYC/BCL2 and MYC/BCL6 double hit lymphomas.

PubMed

Huang, Wenting; Medeiros, L Jeffrey; Lin, Pei; Wang, Wei; Tang, Guilin; Khoury, Joseph; Konoplev, Sergej; Yin, C Cameron; Xu, Jie; Oki, Yasuhiro; Li, Shaoying

2018-05-21

High-grade B-cell lymphomas with MYC, BCL2, and BCL6 rearrangements (triple hit lymphoma) are uncommon. We studied the clinicopathologic features of 40 patients with triple hit lymphoma and compared them to 157 patients with MYC/BCL2 double hit lymphoma and 13 patients with MYC/BCL6 double hit lymphoma. The triple hit lymphoma group included 25 men and 15 women with a median age of 61 years (range, 34-85). Nine patients had a history of B-cell lymphoma. Histologically, 23 (58%) cases were diffuse large B-cell lymphoma and 17 cases had features of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma. Most cases of triple hit lymphoma were positive for CD10 (100%), BCL2 (95%), BCL6 (82%), MYC (74%), and 71% with MYC and BCL2 coexpression. P53 was overexpressed in 29% of triple hit lymphoma cases. The clinicopathological features of triple hit lymphoma patients were similar to patients with MYC/BCL2 and MYC/BCL6 double hit lymphoma, except that triple hit lymphoma cases were more often CD10 positive compared with MYC/BCL6 double hit lymphoma (p < 0.05). Induction chemotherapy used was similar for patients with triple hit lymphoma and double hit lymphoma and overall survival in triple hit lymphoma patients was 17.6 months, similar to the overall survival of patients with double hit lymphoma (p = 0.67). Patients with triple hit lymphoma showing P53 overexpression had significantly worse overall survival compared with those without P53 overexpression (p = 0.04). On the other hand, double expressor status and prior history of B-cell lymphoma did not correlate with overall survival. In conclusion, most patients with triple hit lymphoma have an aggressive clinical course and poor prognosis and these tumors have a germinal center B-cell immunophenotype, similar to patients with double hit lymphomas. P53 expression is a poor prognostic factor in patients with triple hit lymphoma.

SHORT COMMUNICATION: Correlation between the Resistance Ratios of Platinum Resistance Thermometers at the Melting Point of Gallium and the Triple Point of Mercury

NASA Astrophysics Data System (ADS)

Singh, Y. P.; Maas, H.; Edler, F.; Zaidi, Z. H.

1994-01-01

A set of resistance ratios (W) for platinum resistance thermometers was obtained at the triple point of Hg and the melting point of Ga in order to study their relationship. It was found that using measured values for one of the fixed points, a linear equation will predict the value of the other. These measurements also indicate that the fixed points of Hg and of Ga are inconsistent by about 1,5 mK in the sense that either the melting point of Ga or the triple point of Hg was assigned too high a value on the ITS-90.

Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence

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

Jensen, P.K.; Lee, Cheng S.; King, J.A.

1997-12-31

The use of capillary electrophoresis equipped with laser-induced tryptophan fluorescence detection is presented for monitoring the refolding pathway of phage P22 tailspike endorhamnosidase. Upon initiation of refolding, tailspike polypeptides rapidly fold into structured monomeric intermediates with a high content of secondary structure. These monomeric species associate to form the triple-chain defined folding intermediates, the protrimers. Conversion of the protrimer into the native, sodium dodecyl sulfate (SDS) resistant tailspike protein is the rate-limiting step in the refolding pathway. Refolding kinetics and yield measured by capillary electrophoresis are in good agreement with those obtained via native gel electrophoresis, SDS polyacrylamide gel electrophoresismore » (SDS-PAGE) and fluorescence spectrophotometry. To enhance separation resolution between protrimer and native protein in capillary electrophoresis, the use of poly(ethylene oxide) is investigated for the introduction of a sieving separation mechanism. The increased viscosity of the electrophoresis buffer may also play a role in resolution enhancement.« less

Hybrid Correlation Energy (HyCE): An Approach Based on Separate Evaluations of Internal and External Components.

PubMed

Ivanic, Joseph; Schmidt, Michael W

2018-06-04

A novel hybrid correlation energy (HyCE) approach is proposed that determines the total correlation energy via distinct computation of its internal and external components. This approach evolved from two related studies. First, rigorous assessment of the accuracies and size extensivities of a number of electron correlation methods, that include perturbation theory (PT2), coupled-cluster (CC), configuration interaction (CI), and coupled electron pair approximation (CEPA), shows that the CEPA(0) variant of the latter and triples-corrected CC methods consistently perform very similarly. These findings were obtained by comparison to near full CI results for four small molecules and by charting recovered correlation energies for six steadily growing chain systems. Second, by generating valence virtual orbitals (VVOs) and utilizing the CEPA(0) method, we were able to partition total correlation energies into internal (or nondynamic) and external (or dynamic) parts for the aforementioned six chain systems and a benchmark test bed of 36 molecules. When using triple-ζ basis sets it was found that per orbital internal correlation energies were appreciably larger than per orbital external energies and that the former showed far more chemical variation than the latter. Additionally, accumulations of external correlation energies were seen to proceed smoothly, and somewhat linearly, as the virtual space is gradually increased. Combination of these two studies led to development of the HyCE approach, whereby the internal and external correlation energies are determined separately by CEPA(0)/VVO and PT2/external calculations, respectively. When applied to the six chain systems and the 36-molecule benchmark test set it was found that HyCE energies followed closely those of triples-corrected CC and CEPA(0) while easily outperforming MP2 and CCSD. The success of the HyCE approach is more notable when considering that its cost is only slightly more than MP2 and significantly cheaper than the CC approaches.

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.

Fluorescence lidar measurements at the archaeological site House of Augustus at Palatino, Rome

NASA Astrophysics Data System (ADS)

Raimondi, Valentina; Alisi, Chiara; Barup, Kerstin; Bracciale, Maria Paola; Broggi, Alessandra; Conti, Cinzia; Hällström, Jenny; Lognoli, David; Palombi, Lorenzo; Santarelli, Maria Laura; Sprocati, Anna Rosa

2013-10-01

Early diagnostics and documentation fulfill an essential role for an effective planning of conservation and restoration of cultural heritage assets. In particular, remote sensing techniques that do not require the use of scaffolds or lifts, such as fluoresence lidar, can provide useful information to obtain an overall assessment of the status of the investigated surfaces and can be exploited to address analytical studies in selected areas. Here we present the results of a joint Italian-Swedish project focused on documenting and recording the status of some sections of the part closed to the public by using fluorescence hyperspectral imaging lidar. The lidar used a tripled-frequency Nd:YAG laser emitting at 355 nm as excitation source and an intensified, gated 512x512-pixel CCD as detector. The lidar had imaging capabilities thanks to a computer-controlled scanning mirror. The fluorescence characteristics of fresco wall paintings were compared to those of fresco fragments found at the same archaeological site and separately examined in the lab using FT-IR and Raman techniques for the identification of pigments. The fluorescence lidar was also used to remotely detect the growth of phototrophic biodeteriogens on the walls. The fluorescence lidar data were compared with results from biological sampling, cultivation and laboratory analysis by molecular techniques.

Relationship between bony tunnel and knee function in patients after patellar dislocation triple surgeries—a CT-based study

NASA Astrophysics Data System (ADS)

Qin, Le; Li, Mei; Yao, Weiwu; Shen, Ji

2017-01-01

We aimed to assess the CT-based bony tunnel valuations and their correlation with knee function after patellar dislocation triple surgeries. A retrospective study was performed on 66 patients (70 knees) who underwent patellar dislocation triple surgeries. The surgery was MPFL reconstruction primarily, combined with lateral retinaculum release and tibial tubercle osteotomy. CT examinations were performed to determine the femoral tunnel position, along with the patellar and femoral tunnel width 3 days and more than 1 year after operation for follow-up. Functional evaluation based on Kujala and Lysholm scores was also implemented. We compared tunnel width of the first and last examinations and correlated femoral tunnel position of the last examination with knee function. At the last follow-up, femoral tunnel position in the anterior-posterior direction was moderately correlated with knee function. Femoral tunnel position in the proximal-distal direction was not associated with postoperative knee function. Patellar and femoral tunnel width increased significantly at the last follow-up. However, no significant functional difference was found between patients with and without femoral tunnel enlargement. Our results suggested that the tunnel malposition in anterior-posterior position based on CT was related to impaired knee function during the follow-ups.

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.

An investigation of the viscoelastic properties and the actin cytoskeletal structure of triple negative breast cancer cells.

PubMed

Hu, Jingjie; Zhou, Yuxiao; Obayemi, John D; Du, Jing; Soboyejo, Winston O

2018-05-30

An improved understanding of the evolution of cell structure and viscoelasticity with cancer malignancy could enable the development of a new generation of biomarkers and methods for cancer diagnosis. Hence, in this study, we present the viscoelastic properties (moduli and viscosities) and the actin cytoskeletal structures of triple negative breast cancer (TNBC) cells with different metastatic potential. These include: MCF-10A normal breast cells (studied as a control); MDA-MB-468 cells (less metastatic TNBC cells), and MDA-MB-231 cells (highly metastatic TNBC cells). A combination of shear assay and digital imaging correlation (DIC) techniques is used to measure the local viscoelastic properties of live breast cells subjected to constant shear stress. The local moduli and viscosities of the nuclei and cytoplasm are characterized using a generalized Maxwell model, which is used to determine the time-dependent creep responses of cells. The nuclei are shown to be stiffer and more viscous than the cytoplasms of the normal breast cells and TNBC cells. The MCF-10A normal breast cells are found to be twice as stiff as the less metastatic MDA-MB-468 breast cancer cells and over ten times stiffer than the highly metastatic MDA-MB-231 breast cancer cells. Similar trends are also observed in the viscosities of the nuclei and the cytoplasms. The measured differences in cell viscoelastic properties are also associated with significant changes in the cell cytoskeletal structure, which is studied using confocal fluorescence microscopy. This reveals significant differences in the levels of actin expression and organization in TNBC cells as they become highly metastatic. Our results suggest that the shear assay measurements of cell viscoelastic properties may be used as effective biomarkers for TNBC diagnosis and screening. Copyright © 2018 Elsevier Ltd. All rights reserved.

Levels of DNA Methylation Vary at CpG Sites across the BRCA1 Promoter, and Differ According to Triple Negative and "BRCA-Like" Status, in Both Blood and Tumour DNA.

PubMed

Daniels, Sarah L; Burghel, George J; Chambers, Philip; Al-Baba, Shadi; Connley, Daniel D; Brock, Ian W; Cramp, Helen E; Dotsenko, Olena; Wilks, Octavia; Wyld, Lynda; Cross, Simon S; Cox, Angela

2016-01-01

Triple negative breast cancer is typically an aggressive and difficult to treat subtype. It is often associated with loss of function of the BRCA1 gene, either through mutation, loss of heterozygosity or methylation. This study aimed to measure methylation of the BRCA1 gene promoter at individual CpG sites in blood, tumour and normal breast tissue, to assess whether levels were correlated between different tissues, and with triple negative receptor status, histopathological scoring for BRCA-like features and BRCA1 protein expression. Blood DNA methylation levels were significantly correlated with tumour methylation at 9 of 11 CpG sites examined (p<0.0007). The levels of tumour DNA methylation were significantly higher in triple negative tumours, and in tumours with high BRCA-like histopathological scores (10 of 11 CpG sites; p<0.01 and p<0.007 respectively). Similar results were observed in blood DNA (6 of 11 CpG sites; p<0.03 and 7 of 11 CpG sites; p<0.02 respectively). This study provides insight into the pattern of CpG methylation across the BRCA1 promoter, and supports previous studies suggesting that tumours with BRCA1 promoter methylation have similar features to those with BRCA1 mutations, and therefore may be suitable for the same targeted therapies.

A study of the second and third order closure models of turbulence for prediction of separated shear flows

NASA Technical Reports Server (NTRS)

Amano, R. S.

1985-01-01

The hybrid model of the Reynolds-stress turbulence closure is tested for the computation of the flows over a step and disk. Here it is attempted to improve the redistributive action of the turbulence energy among the Reynolds stresses. By evaluating the existing models for the pressure-strain correlation, better coefficients are obtained for the prediction of separating shear flows. Furthermore, the diffusion rate of the Reynolds stresses is reevaluated adopting several algebraic correlations for the triple-velocity products. The models of Cormack et al., Daly-Harlow, Hanjalic-Launder, and Shir were tested for the reattaching shear flows. It was generally observed that all these algebraic models give considerably low values of the triple-velocity products. This is attributed to the fact that none of the algebraic models can take the convective effect of the triple-velocity products into account in the separating shear flows, thus resulting in much lower diffusion rate than Reynolds stresses. In order to improve the evaluation of these quantities correction factors are introduced based on the comparison with some experimental data.

Readily functionalized AAA-DDD triply hydrogen-bonded motifs.

PubMed

Tong, Feng; Linares-Mendez, Iamnica J; Han, Yi-Fei; Wisner, James A; Wang, Hong-Bo

2018-04-25

Herein we present a new, readily functionalized AAA-DDD hydrogen bond array. A novel AAA monomeric unit (3a-b) was obtained from a two-step synthetic procedure starting with 2-aminonicotinaldehyde via microwave radiation (overall yield of 52-66%). 1H NMR and fluorescence spectroscopy confirmed the complexation event with a calculated association constant of 1.57 × 107 M-1. Likewise, the usefulness of this triple hydrogen bond motif in supramolecular polymerization was demonstrated through viscosity measurements in a crosslinked supramolecular alternating copolymer.

Triple Resonance Solid State NMR Experiments with Reduced Dimensionality Evolution Periods

NASA Astrophysics Data System (ADS)

Astrof, Nathan S.; Lyon, Charles E.; Griffin, Robert G.

2001-10-01

Two solid state NMR triple resonance experiments which utilize the simultaneous incrementation of two chemical shift evolution periods to obtain a spectrum with reduced dimensionality are described. The CON CA experiment establishes the correlation of 13Ci-1 to 13Cαi and 15Ni by simultaneously encoding the 13COi-1 and 15Ni chemical shifts. The CAN COCA experiment establishes the correlation 13Cai and 15COi to 13Cαi-1 and 15Ni-1 within a single experiment by simultaneous encoding of the 13Cαi and 15Ni chemical shifts. This experiment establishes sequential amino acid correlations in close analogy to the solution state HNCA experiment. Reduced dimensionality 2D experiments are a practical alternative to recording multiple 3D data sets for the purpose of obtaining sequence-specific resonance assignments of peptides and proteins in the solid state.

Anti-E1E2 antibodies do predict response to triple therapy in treatment-experienced Hepatitis C Virus-cirrhosis cases.

PubMed

Petit, Marie-Anne; Berthillon, Pascale; Pradat, Pierre; Arnaud, Clémence; Bordes, Isabelle; Virlogeux, Victor; Maynard, Marianne; Bailly, François; Zoulim, Fabien; Chemin, Isabelle; Trépo, Christian

2015-12-01

We previously showed that pre-treatment serum anti-E1E2 predicted hepatitis C virus (HCV) RNA viral kinetics (VKs) and treatment outcome in patients with chronic hepatitis C receiving pegylated interferon/ribavirin (Peg-IFN/RBV) double therapy. Here, we determined whether baseline anti-E1E2 was correlated with the on-treatment VK and could predict virological outcome in treatment-experienced HCV-infected cirrhotic patients receiving protease inhibitor-based triple therapy. Sera from 19 patients with HCV genotype 1 infection and compensated cirrhosis who failed to respond to a prior course of Peg-IFN/RBV were selected at time 0 before starting triple therapy with boceprevir or telaprevir. We assessed patients with sustained viral response 12 weeks after the end of triple therapy (SVR12) by analyzing VKs at weeks 4, 12, 24, 36, 48 (end of treatment) and 60. Patients baseline characteristics were similar to the well-defined CUPIC cohort (age, HCV subtype, baseline viremia, and treatment history). Among the 19 patients, 11 achieved an SVR12. Fifteen patients were positive for pre-treatment anti-E1E2 and all of them achieved SVR12. Moreover, anti-E1E2 and SVR12 correlated with prior response to IFN/RBV therapy (relapse, partial or null response). Baseline anti-E1E2 could be considered as a new biomarker to predict SVR12 after triple therapy in this most difficult-to-treat population. These results warrant further validation on larger cohorts including patients receiving highly effective direct-acting antivirals to explore whether this test could help in better defining treatment duration for these very costly molecules. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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.

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

PubMed

Lamb, D C; Müller, B K; Bräuchle, C

2005-10-01

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

Triple Gene Analysis Using Samples Obtained by Endobronchial Ultrasound-guided Transbronchial Needle Aspiration

PubMed Central

Lee, Kyungjong; Um, Sang-Won; Jeong, Byeong-Ho; Yang, Jung Wook; Choi, Yoon-La; Han, Joungho; Kim, Hojoong; Kwon, O Jung

2016-01-01

Objective A mutational analysis of tumor tissue samples is an important part of advanced lung cancer treatment strategies. This study evaluated the efficacy of a triple gene analysis using samples obtained via endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). Methods Either metastatic lymph nodes or primary lung mass samples obtained by EBUS-TBNA were collected between May 2011 and May 2013. We consecutively analyzed epidermal growth factor receptor (EGFR), V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), and anaplastic lymphoma kinase (ALK) fusion genes using remnant tissue samples. Results A total of 109 patients were diagnosed with non-small cell lung cancer (NSCLC). Of these, 70% were adenocarcinoma, 27% squamous cell carcinoma with NSCLC, and 3% were related to other types of lung cancer. EGFR mutations were detected in 23 cases (21.1%), KRAS mutations in 13 cases (11.9%), and ALK fusion genes in 5 cases (4.9%). The ALK fusion genes could not be analyzed in four cases because of insufficient tissue samples remaining after routine histochemistry and an EGFR/KRAS mutation analysis. We found that small biopsy samples from EBUS-TBNA were adequate for performing a triple gene analysis in 97 patients (96%). ALK fusion protein immunohistochemistry (IHC) was 100% consistent with fluorescence in situ hybridization (FISH). Conclusion Small samples obtained by EBUS-TBNA were found to be sufficient for performing a triple gene analysis following routine histology and IHC. ALK IHC showed a very good concordance with FISH for detecting ALK fusion genes. PMID:27803402

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.

Transition from slab to slabless: Results from the 1993 Mendocino triple junction seismic experiment

USGS Publications Warehouse

Beaudoin, B.C.; Godfrey, N.J.; Klemperer, S.L.; Lendl, C.; Trehu, A.M.; Henstock, T.J.; Levander, A.; Holl, J.E.; Meltzer, A.S.; Luetgert, J.H.; Mooney, W.D.

1996-01-01

Three seismic refraction-reflection profiles, part of the Mendocino triple junction seismic experiment, allow us to compare and contrast crust and upper mantle of the North American margin before and after it is modified by passage of the Mendocino triple junction. Upper crustal velocity models reveal an asymmetric Great Valley basin overlying Sierran or ophiolitic rocks at the latitude of Fort Bragg, California, and overlying Sierran or Klamath rocks near Redding, California. In addition, the upper crustal velocity structure indicates that Franciscan rocks underlie the Klamath terrane east of Eureka, California. The Franciscan complex is, on average, laterally homogeneous and is thickest in the triple junction region. North of the triple junction, the Gorda slab can be traced 150 km inboard from the Cascadia subduction zone. South of the triple junction, strong precritical reflections indicate partial melt and/or metamorphic fluids at the base of the crust or in the upper mantle. Breaks in these reflections are correlated with the Maacama and Bartlett Springs faults, suggesting that these faults extend at least to the mantle. We interpret our data to indicate tectonic thickening of the Franciscan complex in response to passage of the Mendocino triple junction and an associated thinning of these rocks south of the triple junction due to assimilation into melt triggered by upwelling asthenosphere. The region of thickened Franciscan complex overlies a zone of increased scattering, intrinsic attenuation, or both, resulting from mechanical mixing of lithologies and/or partial melt beneath the onshore projection of the Mendocino fracture zone. Our data reveal that we have crossed the southern edge of the Gorda slab and that this edge and/or the overlying North American crust may have fragmented because of the change in stress presented by the edge.

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.

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.

Activatable albumin-photosensitizer nanoassemblies for triple-modal imaging and thermal-modulated photodynamic therapy of cancer.

PubMed

Hu, Dehong; Sheng, Zonghai; Gao, Guanhui; Siu, Fungming; Liu, Chengbo; Wan, Qian; Gong, Ping; Zheng, Hairong; Ma, Yifan; Cai, Lintao

2016-07-01

Photodynamic therapy (PDT) is a noninvasive and effective approach for cancer treatment. The main bottlenecks of clinical PDT are poor selectivity of photosensitizer and inadequate oxygen supply resulting in serious side effects and low therapeutic efficiency. Herein, a thermal-modulated reactive oxygen species (ROS) strategy using activatable human serum albumin-chlorin e6 nanoassemblies (HSA-Ce6 NAs) for promoting PDT against cancer is developed. Through intermolecular disulfide bond crosslinking and hydrophobic interaction, Ce6 photosensitizer is effectively loaded into the HSA NAs, and the obtained HSA-Ce6 NAs exhibit excellent reduction response, as well as enhanced tumor accumulation and retention. By the precision control of the overall body temperature instead of local tumor temperature increasing from 37 °C to 43 °C, the photosensitization reaction rate of HSA-Ce6 NAs increases 20%, and the oxygen saturation of tumor tissue raise 52%, significantly enhancing the generation of ROS for promoting PDT. Meanwhile, the intrinsic fluorescence and photoacoustic properties, and the chelating characteristic of porphyrin ring can endow the HSA-Ce6 NAs with fluorescence, photoacoustic and magnetic resonance triple-modal imaging functions. Upon irradiation of low-energy near-infrared laser, the tumors are completely suppressed without tumor recurrence and therapy-induced side effects. The robust thermal-modulated ROS strategy combined with albumin-based activatable nanophotosensitizer is highly potential for multi-modal imaging-guided PDT and clinical translation. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Levels of DNA Methylation Vary at CpG Sites across the BRCA1 Promoter, and Differ According to Triple Negative and “BRCA-Like” Status, in Both Blood and Tumour DNA

PubMed Central

Burghel, George J.; Chambers, Philip; Al-Baba, Shadi; Connley, Daniel D.; Brock, Ian W.; Cramp, Helen E.; Dotsenko, Olena; Wilks, Octavia; Wyld, Lynda; Cross, Simon S.; Cox, Angela

2016-01-01

Triple negative breast cancer is typically an aggressive and difficult to treat subtype. It is often associated with loss of function of the BRCA1 gene, either through mutation, loss of heterozygosity or methylation. This study aimed to measure methylation of the BRCA1 gene promoter at individual CpG sites in blood, tumour and normal breast tissue, to assess whether levels were correlated between different tissues, and with triple negative receptor status, histopathological scoring for BRCA-like features and BRCA1 protein expression. Blood DNA methylation levels were significantly correlated with tumour methylation at 9 of 11 CpG sites examined (p<0.0007). The levels of tumour DNA methylation were significantly higher in triple negative tumours, and in tumours with high BRCA-like histopathological scores (10 of 11 CpG sites; p<0.01 and p<0.007 respectively). Similar results were observed in blood DNA (6 of 11 CpG sites; p<0.03 and 7 of 11 CpG sites; p<0.02 respectively). This study provides insight into the pattern of CpG methylation across the BRCA1 promoter, and supports previous studies suggesting that tumours with BRCA1 promoter methylation have similar features to those with BRCA1 mutations, and therefore may be suitable for the same targeted therapies. PMID:27463681

Triple-helix molecular switch-based aptasensors and DNA sensors.

PubMed

Bagheri, Elnaz; Abnous, Khalil; Alibolandi, Mona; Ramezani, Mohammad; Taghdisi, Seyed Mohammad

2018-07-15

Utilization of traditional analytical techniques is limited because they are generally time-consuming and require high consumption of reagents, complicated sample preparation and expensive equipment. Therefore, it is of great interest to achieve sensitive, rapid and simple detection methods. It is believed that nucleic acids assays, especially aptamers, are very important in modern life sciences for target detection and biological analysis. Aptamers and DNA-based sensors have been widely used for the design of various sensors owing to their unique features. In recent years, triple-helix molecular switch (THMS)-based aptasensors and DNA sensors have been broadly utilized for the detection and analysis of different targets. The THMS relies on the formation of DNA triplex via Watson-Crick and Hoogsteen base pairings under optimal conditions. This review focuses on recent progresses in the development and applications of electrochemical, colorimetric, fluorescence and SERS aptasensors and DNA sensors, which are based on THMS. Also, the advantages and drawbacks of these methods are discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

Efficacy of assessing circulating cell-free DNA using a simple fluorescence assay in patients with triple-negative breast cancer receiving neoadjuvant chemotherapy: a prospective observational study

PubMed Central

Park, Kwonoh; Woo, Miyoung; Kim, Jeong Eun; Ahn, Jin-Hee; Jung, Kyung Hae; Roh, Jin; Gong, Gyungyub; Kim, Sung-Bae

2018-01-01

This study aims to assess cell-free DNA (CFD) by a fluorescence assay as a biomarker for early prediction of a pathologic complete response (pCR) and relapse in patients with triple-negative breast cancer (TNBC) undergoing neoadjuvant chemotherapy. Patients with clinical stage II or III TNBC scheduled for neoadjuvant chemotherapy were prospectively enrolled. All patients underwent four cycles of Adriamycin plus cyclophosphamide (AC), followed by four cycles of cisplatin or docetaxel chemotherapy and surgery. Blood samples were obtained before the initial chemotherapy (baseline-CFD) and after four AC neoadjuvant chemotherapy cycles (AC-CFD) to evaluate CFD levels. In total, 72 patients who met the inclusion criteria were enrolled. The mean baseline-CFD and AC-CFD levels were 239 ± 68 and 210 ± 66 ng/mL, respectively, with a significant decline in the CFD levels after AC neoadjuvant chemotherapy (P = 0.001). In the 33.6-month median follow-up, 18 cases of relapse were reported. A ROC curve analysis of baseline-CFD was performed to determine the predictive value for relapse, and an area under the curve of 0.62 (95% CI, 0.46–0.78) at 264 ng/mL was obtained. Patients with baseline-CFD >264 ng/mL were at a higher risk of relapse than those with baseline-CFD ≤264 ng/mL (HR, 2.84; 95% CI, 1.11–7.24; P = 0.029). Multivariate analysis established baseline-CFD as an independent predicting factor for relapse (HR, 3.74; 95% CI, 1.32–10.53; P = 0.013). In conclusion, baseline-CFD measured by a fluorescence assay might be a potential biomarker to predict relapse, which could be useful for risk stratification of TNBC. PMID:29423090

UV laser interaction with a fluorescent dye solution studied using pulsed digital holography.

PubMed

Amer, Eynas; Gren, Per; Sjödahl, Mikael

2013-10-21

A frequency tripled Q-switched Nd-YAG laser (wavelength 355 nm, pulse duration 12 ns) has been used to pump Coumarin 153 dye solved in ethanol. Simultaneously, a frequency doubled pulse (532 nm) from the same laser is used to probe the solvent perpendicularly resulting in a gain through stimulated laser induced fluorescence (LIF) emission. The resulting gain of the probe beam is recorded using digital holography by blending it with a reference beam on the detector. Two digital holograms without and with the pump beam were recorded. Intensity maps were calculated from the recorded digital holograms and used to calculate the gain of the probe beam due to the stimulated LIF. In addition numerical data of the local temperature rise was calculated from the corresponding phase maps using Radon inversion. It was concluded that about 15% of the pump beam energy is transferred to the dye solution as heat while the rest is consumed in the radiative process. The results show that pulsed digital holography is a promising technique for quantitative study of fluorescent species.

TNK2 Tyrosine Kinase as a Novel Therapeutic Target in Triple-Negative Breast Cancer

DTIC Science & Technology

2017-10-01

performed global phosphotyrosine profiling for a panel of 25 TNBC cell lines. When we correlated protein phosphorylation levels with cellular oncogenic...levels and activation correlate with clinical and pathological features of TNBC? Aim 2: What is the value of TNK2 as a therapeutic target in vitro and

A Computational Study on the Ground and Excited States of Nickel Silicide.

PubMed

Schoendorff, George; Morris, Alexis R; Hu, Emily D; Wilson, Angela K

2015-09-17

Nickel silicide has been studied with a range of computational methods to determine the nature of the Ni-Si bond. Additionally, the physical effects that need to be addressed within calculations to predict the equilibrium bond length and bond dissociation energy within experimental error have been determined. The ground state is predicted to be a (1)Σ(+) state with a bond order of 2.41 corresponding to a triple bond with weak π bonds. It is shown that calculation of the ground state equilibrium geometry requires a polarized basis set and treatment of dynamic correlation including up to triple excitations with CR-CCSD(T)L resulting in an equilibrium bond length of only 0.012 Å shorter than the experimental bond length. Previous calculations of the bond dissociation energy resulted in energies that were only 34.8% to 76.5% of the experimental bond dissociation energy. It is shown here that use of polarized basis sets, treatment of triple excitations, correlation of the valence and subvalence electrons, and a Λ coupled cluster approach is required to obtain a bond dissociation energy that deviates as little as 1% from experiment.

The Role of Novel Substituted Diindolyl Methane Analogues in the Treatment of Triple Negative and ErbB2 Positive Breast Cancer

DTIC Science & Technology

2017-07-01

volume compared to control group . DPL= DIM Liposomes, CDPL: CREKA peptide conjugated DIM liposomes. Collaboration and training: a) Several post...treated with 20 mM DIM-C-pPhCO2Me for 24 h and the number of cells were then counted. The control (untreated) groups (set at 100%) in studies on the...cells was determined by the amount of green fluorescence observed in the treatment groups relative and normalized to the control group . Western blot

Characterization of humic acids by two-dimensional correlation fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Nakashima, K.; Xing, Shaoyong; Gong, Yongkuan; Miyajima, Toru

2008-07-01

We have investigated interaction between humic acids and heavy metal ions by fluorescence spectroscopy. The humic acids examined are Aldrich humic acid (AHA) and Dando humic acid (DHA), and heavy metal ions are Cu 2+ and Pb 2+. The binding constants between the humic acids and the heavy metal ions are obtained by a conventional fluorescence quenching technique. The two prominent bands in the fluorescence spectra of the humic acids give different binding constants, implying that the two bands are originated from different fluorescent species in the matrices of the humic acids. This was confirmed by two-dimensional correlation analysis based on the quenching perturbation on the fluorescence spectra. Two prominent cross peaks corresponding to the two fluorescence bands are obtained in the asynchronous maps, indicating that the two fluorescence bands belong to different species. The order of the response of the two fluorescence bands to the quenching perturbation is also elucidated based on Noda's rule.

New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.

PubMed

Malcova, Ivana; Farkasovsky, Marian; Senohrabkova, Lenka; Vasicova, Pavla; Hasek, Jiri

2016-05-01

Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Reference Correlation of the Thermal Conductivity of Carbon Dioxide from the Triple Point to 1100 K and up to 200 MPa

PubMed Central

Huber, M. L.; Sykioti, E. A.; Assael, M. J.; Perkins, R. A.

2016-01-01

This paper contains new, representative reference equations for the thermal conductivity of carbon dioxide. The equations are based in part upon a body of experimental data that has been critically assessed for internal consistency and for agreement with theory whenever possible. In the case of the dilute-gas thermal conductivity, we incorporated recent theoretical calculations to extend the temperature range of the experimental data. Moreover, in the critical region, the experimentally observed enhancement of the thermal conductivity is well represented by theoretically based equations containing just one adjustable parameter. The correlations are applicable for the temperature range from the triple point to 1100 K and pressures up to 200 MPa. The overall uncertainty (at the 95% confidence level) of the proposed correlation varies depending on the state point from a low of 1% at very low pressures below 0.1 MPa between 300 K and 700 K, to 5% at the higher pressures of the range of validity. PMID:27064300

Loss of PTEN expression is associated with aggressive behavior and poor prognosis in Middle Eastern triple-negative breast cancer.

PubMed

Beg, Shaham; Siraj, Abdul K; Prabhakaran, Sarita; Jehan, Zeenath; Ajarim, Dahish; Al-Dayel, Fouad; Tulbah, Asma; Al-Kuraya, Khawla S

2015-06-01

PTEN is a tumor suppressor that negatively regulates the PI3 K-AKT signaling pathway which is involved in the pathogenesis of many different tumor types and serves as a prognostic marker in breast cancer. However, the significance of the role of PTEN in Middle Eastern ethnic breast cancer has not been explored especially with the fact that breast cancer originating from this ethnic population tend to behave more aggressively than breast cancer in the west. In this study, we analyzed PTEN alteration in a tissue microarray format containing more than 1000 primary breast cancers with clinical follow-up data. Tissue Microarray sections were analyzed for protein expression and copy number change using immunohistochemistry and fluorescence in situ hybridization. Loss of PTEN immunostaining was observed in 77 % of the cases. PTEN loss was significantly associated with large tumor size (p = 0.0030), high grade (p = 0.0281), tumor recurrence (p = 0.0333), and triple-negative breast cancers (p = 0.0086). PTEN loss in triple-negative breast cancers was significantly associated with rapid tumor cell proliferation (p = 0.0396) and poor prognosis (p = 0.0408). PTEN deletion was found only in 60 cases (6.4 %). Loss of PTEN protein expression occurs at high frequency in Middle Eastern breast cancer. PTEN inactivation may potentially lead to an aggressive behavior of tumor cells through stimulation of tumor cell proliferation. Furthermore, PTEN signaling pathway might be used as potential therapeutic target in triple-negative breast cancers since loss of its expression is shown to be significantly associated with this aggressive subtype of breast cancer.

Clinicopathology Figures and Long-term Effects of Tamoxifen Plus Radiation on Survival of Women with Invasive Ductal Carcinoma and Triple Negative Breast Cancer.

PubMed

Payandeh, Mehrdad; Sadeghi, Masoud; Sadeghi, Edris; Aeinfar, Mehrnoush

2015-01-01

Triple negative breast cancer (TNBC), characterized as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 Her2 negative and accounting for 10-17% of all breast carcinomas, is only partially responsive to chemotherapy and suffers from a lack of clinically established targeted therapies. The aim of the current study was to evaluate the patterns of treatment and clinicopathology figures in Kurdish patients with triple-negative breast cancer, and to compare these to other reports. Between 2001 and 2014, 950 breast cancer patients were referred to our clinic. There were 74 female patients with TNBC, including 70 patients was invasive ductal carcinoma entered into our study. ER and PR positivity was defined as positive immunohistochemical staining in more than 10% of tumor cells. Immunohistochemistry assay with anti-HER2 antibodies was used to identify HER negative (0 and 1+) or positive (2+ and 3+). HER2 gene amplification was determined by fluorescent in situ hybridization (FISH). Overall survival (OS) was plotted with GraphPad Prism 5 Software using Kaplan-Meier and log-rank tests for comparison of results. The mean age in the first diagnosis for 70 patients with triple TNBC and invasive ductal carcinoma was 49.6 years that range of age was 27-82 years. All of the patients were female. Of 70 patients, 23 patients had metastasis. Thirty-two patients (45.7%) were treated with tamoxifen and 39 (55.7%) with radiotherapy. Three-year, 5-year and 10-year OS rates for all patients were 82%, 72% and 64%, respectively. The OS in our West Iran TNBC patients is less than reported elsewhere. However, treatment with combination of tamoxifen plus radiation increases the OS and reduces the mortality rate.

Modeling of nanotherapeutics delivery based on tumor perfusion

PubMed Central

van de Ven, Anne L.; Abdollahi, Behnaz; Martinez, Carlos J.; Burey, Lacey A.; Landis, Melissa D.; Chang, Jenny C.; Ferrari, Mauro; Frieboes, Hermann B.

2013-01-01

Heterogeneities in the perfusion of solid tumors prevent optimal delivery of nanotherapeutics. Clinical imaging protocols to obtain patient-specific data have proven difficult to implement. It is challenging to determine which perfusion features hold greater prognostic value and to relate measurements to vessel structure and function. With the advent of systemically administered nanotherapeutics, whose delivery is dependent on overcoming diffusive and convective barriers to transport, such knowledge is increasingly important. We describe a framework for the automated evaluation of vascular perfusion curves measured at the single vessel level. Primary tumor fragments, collected from triple-negative breast cancer patients and grown as xenografts in mice, were injected with fluorescence contrast and monitored using intravital microscopy. The time to arterial peak and venous delay, two features whose probability distributions were measured directly from time-series curves, were analyzed using a Fuzzy C-mean (FCM) supervised classifier in order to rank individual tumors according to their perfusion characteristics. The resulting rankings correlated inversely with experimental nanoparticle accumulation measurements, enabling modeling of nanotherapeutics delivery without requiring any underlying assumptions about tissue structure or function, or heterogeneities contained within. With additional calibration, these methodologies may enable the study of nanotherapeutics delivery strategies in a variety of tumor models. PMID:24039540

Modeling of nanotherapeutics delivery based on tumor perfusion

NASA Astrophysics Data System (ADS)

van de Ven, Anne L.; Abdollahi, Behnaz; Martinez, Carlos J.; Burey, Lacey A.; Landis, Melissa D.; Chang, Jenny C.; Ferrari, Mauro; Frieboes, Hermann B.

2013-05-01

Heterogeneities in the perfusion of solid tumors prevent optimal delivery of nanotherapeutics. Clinical imaging protocols for obtaining patient-specific data have proven difficult to implement. It is challenging to determine which perfusion features hold greater prognostic value and to relate measurements to vessel structure and function. With the advent of systemically administered nanotherapeutics whose delivery is dependent on overcoming diffusive and convective barriers to transport, such knowledge is increasingly important. We describe a framework for the automated evaluation of vascular perfusion curves measured at the single vessel level. Primary tumor fragments, collected from triple-negative breast cancer patients and grown as xenografts in mice, were injected with fluorescence contrast and monitored using intravital microscopy. The time to arterial peak and venous delay, two features whose probability distributions were measured directly from time-series curves, were analyzed using a fuzzy c-mean supervised classifier in order to rank individual tumors according to their perfusion characteristics. The resulting rankings correlated inversely with experimental nanoparticle accumulation measurements, enabling the modeling of nanotherapeutics delivery without requiring any underlying assumptions about tissue structure or function, or heterogeneities contained therein. With additional calibration, these methodologies may enable the investigation of nanotherapeutics delivery strategies in a variety of tumor models.

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

PubMed

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

2005-05-01

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

Auxiliary basis sets for density-fitting second-order Møller-Plesset perturbation theory: weighted core-valence correlation consistent basis sets for the 4d elements Y-Pd.

PubMed

Hill, J Grant

2013-09-30

Auxiliary basis sets (ABS) specifically matched to the cc-pwCVnZ-PP and aug-cc-pwCVnZ-PP orbital basis sets (OBS) have been developed and optimized for the 4d elements Y-Pd at the second-order Møller-Plesset perturbation theory level. Calculation of the core-valence electron correlation energies for small to medium sized transition metal complexes demonstrates that the error due to the use of these new sets in density fitting is three to four orders of magnitude smaller than that due to the OBS incompleteness, and hence is considered negligible. Utilizing the ABSs in the resolution-of-the-identity component of explicitly correlated calculations is also investigated, where it is shown that i-type functions are important to produce well-controlled errors in both integrals and correlation energy. Benchmarking at the explicitly correlated coupled cluster with single, double, and perturbative triple excitations level indicates impressive convergence with respect to basis set size for the spectroscopic constants of 4d monofluorides; explicitly correlated double-ζ calculations produce results close to conventional quadruple-ζ, and triple-ζ is within chemical accuracy of the complete basis set limit. Copyright © 2013 Wiley Periodicals, Inc.

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.

A comparative study of the adhesion of biosynthesized gold and conjugated gold/prodigiosin nanoparticles to triple negative breast cancer cells.

PubMed

Dozie-Nwachukwu, S O; Obayemi, J D; Danyuo, Y; Anuku, N; Odusanya, O S; Malatesta, K; Soboyejo, W O

2017-08-17

This paper explores the adhesion of biosynthesized gold nanoparticles (AuNPs) and gold (Au) nanoparticle/prodigiosin (PG) drug nanoparticles to breast cancer cells (MDA-MB-231 cells). The AuNPs were synthesized in a record time (less than 30 s) from Nauclea latifolia leaf extracts, while the PG was produced via bacterial synthesis with Serratia marcescens sp. The size distributions and shapes of the resulting AuNPs were characterized using transmission electron microscopy (TEM), while the resulting hydrodynamic diameters and polydispersity indices were studied using dynamic light scattering (DLS). Atomic Force Microscopy (AFM) was used to study the adhesion between the synthesized gold nanoparticles (AuNPs)/LHRH-conjugated AuNPs and triple negative breast cancer cells (MDA-MB-231 cells), as well as the adhesion between LHRH-conjugated AuNP/PG drug and MDA-MB-231 breast cancer cells. The adhesion forces between LHRH-conjugated AuNPs and breast cancer cells are shown to be five times greater than those between AuNPs and normal breast cells. The increase in adhesion is shown to be due to the over-expression of LHRH receptors on the surfaces of MDA-MB-231 breast cancer cells, which was revealed by confocal immuno-fluorescence microscopy. The implications of the results are then discussed for the selective and specific targeting and treatment of triple negative breast cancer.

Scalable ion-photon quantum interface based on integrated diffractive mirrors

NASA Astrophysics Data System (ADS)

Ghadimi, Moji; Blūms, Valdis; Norton, Benjamin G.; Fisher, Paul M.; Connell, Steven C.; Amini, Jason M.; Volin, Curtis; Hayden, Harley; Pai, Chien-Shing; Kielpinski, David; Lobino, Mirko; Streed, Erik W.

2017-12-01

Quantum networking links quantum processors through remote entanglement for distributed quantum information processing and secure long-range communication. Trapped ions are a leading quantum information processing platform, having demonstrated universal small-scale processors and roadmaps for large-scale implementation. Overall rates of ion-photon entanglement generation, essential for remote trapped ion entanglement, are limited by coupling efficiency into single mode fibers and scaling to many ions. Here, we show a microfabricated trap with integrated diffractive mirrors that couples 4.1(6)% of the fluorescence from a 174Yb+ ion into a single mode fiber, nearly triple the demonstrated bulk optics efficiency. The integrated optic collects 5.8(8)% of the π transition fluorescence, images the ion with sub-wavelength resolution, and couples 71(5)% of the collected light into the fiber. Our technology is suitable for entangling multiple ions in parallel and overcomes mode quality limitations of existing integrated optical interconnects.

Evaluation of optimal DNA staining for triggering by scanning fluorescence microscopy (SFM)

NASA Astrophysics Data System (ADS)

Mittag, Anja; Marecka, Monika; Pierzchalski, Arkadiusz; Malkusch, Wolf; Bocsi, József; Tárnok, Attila

2009-02-01

In imaging and flow cytometry, DNA staining is a common trigger signal for cell identification. Selection of the proper DNA dye is restricted by the hardware configuration of the instrument. The Zeiss Imaging Solutions GmbH (München, Germany) introduced a new automated scanning fluorescence microscope - SFM (Axio Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of the study was to select optimal DNA dyes as trigger signal in leukocyte detection and subsequent cytometric analysis of double-labeled leukocytes by SFM. Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI, 7-AAD, and TOPRO-3) were tested and found to be suitable for the implemented filtersets (fs) of the SFM (fs: 49, fs: 44, fs: 20). EDTA blood was stained after erythrocyte lysis with DNA dye. Cells were transferred on microscopic slides and embedded in fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping and used in combination with Hoechst. Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could be analyzed without difficulty. These results were confirmed by FCM measurements. DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as primary parameter.

Spatial fluorescence cross-correlation spectroscopy between core and ring pinholes

NASA Astrophysics Data System (ADS)

Blancquaert, Yoann; Delon, Antoine; Derouard, Jacques; Jaffiol, Rodolphe

2006-04-01

Fluorescence Correlation Spectroscopy (FCS) is an attractive method to measure molecular concentration, mobility parameters and chemical kinetics. However its ability to descriminate different diffusing species needs to be improved. Recently, we have proposed a simplified spatial Fluorescence cross Correlation Spectroscopy (sFCCS) method, allowing, with only one focused laser beam to obtain two confocal volumes spatially shifted. Now, we present a new sFCCS optical geometry where the two pinholes, a ring and core, are encapsulated one in the other. In this approach all physical and chemical processes that occur in a single volume, like singlet-triplet dynamics and photobleaching, can be eliminated; moreover, this new optical geometry optimises the collection of fluorescence. The first cross Correlation curves for Rhodamine 6G (Rh6G) in Ethanol are presented, in addition to the effect of the size of fluorescent particules (nano-beads, diameters : 20, 100 and 200 nm). The relative simplicity of the method leads us to propose sFCCS as an appropriate method for the determination of diffusion parameters of fluorophores in solution or cells. Nevertheless, progresses in the ingeniering of the optical Molecular Detection Efficiency volumes are highly desirable, in order to improve the descrimination between the cross correlated volumes.

Keratin 17 is overexpressed and predicts poor survival in estrogen receptor-negative/human epidermal growth factor receptor-2-negative breast cancer.

PubMed

Merkin, Ross D; Vanner, Elizabeth A; Romeiser, Jamie L; Shroyer, A Laurie W; Escobar-Hoyos, Luisa F; Li, Jinyu; Powers, Robert S; Burke, Stephanie; Shroyer, Kenneth R

2017-04-01

Clinicopathological features of breast cancer have limited accuracy to predict survival. By immunohistochemistry (IHC), keratin 17 (K17) expression has been correlated with triple-negative status (estrogen receptor [ER]/progesterone receptor/human epidermal growth factor receptor-2 [HER2] negative) and decreased survival, but K17 messenger RNA (mRNA) expression has not been evaluated in breast cancer. K17 is a potential prognostic cancer biomarker, targeting p27, and driving cell cycle progression. This study compared K17 protein and mRNA expression to ER/progesterone receptor/HER2 receptor status and event-free survival. K17 IHC was performed on 164 invasive breast cancers and K17 mRNA was evaluated in 1097 breast cancers. The mRNA status of other keratins (16/14/9) was evaluated in 113 ER - /HER2 - ductal carcinomas. IHC demonstrated intense cytoplasmic and membranous K17 localization in myoepithelial cells of benign ducts and lobules and tumor cells of ductal carcinoma in situ. In ductal carcinomas, K17 protein was detected in most triple-negative tumors (28/34, 82%), some non-triple-negative tumors (52/112, 46%), but never in lobular carcinomas (0/15). In ductal carcinomas, high K17 mRNA was associated with reduced 5-year event-free survival in advanced tumor stage (n = 149, hazard ratio [HR] = 3.68, P = .018), and large (n = 73, HR = 3.95, P = .047), triple-negative (n = 103, HR = 2.73, P = .073), and ER - /HER2 - (n = 113, HR = 2.99, P = .049) tumors. There were significant correlations among keratins 17, 16, 14, and 9 mRNA levels suggesting these keratins (all encoded on chromosome 17) could be coordinately expressed in breast cancer. Thus, K17 is expressed in a subset of triple-negative breast cancers, and is a marker of poor prognosis in patients with advanced stage and ER - /HER2 - breast cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Mütze, Jörg; Ohrt, Thomas; Petrášek, Zdeněk; Schwille, Petra

In this manuscript, we describe the application of Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and scanning FCS (sFCS) to two in vivo systems. In the first part, we describe the application of two-photon standard and scanning FCS in Caenorhabditis elegans embryos. The differentiation of a single fertilized egg into a complex organism in C. elegans is regulated by a number of protein-dependent processes. The oocyte divides asymmetrically into two daughter cells of different developmental fate. Two of the involved proteins, PAR-2 and NMY-2, are studied. The second investigated system is the mechanism of RNA interference in human cells. An EGFP based cell line that allows to study the dynamics and localization of the RNA-induced silencing complex (RISC) with FCS in vivo is created, which has so far been inaccessible with other experimental methods. Furthermore, Fluorescence Cross-Correlation Spectroscopy is employed to highlight the asymmetric incorporation of labeled siRNAs into RISC.

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

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.

Confinement effects on glass transition temperature, transition breadth, and expansivity: comparison of ellipsometry and fluorescence measurements on polystyrene films.

PubMed

Kim, S; Hewlett, S A; Roth, C B; Torkelson, J M

2009-09-01

Using ellipsometry, we characterized the nanoconfinement effect on the glass transition temperature (T (g)of supported polystyrene (PS) films employing two methods: the intersection of fits to the temperature (Tdependences of rubbery- and glassy-state thicknesses, and the transition mid-point between rubbery- and glassy-state expansivities. The results demonstrate a strong effect of thickness: T(g) (bulk) - T(g)(23 nm) = 10 degrees C. The T -range needed for accurate measurement increases significantly with decreasing thickness, an effect that arises from the broadening of the transition with confinement and a region below T (g) where expansivity slowly decreases with decreasing T . As determined from expansivities, the T (g) breadth triples in going from bulk films to a 21-nm-thick film; this broadening of the transition may be a more dramatic effect of confinement than the T (g) reduction itself. In contrast, there is little effect of confinement on the rubbery- and glassy-state expansivities. Compared with ellipsometry, T (g) 's from fluorescence agree well in bulk films but yield lower values in nanoconfined films: T (g)(bulk) - T (g)(23 nm) = 15( degrees ) C via fluorescence. This small difference in the T (g) confinement effect reflects differences in how fluorescence and ellipsometry report "average T (g) " with confinement. With decreasing nanoscale thickness, fluorescence may slightly overweight the contribution of the free-surface layer while ellipsometry may evenly weight or underweight its contribution.

Confinement effects on glass transition temperature, transition breadth, and expansivity: Comparison of ellipsometry and fluorescence measurements on polystyrene films

NASA Astrophysics Data System (ADS)

Kim, S.; Hewlett, S. A.; Roth, C. B.; Torkelson, J. M.

2009-09-01

Using ellipsometry, we characterized the nanoconfinement effect on the glass transition temperature (T gof supported polystyrene (PS) films employing two methods: the intersection of fits to the temperature (Tdependences of rubbery- and glassy-state thicknesses, and the transition mid-point between rubbery- and glassy-state expansivities. The results demonstrate a strong effect of thickness: ensuremath Tg(bulk)-Tg(23{ nm})= 10 circ C. The T -range needed for accurate measurement increases significantly with decreasing thickness, an effect that arises from the broadening of the transition with confinement and a region below T g where expansivity slowly decreases with decreasing T . As determined from expansivities, the T g breadth triples in going from bulk films to a 21-nm-thick film; this broadening of the transition may be a more dramatic effect of confinement than the T g reduction itself. In contrast, there is little effect of confinement on the rubbery- and glassy-state expansivities. Compared with ellipsometry, T g ’s from fluorescence agree well in bulk films but yield lower values in nanoconfined films: T g(bulk) - T g(23 nm) = 15° C via fluorescence. This small difference in the T g confinement effect reflects differences in how fluorescence and ellipsometry report “average T g ” with confinement. With decreasing nanoscale thickness, fluorescence may slightly overweight the contribution of the free-surface layer while ellipsometry may evenly weight or underweight its contribution. in here

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.

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

Unimolecular Thermal Fragmentation of Ortho-Benzyne

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

Zhang, X.; Maccarone, A. T.; Nimlos, M. R.

2007-01-01

The ortho-benzyne diradical, o-C{sub 6}H{sub 4} has been produced with a supersonic nozzle and its subsequent thermal decomposition has been studied. As the temperature of the nozzle is increased, the benzyne molecule fragments: o-C{sub 6}H{sub 4} + {Delta} {yields} products. The thermal dissociation products were identified by three experimental methods: (i) time-of-flight photoionization mass spectrometry, (ii) matrix-isolation Fourier transform infrared absorption spectroscopy, and (iii) chemical ionization mass spectrometry. At the threshold dissociation temperature, o-benzyne cleanly decomposes into acetylene and diacetylene via an apparent retro-Diels-Alder process: o-C{sub 6}H{sub 4} + {Delta} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH. The experimentalmore » {Delta}{sub rxn}H{sub 298}(o-C{sub 6}H{sub 4} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH) is found to be 57 {+-} 3 kcal mol{sup -1}. Further experiments with the substituted benzyne, 3,6-(CH{sub 3}){sub 2}-o-C{sub 6}H{sub 2}, are consistent with a retro-Diels-Alder fragmentation. But at higher nozzle temperatures, the cracking pattern becomes more complicated. To interpret these experiments, the retro-Diels-Alder fragmentation of o-benzyne has been investigated by rigorous ab initio electronic structure computations. These calculations used basis sets as large as [C(7s6p5d4f3g2h1i)/H(6s5p4d3f2g1h)] (cc-pV6Z) and electron correlation treatments as extensive as full coupled cluster through triple excitations (CCSDT), in cases with a perturbative term for connected quadruples [CCSDT(Q)]. Focal point extrapolations of the computational data yield a 0 K barrier for the concerted, C{sub 2v}-symmetric decomposition of o-benzyne, E{sub b}(o-C{sub 6}H{sub 4} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH) = 88.0 {+-} 0.5 kcal mol{sup -1}. A barrier of this magnitude is consistent with the experimental results. A careful assessment of the thermochemistry for the high temperature fragmentation of benzene is presented: C{sub 6}H{sub 6} {yields} H+[C{sub 6}H{sub 5}] {yields} H+[o-C{sub 6}H{sub 4}] {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH. Benzyne may be an important intermediate in the thermal decomposition of many alkylbenzenes (arenes). High engine temperatures above 1500 K may crack these alkylbenzenes to a mixture of alkyl radicals and phenyl radicals. The phenyl radicals will then dissociate first to benzyne and then to acetylene and diacetylene.« less

Unimolecular thermal fragmentation of ortho-benzene.

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

Zhang, X.; Maccarone, A. T.; Nimlos, M. R.

2007-01-01

The ortho-benzyne diradical, o-C{sub 6}H{sub 4} has been produced with a supersonic nozzle and its subsequent thermal decomposition has been studied. As the temperature of the nozzle is increased, the benzyne molecule fragments o-C{sub 6}H{sub 4}{sup +} {Delta} {yields} products. The thermal dissociation products were identified by three experimental methods: (i) time-of-flight photoionization mass spectrometry, (ii) matrix-isolation Fourier transform infrared absorption spectroscopy, and (iii) chemical ionization mass spectrometry. At the threshold dissociation temperature, o-benzyne cleanly decomposes into acetylene and diacetylene via an apparent retro-Diels-Alder process: o-C{sub 6}H{sub 4}{sup +}{Delta}{yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH. The experimental {Delta}{sub rxn}H{submore » 298}(o-C{sub 6}H{sub 4} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH) is found to be 57 {+-} 3 kcal mol{sup -1}. Further experiments with the substituted benzyne, 3,6-(CH{sub 3}){sub 2}-o-C{sub 6}H{sub 2}, are consistent with a retro-Diels-Alder fragmentation. But at higher nozzle temperatures, the cracking pattern becomes more complicated. To interpret these experiments, the retro-Diels-Alder fragmentation of o-benzyne has been investigated by rigorous ab initio electronic structure computations. These calculations used basis sets as large as [C(7s6p5d4f3g2h1i)/H(6s5p4d3f2g1h)] (cc-pV6Z) and electron correlation treatments as extensive as full coupled cluster through triple excitations (CCSDT), in cases with a perturbative term for connected quadruples [CCSDT(Q)]. Focal point extrapolations of the computational data yield a 0 K barrier for the concerted, C{sub 2v}-symmetric decomposition of o-benzyne, E{sub b}(o-C{sub 6}H{sub 4} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH) = 88.0 {+-} 0.5 kcal mol{sup -1}. A barrier of this magnitude is consistent with the experimental results. A careful assessment of the thermochemistry for the high temperature fragmentation of benzene is presented: C{sub 6}H{sub 6} {yields} H+[C{sub 6}H{sub 5}] {yields} H+[o-C{sub 6}H{sub 4}] {yields} HC {triple_bond} CH-HC {triple_bond} C-C {triple_bond} CH. Benzyne may be an important intermediate in the thermal decomposition of many alkylbenzenes (arenes). High engine temperatures above 1500 K may crack these alkylbenzenes to a mixture of alkyl radicals and phenyl radicals. The phenyl radicals will then dissociate first to benzyne and then to acetylene and diacetylene.« less

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.

[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.

Triple collocation-based estimation of spatially correlated observation error covariance in remote sensing soil moisture data assimilation

NASA Astrophysics Data System (ADS)

Wu, Kai; Shu, Hong; Nie, Lei; Jiao, Zhenhang

2018-01-01

Spatially correlated errors are typically ignored in data assimilation, thus degenerating the observation error covariance R to a diagonal matrix. We argue that a nondiagonal R carries more observation information making assimilation results more accurate. A method, denoted TC_Cov, was proposed for soil moisture data assimilation to estimate spatially correlated observation error covariance based on triple collocation (TC). Assimilation experiments were carried out to test the performance of TC_Cov. AMSR-E soil moisture was assimilated with a diagonal R matrix computed using the TC and assimilated using a nondiagonal R matrix, as estimated by proposed TC_Cov. The ensemble Kalman filter was considered as the assimilation method. Our assimilation results were validated against climate change initiative data and ground-based soil moisture measurements using the Pearson correlation coefficient and unbiased root mean square difference metrics. These experiments confirmed that deterioration of diagonal R assimilation results occurred when model simulation is more accurate than observation data. Furthermore, nondiagonal R achieved higher correlation coefficient and lower ubRMSD values over diagonal R in experiments and demonstrated the effectiveness of TC_Cov to estimate richly structuralized R in data assimilation. In sum, compared with diagonal R, nondiagonal R may relieve the detrimental effects of assimilation when simulated model results outperform observation data.

Correlated multielectron dynamics in mid-infrared laser pulse interactions with neon atoms.

PubMed

Tang, Qingbin; Huang, Cheng; Zhou, Yueming; Lu, Peixiang

2013-09-09

The multielectron dynamics in nonsequential triple ionization (NSTI) of neon atoms driven by mid-infrared (MIR) laser pulses is investigated with the three-dimensional classical ensemble model. In consistent with the experimental result, our numerical result shows that in the MIR regime, the triply charged ion longitudinal momentum spectrum exhibits a pronounced double-hump structure at low laser intensity. Back analysis reveals that as the intensity increases, the responsible triple ionization channels transform from direct (e, 3e) channel to the various mixed channels. This transformation of the NSTI channels leads to the results that the shape of ion momentum spectra becomes narrow and the distinct maxima shift towards low momenta with the increase of the laser intensity. By tracing the triply ionized trajectories, the various ionization channels at different laser intensities are clearly identified and these results provide an insight into the complex dynamics of the correlated three electrons in NSTI.

Streamwise evolution of statistical events and the triple correlation in a model wind turbine array

NASA Astrophysics Data System (ADS)

Viestenz, Kyle; Cal, Raúl Bayoán

2013-11-01

Hot-wire anemometry data, obtained from a wind tunnel experiment containing a 3 × 3 wind turbine array, are used to conditionally average the Reynolds stresses. Nine profiles at the centerline behind the array are analyzed to characterize the turbulent velocity statistics of the wake flow. Quadrant analysis yields statistical events occurring in the wake of the wind farm, where quadrants 2 and 4 produce ejections and sweeps, respectively. A balance between these quadrants is expressed via the ΔSo parameter, which attains a maximum value at the bottom tip and changes sign near the top tip of the rotor. These are then associated to the triple correlation term present in the turbulent kinetic energy equation of the fluctuations. The development of these various quantities is assessed in light of wake remediation, energy transport and possess significance in closure models. National Science Foundation: ECCS-1032647.

Assessment of parental tooth-brushing following instruction with single-headed and triple-headed toothbrushes.

PubMed

Telishevesky, Yoel S; Levin, Liran; Ashkenazi, Malka

2012-01-01

The purpose of this study was to evaluate the effect of toothbrush design on the ability of parents to effectively brush their children's teeth. Parents of children (mean age=5.1±0.75 years old) from 4 kindergarten schools were randomly assigned to receive instruction in brushing their children's teeth using a manual single-headed toothbrush (2 schools) or a triple-headed toothbrush (2 schools). The parents' ability to brush their children's teeth was evaluated according to a novel toothbrush performing skill index (Ashkenazi Index), based on 2 criteria: (1) placement of the toothbrush on each tooth segment to be brushed ("reach"); and (2) completion of enough strokes on each segment ("stay"). One month after instruction, tooth-brushing ability was re-evaluated and plaque index of the children's teeth was assessed. One month after instruction, parents using the triple-headed toothbrush received significantly higher scores on the tooth-brushing performance index (~86%), than did those in the single-headed group (~61%; P=.001). The plaque index was significantly higher in the single-headed group (0.97±0.38) vs the triple-headed group (0.72±0.29; P<.01). The tooth-brushing performance index correlated negatively with the plaque index (P<.01). A triple-headed toothbrush promotes more consistent tooth-brushing by parents than does a single-headed toothbrush.

MRI evaluation of residual breast cancer after neoadjuvant chemotherapy: influence of patient, tumor and chemotherapy characteristics on the correlation with pathological response.

PubMed

Diguisto, Caroline; Ouldamer, Lobna; Arbion, Flavie; Vildé, Anne; Body, Gilles

2015-01-01

The aim of this study was to evaluate the correlation between the residual tumor measured on magnetic resonance imaging and pathological results and to assess whether this correlation varies according to patient, tumor or chemotherapy characteristics. The study population included women treated for breast cancer with indication of neoadjuvant chemotherapy in our tertiary breast cancer Unit between January 2008 and December 2011. Factors related to patients, tumor and chemotherapy were studied. Pearson's correlation coefficient between the size of the tumor on MRI and pathological response was calculated for the entire population. It was also calculated according to patient, tumor and chemotherapy characteristics. During the study period, 107 consecutive women were included. The size of residual tumor on the MRI significantly correlated with the size on pathological result with a Pearson correlation coefficient of 0.52 (p<0.001). The correlation was stronger for women aged 50 years and older (r=0.64, p<0.001) and for post-menopausal women (r=0.61, p<0.001). The correlation was stronger for those with triple-negative tumors (r=0.69, p=0.002) but weaker for those with tumors with a ductal carcinoma in situ component (r =0.18, p=0.42). The size of breast cancer obtained by MRI is significantly correlated to the pathological size of the tumor. This correlation was stronger among women aged 50 years and more, among post-menopausal women, and among women who had triple-negative tumors. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

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

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

2014-10-15

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

Plasma-assisted quadruple-channel optosensing of proteins and cells with Mn-doped ZnS quantum dots.

PubMed

Li, Chenghui; Wu, Peng; Hou, Xiandeng

2016-02-21

Information extraction from nano-bio-systems is crucial for understanding their inner molecular level interactions and can help in the development of multidimensional/multimodal sensing devices to realize novel or expanded functionalities. The intrinsic fluorescence (IF) of proteins has long been considered as an effective tool for studying protein structures and dynamics, but not for protein recognition analysis partially because it generally contributes to the fluorescence background in bioanalysis. Here we explored the use of IF as the fourth channel optical input for a multidimensional optosensing device, together with the triple-channel optical output of Mn-doped ZnS QDs (fluorescence from ZnS host, phosphorescence from Mn(2+) dopant, and Rayleigh light scattering from the QDs), to dramatically improve the protein recognition and discrimination resolution. To further increase the cross-reactivity of the multidimensional optosensing device, plasma modification of proteins was explored to enhance the IF difference as well as their interactions with Mn-doped ZnS QDs. Such a sensor device was demonstrated for highly discriminative and precise identification of proteins in human serum and urine samples, and for cancer and normal cells as well.

RHB-104 triple antibiotics combination in culture is bactericidal and should be effective for treatment of Crohn's disease associated with Mycobacterium paratuberculosis.

PubMed

Alcedo, Karel P; Thanigachalam, Saisathya; Naser, Saleh A

2016-01-01

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated as an etiological agent of Crohn's disease (CD), a debilitating chronic inflammatory bowel disease. Clarithromycin (CLA), clofazimine (CLO), rifabutin (RIF) and other antibiotics have been used individually or in combinations with other drugs to treat mycobacterial diseases including CD. The treatment has varied by regimen, dosage, and duration, resulting in conflicting outcomes and additional suffering to the patients. RHB-104, a drug formula with active ingredients composed of (63.3 %) CLA, (6.7 %) CLO, and (30 %) RIF, has been recently subjected to investigation in an FDA approved Phase III clinical trial to treat patients with moderate to severe CD. In this study, we determined the efficacy of RHB-104 active ingredients against MAP strains isolated from the blood, tissue, and milk of CD patients. Based on fluorescence quenching technology using the Bactec MGIT Para-TB medium, we determined the minimum inhibitory concentration (MIC) of CLA, CLO, RIF individually and in dual and triple combinations against 16 MAP clinical strains and 19 other mycobacteria. The MIC of all drugs against 35 different mycobacteria ranged between 0.25-20 μg/mL. However, the MIC of RHB-104 active ingredients regimen was the lowest at 0.25-10 μg/mL compared to the MIC of the other drugs at 0.5-20 μg/mL. The components of RHB-104 active ingredients at their individual concentrations or in dual combinations were not effective against all microorganisms compared to the triple combinations at MIC level. The MIC of CLA-CLO, CLA-RIF, and CLO-RIF regimens ranged between 0.5-1.25 μg/mL compared to 0.25 μg/mL of bactericidal effect of the triple combination. The data clearly demonstrated that lower concentrations of the triple combination of RHB-104 active ingredients provided synergistic anti-MAP growth activity compared to individual or dual combinations of the drugs. Consequently, this is favorable and should lead to tolerable dosage that is desirable for long-term treatment of CD and Mycobacterium avium complex disease.

[Quercetin ameliorates inflammation in CA1 hippocampal region in aged triple transgenic Alzheimer´s disease mice model.

PubMed

Vargas-Restrepo, Felipe; Sabogal-Guáqueta, Angélica María; Cardona-Gómez, Gloria Patricia

2018-05-01

Alzheimer's disease is the most common form of dementia. It is characterized by histopathological hallmarks such as senile plaques and neurofibrillary tangles, as well as a concomitant activation of microglial cells and astrocytes that release pro-inflammatory mediators such as IL-1β, iNOS, and COX-2, leading to neuronal dysfunction and death. To evaluate the effect of quercetin on the inflammatory response in the CA1 area of the hippocampus in a 3xTg-AD male and female mice model. Animals were injected intraperitoneally with quercetin every 48 hours during three months, and we conducted histological and biochemical studies. We found that in quercetin-treated 3xTg-AD mice, reactive microglia and fluorescence intensity of Aβ aggregates significantly decreased. GFAP, iNOS, and COX-2 immunoreactivity also decreased and we observed a clear tendency in the reduction of IL-1β in hippocampal lysates. Our work suggests an anti-inflammatory effect of quercetin in the CA1 hippocampal region of aged triple transgenic Alzheimer's disease mice.

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.

Reference correlation of the thermal conductivity of carbon dioxide from the triple point to 1100 K and up to 200 MPa

DOE PAGES

Huber, M. L.; Sykioti, E. A.; Assael, M. J.; ...

2016-02-25

This article contains new, representative reference equations for the thermal conductivity of carbon dioxide. The equations are based in part upon a body of experimental data that has been critically assessed for internal consistency and for agreement with theory whenever possible. In the case of the dilute-gas thermal conductivity, we incorporated recent theoretical calculations to extend the temperature range of the experimental data. Moreover, in the critical region, the experimentally observed enhancement of the thermal conductivity is well represented by theoretically based equations containing just one adjustable parameter. The correlation is applicable for the temperature range from the triple pointmore » to 1100 K and pressures up to 200 MPa. Lastly, the overall uncertainty (at the 95% confidence level) of the proposed correlation varies depending on the state point from a low of 1% at very low pressures below 0.1 MPa between 300 and 700 K, to 5% at the higher pressures of the range of validity.« less

Mitochondrial Molecular Abnormalities Revealed by Proteomic Analysis of Hippocampal Organelles of Mice Triple Transgenic for Alzheimer Disease

PubMed Central

Yu, Haitao; Lin, Xuemei; Wang, Dian; Zhang, Zaijun; Guo, Yi; Ren, Xiaohu; Xu, Benhong; Yuan, Jianhui; Liu, Jianjun; Spencer, Peter S.; Wang, Jian-Zhi; Yang, Xifei

2018-01-01

Mitochondrial dysfunction is implicated in the pathogenesis of Alzheimer’s disease (AD). However, the precise mitochondrial molecular deficits in AD remain poorly understood. Mitochondrial and nuclear proteomic analysis in mature male triple transgenic AD mice (PS1M146V/APPSwe/TauP301L) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF-MS/MS, bio-informatics analysis and immunofluorescent staining were performed in this study. In addition to impaired spatial memory impairment and intracellular accumulation of amyloid 1–42 (Aβ1–42) in the 3xTg-AD mice, a well-accepted mouse model of the human disease, we also found significantly increased DNA oxidative damage in entorhinal cortex, hippocampal CA1, CA3 and dental gyrus (DG), as evidenced by the positive staining of 8-hydroxyguanosine, a biomarker of mild cognitive impairment early in AD. We identified significant differences in 27 hippocampal mitochondrial proteins (11 increased and 16 decreased), and 37 hippocampal nuclear proteins (12 increased and 25 decreased) in 3xTg-AD mice compared with the wild-type (WT) mice. Differentially expressed mitochondrial and nuclear proteins were mainly involved in energy metabolism (>55%), synapses, DNA damage, apoptosis and oxidative stress. Two proteins were differentially expressed in both hippocampal mitochondria and nuclei, namely electron transport chain (ETC)-related protein ATP synthase subunit d (ATP5H) was significantly decreased, and apoptosis-related dynamin-1 (DYN1), a pre-synaptic and mitochondrial division-regulated protein that was significantly increased. In sum, perturbations of hippocampus mitochondrial energy metabolism-related proteins responsible for ATP generation via oxidation phosphorylation (OXPHOS), especially nuclear-encoded OXPHOS proteins, correlated with the amyloid-associated cognitive deficits of this murine AD model. The molecular changes in respiratory chain-related proteins and DYN1 may represent novel biomarkers of AD. PMID:29593495

Effect of H. pylori density by histopathology on its complications and eradication therapy.

PubMed

Shah, Dharmesh K; Jain, Samit S; Mohite, Ashok; Amarapurkar, Anjali D; Contractor, Q Q; Rathi, Pravin M

2015-01-01

Helicobacter pylori (H. pylori) infection causes chronic gastritis and is a major risk factor for duodenal and gastric ulceration, gastric adenocarcinoma, and primary gastric lymphoma. Increased gastric bacterial density may lead to increased levels of inflammation and epithelial injury. 1) To study the effect of H. pylori density by histological changes in stomach. 2) To study the effect of H. pylori density on the efficacy of standard triple drug eradication treatment. 3) To study the effect of H. pylori density on the complication related to H. pylori. All the patients visiting gastroenterology OPD with the symptoms of dyspepsia not responding to proton pump inhibitor or having alarm symptoms were subjected to upper GI endoscopy and biopsy. If H. pylori was present they were included in the study. The patients were given standard 14 day triple antibiotic combination for H. pylori eradication. H. pylori eradication was confirmed by urea breath test after six weeks of completion of treatment. Out of 250 patients screened, 120 patients enrolled in the study. On clinical history 41.5% patients had symptoms of heart burn where as 63.3% patients had dyspeptic symptoms. Success rate of anti H. pylori triple drug therapy was 80%. Rate of eradication was significantly lower among the patients with higher H. pylori density (p < 0.05) on histopathology by Sydney classification. Duodenal ulcer, Gastric ulcer and gastric erosion were noted in higher frequencies among the patients with higher H. pylori density (p < 0.05). H. pylori density by histopathology correlates with the complication related to H. pylori i.e. duodenal ulcer, reflux esophagitis and antral erosions. It also correlates with the success of the standard triple drug eradication treatment.

Dielectric controlled excited state relaxation pathways of a representative push-pull stilbene: a mechanistic study using femtosecond fluorescence up-conversion technique.

PubMed

Rafiq, Shahnawaz; Sen, Pratik

2013-02-28

Femtosecond fluorescence up-conversion technique was employed to reinvestigate the intriguing dependence of fluorescence quantum yield of trans-4-dimethylamino-4(')-nitrostilbene (DNS) on dielectric properties of the media. In polar solvents, such as methanol and acetonitrile, the two time components of the fluorescence transients were assigned to intramolecular charge transfer (ICT) dynamics and to the depletion of the ICT state to the ground state via internal conversion along the torsional coordinate of nitro moiety. The viscosity independence of the first time component indicates the absence of any torsional coordinate in the charge transfer process. In slightly polar solvent (carbon tetrachloride) the fluorescence transients show a triple exponential behavior. The first time component was assigned to the formation of the ICT state on a 2 ps time scale. Second time component was assigned to the relaxation of the ICT state via two torsion controlled channels. First channel involves the torsional motion about the central double bond leading to the trans-cis isomerization via a conical intersection or avoided crossing. The other channel contributing to the depopulation of ICT state involves the torsional coordinates of dimethylanilino and∕or nitrophenyl moieties and leads to the formation of a conformationally relaxed state, which subsequently relaxes back to the ground state radiatively, and is responsible for the high fluorescence quantum yield of DNS in slightly polar solvents such as carbon tetrachloride, toluene, etc. The excited singlet state which is having a dominant π-π∗ character may also decay via intersystem crossing to the n-π∗ triplet manifold and thus accounts for the observed triplet yield of the molecule in slightly polar solvents.

Dielectric controlled excited state relaxation pathways of a representative push-pull stilbene: A mechanistic study using femtosecond fluorescence up-conversion technique

NASA Astrophysics Data System (ADS)

Rafiq, Shahnawaz; Sen, Pratik

2013-02-01

Femtosecond fluorescence up-conversion technique was employed to reinvestigate the intriguing dependence of fluorescence quantum yield of trans-4-dimethylamino-4'-nitrostilbene (DNS) on dielectric properties of the media. In polar solvents, such as methanol and acetonitrile, the two time components of the fluorescence transients were assigned to intramolecular charge transfer (ICT) dynamics and to the depletion of the ICT state to the ground state via internal conversion along the torsional coordinate of nitro moiety. The viscosity independence of the first time component indicates the absence of any torsional coordinate in the charge transfer process. In slightly polar solvent (carbon tetrachloride) the fluorescence transients show a triple exponential behavior. The first time component was assigned to the formation of the ICT state on a 2 ps time scale. Second time component was assigned to the relaxation of the ICT state via two torsion controlled channels. First channel involves the torsional motion about the central double bond leading to the trans-cis isomerization via a conical intersection or avoided crossing. The other channel contributing to the depopulation of ICT state involves the torsional coordinates of dimethylanilino and/or nitrophenyl moieties and leads to the formation of a conformationally relaxed state, which subsequently relaxes back to the ground state radiatively, and is responsible for the high fluorescence quantum yield of DNS in slightly polar solvents such as carbon tetrachloride, toluene, etc. The excited singlet state which is having a dominant π-π* character may also decay via intersystem crossing to the n-π* triplet manifold and thus accounts for the observed triplet yield of the molecule in slightly polar solvents.

Noninvasive in vivo multispectral optoacoustic imaging of apoptosis in triple negative breast cancer using indocyanine green conjugated phosphatidylserine monoclonal antibody

NASA Astrophysics Data System (ADS)

Kannadorai, Ravi Kumar; Udumala, Sunil Kumar; Sidney, Yu Wing Kwong

2016-12-01

Noninvasive and nonradioactive imaging modality to track and image apoptosis during chemotherapy of triple negative breast cancer is much needed for an effective treatment plan. Phosphatidylserine (PS) is a biomarker transiently exposed on the outer surface of the cells during apoptosis. Its externalization occurs within a few hours of an apoptotic stimulus by a chemotherapy drug and leads to presentation of millions of phospholipid molecules per apoptotic cell on the cell surface. This makes PS an abundant and accessible target for apoptosis imaging. In the current work, we show that PS monoclonal antibody tagged with indocyanine green (ICG) can help to track and image apoptosis using multispectral optoacoustic tomography in vivo. When compared to saline control, the doxorubicin treated group showed a significant increase in uptake of ICG-PS monoclonal antibody in triple negative breast tumor xenografted in NCr nude female mice. Day 5 posttreatment had the highest optoacoustic signal in the tumor region, indicating maximum apoptosis and the tumor subsequently shrank. Since multispectral optoacoustic imaging does not involve the use of radioactivity, the longer the circulatory time of the PS antibody can be exploited to monitor apoptosis over a period of time without multiple injections of commonly used imaging probes such as Tc-99m Annexin V or F-18 ML10. The proposed apoptosis imaging technique involving multispectral optoacoustic tomography, monoclonal antibody, and near-infrared absorbing fluorescent marker can be an effective tool for imaging apoptosis and treatment planning.

Functional disubstituted polyacetylenes: Synthesis, liquid crystallinity, light emission, and fluorescent photopatterning of biphenyl-containing poly(1-phenyl-octyne)s with different functional bridges.

PubMed

Lam, Jacky W Y; Qin, Anjun; Dong, Yuping; Lai, Lo Ming; Häussler, Matthias; Dong, Yongqiang; Tang, Ben Zhong

2006-11-02

Biphenyl (Biph)-containing 1-phenyl-1-octynes and their polymers are synthesized, and the effects of functional bridge groups on the mesomorphic and optical properties of the polymers are studied. The nonmesomorphic disubstituted acetylene monomers (C6H13)C[triple bond]C(C6H4)O(CH2)12O-Biph-OC7H15 (1), (C6H13)C[triple bond]C(C6H4)O(CH2)11OOC-Biph-OC7H15 (2), and (C6H13)C[triple bond]C(C6H4)CO2(CH2)12OOC-Biph-OC7H15 (3) are prepared by multistep reaction routes and converted into their corresponding polymers P1-P3 by a WCl6-Ph4Sn catalyst. The structures and properties of the polymers are characterized and evaluated by NMR, TGA, DSC, POM, XRD, UV, and PL analyses. The mesogenic pendants have endowed the polymers with high thermal stability (> or =400 degrees C). While P1 exhibits no liquid crystallinity, P2 and P3 form enantiotropic S(A) phase with a monolayer structure. Upon photoexcitation, the polymers emit blue and blue-green lights of 460 and 480 nm, respectively, in THF with quantum efficiencies larger than 30%. UV irradiation of a thin film of P2 through a mask oxidizes and quenches the light emission of the exposed regions, generating a two-dimensional luminescent photoimage.

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.

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.

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.

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.

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.

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

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.

Core-core and core-valence correlation energy atomic and molecular benchmarks for Li through Ar

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

Ranasinghe, Duminda S.; Frisch, Michael J.; Petersson, George A., E-mail: gpetersson@wesleyan.edu

2015-12-07

We have established benchmark core-core, core-valence, and valence-valence absolute coupled-cluster single double (triple) correlation energies (±0.1%) for 210 species covering the first- and second-rows of the periodic table. These species provide 194 energy differences (±0.03 mE{sub h}) including ionization potentials, electron affinities, and total atomization energies. These results can be used for calibration of less expensive methodologies for practical routine determination of core-core and core-valence correlation energies.

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.

Basis set and electron correlation effects on the polarizability and second hyperpolarizability of model open-shell π-conjugated systems

NASA Astrophysics Data System (ADS)

Champagne, Benoı̂t; Botek, Edith; Nakano, Masayoshi; Nitta, Tomoshige; Yamaguchi, Kizashi

2005-03-01

The basis set and electron correlation effects on the static polarizability (α) and second hyperpolarizability (γ) are investigated ab initio for two model open-shell π-conjugated systems, the C5H7 radical and the C6H8 radical cation in their doublet state. Basis set investigations evidence that the linear and nonlinear responses of the radical cation necessitate the use of a less extended basis set than its neutral analog. Indeed, double-zeta-type basis sets supplemented by a set of d polarization functions but no diffuse functions already provide accurate (hyper)polarizabilities for C6H8 whereas diffuse functions are compulsory for C5H7, in particular, p diffuse functions. In addition to the 6-31G*+pd basis set, basis sets resulting from removing not necessary diffuse functions from the augmented correlation consistent polarized valence double zeta basis set have been shown to provide (hyper)polarizability values of similar quality as more extended basis sets such as augmented correlation consistent polarized valence triple zeta and doubly augmented correlation consistent polarized valence double zeta. Using the selected atomic basis sets, the (hyper)polarizabilities of these two model compounds are calculated at different levels of approximation in order to assess the impact of including electron correlation. As a function of the method of calculation antiparallel and parallel variations have been demonstrated for α and γ of the two model compounds, respectively. For the polarizability, the unrestricted Hartree-Fock and unrestricted second-order Møller-Plesset methods bracket the reference value obtained at the unrestricted coupled cluster singles and doubles with a perturbative inclusion of the triples level whereas the projected unrestricted second-order Møller-Plesset results are in much closer agreement with the unrestricted coupled cluster singles and doubles with a perturbative inclusion of the triples values than the projected unrestricted Hartree-Fock results. Moreover, the differences between the restricted open-shell Hartree-Fock and restricted open-shell second-order Møller-Plesset methods are small. In what concerns the second hyperpolarizability, the unrestricted Hartree-Fock and unrestricted second-order Møller-Plesset values remain of similar quality while using spin-projected schemes fails for the charged system but performs nicely for the neutral one. The restricted open-shell schemes, and especially the restricted open-shell second-order Møller-Plesset method, provide for both compounds γ values close to the results obtained at the unrestricted coupled cluster level including singles and doubles with a perturbative inclusion of the triples. Thus, to obtain well-converged α and γ values at low-order electron correlation levels, the removal of spin contamination is a necessary but not a sufficient condition. Density-functional theory calculations of α and γ have also been carried out using several exchange-correlation functionals. Those employing hybrid exchange-correlation functionals have been shown to reproduce fairly well the reference coupled cluster polarizability and second hyperpolarizability values. In addition, inclusion of Hartree-Fock exchange is of major importance for determining accurate polarizability whereas for the second hyperpolarizability the gradient corrections are large.

FluoRender: joint freehand segmentation and visualization for many-channel fluorescence data analysis.

PubMed

Wan, Yong; Otsuna, Hideo; Holman, Holly A; Bagley, Brig; Ito, Masayoshi; Lewis, A Kelsey; Colasanto, Mary; Kardon, Gabrielle; Ito, Kei; Hansen, Charles

2017-05-26

Image segmentation and registration techniques have enabled biologists to place large amounts of volume data from fluorescence microscopy, morphed three-dimensionally, onto a common spatial frame. Existing tools built on volume visualization pipelines for single channel or red-green-blue (RGB) channels have become inadequate for the new challenges of fluorescence microscopy. For a three-dimensional atlas of the insect nervous system, hundreds of volume channels are rendered simultaneously, whereas fluorescence intensity values from each channel need to be preserved for versatile adjustment and analysis. Although several existing tools have incorporated support of multichannel data using various strategies, the lack of a flexible design has made true many-channel visualization and analysis unavailable. The most common practice for many-channel volume data presentation is still converting and rendering pseudosurfaces, which are inaccurate for both qualitative and quantitative evaluations. Here, we present an alternative design strategy that accommodates the visualization and analysis of about 100 volume channels, each of which can be interactively adjusted, selected, and segmented using freehand tools. Our multichannel visualization includes a multilevel streaming pipeline plus a triple-buffer compositing technique. Our method also preserves original fluorescence intensity values on graphics hardware, a crucial feature that allows graphics-processing-unit (GPU)-based processing for interactive data analysis, such as freehand segmentation. We have implemented the design strategies as a thorough restructuring of our original tool, FluoRender. The redesign of FluoRender not only maintains the existing multichannel capabilities for a greatly extended number of volume channels, but also enables new analysis functions for many-channel data from emerging biomedical-imaging techniques.

Engineering Remotely Triggered Liposomes to Target Triple Negative Breast Cancer

PubMed Central

Sneider, Alexandra; Jadia, Rahul; Piel, Brandon; VanDyke, Derek; Tsiros, Christopher; Rai, Prakash

2017-01-01

Triple Negative Breast Cancer (TNBC) continues to present a challenge in the clinic, as there is still no approved targeted therapy. TNBC is the worst sub-type of breast cancer in terms of prognosis and exhibits a deficiency in estrogen, progesterone, and human epidermal growth factor 2 (HER2) receptors. One possible option for the treatment of TNBC is chemotherapy. The issue with many chemotherapy drugs is that their effectiveness is diminished due to poor water solubility, and the method of administration directly or with a co-solvent intravenously can lead to an increase in toxicity. The issues of drug solubility can be avoided by using liposomes as a drug delivery carrier. Liposomes are engineered, biological nanoconstructs that possess the ability to encapsulate both hydrophobic and hydrophilic drugs and have been clinically approved to treat cancer. Specific targeting of cancer cell receptors through the use of ligands conjugated to the surface of drug-loaded liposomes could lessen damage to normal, healthy tissue. This study focuses on polyethylene glycol (PEG)-coated, folate conjugated, benzoporphyrin derivative (BPD)-loaded liposomes for treatment via photodynamic therapy (PDT). The folate receptor is over expressed on TNBC cells so these liposomes are targeted for greater uptake into cancer cells. PDT involves remotely irradiating light at 690 nm to trigger BPD, a hydrophobic photosensitive drug, to form reactive oxygen species that cause tumor cell death. BPD also displays a fluorescence signal when excited by light making it possible to image the fluorescence prior to PDT and for theranostics. In this study, free BPD, non-targeted and folate-targeted PEGylated BPD-loaded liposomes were introduced to a metastatic breast cancer cell line (MDA-MB-231) in vitro. The liposomes were reproducibly synthesized and characterized for size, polydispersity index (PDI), zeta potential, stability, and BPD release kinetics. Folate competition tests, fluorescence confocal imaging, and MTT assay were used to observe and quantify targeting effectiveness. The toxicity of BPD before and after PDT in monolayer and 3D in vitro cultures with TNBC cells was observed. This study may contribute to a novel nanoparticle-mediated approach to target TNBC using PDT. PMID:28174679

Apolar Distal Pocket Mutants of Yeast Cytochrome c Peroxidase: Hydrogen Peroxide Reactivity and Cyanide Binding of the TriAla, TriVal, and TriLeu Variants

PubMed Central

Bidwai, Anil K.; Meyen, Cassandra; Kilheeney, Heather; Wroblewski, Damian; Vitello, Lidia B.; Erman, James E.

2012-01-01

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H2O2 compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H2O2 and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H2O2 and HCN while features that inhibit substrate ionization slow the reactions. PMID:23022490

Quantitative evaluation of cross correlation between two finite-length time series with applications to single-molecule FRET.

PubMed

Hanson, Jeffery A; Yang, Haw

2008-11-06

The statistical properties of the cross correlation between two time series has been studied. An analytical expression for the cross correlation function's variance has been derived. On the basis of these results, a statistically robust method has been proposed to detect the existence and determine the direction of cross correlation between two time series. The proposed method has been characterized by computer simulations. Applications to single-molecule fluorescence spectroscopy are discussed. The results may also find immediate applications in fluorescence correlation spectroscopy (FCS) and its variants.

Titanium and Oxygen Isotope Compositions of Individual Chondrules from Ordinary Chondrites

NASA Astrophysics Data System (ADS)

Bauer, K. K.; Schönbächler, M.; Fehr, M. A.; Vennemann, T.; Chaumard, N.; Zanda, B.

2016-08-01

We measured Ti and triple-O isotope compositions of individual chondrules (characterized by CT scanning) from ordinary chondrites. We will discuss correlations between Ti and ∆17O and their implication for the origin of nucleosynthetic anomalies.

Spin and charge controlled by antisymmetric spin-orbit coupling in a triangular-triple-quantum-dot Kondo system

NASA Astrophysics Data System (ADS)

Koga, M.; Matsumoto, M.; Kusunose, H.

2018-05-01

We study a local antisymmetric spin-orbit (ASO) coupling effect on a triangular-triple-quantum-dot (TTQD) system as a theoretical proposal for a new application of the Kondo physics to nanoscale devices. The electric polarization induced by the Kondo effect is strongly correlated with the spin configurations and molecular orbital degrees of freedom in the TTQD. In particular, an abrupt sign reversal of the emergent electric polarization is associated with a quantum critical point in a magnetic field, which can also be controlled by the ASO coupling that changes the mixing weight of different orbital components in the TTQD ground state.

Apple S-RNase interacts with an actin-binding protein, MdMVG, to reduce pollen tube growth by inhibiting its actin-severing activity at the early stage of self-pollination induction.

PubMed

Yang, Qing; Meng, Dong; Gu, Zhaoyu; Li, Wei; Chen, Qiuju; Li, Yang; Yuan, Hui; Yu, Jie; Liu, Chunsheng; Li, Tianzhong

2018-04-18

In S-RNase-mediated self-incompatibility, S-RNase secreted from the style destroys the actin cytoskeleton of the self-pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S-RNase does not bind or sever filamentous actin (F-actin). In apple (Malus domestica), we identified an actin-binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S-RNase and directly binds and severs F-actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S-RNase inhibits the F-actin-severing activity of MdMVG in vitro. In vivo, the addition of S-RNase to self-pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self-pollination induction in the presence of MdMVG overexpression. By generating 25 single-, double- and triple-point mutations in the amino acid motif E-E-K-E-K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple-point mutant, MdMVG (E167A/E171A/K185A) , that no longer has F-actin-severing activity or interacts with any of the four S-haplotype S-RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S-RNases. We conclude that apple S-RNase interacts with MdMVG to reduce self-pollen tube growth by inhibiting its F-actin-severing activity. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Folate Receptor Targeted Polymeric Micellar Nanocarriers for Delivery of Orlistat as a Repurposed Drug against Triple Negative Breast Cancer

PubMed Central

Paulmurugan, Ramasamy; Bhethanabotla, Rohith; Mishra, Kaushik; Devulapally, Rammohan; Foygel, Kira; Sekar, Thillai V; Ananta, Jeyarama S; Massoud, Tarik F; Joy, Abraham

2015-01-01

Triple negative breast cancer (TNBC) is a recalcitrant malignancy with no available targeted therapy. Off target effects and poor bioavailability of the FDA approved anti-obesity drug orlistat hinder its clinical translation as a repurposed new drug against TNBC. Here we demonstrate a newly engineered drug formulation for packaging orlistat tailored to TNBC treatment. We synthesized TNBC-specific folate receptor targeted micellar nanoparticles (NPs) carrying orlistat, which improved the solubility (70-80 μg/ml) of this water insoluble drug. The targeted NPs also improved the delivery and bioavailability of orlistat to MDA-MB-231 cells in culture and to tumor xenografts in nude mouse model. We prepared HEA-EHA copolymer micellar NPs by copolymerization of 2-hydroxyethylacrylate (HEA) and 2-ethylhexylacrylate (EHA), and functionalized them with folic acid and an imaging dye. Fluorescence activated cell sorting (FACS) analysis of TNBC cells indicated a dose dependent increase in apoptotic populations in cells treated with free orlistat, orlistat NPs, and folate-receptor targeted Fol-HEA-EHA-orlistat NPs in which Fol-HEA-EHA-orlistat NPs showed significantly higher cytotoxicity than free orlistat. In vitro analysis data demonstrated significant apoptosis at nanomolar concentrations in cells activated through caspase 3 and PARP inhibition. In vivo analysis demonstrated significant antitumor effects in living mice after targeted treatment of tumors, and confirmed by fluorescence imaging. Moreover, Folate receptor targeted Fol-DyLight747-orlistat NPs treated mice exhibited significantly higher reduction in tumor volume compared to control group. Taken together, these results indicate that orlistat packaged in HEA-b-EHA micellar NPs is a highly promising new drug formulation for TNBC therapy. PMID:26553061

Bodipy-C60 triple hydrogen bonding assemblies as heavy atom-free triplet photosensitizers: preparation and study of the singlet/triplet energy transfer.

PubMed

Guo, Song; Xu, Liang; Xu, Kejing; Zhao, Jianzhang; Küçüköz, Betül; Karatay, Ahmet; Yaglioglu, Halime Gul; Hayvali, Mustafa; Elmali, Ayhan

2015-07-01

Supramolecular triplet photosensitizers based on hydrogen bonding-mediated molecular assemblies were prepared. Three thymine-containing visible light-harvesting Bodipy derivatives ( B-1 , B-2 and B-3 , which show absorption at 505 nm, 630 nm and 593 nm, respectively) were used as H-bonding modules, and 1,6-diaminopyridine-appended C 60 was used as the complementary hydrogen bonding module ( C-1 ), in which the C 60 part acts as a spin converter for triplet formation. Visible light-harvesting antennae with methylated thymine were prepared as references ( B-1-Me , B-2-Me and B-3-Me ), which are unable to form strong H-bonds with C-1 . Triple H-bonds are formed between each Bodipy antenna ( B-1 , B-2 and B-3 ) and the C 60 module ( C-1 ). The photophysical properties of the H-bonding assemblies and the reference non-hydrogen bond-forming mixtures were studied using steady state UV/vis absorption spectroscopy, fluorescence emission spectroscopy, electrochemical characterization, and nanosecond transient absorption spectroscopy. Singlet energy transfer from the Bodipy antenna to the C 60 module was confirmed by fluorescence quenching studies. The intersystem crossing of the latter produced the triplet excited state. The nanosecond transient absorption spectroscopy showed that the triplet state is either localized on the C 60 module (for assembly B-1·C-1 ), or on the styryl-Bodipy antenna (for assemblies B-2·C-1 and B-3·C-1 ). Intra-assembly forward-backward (ping-pong) singlet/triplet energy transfer was proposed. In contrast to the H-bonding assemblies, slow triplet energy transfer was observed for the non-hydrogen bonding mixtures. As a proof of concept, these supramolecular assemblies were used as triplet photosensitizers for triplet-triplet annihilation upconversion.

Identification and prognostic value of anterior gradient protein 2 expression in breast cancer based on tissue microarray.

PubMed

Guo, Jilong; Gong, Guohua; Zhang, Bin

2017-07-01

Breast cancer has attracted substantial attention as one of the major cancers causing death in women. It is crucial to find potential biomarkers of prognostic value in breast cancer. In this study, the expression pattern of anterior gradient protein 2 in breast cancer was identified based on the main molecular subgroups. Through analysis of 69 samples from the Gene Expression Omnibus database, we found that anterior gradient protein 2 expression was significantly higher in non-triple-negative breast cancer tissues compared with normal tissues and triple-negative breast cancer tissues (p < 0.05). The data from a total of 622 patients from The Cancer Genome Atlas were analysed. The data from The Cancer Genome Atlas and results from quantitative reverse transcription polymerase chain reaction also verified the anterior gradient protein 2 expression pattern. Furthermore, we performed immunohistochemical analysis. The quantification results revealed that anterior gradient protein 2 is highly expressed in non-triple-negative breast cancer (grade 3 excluded) and grade 1 + 2 (triple-negative breast cancer excluded) tumours compared with normal tissues. Anterior gradient protein 2 was significantly highly expressed in non-triple-negative breast cancer (grade 3 excluded) and non-triple-negative breast cancer tissues compared with triple-negative breast cancer tissues (p < 0.01). In addition, anterior gradient protein 2 was significantly highly expressed in grade 1 + 2 (triple-negative breast cancer excluded) and grade 1 + 2 tissues compared with grade 3 tissues (p < 0.05). Analysis by Fisher's exact test revealed that anterior gradient protein 2 expression was significantly associated with histologic type, histological grade, oestrogen status and progesterone status. Univariate analysis of clinicopathological variables showed that anterior gradient protein 2 expression, tumour size and lymph node status were significantly correlated with overall survival in patients with grade 1 and 2 tumours. Cox multivariate analysis revealed anterior gradient protein 2 as a putative independent indicator of unfavourable outcomes (p = 0.031). All these data clearly showed that anterior gradient protein 2 is highly expressed in breast cancer and can be regarded as a putative biomarker for breast cancer prognosis.

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.

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.

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.

Nanopatterned organic semiconductors for visible light communications

NASA Astrophysics Data System (ADS)

Yang, Xilu; Dong, Yurong; Zeng, Pan; Yu, Yan; Xie, Yujun; Gong, Junyi; Shi, Meng; Liang, Rongqing; Ou, Qiongrong; Chi, Nan; Zhang, Shuyu

2018-03-01

Visible light communication (VLC) is becoming an important and promising supplement to the existing Wi-Fi network for the coming 5G communications. Organic light-emitting semiconductors present much fast fluorescent decay rates compared to those of conventional colour-converting phosphors, therefore capable of achieving much higher bandwidths. Here we explore how nanopatterned organic semiconductors can further enhance the data rates of VLC links by improving bandwidths and signal-to-noise ratios (SNRs) and by supporting spatial multiplexing. We first demonstrate a colour-converting VLC system based on nanopatterned hyperbolic metamaterials (HMM), the bandwidth of which is enhanced by 50%. With regard to enhancing SNRs, we achieve a tripling of optical gain by integrating a nanopatterned luminescent concentrator to a signal receiver. In addition, we demonstrate highly directional fluorescent VLC antennas based on nanoimprinted polymer films, paving the way to achieving parallel VLC communications via spatialmultiplexing. These results indicate nanopatterned organic semiconductors provide a promising route to high speed VLC links.

A coupled cluster theory with iterative inclusion of triple excitations and associated equation of motion formulation for excitation energy and ionization potential

NASA Astrophysics Data System (ADS)

Maitra, Rahul; Akinaga, Yoshinobu; Nakajima, Takahito

2017-08-01

A single reference coupled cluster theory that is capable of including the effect of connected triple excitations has been developed and implemented. This is achieved by regrouping the terms appearing in perturbation theory and parametrizing through two different sets of exponential operators: while one of the exponentials, involving general substitution operators, annihilates the ground state but has a non-vanishing effect when it acts on the excited determinant, the other is the regular single and double excitation operator in the sense of conventional coupled cluster theory, which acts on the Hartree-Fock ground state. The two sets of operators are solved as coupled non-linear equations in an iterative manner without significant increase in computational cost than the conventional coupled cluster theory with singles and doubles excitations. A number of physically motivated and computationally advantageous sufficiency conditions are invoked to arrive at the working equations and have been applied to determine the ground state energies of a number of small prototypical systems having weak multi-reference character. With the knowledge of the correlated ground state, we have reconstructed the triple excitation operator and have performed equation of motion with coupled cluster singles, doubles, and triples to obtain the ionization potential and excitation energies of these molecules as well. Our results suggest that this is quite a reasonable scheme to capture the effect of connected triple excitations as long as the ground state remains weakly multi-reference.

Non-iterative triple excitations in equation-of-motion coupled-cluster theory for electron attachment with applications to bound and temporary anions.

PubMed

Jagau, Thomas-C

2018-01-14

The impact of residual electron correlation beyond the equation-of-motion coupled-cluster singles and doubles (EOM-CCSD) approximation on positions and widths of electronic resonances is investigated. To establish a method that accomplishes this task in an economical manner, several approaches proposed for the approximate treatment of triple excitations are reviewed with respect to their performance in the electron attachment (EA) variant of EOM-CC theory. The recently introduced EOM-CCSD(T)(a)* method [D. A. Matthews and J. F. Stanton, J. Chem. Phys. 145, 124102 (2016)], which includes non-iterative corrections to the reference and the target states, reliably reproduces vertical attachment energies from EOM-EA-CC calculations with single, double, and full triple excitations in contrast to schemes in which non-iterative corrections are applied only to the target states. Applications of EOM-EA-CCSD(T)(a)* augmented by a complex absorbing potential (CAP) to several temporary anions illustrate that shape resonances are well described by EOM-EA-CCSD, but that residual electron correlation often makes a non-negligible impact on their positions and widths. The positions of Feshbach resonances, on the other hand, are significantly improved when going from CAP-EOM-EA-CCSD to CAP-EOM-EA-CCSD(T)(a)*, but the correct energetic order of the relevant electronic states is still not achieved.

Non-iterative triple excitations in equation-of-motion coupled-cluster theory for electron attachment with applications to bound and temporary anions

NASA Astrophysics Data System (ADS)

Jagau, Thomas-C.

2018-01-01

The impact of residual electron correlation beyond the equation-of-motion coupled-cluster singles and doubles (EOM-CCSD) approximation on positions and widths of electronic resonances is investigated. To establish a method that accomplishes this task in an economical manner, several approaches proposed for the approximate treatment of triple excitations are reviewed with respect to their performance in the electron attachment (EA) variant of EOM-CC theory. The recently introduced EOM-CCSD(T)(a)* method [D. A. Matthews and J. F. Stanton, J. Chem. Phys. 145, 124102 (2016)], which includes non-iterative corrections to the reference and the target states, reliably reproduces vertical attachment energies from EOM-EA-CC calculations with single, double, and full triple excitations in contrast to schemes in which non-iterative corrections are applied only to the target states. Applications of EOM-EA-CCSD(T)(a)* augmented by a complex absorbing potential (CAP) to several temporary anions illustrate that shape resonances are well described by EOM-EA-CCSD, but that residual electron correlation often makes a non-negligible impact on their positions and widths. The positions of Feshbach resonances, on the other hand, are significantly improved when going from CAP-EOM-EA-CCSD to CAP-EOM-EA-CCSD(T)(a)*, but the correct energetic order of the relevant electronic states is still not achieved.

Guided Search for Triple Conjunctions

PubMed Central

Nordfang, Maria; Wolfe, Jeremy M

2017-01-01

A key tenet of Feature Integration Theory and related theories such as Guided Search (GS) is that the binding of basic features requires attention. This would seem to predict that conjunctions of features of objects that have not been attended should not influence search. However, Found (1998) reported that an irrelevant feature (size) improved the efficiency of search for a color × orientation conjunction if it was correlated with the other two features across the display compared to the case where size was not correlated with color and orientation features. We examine this issue with somewhat different stimuli. We use triple conjunctions of color, orientation and shape (e.g. search for a red, vertical, oval-shaped item). This allows us to manipulate the number of features that each distractor shares with the target (Sharing) and it allows us to vary the total number of distractor types (and, thus, the number of groups of identical items; Grouping). We find these triple conjunction searches are generally very efficient – producing very shallow reaction time (RT) × set size slopes, consistent with strong guidance by basic features. Nevertheless, both of these variables, Sharing and Grouping modulate performance. These influences are not predicted by previous accounts of GS. However, both can be accommodated in a GS framework. Alternatively, it is possible, if not necessary, to see these effects as evidence for “preattentive binding” of conjunctions. PMID:25005070

Guided search for triple conjunctions.

PubMed

Nordfang, Maria; Wolfe, Jeremy M

2014-08-01

A key tenet of feature integration theory and of related theories such as guided search (GS) is that the binding of basic features requires attention. This would seem to predict that conjunctions of features of objects that have not been attended should not influence search. However, Found (1998) reported that an irrelevant feature (size) improved the efficiency of search for a Color × Orientation conjunction if it was correlated with the other two features across the display, as compared to the case in which size was not correlated with color and orientation features. We examined this issue with somewhat different stimuli. We used triple conjunctions of color, orientation, and shape (e.g., search for a red, vertical, oval-shaped item). This allowed us to manipulate the number of features that each distractor shared with the target (sharing) and it allowed us to vary the total number of distractor types (and, thus, the number of groups of identical items: grouping). We found that these triple conjunction searches were generally very efficient--producing very shallow Reaction Time × Set Size slopes, consistent with strong guidance by basic features. Nevertheless, both of the variables, sharing and grouping, modulated performance. These influences were not predicted by previous accounts of GS; however, both can be accommodated in a GS framework. Alternatively, it is possible, though not necessary, to see these effects as evidence for "preattentive binding" of conjunctions.

"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.

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.

Assessment of SMAP soil moisture for global simulation of gross primary production

NASA Astrophysics Data System (ADS)

He, Liming; Chen, Jing M.; Liu, Jane; Bélair, Stéphane; Luo, Xiangzhong

2017-07-01

In this study, high-quality soil moisture data derived from the Soil Moisture Active Passive (SMAP) satellite measurements are evaluated from a perspective of improving the estimation of the global gross primary production (GPP) using a process-based ecosystem model, namely, the Boreal Ecosystem Productivity Simulator (BEPS). The SMAP soil moisture data are assimilated into BEPS using an ensemble Kalman filter. The correlation coefficient (r) between simulated GPP from the sunlit leaves and Sun-induced chlorophyll fluorescence (SIF) measured by Global Ozone Monitoring Experiment-2 is used as an indicator to evaluate the performance of the GPP simulation. Areas with SMAP data in low quality (i.e., forests), or with SIF in low magnitude (e.g., deserts), or both are excluded from the analysis. With the assimilated SMAP data, the r value is enhanced for Africa, Asia, and North America by 0.016, 0.013, and 0.013, respectively (p < 0.05). Significant improvement in r appears in single-cropping agricultural land where the irrigation is not considered in the model but well captured by SMAP (e.g., 0.09 in North America, p < 0.05). With the assimilation of SMAP, areas with weak model performances are identified in double or triple cropping cropland (e.g., part of North China Plain) and/or mountainous area (e.g., Spain and Turkey). The correlation coefficient is enhanced by 0.01 in global average for shrub, grass, and cropland. This enhancement is small and insignificant because nonwater-stressed areas are included.

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.

Integrated MicroRNA and mRNA Signatures Associated with Survival in Triple Negative Breast Cancer

PubMed Central

Lovat, Francesca; Carasi, Stefania; Pulvirenti, Alfredo; Ferro, Alfredo; Alder, Hansjuerg; He, Gang; Vecchione, Andrea; Croce, Carlo M.; Shapiro, Charles L.; Huebner, Kay

2013-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways. Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis. PMID:23405235

Integrated microRNA and mRNA signatures associated with survival in triple negative breast cancer.

PubMed

Cascione, Luciano; Gasparini, Pierluigi; Lovat, Francesca; Carasi, Stefania; Pulvirenti, Alfredo; Ferro, Alfredo; Alder, Hansjuerg; He, Gang; Vecchione, Andrea; Croce, Carlo M; Shapiro, Charles L; Huebner, Kay

2013-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways.Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis.

Exploring the kinetics of gelation and final architecture of enzymatically cross-linked chitosan/gelatin gels.

PubMed

da Silva, Marcelo A; Bode, Franziska; Grillo, Isabelle; Dreiss, Cécile A

2015-04-13

Small-angle neutron scattering (SANS) was used to characterize the nanoscale structure of enzymatically cross-linked chitosan/gelatin hydrogels obtained from two protocols: a pure chemical cross-linking process (C), which uses the natural enzyme microbial transglutaminase, and a physical-co-chemical (PC) hybrid process, where covalent cross-linking is combined with the temperature-triggered gelation of gelatin, occurring through the formation of triple-helices. SANS measurements on the final and evolving networks provide a correlation length (ξ), which reflects the average size of expanding clusters. Their growth in PC gels is restricted by the triple-helices (ξ ∼ 10s of Å), while ξ in pure chemical gels increases with cross-linker concentration (∼100s of Å). In addition, the shear elastic modulus in PC gels is higher than in pure C gels. Our results thus demonstrate that gelatin triple helices provide a template to guide the cross-linking process; overall, this work provides important structural insight to improve the design of biopolymer-based gels.

NHERF1 inhibits proliferation of triple-negative breast cancer cells by suppressing GPER signaling.

PubMed

Wang, Yan; Peng, Zhiqiang; Meng, Ran; Tao, Tao; Wang, Qiqi; Zhao, Chunjuan; Liu, Hua; Song, Ran; Zheng, Junfang; Qin, Qiong; He, Junqi

2017-07-01

G protein-coupled estrogen receptor (GPER) signaling is activated in triple-negative breast cancer (TNBC); however, the detailed mechanisms of its regulation remain unclear. The present study aimed to elucidate the molecular mechanisms involved in GPER activation in TNBC. In MDA-MB-231 cells, a TNBC cell line, NHERF1 interaction with GPER was verified by co-immunoprecipitation and immunofluorescent staining assays. Overexpression of NHERF1 in MDA-MB-231 cells inhibited GPER-mediated proliferation and phosphorylation of ERK1/2 and Akt. Furthermore, NHERF1 expression levels were negatively correlated with the gene signatures of GPER activation, ERK1/2 and Akt signaling, and cell proliferation in early stage of TNBC tumors from the TCGA data set. Taken together, NHERF1 inhibited the activation of GPER-mediated signaling and suppressed the proliferation of triple-negative breast cancer cells. Loss of NHERF1 expression may play a pivotal role in the early stage of TNBC carcinogenesis.

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.

Constrained Analysis of Fluorescence Anisotropy Decay:Application to Experimental Protein Dynamics

PubMed Central

Feinstein, Efraim; Deikus, Gintaras; Rusinova, Elena; Rachofsky, Edward L.; Ross, J. B. Alexander; Laws, William R.

2003-01-01

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay. PMID:12524313

[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.

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.

State-resolved three-dimensional electron-momentum correlation in nonsequential double ionization of benzene

NASA Astrophysics Data System (ADS)

Winney, Alexander H.; Lin, Yun Fei; Lee, Suk Kyoung; Adhikari, Pradip; Li, Wen

2016-03-01

We report state-resolved electron-momentum correlation measurement of strong-field nonsequential double ionization in benzene. With a novel coincidence detection apparatus, highly efficient triple coincidence (electron-electron dication) and quadruple coincidence (electron-electron-cation-cation) are used to resolve the final ionic states and to characterize three-dimensional (3D) electron-momentum correlation. The primary states associated with dissociative and nondissociative dications are assigned. A 3D momentum anticorrelation is observed for the electrons in coincidence with dissociative benzene dication states whereas such a correlation is absent for nondissociative dication states.

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

Selective and Orthogonal Post-Polymerization Modification using Sulfur(VI) Fluoride Exchange (SuFEx) and Copper-Catalyzed Azide–Alkyne Cycloaddition (CuAAC) Reactions

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

Oakdale, James S.; Kwisnek, Luke; Fokin, Valery V.

2016-06-10

Functional polystyrenes and polyacrylamides, containing combinations of fluorosulfate, aromatic silyl ether, and azide side chains, were used as scaffolds to demonstrate the postpolymerization modification capabilities of sulfur(VI) fluoride exchange (SuFEx) and CuAAC chemistries. Fluorescent dyes bearing appropriate functional groups were sequentially attached to the backbone of the copolymers, quantitatively and selectively addressing their reactive partners. Furthermore, this combined SuFEx and CuAAC approach proved to be robust and versatile, allowing for a rare accomplishment: triple orthogonal functionalization of a copolymer under essentially ambient conditions without protecting groups.

Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

PubMed Central

Suchanski, Jaroslaw; Olbromski, Mateusz; Gomulkiewicz, Agnieszka; Owczarek, Tomasz; Kruczak, Anna; Ambicka, Aleksandra; Rys, Janusz; Ugorski, Maciej; Podhorska-Okolow, Marzena; Dziegiel, Piotr

2015-01-01

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases. PMID:25933064

Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals. A novel labeling method in study of membranes and surface systems.

PubMed

Papper, V; Medvedeva, N; Fishov, I; Likhtenshtein, G I

2000-01-01

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the "triple" cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer's triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10(-12) mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10(-5) M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10(-3) microL or less.

Triple assessment of sentinel lymph node metastasis in early breast cancer using preoperative CTLG, intraoperative fluorescence navigation and OSNA.

PubMed

Mokhtar, Mohamed; Tadokoro, Yukiko; Nakagawa, Misako; Morimoto, Masami; Takechi, Hirokazu; Kondo, Kazuya; Tangoku, Akira

2016-03-01

Sentinel lymph node biopsy (SLNB) became a standard surgical procedure for patients with early breast cancer; however, the optimal method of sentinel lymph node (SLN) identification remains controversial. The current study presents the protocol of our institution for preoperative and intraoperative SLN detection. Fifty female patients with early breast cancer and clinically node-negative axilla were enrolled in this study. All patients underwent preoperative CT lymphography (CTLG), intraoperative SLNB using fluorescence navigation, intraoperative one-step nucleic acid amplification (OSNA) and postoperative hematoxylin and eosin histopathological analysis. Prediction of metastasis by CTLG and detection of metastasis by OSNA were compared to results of histopathology as standard reference. SLN were identified by preoperative CTLG and intraoperative SLNB with fluorescence navigation in all patients, the identification rate was 100 %. SLN metastases were detected as positive by OSNA in 9 patients (18 %), 4 were (++), 4 were (+) and 1 was (+I). SLN metastases were detected as positive by histopathology in 10 patients (20 %). The concordance rate between OSNA and permanent sections was 90 %. The negative predictive value of CTLG was 80 %. Use of CTLG and fluorescence navigation made performing SLNB with high accuracy possible in institutions that cannot use the radioisotope method. OSNA provided accurate intraoperative method, allowing for completion of axillary node dissection during surgery and avoidance of second surgical procedure in patients with positive SLNs, thereby reducing patient distress and, finally, saving hospital costs.

Spatial Distribution of Resonance in the Velocity Field for Transonic Flow over a Rectangular Cavity

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

Beresh, Steven J.; Wagner, Justin L.; Casper, Katya M.

Pulse-burst particle image velocimetry (PIV) has been used to acquire time-resolved data at 37.5 kHz of the flow over a finite-width rectangular cavity at Mach 0.8. Power spectra of the PIV data reveal four resonance modes that match the frequencies detected simultaneously using high-frequency wall pressure sensors but whose magnitudes exhibit spatial dependence throughout the cavity. Spatio-temporal cross-correlations of velocity to pressure were calculated after bandpass filtering for specific resonance frequencies. Cross-correlation magnitudes express the distribution of resonance energy, revealing local maxima and minima at the edges of the shear layer attributable to wave interference between downstream- and upstream-propagating disturbances.more » Turbulence intensities were calculated using a triple decomposition and are greatest in the core of the shear layer for higher modes, where resonant energies ordinarily are lower. Most of the energy for the lowest mode lies in the recirculation region and results principally from turbulence rather than resonance. Together, the velocity-pressure cross-correlations and the triple-decomposition turbulence intensities explain the sources of energy identified in the spatial distributions of power spectra amplitudes.« less

Spatial Distribution of Resonance in the Velocity Field for Transonic Flow over a Rectangular Cavity

DOE PAGES

Beresh, Steven J.; Wagner, Justin L.; Casper, Katya M.; ...

2017-07-27

Pulse-burst particle image velocimetry (PIV) has been used to acquire time-resolved data at 37.5 kHz of the flow over a finite-width rectangular cavity at Mach 0.8. Power spectra of the PIV data reveal four resonance modes that match the frequencies detected simultaneously using high-frequency wall pressure sensors but whose magnitudes exhibit spatial dependence throughout the cavity. Spatio-temporal cross-correlations of velocity to pressure were calculated after bandpass filtering for specific resonance frequencies. Cross-correlation magnitudes express the distribution of resonance energy, revealing local maxima and minima at the edges of the shear layer attributable to wave interference between downstream- and upstream-propagating disturbances.more » Turbulence intensities were calculated using a triple decomposition and are greatest in the core of the shear layer for higher modes, where resonant energies ordinarily are lower. Most of the energy for the lowest mode lies in the recirculation region and results principally from turbulence rather than resonance. Together, the velocity-pressure cross-correlations and the triple-decomposition turbulence intensities explain the sources of energy identified in the spatial distributions of power spectra amplitudes.« less

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.

Interaction study of collagen and sericin in blending solution.

PubMed

Duan, Lian; Yuan, Jingjie; Yang, Xiao; Cheng, Xinjian; Li, Jiao

2016-12-01

The interactions of collagen and sericin were studied by fluorescence spectra, ultraviolet spectra, FTIR spectra and dynamic light scattering. The fluorescence quenching in emission spectra and red-shift (283-330nm) in synchronous fluorescence spectra suggested the Tyr of collagen and sericin overlapped with a distance of 3Å, generating excimer. The overlapped Tyr of collagen and sericin decreased the hydrophobicity of collagen, which resulted in the red-shifts (233-240nm) in ultraviolet spectra. Moreover, the red-shifts of amide bands of collagen in FTIR spectra indicated the hydrogen bonds of collagen were weaken and it could also be explained by the overlapped Tyr. The results of 2D-FTIR spectra demonstrated the backbone of collagen molecule was varied and the most susceptible structure of collagen was the triple helix with the presence of sericin. Based on dynamic light scattering, we conjectured large pure collagen aggregates were replaced by hybrid aggregates of collagen and sericin particles after the addition of sericin. With ascending sericin ratio, the diameters of the hybrid aggregates increased and attained maximum with 60% ratio of sericin, which were on account of the increasing excimer number. The results of DSC demonstrated the presence of sericin enhanced the thermal stability of collagen. Copyright © 2016 Elsevier B.V. All rights reserved.

Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics.

PubMed

Földes-Papp, Zeno; Baumann, Gerd; Demel, Ulrike; Tilz, Gernot P

2004-04-01

Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.

Hierachical assembly of collagen mimetic peptides into biofunctional materials

NASA Astrophysics Data System (ADS)

Gleaton, Jeremy W.

Collagen is a remarkably strong and prevalent protein distributed throughout nature and as such, collagen is an ideal material for a variety of medical applications. Research efforts for the development of synthetic collagen biomaterials is an area of rapid growth. Here we present two methods for the assembly of collagen mimetic peptides (CMPs). The initial approach prompts assembly of CMPs which contain modifications for metal ion-triggered assembly. Hierarchical assembly into triple helices, followed by formation of disks via hydrophobic interactions has been demonstrated. Metal-ion mediated assembly of these disks, using iron (II)-bipyrdine interactions, has been shown to form micron-sized cages. The nature of the final structures that form depends on the number of bipyridine moieties incorporated into the CMP. These hollow spheres encapsulate a range of molecular weight fluorescently labeled dextrans. Furthermore, they demonstrate a time dependent release of contents under a variety of thermal conditions. The second approach assembles CMPs via the copper-catalyzed alkyne-azide cycloaddition (CuAAC) and the strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. CMPs that incorporate the unnatural amino acids L-propargylglycine and L-azidolysine form triple helices and demonstrate higher order assembly when reacted via CuAAC. Reaction of the alkyne/azide modified CMPs under CuAAC conditions was found to produce an crosslinked 3-dimensional network. Moreover, we demonstrate that polymers, such as, PEG, can be reacted with alkyne and azide CMP triple helices via CuAAC and SPAAC. This designed covalent CMP chemistry allows for high flexibility in integrating various chemical cues, such as cell growth and differentiation within the higher order structures.

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

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.

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.

Novel cell-penetrating peptide-loaded nanobubbles synergized with ultrasound irradiation enhance EGFR siRNA delivery for triple negative Breast cancer therapy.

PubMed

Jing, Hui; Cheng, Wen; Li, Shouqiang; Wu, Bolin; Leng, Xiaoping; Xu, Shouping; Tian, Jiawei

2016-10-01

The lack of safe and effective gene delivery strategies remains a bottleneck for cancer gene therapy. Here, we describe the synthesis, characterization, and application of cell-penetrating peptide (CPP)-loaded nanobubbles (NBs), which are characterized by their safety, strong penetrating power and high gene loading capability for gene delivery. An epidermal growth factor receptor (EGFR)-targeted small interfering RNA (siEGFR) was transfected into triple negative breast cancer (TNBC) cells via prepared CPP-NBs synergized with ultrasound-targeted microbubble destruction (UTMD) technology. Fluorescence microscopy showed that siEGFR and CPP were loaded on the shells of the NBs. The transfection efficiency and cell proliferation levels were evaluated by FACS and MTT assays, respectively. In addition, in vivo experiments showed that the expression of EGFR mRNA and protein could be efficiently downregulated and that the growth of a xenograft tumor derived from TNBC cells could be inhibited. Our results indicate that CPP-NBs carrying siEGFR could potentially be used as a promising non-viral gene vector that can be synergized with UTMD technology for efficient TNBC therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation

PubMed Central

Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D.C.

2013-01-01

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments. PMID:23746509

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.

Effect of aqueous ethanol on the triple helical structure of collagen.

PubMed

Gopinath, Arun; Reddy, Samala Murali Mohan; Madhan, Balaraman; Shanmguam, Ganesh; Rao, Jonnalagadda Raghava

2014-12-01

Collagen, the most abundant protein in mammals, is widely used for making biomaterials. Recently, organic solvents have been used to fabricate collagen-based biomaterials for biological applications. It is therefore necessary to understand the behavior of collagen in the presence of organic solvents at low (≤50%, v/v) and high (≥90%, v/v) concentrations. This study was conducted to examine how collagen reacts when exposed to low and high concentrations of ethanol, one of the solvents used to make collagen-based biomaterials. Solubility testing indicated that collagen remains in solution at low concentrations (≤50%, v/v) of ethanol but precipitates (gel-like) thereafter, irrespective of the method of addition of ethanol (single shot or gradual addition); this behavior is different from that observed recently with acetonitrile. Collagen retains its triple helix in the presence of ethanol but becomes thermodynamically unstable, with substantially reduced melting temperature, with increasing concentration of ethanol. It was also found that the CD ellipticity at 222 nm, characteristic of the triple-helical structure, does not correlate with the thermal stability of collagen. Time-dependent experiments reveal that the collagen triple helix is kinetically stable in the presence of 0-40% (v/v) ethanol at low temperature (5 °C) but highly unstable in the presence of ethanol at elevated temperature (~34 °C). These results indicate that when ethanol is used to process collagen-based biomaterials, such factors as temperature and duration should be done taking into account, to prevent extensive damage to the triple-helical structure of collagen.

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).

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

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.

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.

Measurement of a solid-state triple point at the metal-insulator transition in VO2.

PubMed

Park, Jae Hyung; Coy, Jim M; Kasirga, T Serkan; Huang, Chunming; Fei, Zaiyao; Hunter, Scott; Cobden, David H

2013-08-22

First-order phase transitions in solids are notoriously challenging to study. The combination of change in unit cell shape, long range of elastic distortion and flow of latent heat leads to large energy barriers resulting in domain structure, hysteresis and cracking. The situation is worse near a triple point, where more than two phases are involved. The well-known metal-insulator transition in vanadium dioxide, a popular candidate for ultrafast optical and electrical switching applications, is a case in point. Even though VO2 is one of the simplest strongly correlated materials, experimental difficulties posed by the first-order nature of the metal-insulator transition as well as the involvement of at least two competing insulating phases have led to persistent controversy about its nature. Here we show that studying single-crystal VO2 nanobeams in a purpose-built nanomechanical strain apparatus allows investigation of this prototypical phase transition with unprecedented control and precision. Our results include the striking finding that the triple point of the metallic phase and two insulating phases is at the transition temperature, Ttr = Tc, which we determine to be 65.0 ± 0.1 °C. The findings have profound implications for the mechanism of the metal-insulator transition in VO2, but they also demonstrate the importance of this approach for mastering phase transitions in many other strongly correlated materials, such as manganites and iron-based superconductors.

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.

The error structure of the SMAP single and dual channel soil moisture retrievals

USDA-ARS?s Scientific Manuscript database

Knowledge of the temporal error structure for remotely-sensed surface soil moisture retrievals can improve our ability to exploit them for hydrology and climate studies. This study employs a triple collocation type analysis to investigate both the total variance and temporal auto-correlation of erro...

The potential of 2D Kalman filtering for soil moisture data assimilation

USDA-ARS?s Scientific Manuscript database

We examine the potential for parameterizing a two-dimensional (2D) land data assimilation system using spatial error auto-correlation statistics gleaned from a triple collocation analysis and the triplet of: (1) active microwave-, (2) passive microwave- and (3) land surface model-based surface soil ...

Nondestructive assessment of collagen hydrogel cross-linking using time-resolved autofluorescence imaging

NASA Astrophysics Data System (ADS)

Sherlock, Benjamin E.; Harvestine, Jenna N.; Mitra, Debika; Haudenschild, Anne; Hu, Jerry; Athanasiou, Kyriacos A.; Leach, J. Kent; Marcu, Laura

2018-03-01

We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable.

High expressions of LDHA and AMPK as prognostic biomarkers for breast cancer.

PubMed

Huang, Xiaojia; Li, Xing; Xie, Xinhua; Ye, Feng; Chen, Bo; Song, Cailu; Tang, Hailin; Xie, Xiaoming

2016-12-01

The purpose of this study was to investigate the potential correlation between lactate dehydrogenase A (LDHA) and AMP-activated protein kinase (AMPK) and their clinicopathologic significance in breast cancer. Western blot and qRT-PCR were used to detect the expression levels of LDHA and AMPK in eight breast cancer lines and eight breast cancer tissues. In addition, LDHA and AMPK were detected by immunohistochemistry (IHC) using breast cancer tissue microarrays (TMAs) of 112 patients. The association between LDHA and AMPK expression levels was statistically analyzed. So were the prognostic roles and clinicopathologic significances in breast cancer. The expression levels of LDHA and AMPK were relatively higher in triple-negative breast cancer (TNBC) cell lines than in non-triple-negative breast cancer (NTNBC) cell lines. LDHA and AMPK were also further up-regulated in TNBC tissues than in NTNBC tissues. Correlation analysis showed a positive correlation between LDHA and AMPK expression levels. Expression of LDHA and AMPK were significantly correlated with TNM stage, distant metastasis, Ki67 status and survival outcomes of patients. Patients with both positive expression of LDHA and AMPK showed shorter overall survival (OS) and disease-free survival (DFS). These findings improve our understanding of the expression pattern of LDHA and AMPK in breast cancer and clarify the role of LDHA and AMPK as promising prognostic biomarkers for breast cancer. Copyright © 2016. Published by Elsevier Ltd.

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation

NASA Astrophysics Data System (ADS)

Zhong, Zhensheng; Yang, Lixia; Zhang, Haiping; Shi, Jiahao; Vandana, J. Jeya; Lam, Do Thuy Uyen Ha; Olsthoorn, René C. L.; Lu, Lanyuan; Chen, Gang

2016-12-01

Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The -1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between -1 frameshifting efficiency and unfolding rate at forces of 15-35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and -1 frameshifting efficiency.

An Accurate ab initio Quartic Force Field and Vibrational Frequencies for CH4 and Isotopomers

NASA Technical Reports Server (NTRS)

Lee, Timothy J.; Martin, Jan M. L.; Taylor, Peter R.

1995-01-01

A very accurate ab initio quartic force field for CH4 and its isotopomers is presented. The quartic force field was determined with the singles and doubles coupled-cluster procedure that includes a quasiperturbative estimate of the effects of connected triple excitations, CCSD(T), using the correlation consistent polarized valence triple zeta, cc-pVTZ, basis set. Improved quadratic force constants were evaluated with the correlation consistent polarized valence quadruple zeta, cc-pVQZ, basis set. Fundamental vibrational frequencies are determined using second-order perturbation theory anharmonic analyses. All fundamentals of CH4 and isotopomers for which accurate experimental values exist and for which there is not a large Fermi resonance, are predicted to within +/- 6 cm(exp -1). It is thus concluded that our predictions for the harmonic frequencies and the anharmonic constants are the most accurate estimates available. It is also shown that using cubic and quartic force constants determined with the correlation consistent polarized double zeta, cc-pVDZ, basis set in conjunction with the cc-pVQZ quadratic force constants and equilibrium geometry leads to accurate predictions for the fundamental vibrational frequencies of methane, suggesting that this approach may be a viable alternative for larger molecules. Using CCSD(T), core correlation is found to reduce the CH4 r(e), by 0.0015 A. Our best estimate for r, is 1.0862 +/- 0.0005 A.

Composite vibrational spectroscopy of the group 12 difluorides: ZnF2, CdF2, and HgF2.

PubMed

Solomonik, Victor G; Smirnov, Alexander N; Navarkin, Ilya S

2016-04-14

The vibrational spectra of group 12 difluorides, MF2 (M = Zn, Cd, Hg), were investigated via coupled cluster singles, doubles, and perturbative triples, CCSD(T), including core correlation, with a series of correlation consistent basis sets ranging in size from triple-zeta through quintuple-zeta quality, which were then extrapolated to the complete basis set (CBS) limit using a variety of extrapolation procedures. The explicitly correlated coupled cluster method, CCSD(T)-F12b, was employed as well. Although exhibiting quite different convergence behavior, the F12b method yielded the CBS limit estimates closely matching more computationally expensive conventional CBS extrapolations. The convergence with respect to basis set size was examined for the contributions entering into composite vibrational spectroscopy, including those from higher-order correlation accounted for through the CCSDT(Q) level of theory, second-order spin-orbit coupling effects assessed within four-component and two-component relativistic formalisms, and vibrational anharmonicity evaluated via a perturbative treatment. Overall, the composite results are in excellent agreement with available experimental values, except for the CdF2 bond-stretching frequencies compared to spectral assignments proposed in a matrix isolation infrared and Raman study of cadmium difluoride vapor species [Loewenschuss et al., J. Chem. Phys. 50, 2502 (1969); Givan and Loewenschuss, J. Chem. Phys. 72, 3809 (1980)]. These assignments are called into question in the light of the composite results.

Composite vibrational spectroscopy of the group 12 difluorides: ZnF2, CdF2, and HgF2

NASA Astrophysics Data System (ADS)

Solomonik, Victor G.; Smirnov, Alexander N.; Navarkin, Ilya S.

2016-04-01

The vibrational spectra of group 12 difluorides, MF2 (M = Zn, Cd, Hg), were investigated via coupled cluster singles, doubles, and perturbative triples, CCSD(T), including core correlation, with a series of correlation consistent basis sets ranging in size from triple-zeta through quintuple-zeta quality, which were then extrapolated to the complete basis set (CBS) limit using a variety of extrapolation procedures. The explicitly correlated coupled cluster method, CCSD(T)-F12b, was employed as well. Although exhibiting quite different convergence behavior, the F12b method yielded the CBS limit estimates closely matching more computationally expensive conventional CBS extrapolations. The convergence with respect to basis set size was examined for the contributions entering into composite vibrational spectroscopy, including those from higher-order correlation accounted for through the CCSDT(Q) level of theory, second-order spin-orbit coupling effects assessed within four-component and two-component relativistic formalisms, and vibrational anharmonicity evaluated via a perturbative treatment. Overall, the composite results are in excellent agreement with available experimental values, except for the CdF2 bond-stretching frequencies compared to spectral assignments proposed in a matrix isolation infrared and Raman study of cadmium difluoride vapor species [Loewenschuss et al., J. Chem. Phys. 50, 2502 (1969); Givan and Loewenschuss, J. Chem. Phys. 72, 3809 (1980)]. These assignments are called into question in the light of the composite results.

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

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.

First-line eradication for Helicobacter pylori-positive gastritis by esomeprazole-based triple therapy is influenced by CYP2C19 genotype

PubMed Central

Saito, Yoshimasa; Serizawa, Hiroshi; Kato, Yukako; Nakano, Masaru; Nakamura, Masahiko; Saito, Hidetsugu; Suzuki, Hidekazu; Kanai, Takanori

2015-01-01

AIM: To evaluate the effect of first line esomeprazole (EPZ)-based triple therapy on Helicobacter pylori (H. pylori) eradication. METHODS: A total of 80 Japanese patients with gastritis who were diagnosed as positive for H. pylori infection by endoscopic biopsy-based or 13C-urea breath tests were included in this study. The average age of the patients was 57.2 years (male/female, 42/38). These patients were treated by first-line eradication therapy with EPZ 40 mg/d, amoxicillin 1500 mg/d, and clarithromycin 400 mg/d for 7 d. All drugs were given twice per day. Correlations between H. pylori eradication, CYP2C19 genotype, and serum pepsinogen (PG) level were analyzed. This study was registered with the UMIN Clinical Trials Registry (UMIN000009642). RESULTS: The H. pylori eradication rates by EPZ-based triple therapy evaluated by intention-to-treat and per protocol were 67.5% and 68.4%, respectively, which were similar to triple therapies with other first-generation proton pump inhibitors (PPIs). The eradication rates in three different CYP2C19 genotypes, described as extensive metabolizer (EM), intermediate metabolizer, and poor metabolizer, were 52.2%, 72.1%, and 84.6%, respectively. The H. pylori eradication rate was significantly lower in EM than non-EM (P < 0.05). The serum PG I level and PG I/II ratio were significantly increased after eradication of H. pylori (P < 0.01), suggesting that gastric atrophy was improved by H. pylori eradication. Thus, first-line eradication by EPZ-based triple therapy for patients with H. pylori-positive gastritis was influenced by CYP2C19 genotype, and the eradication rate was on the same level with other first-generation PPIs in the Japanese population. CONCLUSION: The results from this study suggest that there is no advantage to EPZ-based triple therapy on H. pylori eradication compared to other first-generation PPIs. PMID:26730167

Incidence and risk factors for breast cancer subtypes in three distinct South-East Asian ethnic groups: Chinese, Malay and natives of Sarawak, Malaysia.

PubMed

Devi, C R Beena; Tang, Tieng Swee; Corbex, Marilys

2012-12-15

We determined the incidences of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) subtypes among breast cancer cases in Sarawak, Malaysia and their correlation with various risk factors in the three ethnic groups: Chinese, Malay and native. Subtype status was ascertained for 1,034 cases of female breast cancer (93% of all cases diagnosed since 2003), and the age-standardized incidence rates (ASRs) of each subtype were inferred. Case-case comparisons across subtypes were performed for reproductive risk factors. We found 48% luminal A (ER+/PR+/HER2-), 29% triple-negative (ER-/PR-/HER2-), 12% triple-positive (ER+/PR+/HER2+) and 11% HER2-overexpressing (ER-/PR-/HER2+) subtypes, with ASRs of 10.6, 6.0, 2.8 and 2.8 per 100,000, respectively. The proportions of subtypes and ASRs differed significantly by ethnic groups: HER2-positive cases were more frequent in Malays (29%; 95% CI [23;35]) than Chinese (22%; [19;26] and natives (21%; [16;26]); triple-negative cases were less frequent among Chinese (23%; [20;27]) than Malays (33%; [27;39]) and natives (37%; [31;43]). The results of the case-case comparison were in accordance with those observed in western case series. Some uncommon associations, such as between triple-negative subtype and older age at menopause (OR, 1.59; p < 0.05), were found. The triple-negative and HER2+ subtypes predominate in our region, with significant differences among ethnic groups. Our results support the idea that the risk factors for different subtypes vary markedly. Westernized populations are more likely to have factors that increase the risk for the luminal A type, while risk factors for the triple-negative type are more frequent in local populations. Copyright © 2012 UICC.

Effects of H{sub 2} enrichment on the propagation characteristics of CH{sub 4}-air triple flames

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

Briones, Alejandro M.; Aggarwal, Suresh K.; Katta, Viswanath R.

The effects of H{sub 2} enrichment on the propagation of laminar CH{sub 4}-air triple flames in axisymmetric coflowing jets are numerically investigated. A comprehensive, time-dependent computational model, which employs a detailed description of chemistry and transport, is used to simulate the transient ignition and flame propagation phenomena. Flames are ignited in a jet-mixing layer far downstream of the burner. Following ignition, a well-defined triple flame is formed that propagates upstream along the stoichiometric mixture fraction line with a nearly constant displacement velocity. As the flame approaches the burner, it transitions to a double flame, and subsequently to a burner-stabilized nonpremixedmore » flame. Predictions are validated using measurements of the displacement flame velocity. As the H{sub 2} concentration in the fuel blend is increased, the displacement flame velocity and local triple flame speed increase progressively due to the enhanced chemical reactivity, diffusivity, and preferential diffusion caused by H{sub 2} addition. In addition, the flammability limits associated with the triple flames are progressively extended with the increase in H{sub 2} concentration. The flame structure and flame dynamics are also markedly modified by H{sub 2} enrichment, which substantially increases the flame curvature and mixture fraction gradient, as well as the hydrodynamic and curvature-induced stretch near the triple point. For all the H{sub 2}-enriched methane-air flames investigated in this study, there is a negative correlation between flame speed and stretch, with the flame speed decreasing almost linearly with stretch, consistent with previous studies. The H{sub 2} addition also modifies the flame sensitivity to stretch, as it decreases the Markstein number (Ma), implying an increased tendency toward diffusive-thermal instability (i.e. Ma {yields} 0). These results are consistent with the previously reported experimental results for outwardly propagating spherical flames burning a mixture of natural gas and hydrogen. (author)« less

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.

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.

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.

Optimization of the linear-scaling local natural orbital CCSD(T) method: Redundancy-free triples correction using Laplace transform.

PubMed

Nagy, Péter R; Kállay, Mihály

2017-06-07

An improved algorithm is presented for the evaluation of the (T) correction as a part of our local natural orbital (LNO) coupled-cluster singles and doubles with perturbative triples [LNO-CCSD(T)] scheme [Z. Rolik et al., J. Chem. Phys. 139, 094105 (2013)]. The new algorithm is an order of magnitude faster than our previous one and removes the bottleneck related to the calculation of the (T) contribution. First, a numerical Laplace transformed expression for the (T) fragment energy is introduced, which requires on average 3 to 4 times fewer floating point operations with negligible compromise in accuracy eliminating the redundancy among the evaluated triples amplitudes. Second, an additional speedup factor of 3 is achieved by the optimization of our canonical (T) algorithm, which is also executed in the local case. These developments can also be integrated into canonical as well as alternative fragmentation-based local CCSD(T) approaches with minor modifications. As it is demonstrated by our benchmark calculations, the evaluation of the new Laplace transformed (T) correction can always be performed if the preceding CCSD iterations are feasible, and the new scheme enables the computation of LNO-CCSD(T) correlation energies with at least triple-zeta quality basis sets for realistic three-dimensional molecules with more than 600 atoms and 12 000 basis functions in a matter of days on a single processor.

Anticancer and Anti-Inflammatory Properties of Ganoderma lucidum Extract Effects on Melanoma and Triple-Negative Breast Cancer Treatment

PubMed Central

Barbieri, Antonio; Quagliariello, Vincenzo; Del Vecchio, Vitale; Falco, Michela; Luciano, Antonio; Amruthraj, Nagoth Joseph; Nasti, Guglielmo; Ottaiano, Alessandro; Berretta, Massimiliano; Iaffaioli, Rosario Vincenzo; Arra, Claudio

2017-01-01

Among the most important traditional medicinal fungi, Ganoderma lucidum has been used as a therapeutic agent for the treatment of numerous diseases, including cancer, in Oriental countries. The aim of this study is to investigate the anti-inflammatory, anticancer and anti-metastatic activities of Ganoderma lucidum extracts in melanoma and triple-negative breast cancer cells. Ganoderma lucidum extracts were prepared by using common organic solvents; MDA-MB 231 and B16-F10 cell lines were adopted as cellular models for triple-negative breast cancer and melanoma and characterized for cell viability, wound-healing assay and measurement of cytokines secreted by cancer cells under pro-inflammatory conditions (incubation with lipopolysaccharide, LPS) and pretreatment with Ganoderma lucidum extract at different concentrations. Our study demonstrates, for the first time, how Ganoderma lucidum extracts can significantly inhibit the release of IL-8, IL-6, MMP-2 and MMP-9 in cancer cells under pro-inflammatory condition. Interestingly, Ganoderma lucidum extracts significantly also decrease the viability of both cancer cells in a time- and concentration-dependent manner, with abilities to reduce cell migration over time, which is correlated with a lower release of matrix metalloproteases. Taken together, these results indicate the possible use of Ganoderma lucidum extract for the therapeutic management of melanoma and human triple-negative breast cancer. PMID:28264501

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models.

PubMed

Grabmüller, Melanie; Schyma, Christian; Euteneuer, Jan; Madea, Burkhard; Courts, Cornelius

2015-09-01

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

Optimization of the linear-scaling local natural orbital CCSD(T) method: Redundancy-free triples correction using Laplace transform

PubMed Central

2017-01-01

An improved algorithm is presented for the evaluation of the (T) correction as a part of our local natural orbital (LNO) coupled-cluster singles and doubles with perturbative triples [LNO-CCSD(T)] scheme [Z. Rolik et al., J. Chem. Phys. 139, 094105 (2013)]. The new algorithm is an order of magnitude faster than our previous one and removes the bottleneck related to the calculation of the (T) contribution. First, a numerical Laplace transformed expression for the (T) fragment energy is introduced, which requires on average 3 to 4 times fewer floating point operations with negligible compromise in accuracy eliminating the redundancy among the evaluated triples amplitudes. Second, an additional speedup factor of 3 is achieved by the optimization of our canonical (T) algorithm, which is also executed in the local case. These developments can also be integrated into canonical as well as alternative fragmentation-based local CCSD(T) approaches with minor modifications. As it is demonstrated by our benchmark calculations, the evaluation of the new Laplace transformed (T) correction can always be performed if the preceding CCSD iterations are feasible, and the new scheme enables the computation of LNO-CCSD(T) correlation energies with at least triple-zeta quality basis sets for realistic three-dimensional molecules with more than 600 atoms and 12 000 basis functions in a matter of days on a single processor. PMID:28576082

Anticancer and Anti-Inflammatory Properties of Ganoderma lucidum Extract Effects on Melanoma and Triple-Negative Breast Cancer Treatment.

PubMed

Barbieri, Antonio; Quagliariello, Vincenzo; Del Vecchio, Vitale; Falco, Michela; Luciano, Antonio; Amruthraj, Nagoth Joseph; Nasti, Guglielmo; Ottaiano, Alessandro; Berretta, Massimiliano; Iaffaioli, Rosario Vincenzo; Arra, Claudio

2017-02-28

Among the most important traditional medicinal fungi, Ganoderma lucidum has been used as a therapeutic agent for the treatment of numerous diseases, including cancer, in Oriental countries. The aim of this study is to investigate the anti-inflammatory, anticancer and anti-metastatic activities of Ganoderma lucidum extracts in melanoma and triple-negative breast cancer cells. Ganoderma lucidum extracts were prepared by using common organic solvents; MDA-MB 231 and B16-F10 cell lines were adopted as cellular models for triple-negative breast cancer and melanoma and characterized for cell viability, wound-healing assay and measurement of cytokines secreted by cancer cells under pro-inflammatory conditions (incubation with lipopolysaccharide, LPS) and pretreatment with Ganoderma lucidum extract at different concentrations. Our study demonstrates, for the first time, how Ganoderma lucidum extracts can significantly inhibit the release of IL-8, IL-6, MMP-2 and MMP-9 in cancer cells under pro-inflammatory condition. Interestingly, Ganoderma lucidum extracts significantly also decrease the viability of both cancer cells in a time- and concentration-dependent manner, with abilities to reduce cell migration over time, which is correlated with a lower release of matrix metalloproteases. Taken together, these results indicate the possible use of Ganoderma lucidum extract for the therapeutic management of melanoma and human triple-negative breast cancer.

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.

Creep deformation and mechanisms in Haynes 230 at 800 °C and 900 °C

NASA Astrophysics Data System (ADS)

Pataky, Garrett J.; Sehitoglu, Huseyin; Maier, Hans J.

2013-11-01

Creep was studied in Haynes 230, a material candidate for the very high temperature reactor's intermediate heat exchanger, at 800 °C and 900 °C. This study focused on the differences between the behavior at the two elevated temperature, and using the microstructure, grain boundary serrations and triple junction strain concentrations were quantitatively identified. There was significant damage in the 900 °C samples and the creep was almost entirely tertiary. In contrast, the 800 °C sample exhibited secondary creep. Using an Arrhenius equation, the minimum creep rate exponents were found to be n ≈ 3 and n ≈ 5 for 900 °C and 800 °C, respectively. The creep mechanisms were identified as solute drag for n ≈ 3 and dislocation climb for n ≈ 5. Strain concentrations were identified at triple junctions and grain boundary serrations using high resolution digital image correlation overlaid on the microstructure. The grain boundary serrations restrict grain boundary sliding which may reduce the creep damage at triple junctions and extend the creep life of Haynes 230 at elevated temperatures.

Shear-rate dependence of the viscosity of the Lennard-Jones liquid at the triple point

NASA Astrophysics Data System (ADS)

Ferrario, M.; Ciccotti, G.; Holian, B. L.; Ryckaert, J. P.

1991-11-01

High-precision molecular-dynamics (MD) data are reported for the shear viscosity η of the Lennard-Jones liquid at its triple point, as a function of the shear rate ɛ˙ for a large system (N=2048). The Green-Kubo (GK) value η(ɛ˙=0)=3.24+/-0.04 is estimated from a run of 3.6×106 steps (40 nsec). We find no numerical evidence of a t-3/2 long-time tail for the GK integrand (stress-stress time-correlation function). From our nonequilibrium MD results, obtained both at small and large values of ɛ˙, a consistent picture emerges that supports an analytical (quadratic at low shear rate) dependence of the viscosity on ɛ˙.

Application of the Intuitionistic Fuzzy InterCriteria Analysis Method with Triples to a Neural Network Preprocessing Procedure

PubMed Central

Atanassova, Vassia; Sotirova, Evdokia; Doukovska, Lyubka; Bureva, Veselina; Mavrov, Deyan; Tomov, Jivko

2017-01-01

The approach of InterCriteria Analysis (ICA) was applied for the aim of reducing the set of variables on the input of a neural network, taking into account the fact that their large number increases the number of neurons in the network, thus making them unusable for hardware implementation. Here, for the first time, with the help of the ICA method, correlations between triples of the input parameters for training of the neural networks were obtained. In this case, we use the approach of ICA for data preprocessing, which may yield reduction of the total time for training the neural networks, hence, the time for the network's processing of data and images. PMID:28874908

Collisional excitation of interstellar PO(X2Π) by He: new ab initio potential energy surfaces and scattering calculations

NASA Astrophysics Data System (ADS)

Lique, François; Jiménez-Serra, Izaskun; Viti, Serena; Marinakis, Sarantos

2018-01-01

We present the first ab initio potential energy surfaces (PESs) for the PO(X2Π)-He van der Waals system. The PESs were obtained using the open-shell partially spin-restricted coupled cluster approach with single, double and perturbative triple excitations [UCCSD(T)]. The augmented correlation-consistent polarized valence triple-zeta (aug-cc-pVTZ) basis set was employed supplemented by mid-bond functions. Integral and differential cross sections for the rotational excitation in PO-He collisions were calculated using the new PES and compared with results in similar systems. Finally, our work presents the first hyperfine-resolved cross sections for this system that are needed for accurate modelling in astrophysical 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.

2014-08-01

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.

Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging.

PubMed

Breitsprecher, Dennis; Jaiswal, Richa; Bombardier, Jeffrey P; Gould, Christopher J; Gelles, Jeff; Goode, Bruce L

2012-06-01

Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single-molecule fluorescence microscopy to image the tumor suppressor adenomatous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers initiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly and then separate but retain independent associations with either end of the growing filament.

Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging

PubMed Central

Breitsprecher, Dennis; Jaiswal, Richa; Bombardier, Jeffrey P.; Gould, Christopher J.; Gelles, Jeff; Goode, Bruce L.

2013-01-01

Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single molecule fluorescence microscopy to image the tumor-suppressor Adenomateous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers intiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly, and then separate but retain independent associations with either end of the growing filament. PMID:22654058

The Turner syndrome in patient with 45X/47XXX mosaic karyotype--case report.

PubMed

Maciejewska-Jeske, Marzena; Czyzyk, Adam; Meczekalski, Blazej

2015-07-01

Turner syndrome (TS) is a gonadal dysgenesis related to partial or total lack of one of the X chromosomes. It this report we describe a young patient presenting some somatic features of TS, who underwent spontaneous puberty and was eumenoorheic up to the age of 23. Using fluorescent in situ hybridization (FISH) mosaic karyotype (45X[131]/47XXX[9]) of TS and triple X syndrome was found. She presented uncommon for TS somatic hemihypotrophy and underwent growth hormone and surgical therapy. The patient was diagnosed with premature ovarian failure when she was 23, with absent follicular reserve. Clinical features of this case and a few published cases will be reviewed briefly.

[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.

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.

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.

Spatiotemporal assessment of spontaneous metastasis formation using multimodal in vivo imaging in HER2+ and triple negative metastatic breast cancer xenograft models in mice.

PubMed

Fricke, Inga B; De Souza, Raquel; Costa Ayub, Lais; Francia, Giulio; Kerbel, Robert; Jaffray, David A; Zheng, Jinzi

2018-01-01

Preclinical breast cancer models recapitulating the clinical course of metastatic disease are crucial for drug development. Highly metastatic cell lines forming spontaneous metastasis following orthotopic implantation were previously developed and characterized regarding their biological and histological characteristics. This study aimed to non-invasively and longitudinally characterize the spatiotemporal pattern of metastasis formation and progression in the MDA-MB-231-derived triple negative LM2-4 and HER2+ LM2-4H2N cell lines, using bioluminescence imaging (BLI), contrast enhanced computed tomography (CT), fluorescence imaging, and 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography ([18F]FDG-PET). LM2-4, LM2-4H2N, and MDA-MB-231 tumors were established in the right inguinal mammary fat pad (MFP) of female SCID mice and resected 14-16 days later. Metastasis formation was monitored using BLI. Metabolic activity of primary and metastatic lesions in mice bearing LM2-4 or LM2-4H2N was assessed by [18F]FDG-PET. Metastatic burden at study endpoint was assessed by CT and fluorescence imaging following intravenous dual-modality liposome agent administration. Comparable temporal metastasis patterns were observed using BLI for the highly metastatic cell lines LM2-4 and LM2-4H2N, while metastasis formed about 10 days later for MDA-MB-231. 21 days post primary tumor resection, metastases were detected in 86% of LM2-4, 69% of LM2-4H2N, and 60% of MDA-MB-231 inoculated mice, predominantly in the axillary region, contralateral MFP, and liver/lung. LM2-4 and LM2-4H2N tumors displayed high metabolism based on [18F]FDG-PET uptake. Lung metastases were detected as the [18F]FDG-PET uptake increased significantly between pre- and post-metastasis scan. Using a liposomal dual-modality agent, CT and fluorescence confirmed BLI detected lesions and identified additional metastatic nodules in the intraperitoneal cavity and lung. The combination of complementary anatomical and functional imaging techniques can provide high sensitivity characterization of metastatic disease spread, progression and overall disease burden. The described models and imaging toolset can be implemented as an effective means for quantitative treatment response evaluation in metastatic breast cancer.

Implementation of a noise reduction circuit for spaceflight IR spectrometers

NASA Technical Reports Server (NTRS)

Ramirez, L.; Hickok, R.; Pain, B.; Staller, C.

1992-01-01

The paper discusses the implementation and analysis of a correlated triple sampling circuit using analog subtractor/integrators. The software and test setup for noise measurements are also described. The correlation circuitry is part of the signal chain for a 256-element InSb line array used in the Visible and Infrared Mapping Spectrometer. Using a focal-plane array (FPA) simulator, system noise measurements of 0.7 DN are obtained. A test setup for FPA/SPE (signal processing electronics) characterization along with noise measurements is demonstrated.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction

PubMed Central

Parashurama, Natesh; Ahn, Byeong-Cheol; Ziv, Keren; Ito, Ken; Paulmurugan, Ramasamy; Willmann, Jürgen K.; Chung, Jaehoon; Ikeno, Fumiaki; Swanson, Julia C.; Merk, Denis R.; Lyons, Jennifer K.; Yerushalmi, David; Teramoto, Tomohiko; Kosuge, Hisanori; Dao, Catherine N.; Ray, Pritha; Patel, Manishkumar; Chang, Ya-fang; Mahmoudi, Morteza; Cohen, Jeff Eric; Goldstone, Andrew Brooks; Habte, Frezghi; Bhaumik, Srabani; Yaghoubi, Shahriar; Robbins, Robert C.; Dash, Rajesh; Yang, Phillip C.; Brinton, Todd J.; Yock, Paul G.; McConnell, Michael V.

2016-01-01

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow–derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. © RSNA, 2016 Online supplemental material is available for this article. PMID:27308957

Reduction of the virtual space for coupled-cluster excitation energies of large molecules and embedded systems

PubMed Central

Send, Robert; Kaila, Ville R. I.; Sundholm, Dage

2011-01-01

We investigate how the reduction of the virtual space affects coupled-cluster excitation energies at the approximate singles and doubles coupled-cluster level (CC2). In this reduced-virtual-space (RVS) approach, all virtual orbitals above a certain energy threshold are omitted in the correlation calculation. The effects of the RVS approach are assessed by calculations on the two lowest excitation energies of 11 biochromophores using different sizes of the virtual space. Our set of biochromophores consists of common model systems for the chromophores of the photoactive yellow protein, the green fluorescent protein, and rhodopsin. The RVS calculations show that most of the high-lying virtual orbitals can be neglected without significantly affecting the accuracy of the obtained excitation energies. Omitting all virtual orbitals above 50 eV in the correlation calculation introduces errors in the excitation energies that are smaller than 0.1 eV . By using a RVS energy threshold of 50 eV , the CC2 calculations using triple-ζ basis sets (TZVP) on protonated Schiff base retinal are accelerated by a factor of 6. We demonstrate the applicability of the RVS approach by performing CC2∕TZVP calculations on the lowest singlet excitation energy of a rhodopsin model consisting of 165 atoms using RVS thresholds between 20 eV and 120 eV. The calculations on the rhodopsin model show that the RVS errors determined in the gas-phase are a very good approximation to the RVS errors in the protein environment. The RVS approach thus renders purely quantum mechanical treatments of chromophores in protein environments feasible and offers an ab initio alternative to quantum mechanics∕molecular mechanics separation schemes. PMID:21663351

Reduction of the virtual space for coupled-cluster excitation energies of large molecules and embedded systems.

PubMed

Send, Robert; Kaila, Ville R I; Sundholm, Dage

2011-06-07

We investigate how the reduction of the virtual space affects coupled-cluster excitation energies at the approximate singles and doubles coupled-cluster level (CC2). In this reduced-virtual-space (RVS) approach, all virtual orbitals above a certain energy threshold are omitted in the correlation calculation. The effects of the RVS approach are assessed by calculations on the two lowest excitation energies of 11 biochromophores using different sizes of the virtual space. Our set of biochromophores consists of common model systems for the chromophores of the photoactive yellow protein, the green fluorescent protein, and rhodopsin. The RVS calculations show that most of the high-lying virtual orbitals can be neglected without significantly affecting the accuracy of the obtained excitation energies. Omitting all virtual orbitals above 50 eV in the correlation calculation introduces errors in the excitation energies that are smaller than 0.1 eV. By using a RVS energy threshold of 50 eV, the CC2 calculations using triple-ζ basis sets (TZVP) on protonated Schiff base retinal are accelerated by a factor of 6. We demonstrate the applicability of the RVS approach by performing CC2/TZVP calculations on the lowest singlet excitation energy of a rhodopsin model consisting of 165 atoms using RVS thresholds between 20 eV and 120 eV. The calculations on the rhodopsin model show that the RVS errors determined in the gas-phase are a very good approximation to the RVS errors in the protein environment. The RVS approach thus renders purely quantum mechanical treatments of chromophores in protein environments feasible and offers an ab initio alternative to quantum mechanics/molecular mechanics separation schemes. © 2011 American Institute of Physics

Analytical energy gradients for explicitly correlated wave functions. I. Explicitly correlated second-order Møller-Plesset perturbation theory

NASA Astrophysics Data System (ADS)

Győrffy, Werner; Knizia, Gerald; Werner, Hans-Joachim

2017-12-01

We present the theory and algorithms for computing analytical energy gradients for explicitly correlated second-order Møller-Plesset perturbation theory (MP2-F12). The main difficulty in F12 gradient theory arises from the large number of two-electron integrals for which effective two-body density matrices and integral derivatives need to be calculated. For efficiency, the density fitting approximation is used for evaluating all two-electron integrals and their derivatives. The accuracies of various previously proposed MP2-F12 approximations [3C, 3C(HY1), 3*C(HY1), and 3*A] are demonstrated by computing equilibrium geometries for a set of molecules containing first- and second-row elements, using double-ζ to quintuple-ζ basis sets. Generally, the convergence of the bond lengths and angles with respect to the basis set size is strongly improved by the F12 treatment, and augmented triple-ζ basis sets are sufficient to closely approach the basis set limit. The results obtained with the different approximations differ only very slightly. This paper is the first step towards analytical gradients for coupled-cluster singles and doubles with perturbative treatment of triple excitations, which will be presented in the second part of this series.

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

Palisade Endings of Extraocular Muscles Develop Postnatally Following Different Time Courses.

PubMed

Blumer, Roland; Streicher, Johannes; Davis-López de Carrizosa, María A; de la Cruz, Rosa R; Pastor, Angel M

2017-10-01

To analyze in a frontal-eyed mammal (cat) the postnatal development of palisade endings in extraocular muscles (EOMs) and to compare the spatiotemporal and quantitative patterns of palisade endings among individual rectus muscles. Cats of different ages ranging from birth to adult stage were studied. EOM whole-mount preparations were fluorescently labeled using six combinations of triple staining and analyzed in the confocal laser scanning microscope. Palisade endings developed postnatally and passed in each rectus muscle through the same, three developmental steps but in a heterochronic sequence and to a different final density per muscle. Specifically, palisade ending development was first completed in the medial rectus and later in the inferior, lateral, and superior rectus. The highest density of palisade endings was observed in the medial rectus and the lowest in the lateral rectus whereas values for the inferior and superior rectus were in between. Palisade endings expressed high levels of growth associated protein 43 during development and were supplied by axons that established motor terminals. Cats open their eyes 7 to 10 days after birth and later develop a complex three-dimensional visuomotor climbing and jumping behavior depending on accurate binocular vision and fine tuning of the ocular movements. Our findings indicate that palisade ending development correlates with important landmarks in visuomotor behavior and provide support for our previous notion that palisade endings play an important role for convergence eye movements in frontal-eyed species.

Different segregation patterns in five carriers due to a pericentric inversion of chromosome 1.

PubMed

Luo, Yuqin; Xu, Chenming; Sun, Yixi; Wang, Liya; Chen, Songchang; Jin, Fan

2014-12-01

Pericentric inversion can produce recombinant gametes; however, meiotic segregation studies on the relationship between the frequency of recombinants and the inverted segment size are rare. Triple-color fluorescence in situ hybridization (FISH) was performed to analyze the meiotic behavior in five inv(1) carriers with different breakpoints. Recombination gametes were absent in Patient 1, whereas the percentages of the recombinants in Patients 2, 3, 4, and 5 were of 9.2%, 15.3%, 17.3%, and 40.9%, respectively. A significant difference was present for the frequencies of the recombinant spermatozoa among the five patients (p < 0.001). For each patient, the frequency of the two types of recombinant gametes (dup(1p)/del(1q) or del(1p)/dup(1q)) did not exhibit a significant difference in comparison with the expected 1:1 ratio (p > 0.05). The meiotic segregation of nine inv(1) carriers (including those presented in this paper) is now available. A significant correlation was discovered between the rate of recombination and the proportion of the chromosome implicated in the inversion (R = 0.9435, p < 0.001). The frequency of the recombinant gametes was directly related to the proportion of the chromosome that was inverted. Sperm-FISH allowed an additional comprehension of the patterns of meiotic segregation and provided accurate genetic counseling.

The Relation between Distance Education Students' Motivation and Satisfaction

ERIC Educational Resources Information Center

Goulimaris, Dimitrios

2015-01-01

The aim of the present study, within the frame of self-determination theory (SDT), was triple: a) to examine the structural validity of the "Situational Motivation Scale" (SIMS) in the field of distance education, b) to investigate the correlation between the subscales of the motivation and satisfaction of students who attend distance…

The Impact of Religion on Women Empowerment as a Millennium Development Goal in Africa

ERIC Educational Resources Information Center

Njoh, Ambe J.; Akiwumi, Fenda A.

2012-01-01

The study examines the impact of religion on women empowerment. Three religions, indigenous African, Islam and Christianity, comprising Africa's triple heritage, are considered. The hypothesized relationships are confirmed. Christianity correlates with each of the four Development Targets specified for women empowerment under the third of the…

An Examination of Triple Jeopardy in Rural Youth Physical Activity Participation

ERIC Educational Resources Information Center

Shores, Kindal A.; Moore, Justin B.; Yin, Zenong

2010-01-01

Purpose: Chances for a healthy life are not equally distributed across society. Instead, genetic, social, and environmental factors help determine the probability that a child will be healthy and active. We investigate the probability that youth will be physically active by examining 3 consistent correlates of physical activity. The individual and…

USING AN ACCURATE MASS, TRIPLE QUADRUPOLE MASS SPECTROMETER AND AN ION CORRELATION PROGRAM TO IDENTIFY COMPOUNDS

EPA Science Inventory

Most compounds are not found in mass spectral libraries and must be identified by other means. Often, compound identities can be deduced from the compositions of the ions in their mass spectra and review of the chemical literature. Confirmation is provided by mass spectra and r...

Kinetics and dynamics of the C(3P) + H2O reaction on a full-dimensional accurate triplet state potential energy surface.

PubMed

Li, Jun; Xie, Changjian; Guo, Hua

2017-08-30

A full dimensional accurate potential energy surface (PES) for the C( 3 P) and H 2 O reaction is developed based on ∼34 000 data points calculated at the level of the explicitly correlated unrestricted coupled cluster method with single, double, and perturbative triple excitations with the augmented correlation-consistent polarized triple zeta basis set (CCSD(T)-F12a/AVTZ). The PES is invariant with respect to the permutation of the two hydrogen atoms and the total root mean square error (RMSE) of the fit is only 0.31 kcal mol -1 . The PES features two barriers in the entrance channel and several potential minima, as well as multiple product channels. The rate coefficients of this reaction calculated using a transition-state theory and quasi-classical trajectory (QCT) method are small near room temperature, consistent with experiments. The reaction dynamics is also investigated with QCT on the new PES, which found that the reactivity is constrained by the entrance barriers and the final product branching is not statistical.

The Critical, Clinical Role of Interferon-Beta in Regulating Cancer Stem Cell Properties in Triple-Negative Breast Cancer.

PubMed

Doherty, Mary R; Jackson, Mark W

2018-05-11

Triple-negative breast cancer (TNBC) the deadliest form of this disease currently lacks a targeted therapy and is characterized by increased risk of metastasis and presence of therapeutically resistant cancer stem cells (CSC). Recent evidence has demonstrated that the presence of an interferon (IFN)/signal transducer of activated transcription 1 (STAT1) gene signature correlates with improved therapeutic response and overall survival in TNBC patients. In agreement with these clinical observations, our recent work has demonstrated, in a cell model of TNBC that CSC have intrinsically repressed IFN signaling. Administration of IFN-β represses CSC properties, inducing a less aggressive non-CSC state. Moreover, an elevated IFN-β gene signature correlated with repressed CSC-related genes and an increased presence of tumor-infiltrating lymphocytes in TNBC specimens. We therefore propose that IFN-β be considered as a potential therapeutic option in the treatment of TNBC, to repress the CSC properties responsible for therapy failure. Future studies aim to improve methods to target delivery of IFN-β to tumors, to maximize therapeutic efficacy while minimizing systemic side effects.

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.

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

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.

Nondestructive assessment of collagen hydrogel cross-linking using time-resolved autofluorescence imaging.

PubMed

Sherlock, Benjamin E; Harvestine, Jenna N; Mitra, Debika; Haudenschild, Anne; Hu, Jerry; Athanasiou, Kyriacos A; Leach, J Kent; Marcu, Laura

2018-03-01

We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

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.

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.

Spin-orbit splitted excited states using explicitly-correlated equation-of-motion coupled-cluster singles and doubles eigenvectors

NASA Astrophysics Data System (ADS)

Bokhan, Denis; Trubnikov, Dmitrii N.; Perera, Ajith; Bartlett, Rodney J.

2018-04-01

An explicitly-correlated method of calculation of excited states with spin-orbit couplings, has been formulated and implemented. Developed approach utilizes left and right eigenvectors of equation-of-motion coupled-cluster model, which is based on the linearly approximated explicitly correlated coupled-cluster singles and doubles [CCSD(F12)] method. The spin-orbit interactions are introduced by using the spin-orbit mean field (SOMF) approximation of the Breit-Pauli Hamiltonian. Numerical tests for several atoms and molecules show good agreement between explicitly-correlated results and the corresponding values, calculated in complete basis set limit (CBS); the highly-accurate excitation energies can be obtained already at triple- ζ level.

Expression of connective tissue growth factor in male breast cancer: clinicopathologic correlations and prognostic value.

PubMed

Lacle, Miangela M; van Diest, Paul J; Goldschmeding, Roel; van der Wall, Elsken; Nguyen, Tri Q

2015-01-01

Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of secreted proteins that are believed to play an important role in the development of neoplasia. In particular, CTGF has been reported to play an important role in mammary tumorigenesis and to have prognostic value in female breast cancer (FBC). The aim of the present study was to investigate clinicopathologic correlations and prognostic value of CTGF in male breast cancer (MBC) and to compare these findings with FBC. For this, we studied CTGF protein expression by immunohistochemistry in 109 MBC cases and 75 FBC cases. In MBC, stromal CTGF expression was seen in the majority of the cases 78% (85/109) with high expression in 31/109 cases (28.4%), but expression in tumor cells was only seen in 9.2% (10/109) of cases. High stromal CTGF expression correlated with high grade and high proliferation index (>15%) assessed by MIB-1 immunohistochemical staining. CTGF expression in tumor epithelial cells did not correlate with any of the clinicopathologic features. In FBC, stromal CTGF expression positively correlated with mitotic count and tumor CTGF expression was associated with triple negative status of the tumor (p = 0.002). Neither stromal nor tumor epithelial cell CTGF expression had prognostic value in MBC and FBC. In conclusion, stromal CTGF expression was seen in a high percentage of MBC and was correlated with high grade and high proliferation index. In view of the important role of the microenvironment in cancer progression, this might suggest that stromal CTGF could be an interesting target for novel therapies and molecular imaging. However, the lack of association with prognosis warrants caution. The potential role of CTGF as a therapeutic target for triple negative FBC deserves to be further studied.

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.

Second harmonic generation quantitative measurements on collagen fibrils through correlation to electron microscopy

NASA Astrophysics Data System (ADS)

Bancelin, S.; Aimé, C.; Gusachenko, I.; Kowalczuk, L.; Latour, G.; Coradin, T.; Schanne-Klein, M.-C.

2015-03-01

Type I collagen is a major structural protein in mammals that shows highly structured macromolecular organizations specific to each tissue. This biopolymer is synthesized as triple helices, which self-assemble into fibrils (Ø =10-300 nm) and further form various 3D organization. In recent years, Second Harmonic Generation (SHG) microscopy has emerged as a powerful technique to probe in situ the fibrillar collagenous network within tissues. However, this optical technique cannot resolve most of the fibrils and is a coherent process, which has impeded quantitative measurements of the fibril diameter so far. In this study, we correlated SHG microscopy with Transmission Electron Microscopy to determine the sensitivity of SHG microscopy and to calibrate SHG signals as a function of the fibril diameter in reconstructed collagen gels. To that end, we synthetized isolated fibrils with various diameters and successfully imaged the very same fibrils with both techniques, down to 30 nm diameter. We observed that SHG signals scaled as the fourth power of the fibril diameter, as expected from analytical and numerical calculations. This calibration was then applied to diabetic rat cornea in which we successfully recovered the diameter of hyperglycemia-induced fibrils in the Descemet's membrane without having to resolve them. Finally we derived the first hyperpolarizability from a single collagen triple helix which validates the bottom-up approach used to calculate the non-linear response at the fibrillar scale and denotes a parallel alignment of triple helices within the fibrils. These results represent a major step towards quantitative SHG imaging of nm-sized collagen fibrils.

Bodipy–C60 triple hydrogen bonding assemblies as heavy atom-free triplet photosensitizers: preparation and study of the singlet/triplet energy transfer† †Electronic supplementary information (ESI) available: Syntheses, structure characterization data, and UV/vis absorption and emission spectra. See DOI: 10.1039/c4sc03865g

PubMed Central

Guo, Song; Xu, Liang; Xu, Kejing; Küçüköz, Betül; Karatay, Ahmet; Yaglioglu, Halime Gul; Hayvali, Mustafa; Elmali, Ayhan

2015-01-01

Supramolecular triplet photosensitizers based on hydrogen bonding-mediated molecular assemblies were prepared. Three thymine-containing visible light-harvesting Bodipy derivatives (B-1, B-2 and B-3, which show absorption at 505 nm, 630 nm and 593 nm, respectively) were used as H-bonding modules, and 1,6-diaminopyridine-appended C60 was used as the complementary hydrogen bonding module (C-1), in which the C60 part acts as a spin converter for triplet formation. Visible light-harvesting antennae with methylated thymine were prepared as references (B-1-Me, B-2-Me and B-3-Me), which are unable to form strong H-bonds with C-1. Triple H-bonds are formed between each Bodipy antenna (B-1, B-2 and B-3) and the C60 module (C-1). The photophysical properties of the H-bonding assemblies and the reference non-hydrogen bond-forming mixtures were studied using steady state UV/vis absorption spectroscopy, fluorescence emission spectroscopy, electrochemical characterization, and nanosecond transient absorption spectroscopy. Singlet energy transfer from the Bodipy antenna to the C60 module was confirmed by fluorescence quenching studies. The intersystem crossing of the latter produced the triplet excited state. The nanosecond transient absorption spectroscopy showed that the triplet state is either localized on the C60 module (for assembly B-1·C-1), or on the styryl-Bodipy antenna (for assemblies B-2·C-1 and B-3·C-1). Intra-assembly forward–backward (ping-pong) singlet/triplet energy transfer was proposed. In contrast to the H-bonding assemblies, slow triplet energy transfer was observed for the non-hydrogen bonding mixtures. As a proof of concept, these supramolecular assemblies were used as triplet photosensitizers for triplet–triplet annihilation upconversion. PMID:29218142

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.

Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

NASA Astrophysics Data System (ADS)

Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

2016-02-01

Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

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.

Solving the Quantum Many-Body Problem via Correlations Measured with a Momentum Microscope

NASA Astrophysics Data System (ADS)

Hodgman, S. S.; Khakimov, R. I.; Lewis-Swan, R. J.; Truscott, A. G.; Kheruntsyan, K. V.

2017-06-01

In quantum many-body theory, all physical observables are described in terms of correlation functions between particle creation or annihilation operators. Measurement of such correlation functions can therefore be regarded as an operational solution to the quantum many-body problem. Here, we demonstrate this paradigm by measuring multiparticle momentum correlations up to third order between ultracold helium atoms in an s -wave scattering halo of colliding Bose-Einstein condensates, using a quantum many-body momentum microscope. Our measurements allow us to extract a key building block of all higher-order correlations in this system—the pairing field amplitude. In addition, we demonstrate a record violation of the classical Cauchy-Schwarz inequality for correlated atom pairs and triples. Measuring multiparticle momentum correlations could provide new insights into effects such as unconventional superconductivity and many-body localization.

Technological aspects of GEM detector design and assembling for soft x-ray application

NASA Astrophysics Data System (ADS)

Kowalska-Strzeciwilk, E.; Chernyshova, M.

2016-09-01

Various types of Micro Pattern Gas Detectors (MPGDs) found applications as tracking detectors in high energy particle physics experiments and as well as imaging detectors, especially for soft X-rays. These detectors offer several advantages like high count rate capability, good spatial and energy resolution, low cost and possibility of constructing large area detectors with very small dead area. Construction, like the triple Gas Electron Multiplier (GEM) detector has become a standard detector, which is widely used for different imaging applications. Some examples of such applications are: monitoring the impurity in plasma, imaging system for mapping of some parameters like pigment distributions using X-ray fluorescence technique[1], proton range radiography system for quality assurance in hadron therapy. Measuring of the Soft X-Ray (SXR) radiation of magnetic fusion plasma is a standard way of accessing valuable information, for example, about particle transport and MHD. The paper is focused on the design of GEM based soft Xray radiation detecting system which is under development. It is dedicated to study soft X-ray emission of plasma radiation with focus on tungsten emission lines energy region. The paper presents the designing, construction and assembling of a prototype of two triple-GEM detectors for soft-X ray application on the WEST device.

Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase

PubMed Central

Mathias, Jonathan R.; Zhang, Zhanying; Saxena, Meera T.

2014-01-01

Abstract Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology. PMID:24428354

Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

PubMed

Mathias, Jonathan R; Zhang, Zhanying; Saxena, Meera T; Mumm, Jeff S

2014-04-01

Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.

Single-indicator-based Multidimensional Sensing: Detection and Identification of Heavy Metal Ions and Understanding the Foundations from Experiment to Simulation

PubMed Central

Leng, Yumin; Qian, Sihua; Wang, Yuhui; Lu, Cheng; Ji, Xiaoxu; Lu, Zhiwen; Lin, Hengwei

2016-01-01

Multidimensional sensing offers advantages in accuracy, diversity and capability for the simultaneous detection and discrimination of multiple analytes, however, the previous reports usually require complicated synthesis/fabrication process and/or need a variety of techniques (or instruments) to acquire signals. Therefore, to take full advantages of this concept, simple designs are highly desirable. Herein, a novel concept is conceived to construct multidimensional sensing platforms based on a single indicator that has capability of showing diverse color/fluorescence responses with the addition of different analytes. Through extracting hidden information from these responses, such as red, green and blue (RGB) alterations, a triple-channel-based multidimensional sensing platform could consequently be fabricated, and the RGB alterations are further applicable to standard statistical methods. As a proof-of-concept study, a triple-channel sensing platform is fabricated solely using dithizone with assistance of cetyltrimethylammonium bromide (CTAB) for hyperchromicity and sensitization, which demonstrates superior capabilities in detection and identification of ten common heavy metal ions at their standard concentrations of wastewater-discharge of China. Moreover, this sensing platform exhibits promising applications in semi-quantitative and even quantitative analysis individuals of these heavy metal ions with high sensitivity as well. Finally, density functional theory calculations are performed to reveal the foundations for this analysis. PMID:27146105

Enhanced cellular uptake of LHRH-conjugated PEG-coated magnetite nanoparticles for specific targeting of triple negative breast cancer cells.

PubMed

Hu, J; Obayemi, J D; Malatesta, K; Košmrlj, A; Soboyejo, W O

2018-07-01

Targeted therapy is an emerging technique in cancer detection and treatment. This paper presents the results of a combined experimental and theoretical study of the specific targeting and entry of luteinizing hormone releasing hormone (LHRH)-conjugated PEG-coated magnetite nanoparticles into triple negative breast cancer (TNBC) cells and normal breast cells. The conjugated nanoparticles structures, cellular uptake of PEG-coated magnetite nanoparticles (MNPs) and LHRH-conjugated PEG-coated magnetite nanoparticles (LHRH-MNPs) into breast cancer cells and normal breast cells were investigated using a combination of transmission electron microscope, optical and confocal fluorescence microscopy techniques. The results show that the presence of LHRH enhances the uptake of LHRH-MNPs into TNBC cells. Nanoparticle entry into breast cancer cells is also studied using a combination of thermodynamics and kinetics models. The trends in the predicted nanoparticle entry times (into TNBC cells) and the size ranges of the engulfed nanoparticles (within the TNBC cells) are shown to be consistent with experimental observations. The implications of the results are then discussed for the specific targeting of TNBCs with LHRH-conjugated PEG-coated magnetite nanoparticles for the early detection and treatment of TNBC. Copyright © 2018. Published by Elsevier B.V.

The set of triple-resonance sequences with a multiple quantum coherence evolution period

NASA Astrophysics Data System (ADS)

Koźmiński, Wiktor; Zhukov, Igor

2004-12-01

The new pulse sequence building block that relies on evolution of heteronuclear multiple quantum coherences is proposed. The particular chemical shifts are obtained in multiple quadrature, using linear combinations of frequencies taken from spectra measured at different quantum levels. The pulse sequences designed in this way consist of small number of RF-pulses, are as short as possible, and could be applied for determination of coupling constants. The examples presented involve 2D correlations H NCO, H NCA, H N(CO) CA, and H(N) COCA via heteronuclear zero and double coherences, as well as 2D H NCOCA technique with simultaneous evolution of triple and three distinct single quantum coherences. Applications of the new sequences are presented for 13C, 15N-labeled ubiquitin.

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.

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

Eriksen, Janus J., E-mail: janusje@chem.au.dk; Jørgensen, Poul; Matthews, Devin A.

The accuracy at which total energies of open-shell atoms and organic radicals may be calculated is assessed for selected coupled cluster perturbative triples expansions, all of which augment the coupled cluster singles and doubles (CCSD) energy by a non-iterative correction for the effect of triple excitations. Namely, the second- through sixth-order models of the recently proposed CCSD(T–n) triples series [J. J. Eriksen et al., J. Chem. Phys. 140, 064108 (2014)] are compared to the acclaimed CCSD(T) model for both unrestricted as well as restricted open-shell Hartree-Fock (UHF/ROHF) reference determinants. By comparing UHF- and ROHF-based statistical results for a test setmore » of 18 modest-sized open-shell species with comparable RHF-based results, no behavioral differences are observed for the higher-order models of the CCSD(T–n) series in their correlated descriptions of closed- and open-shell species. In particular, we find that the convergence rate throughout the series towards the coupled cluster singles, doubles, and triples (CCSDT) solution is identical for the two cases. For the CCSD(T) model, on the other hand, not only its numerical consistency, but also its established, yet fortuitous cancellation of errors breaks down in the transition from closed- to open-shell systems. The higher-order CCSD(T–n) models (orders n > 3) thus offer a consistent and significant improvement in accuracy relative to CCSDT over the CCSD(T) model, equally for RHF, UHF, and ROHF reference determinants, albeit at an increased computational cost.« less

Simultaneous determination of triple therapy for Helicobacter pylori in human plasma by reversed phase chromatography with online wavelength switching

NASA Astrophysics Data System (ADS)

Ahmed, Sameh; Atia, Noha N.

2015-02-01

The infection of gastric mucosa by Helicobacter pylori (HP) is an essential cofactor in the aetiology of gastroduodenal ulcer and gastric carcinoma. Because of the bacterial resistance, combination therapy containing omeprazole (OME), tinidazole (TNZ) and clarithromycin (CLA) is commonly used for eradication of HP. However, the simultaneous determination of the triple therapy in human plasma was not reported. A simple, reproducible, and selective HPLC method was developed for the simultaneous determination of the triple therapy mixture used for management of HP infections in human plasma. An HPLC procedure based on a liquid-liquid extraction, enrichment of the analytes and subsequent reversed-phase chromatography with UV detection was used. To enable sensitive and selective detection, the method involved the use of online wavelength switching detection, with two different detection wavelengths; 280 nm for detection of OME and TNZ and 210 nm for detection of CLA. Separations were performed on C18 analytical column with acetonitrile-10 mM phosphate buffer of pH = 3.0 at flow rate of 1.0 mL min-1. The linear ranges in human plasma were 0.05-10 μg mL-1 with correlation coefficients >0.9990. The detection limits in human plasma were 0.02-0.07 μg mL-1. Validation parameters were assessed in compliance with US-FDA guidelines. The method proved to be valuable for the therapeutic drug monitoring after oral administration of triple therapy tablets.

Study of resonances produced in light nuclei through two and multi particle correlations

NASA Astrophysics Data System (ADS)

Quattrocchi, L.; Acosta, L.; Amorini, F.; Anzalone, A.; Auditore, L.; Berceanu, I.; Cardella, G.; Chbihi, A.; De Filippo, E.; De Luca, S.; Dell' Aquila, D.; Francalanza, L.; Gnoffo, B.; Grzeszczuk, A.; Lanzalone, G.; Lombardo, I.; Martel, I.; Martorana, N. S.; Minniti, T.; Norella, S.; Pagano, A.; Pagano, E. V.; Papa, M.; Pirrone, S.; Politi, G.; Porto, F.; Rizzo, F.; Rosato, E.; Russotto, P.; Trifirò, A.; Trimarchi, M.; Verde, G.; Veselsky, M.; Vigilante, M.

2017-06-01

CORRELATION experiment has been performed at INFN-LNS of Catania, using the 4π multi-detector CHIMERA, with the aim of exploring correlations between two and multi light particle produced in 12C+24Mg collisions at 35 AMeV. Particular attention has been paid to the decay mechanisms of Hoyle state, an excited resonant state of 12C produced via the triple-α process and characterized by a pronounced molecular like structure with three α particles. The study of the Hoyle state is essential for nucleosynthesis, but it also represents a clearly isolated state that can be studied as a three-α cluster system.

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.

Image correlation microscopy for uniform illumination.

PubMed

Gaborski, T R; Sealander, M N; Ehrenberg, M; Waugh, R E; McGrath, J L

2010-01-01

Image cross-correlation microscopy is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. Image cross-correlation microscopy has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy. In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy. Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning image cross-correlation microscopy, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function. Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function depends strongly on particle size and not particle shape. In this report, we establish the relationships between the spatial autocorrelation function feature size, temporal autocorrelation function characteristic time and the diffusion coefficient for uniform illumination image correlation microscopy using analytical, Monte Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate uniform illumination image correlation microscopy analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils.

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

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

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.

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.

[Qualitative and quantitative analysis of amygdalin and its metabolite prunasin in plasma by ultra-high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry].

PubMed

Gao, Meng; Wang, Yuesheng; Wei, Huizhen; Ouyang, Hui; He, Mingzhen; Zeng, Lianqing; Shen, Fengyun; Guo, Qiang; Rao, Yi

2014-06-01

A method was developed for the determination of amygdalin and its metabolite prunasin in rat plasma after intragastric administration of Maxing shigan decoction. The analytes were identified by ultra-high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry and quantitatively determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry. After purified by liquid-liquid extraction, the qualitative analysis of amygdalin and prunasin in the plasma sample was performed on a Shim-pack XR-ODS III HPLC column (75 mm x 2.0 mm, 1.6 microm), using acetonitrile-0.1% (v/v) formic acid aqueous solution. The detection was performed on a Triple TOF 5600 quadrupole time of flight mass spectrometer. The quantitative analysis of amygdalin and prunasin in the plasma sample was performed by separation on an Agilent C18 HPLC column (50 mm x 2.1 mm, 1.7 microm), using acetonitrile-0.1% (v/v) formic acid aqueous solution. The detection was performed on an AB Q-TRAP 4500 triple quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operated in negative ion mode and multiple-reaction monitoring (MRM) mode. The qualitative analysis results showed that amygdalin and its metabolite prunasin were detected in the plasma sample. The quantitative analysis results showed that the linear range of amygdalin was 1.05-4 200 ng/mL with the correlation coefficient of 0.999 0 and the linear range of prunasin was 1.25-2 490 ng/mL with the correlation coefficient of 0.997 0. The method had a good precision with the relative standard deviations (RSDs) lower than 9.20% and the overall recoveries varied from 82.33% to 95.25%. The limits of detection (LODs) of amygdalin and prunasin were 0.50 ng/mL. With good reproducibility, the method is simple, fast and effective for the qualitative and quantitative analysis of the amygdalin and prunasin in plasma sample of rats which were administered by Maxing shigan decoction.

A practical theoretical formalism for atomic multielectron processes: direct multiple ionization by a single auger decay or by impact of a single electron or photon

NASA Astrophysics Data System (ADS)

Liu, Pengfei; Zeng, Jiaolong; Yuan, Jianmin

2018-04-01

Multiple electron processes occur widely in atoms, molecules, clusters, and condensed matters when they are interacting with energetic particles or intense laser fields. Direct multielectron processes (DMEP) are the most complicated among the general multiple electron processes and are the most difficult to describe theoretically. In this work, a unified and accurate theoretical formalism is proposed on the DMEP of atoms including the multiple auger decay and multiple ionization by an impact of a single electron or a single photon based on the atomic collision theory described by a correlated many-body Green's function. Such a practical treatment is made possible by taking consideration of the different coherence features of the atoms (matter waves) in the initial and final states. We first explain how the coherence characteristics of the ejected continuum electrons is largely destructed, by taking the electron impact direct double ionization process as an example. The direct double ionization process is completely different from the single ionization where the complete interference can be maintained. The detailed expressions are obtained for the energy correlations among the continuum electrons and energy resolved differential and integral cross sections according to the separation of knock-out (KO) and shake-off (SO) mechanisms for the electron impact direct double ionization, direct double and triple auger decay, and double and triple photoionization (TPI) processes. Extension to higher order DMEP than triple ionization is straight forward by adding contributions of the following KO and SO processes. The approach is applied to investigate the electron impact double ionization processes of C+, N+, and O+, the direct double and triple auger decay of the K-shell excited states of C+ 1s2{s}22{p}2{}2D and {}2P, and the double and TPI of lithium. Comparisons with the experimental and other theoretical investigations wherever available in the literature show that our theoretical formalism is accurate and effective in treating the atomic multielectron processes.

Liquid Dynamics from Neutron Spectrometry

DOE R&D Accomplishments Database

Brockhouse, Bertram N.; Bergsma, J.; Dasannacharya, B. A.; Pope, N. K.

1962-10-01

Recent experiments carried out at Chalk River on the dynamics of liquids using neutron inelastic scattering are reviewed, including one by Sakamoto et al., in which the Van Hove self-correlation functions in water at 25 and 75 deg C were determined, and another in which the correlation functions in liquid argon near its triple point were studied. The possible occurrence of short wavelength phonons in classical liquids is discussed, in analogy with their existence in the quantum liquid He4, and in connection with incomplete experiments on liquid tin. (auth)

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.

Is HO3 minimum cis or trans? An analytic full-dimensional ab initio isomerization path.

PubMed

Varandas, A J C

2011-05-28

The minimum energy path for isomerization of HO(3) has been explored in detail using accurate high-level ab initio methods and techniques for extrapolation to the complete basis set limit. In agreement with other reports, the best estimates from both valence-only and all-electron single-reference methods here utilized predict the minimum of the cis-HO(3) isomer to be deeper than the trans-HO(3) one. They also show that the energy varies by less than 1 kcal mol(-1) or so over the full isomerization path. A similar result is found from valence-only multireference configuration interaction calculations with the size-extensive Davidson correction and a correlation consistent triple-zeta basis, which predict the energy difference between the two isomers to be of only Δ = -0.1 kcal mol(-1). However, single-point multireference calculations carried out at the optimum triple-zeta geometry with basis sets of the correlation consistent family but cardinal numbers up to X = 6 lead upon a dual-level extrapolation to the complete basis set limit of Δ = (0.12 ± 0.05) kcal mol(-1). In turn, extrapolations with the all-electron single-reference coupled-cluster method including the perturbative triples correction yield values of Δ = -0.19 and -0.03 kcal mol(-1) when done from triple-quadruple and quadruple-quintuple zeta pairs with two basis sets of increasing quality, namely cc-cpVXZ and aug-cc-pVXZ. Yet, if added a value of 0.25 kcal mol(-1) that accounts for the effect of triple and perturbative quadruple excitations with the VTZ basis set, one obtains a coupled cluster estimate of Δ = (0.14 ± 0.08) kcal mol(-1). It is then shown for the first time from systematic ab initio calculations that the trans-HO(3) isomer is more stable than the cis one, in agreement with the available experimental evidence. Inclusion of the best reported zero-point energy difference (0.382 kcal mol(-1)) from multireference configuration interaction calculations enhances further the relative stability to ΔE(ZPE) = (0.51 ± 0.08) kcal mol(-1). A scheme is also suggested to model the full-dimensional isomerization potential-energy surface using a quadratic expansion that is parametrically represented by a Fourier analysis in the torsion angle. The method illustrated at the raw and complete basis-set limit coupled-cluster levels can provide a valuable tool for a future analysis of the available (incomplete thus far) experimental rovibrational data. This journal is © the Owner Societies 2011

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).

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells

PubMed Central

Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

2008-01-01

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large ∼3 MDa complex in the cytoplasm and a 20-fold smaller complex of ∼158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments. PMID:18842624

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

PubMed

Ohrt, Thomas; Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

2008-11-01

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

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.

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.

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.

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.

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.

The Correlation Between PARP1 and BRCA1 in AR Positive Triple-negative Breast Cancer.

PubMed

Luo, Jiayan; Jin, Juan; Yang, Fang; Sun, Zijia; Zhang, Wenwen; Shi, Yaqin; Xu, Jing; Guan, Xiaoxiang

2016-01-01

Triple-negative breast cancer (TNBC) lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression and thus cannot benefit from conventional hormonal or anti-HER2 targeted therapies. Anti-androgen therapy has shown a certain effect on androgen receptor (AR) positive TNBC. The emerging researches have proved that poly (ADP-ribose) polymerase (PARP) inhibitor is effective in BRCA1-deficient breast cancers. We demonstrated that combination of AR antagonist (bicalutamide) and PARP inhibitor (ABT-888) could inhibit cell viability and induce cell apoptosis significantly whatever in vitro or in vivo setting in AR-positive TNBC. Previous studies have proved that both BRCA1 and PARP1 have close connections with AR in prostate cancer. We explored the correlation among AR, PARP1 and BRCA1 in TNBC for the first time. After BRCA1 overexpression, the expression of AR and PARP1 were decreased in mRNA and protein levels. Additionally, AR positively regulated PARP1 while PARP1 also up-regulated AR expression in vitro. We also confirmed BRCA1 expression was negatively correlated with AR and PARP1 in TNBC patients using a tissue microarray with TNBC patient samples. These results suggest that the combination of bicalutamide and PARP inhibitor may be a potential strategy for TNBC patients and merits further evaluation.

Alternative formulation of explicitly correlated third-order Møller-Plesset perturbation theory

NASA Astrophysics Data System (ADS)

Ohnishi, Yu-ya; Ten-no, Seiichiro

2013-09-01

The second-order wave operator in the explicitly correlated wave function theory has been newly defined as an extension of the conventional s- and p-wave (SP) ansatz (also referred to as the FIXED amplitude ansatz) based on the linked-diagram theorem. The newly defined second-order wave operator has been applied to the calculation of the F12 correction to the third-order many-body perturbation (MP3) energy. In addition to this new wave operator, the F12 correction with the conventional first-order wave operator has been derived and calculated. Among three components of the MP3 correlation energy, the particle ladder contribution, which has shown the slowest convergence with respect to the basis set size, is fairly ameliorated by employing these F12 corrections. Both the newly defined and conventional formalisms of the F12 corrections exhibit a similar recovery of over 90% of the complete basis set limit of the particle ladder contribution of the MP3 correlation energy with a triple-zeta quality basis set for the neon atom, while the amount is about 75% without the F12 correction. The corrections to the ring term are small but the corrected energy has shown similar recovery as the particle ladder term. The hole ladder term has shown a rapid convergence even without the F12 corrections. Owing to these balanced recoveries, the deviation of the total MP3 correlation energy from the complete basis set limit has been calculated to be about 1 kcal/mol with the triple-zeta quality basis set, which is more than five times smaller than the error without the F12 correction.

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

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...

Multimodal investigation of triple network connectivity in patients with 22q11DS and association with executive functions.

PubMed

Padula, Maria C; Schaer, Marie; Scariati, Elisa; Maeder, Johanna; Schneider, Maude; Eliez, Stephan

2017-04-01

Large-scale brain networks play a prominent role in cognitive abilities and their activity is impaired in psychiatric disorders, such as schizophrenia. Patients with 22q11.2 deletion syndrome (22q11DS) are at high risk of developing schizophrenia and present similar cognitive impairments, including executive functions deficits. Thus, 22q11DS represents a model for the study of neural biomarkers associated with schizophrenia. In this study, we investigated structural and functional connectivity within and between the Default Mode (DMN), the Central Executive (CEN), and the Saliency network (SN) in 22q11DS using resting-state fMRI and DTI. Furthermore, we investigated if triple network impairments were related to executive dysfunctions or the presence of psychotic symptoms. Sixty-three patients with 22q11DS and sixty-eighty controls (age 6-33 years) were included in the study. Structural connectivity between main nodes of DMN, CEN, and SN was computed using probabilistic tractography. Functional connectivity was computed as the partial correlation between the time courses extracted from each node. Structural and functional connectivity measures were then correlated to executive functions and psychotic symptom scores. Our results showed mainly reduced structural connectivity within the CEN, DMN, and SN, in patients with 22q11DS compared with controls as well as reduced between-network connectivity. Functional connectivity appeared to be more preserved, with impairments being evident only within the DMN. Structural connectivity impairments were also related to executive dysfunctions. These findings show an association between triple network structural alterations and executive deficits in patients with the microdeletion, suggesting that 22q11DS and schizophrenia share common psychopathological mechanisms. Hum Brain Mapp 38:2177-2189, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

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.

VizieR Online Data Catalog: Cyanomethyl anion and its deuterated derivatives (Liton+, 2014)

NASA Astrophysics Data System (ADS)

Liton, M.; Das., A.; Chakrabarti, S. K.

2013-11-01

We performed detailed quantum chemical simulations to present the spectral properties (infrared, electronic, and rotational) of various forms of the cyanomethyl radical. Moller-Plesset perturbation theory along with the triple-zeta, correlation-consistent basis set is used to obtain different spectroscopic constants of CH2CN-, CHDCN-, and CD2CN- in the gas phase. (4 data files).

VizieR Online Data Catalog: Cyanomethyl anion and its deuterated derivatives (Majumdar+, 2014)

NASA Astrophysics Data System (ADS)

Majumdar, L.; Das., A.; Chakrabarti, S. K.

2013-11-01

We performed detailed quantum chemical simulations to present the spectral properties (infrared, electronic, and rotational) of various forms of the cyanomethyl radical. Moller-Plesset perturbation theory along with the triple-zeta, correlation-consistent basis set is used to obtain different spectroscopic constants of CH2CN-, CHDCN-, and CD2CN- in the gas phase. (4 data files).

Collisional rates based on the first potential energy surface of the NeH+ -He system

NASA Astrophysics Data System (ADS)

Bop, Cheikh T.; Hammami, K.; Faye, N. A. B.

2017-09-01

The potential energy surface is computed at the explicitly correlated coupled cluster with simple, second and perturbative triple excitation method (CCSD(T)-F12) in connection with the augmented-correlation consistent-polarized valence triple zeta (aug-cc-pVTZ) Gaussian basis set for the NeH+ -He system. The calculations were performed by first taking into account the vibration of the molecule and then averaging the so-obtained three-dimensional potential. From this average interaction potential, cross-sections among the 11 first rotational levels of NeH+ induced by collision with He are calculated for energies up to 4000 cm-1 using the quantum mechanical close coupling (CC) approach. Collisional rate coefficients are obtained by thermally averaging these cross-sections at low temperature (T ≤ 300 K). The propensity rules of the rotational transitions obtained in this paper are discussed and compared with those of HeH+ and ArH+ in collision with electron. This work may be helpful for the eventual investigations, both theoretical and experimental, focused to detect the key cationic noble gas hydride NeH+ in the interstellar and circumstellar media as well as in laboratory experiments.

Imaging Spectrophotometry of the Jet/ISM Interaction in IC5063

NASA Technical Reports Server (NTRS)

Cecil, G.; Schuft, B.; Morse, J.; Bland-Hawthorn, J.

2004-01-01

IC5063 is a somewhat dusty z=0.0110 S0 galaxy with a Seyfert 2 nucleus. It has a triple radio source that spans 3 arcsec, mostly blueshifted H I absorption that spans 700 km/s, and ionization cones that extend for more than 2 arcmins. We obtained fully sampled [O III]\\lambda5007 grids at 0."9 and 70 km/s FWHM resolution using the Rutgers Fabry-Perot system on the Blanco 4m telescope. Complementary long-slit spectra using the RC spectrograph on the Blanco, and Taurus Tunable Filter spectral images in H\\alpha and [N II]\\lambda6583, were also obtained to assess gaseous ionization conditions. We present the results of our analysis, and correlate spectral structures to those visible in archival WFPC2 images. We find that, in the region near the radio triple, gaseous ionization and line velocity width is tightly correlated, in excellent quantitative agreement with the high-velocity shock regime in the diagnostic emission-line ratio diagrams of Dopita & Sutherland. We separate kinematically gas in normal disk rotation that is illuminated by the AGN in the ionization cones from that agitated mechanically by the jet, and assess the energy input from both processes.

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

Gold Nanoantenna-Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer.

PubMed

Ou, Yu-Chuan; Webb, Joseph A; Faley, Shannon; Shae, Daniel; Talbert, Eric M; Lin, Sharon; Cutright, Camden C; Wilson, John T; Bellan, Leon M; Bardhan, Rizia

2016-08-31

In this work, we demonstrate controlled drug delivery from low-temperature-sensitive liposomes (LTSLs) mediated by photothermal heating from multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. The unique geometry of MGNs enables the generation of mild hyperthermia (∼42 °C) by converting near-infrared light to heat and effectively delivering doxorubicin (DOX) from the LTSLs in breast cancer cells. We confirmed the cellular uptake of MGNs by using both fluorescence confocal Z-stack imaging and transmission electron microscopy (TEM) imaging. We performed a cellular viability assay and live/dead cell fluorescence imaging of the combined therapeutic effects of MGNs with DOX-loaded LTSLs (DOX-LTSLs) and compared them with free DOX and DOX-loaded non-temperature-sensitive liposomes (DOX-NTSLs). Imaging of fluorescent live/dead cell indicators and MTT assay outcomes both demonstrated significant decreases in cellular viability when cells were treated with the combination therapy. Because of the high phase-transition temperature of NTSLs, no drug delivery was observed from the DOX-NTSLs. Notably, even at a low DOX concentration of 0.5 μg/mL, the combination treatment resulted in a higher (33%) cell death relative to free DOX (17% cell death). The results of our work demonstrate that the synergistic therapeutic effect of photothermal hyperthermia of MGNs with drug delivery from the LTSLs can successfully eradicate aggressive breast cancer cells with higher efficacy than free DOX by providing a controlled light-activated approach and minimizing off-target toxicity.

Gold Nanoantenna-Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer

PubMed Central

2016-01-01

In this work, we demonstrate controlled drug delivery from low-temperature-sensitive liposomes (LTSLs) mediated by photothermal heating from multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. The unique geometry of MGNs enables the generation of mild hyperthermia (∼42 °C) by converting near-infrared light to heat and effectively delivering doxorubicin (DOX) from the LTSLs in breast cancer cells. We confirmed the cellular uptake of MGNs by using both fluorescence confocal Z-stack imaging and transmission electron microscopy (TEM) imaging. We performed a cellular viability assay and live/dead cell fluorescence imaging of the combined therapeutic effects of MGNs with DOX-loaded LTSLs (DOX-LTSLs) and compared them with free DOX and DOX-loaded non-temperature-sensitive liposomes (DOX-NTSLs). Imaging of fluorescent live/dead cell indicators and MTT assay outcomes both demonstrated significant decreases in cellular viability when cells were treated with the combination therapy. Because of the high phase-transition temperature of NTSLs, no drug delivery was observed from the DOX-NTSLs. Notably, even at a low DOX concentration of 0.5 μg/mL, the combination treatment resulted in a higher (33%) cell death relative to free DOX (17% cell death). The results of our work demonstrate that the synergistic therapeutic effect of photothermal hyperthermia of MGNs with drug delivery from the LTSLs can successfully eradicate aggressive breast cancer cells with higher efficacy than free DOX by providing a controlled light-activated approach and minimizing off-target toxicity. PMID:27656689

Fluorescence lifetime measurements of native and glycated human serum albumin and bovine serum albumin

NASA Astrophysics Data System (ADS)

Joshi, Narahari V.; Joshi, Virgina O. d.; Contreras, Silvia; Gil, Herminia; Medina, Honorio; Siemiarczuk, Aleksander

1999-05-01

Nonenzymatic glycation, also known as Maillard reaction, plays an important role in the secondary complications of the diabetic pathology and aging, therefore, human serum albumin (HSA) and bovine serum albumin (BSA) were glycated by a conventional method in our laboratory using glucose as the glycating agent. Fluorescence lifetime measurements were carried out with a laser strobe fluorometer equipped with a nitrogen/dye laser and a frequency doubler as a pulsed excitation source. The samples were excited at 295 nm and the emission spectra were recorded at 345 nm. The obtained decay curves were tried for double and triple exponential functions. It has been found that the shorter lifetime increases for glycated proteins as compared with that of the native ones. For example, in the case of glycated BSA the lifetime increased from 1.36 ns to 2.30 ns. Similarly, for HSA, the lifetime increases from 1.58 ns to 2.26 ns. Meanwhile, the longer lifetime changed very slightly for both proteins (from 6.52 ns to 6.72 ns). The increase in the lifetime can be associated with the environmental effect; originated from the attachment of glucose to some lysine residues. A good example is Trp 214 which is in the cage of Lys 225, Lys 212, Lys 233, Lys 205, Lys 500, Lys 199 and Lys 195. If fluorescence lifetime technique is calibrated and properly used it could be employed for assessing glycation of proteins.

Interaction of triplet sensitizers with chlorophyll: Formation of singlet chlorophyll

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

Bohne, C.; Scaiano, J.C.

1989-03-29

The interaction of several triplet sensitizers with chlorophyll a (Chla) has been examined using laser techniques. For the carbonyl sensitizers (with triplet energies > 53 kcal/mol) it was possible to measure the quenching rate constants; these were systematically {>=} 10{sup 10} M{sup {minus}1} s{sup {minus}1}. In the cases of acetone, benzophenone, and p-methoxyacetophenone the quenching process leads to the formation of the fluorescent singlet state of Chla. For benzophenone (k{sub q} = 2.4 {times} 10{sup 10} M{sup {minus}1} s{sup {minus}1}) approximately 3% of the quenching events lead to the formation of excited Chla. Several sensitizers (decafluorobenzophenone, benzil, and fluorenone) domore » not induce Chla fluorescence (or do it very inefficiently) in spite of having triplet energies above the S{sub 1} level of Chla. In light of their results the most probable mechanism involves energy transfer from the triplet sensitizer to an upper triple state of Chla ({sup 3}Chla**) which can undergo reverse intersystem crossing to the singlet manifold of Chla and thus induce fluorescence. The inefficient sensitizers are those where electron transfer between the excited singlet of Chla or {sup 3}Chla** and ground-state sensitizers is energetically favorable, leading to rapid in-cage quenching of the initially formed excited states of Chla. Formation of radical-ion pair between the triplet sensitizer and Chla followed by the generation of singlet Chla in the recombination of the radical ions could not be completely discarded.« less

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.

A Noninvasive In Vitro Monitoring System Reporting Skeletal Muscle Differentiation.

PubMed

Öztürk-Kaloglu, Deniz; Hercher, David; Heher, Philipp; Posa-Markaryan, Katja; Sperger, Simon; Zimmermann, Alice; Wolbank, Susanne; Redl, Heinz; Hacobian, Ara

2017-01-01

Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.

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...

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

Co-targeting the HER and IGF/insulin receptor axis in breast cancer, with triple targeting with endocrine therapy for hormone-sensitive disease.

PubMed

Chakraborty, Ashok; Hatzis, Christos; DiGiovanna, Michael P

2017-05-01

Interactions between HER2, estrogen receptor (ER), and insulin-like growth factor I receptor (IGF1R) are implicated in resistance to monotherapies targeting these receptors. We have previously shown in pre-clinical studies synergistic anti-tumor effects for co-targeting each pairwise combination of HER2, IGF1R, and ER. Strikingly, synergy for HER2/IGF1R targeting occurred not only in a HER2+ model, but also in a HER2-normal model. The purpose of the current study was therefore to determine the generalizability of synergistic anti-tumor effects of co-targeting HER2/IGF1R, the anti-tumor activity of triple-targeting HER2/IGF1R/ER in hormone-dependent cell lines, and the effect of using the multi-targeting drugs neratinib (pan-HER) and BMS-754807 (dual IGF1R/insulin receptor). Proliferation and apoptosis assays were performed in a large panel of cell lines representing varying receptor expression levels. Mechanistic effects were studied using phospho-protein immunoblotting. Analyses of drug interaction effects were performed using linear mixed-effects regression models. Enhanced anti-proliferative effects of HER/IGF-insulin co-targeting were seen in most, though not all, cell lines, including HER2-normal lines. For ER+ lines, triple targeting with inclusion of anti-estrogen generally resulted in the greatest anti-tumor effects. Double or triple targeting generally resulted in marked increases in apoptosis in the sensitive lines. Mechanistic studies demonstrated that the synergy between drugs was correlated with maximal inhibition of Akt and ERK pathway signaling. Dual HER/IGF-insulin targeting, and triple targeting with inclusion of anti-estrogen drugs, shows striking anti-tumor activity across breast cancer types, and drugs with broader receptor specificity may be more effective than single receptor selective drugs, particularly for ER- cells.

GPS satellite clock determination in case of inter-frequency clock biases for triple-frequency precise point positioning

NASA Astrophysics Data System (ADS)

Guo, Jiang; Geng, Jianghui

2017-12-01

Significant time-varying inter-frequency clock biases (IFCBs) within GPS observations prevent the application of the legacy L1/L2 ionosphere-free clock products on L5 signals. Conventional approaches overcoming this problem are to estimate L1/L5 ionosphere-free clocks in addition to their L1/L2 counterparts or to compute IFCBs between the L1/L2 and L1/L5 clocks which are later modeled through a harmonic analysis. In contrast, we start from the undifferenced uncombined GNSS model and propose an alternative approach where a second satellite clock parameter dedicated to the L5 signals is estimated along with the legacy L1/L2 clock. In this manner, we do not need to rely on the correlated L1/L2 and L1/L5 ionosphere-free observables which complicates triple-frequency GPS stochastic models, or account for the unfavorable time-varying hardware biases in undifferenced GPS functional models since they can be absorbed by the L5 clocks. An extra advantage over the ionosphere-free model is that external ionosphere constraints can potentially be introduced to improve PPP. With 27 days of triple-frequency GPS data from globally distributed stations, we find that the RMS of the positioning differences between our GPS model and all conventional models is below 1 mm for all east, north and up components, demonstrating the effectiveness of our model in addressing triple-frequency observations and time-varying IFCBs. Moreover, we can combine the L1/L2 and L5 clocks derived from our model to calculate precisely the L1/L5 clocks which in practice only depart from their legacy counterparts by less than 0.006 ns in RMS. Our triple-frequency GPS model proves convenient and efficient in combating time-varying IFCBs and can be generalized to more than three frequency signals for satellite clock determination.

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.

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.

Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and c-JUN to Bypass PUMILIO Posttranscriptional Regulation

PubMed Central

Miles, Wayne O.; Lembo, Antonio; Volorio, Angela; Brachtel, Elena; Tian, Bin; Sgroi, Dennis; Provero, Paolo; Dyson, Nicholas

2017-01-01

Alternative polyadenylation (APA) is a process that changes the posttranscriptional regulation and translation potential of mRNAs via addition or deletion of 3′ untranslated region (3′ UTR) sequences. To identify posttranscriptional-regulatory events affected by APA in breast tumors, tumor datasets were analyzed for recurrent APA events. Motif mapping of the changed 3′ UTR regions found that APA-mediated removal of Pumilio regulatory elements (PRE) was unusually common. Breast tumor subtype–specific APA profiling identified triple-negative breast tumors as having the highest levels of APA. To determine the frequency of these events, an independent cohort of triple-negative breast tumors and normal breast tissue was analyzed for APA. APA-mediated shortening of NRAS and c-JUN was seen frequently, and this correlated with changes in the expression of downstream targets. mRNA stability and luciferase assays demonstrated APA-dependent alterations in RNA and protein levels of affected candidate genes. Examination of clinical parameters of these tumors found those with APA of NRAS and c-JUN to be smaller and less proliferative, but more invasive than non-APA tumors. RT-PCR profiling identified elevated levels of polyadenylation factor CSTF3 in tumors with APA. Overexpression of CSTF3 was common in triple-negative breast cancer cell lines, and elevated CSTF3 levels were sufficient to induce APA of NRAS and c-JUN. Our results support the hypothesis that PRE-containing mRNAs are disproportionately affected by APA, primarily due to high sequence similarity in the motifs utilized by polyadenylation machinery and the PUM complex. PMID:27758885

Evolution of the Gorda Escarpment, San Andreas fault and Mendocino triple junction from multichannel seismic data collected across the northern Vizcaino block, offshore northern California

USGS Publications Warehouse

Godfrey, N.J.; Meltzer, A.S.; Klemperer, S.L.; Trehu, A.M.; Leitner, B.; Clarke, S.H.; Ondrus, A.

1998-01-01

The Gorda Escarpment is a north facing scarp immediately south of the Mendocino transform fault (the Gorda/Juan de Fuca-Pacific plate boundary) between 126??W and the Mendocino triple junction. It elevates the seafloor at the northern edge of the Vizcaino block, part of the Pacific plate, ??? 1.5 km above the seafloor of the Gorda/Juan de Fuca plate to the north. Stratigraphy interpreted from multichannel seismic data across and close to the Gorda Escarpment suggests that the escarpment is a relatively recent pop-up feature caused by north-south compression across the plate boundary. Close to 126??W. the Vizcaino block acoustic basement shallows and is overlain by sediments that thin north toward the Gorda Escarpment. These sediments are tilted south and truncated at the seafloor. By contrast, in a localized region at the eastern end of the Gorda Escarpment, close to the Mendocino triple junction, the top of acoustic basement dips north and is overlain by a 2-km-thick wedge of pre-11 Ma sedimentary rocks that thickens north, toward the Gorda Escarpment. This wedge of sediments is restricted to the northeast corner of the Vizcaino block. Unless the wedge of sediments was a preexisting feature on the Vizcaino block before it was transferred from the North American to the Pacific plate, the strong spatial correlation between the sedimentary wedge and the triple junction suggests the entire Vizcaino block, with the San Andreas at its eastern boundary, has been part of the Pacific plate since significantly before 11 Ma.

Detection of doublecortin domain-containing 2 (DCDC2), a new candidate tumor suppressor gene of hepatocellular carcinoma, by triple combination array analysis

PubMed Central

2013-01-01

Background To detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis. Methods A surgical specimen obtained from a 68-year-old female HCC patient was analyzed by triple combination array, which identified doublecortin domain-containing 2 (DCDC2) as a candidate tumor suppressor gene of HCC. Subsequently, samples from 48 HCC patients were evaluated for their DCDC2 methylation and expression status using methylation specific PCR (MSP) and semi-quantitative reverse transcriptase (RT) PCR, respectively. Then, we investigated the relationship between clinicopathological factors and methylation status of DCDC2. Results DCDC2 was revealed to be hypermethylated (methylation value 0.846, range 0–1.0) in cancer tissue, compared with adjacent normal tissue (0.212) by methylation array in the 68-year-old female patient. Expression array showed decreased expression of DCDC2 in cancerous tissue. SNP array showed that the copy number of chromosome 6p22.1, in which DCDC2 resides, was normal. MSP revealed hypermethylation of the promoter region of DCDC2 in 41 of the tumor samples. DCDC2 expression was significantly decreased in the cases with methylation (P = 0.048). Furthermore, the methylated cases revealed worse prognosis for overall survival than unmethylated cases (P = 0.048). Conclusions The present study indicates that triple combination array is an effective method to detect novel genes related to HCC. We propose that DCDC2 is a tumor suppressor gene of HCC. PMID:24034596

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.

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.

New correlation for the temperature-dependent viscosity for saturated liquids

NASA Astrophysics Data System (ADS)

Tian, Jianxiang; Zhang, Laibin

2016-11-01

Based on the recent progress on both the temperature dependence of surface tension [H. L. Yi, J. X. Tian, A. Mulero and I. Cachading, J. Therm. Anal. Calorim. 126 (2016) 1603, and the correlation between surface tension and viscosity of liquids [J. X. Tian and A. Mulero, Ind. Eng. Chem. Res. 53 (2014) 9499], we derived a new multiple parameter correlation to describe the temperature-dependent viscosity of liquids. This correlation is verified by comparing with data from NIST Webbook for 35 saturated liquids including refrigerants, hydrocarbons and others, in a wide temperature range from the triple point temperature to the one very near to the critical temperature. Results show that this correlation predicts the NIST data with high accuracy with absolute average deviation (AAD) less than 1% for 21 liquids and more than 3% for only four liquids, and is clearly better than the popularly used Vogel-Fulcher-Tamman (VFT) correlation.

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

KEY COMPARISON: Final Report on CCT-K7: Key comparison of water triple point cells

NASA Astrophysics Data System (ADS)

Stock, M.; Solve, S.; del Campo, D.; Chimenti, V.; Méndez-Lango, E.; Liedberg, H.; Steur, P. P. M.; Marcarino, P.; Dematteis, R.; Filipe, E.; Lobo, I.; Kang, K. H.; Gam, K. S.; Kim, Y.-G.; Renaot, E.; Bonnier, G.; Valin, M.; White, R.; Dransfield, T. D.; Duan, Y.; Xiaoke, Y.; Strouse, G.; Ballico, M.; Sukkar, D.; Arai, M.; Mans, A.; de Groot, M.; Kerkhof, O.; Rusby, R.; Gray, J.; Head, D.; Hill, K.; Tegeler, E.; Noatsch, U.; Duris, S.; Kho, H. Y.; Ugur, S.; Pokhodun, A.; Gerasimov, S. F.

2006-01-01

The triple point of water serves to define the kelvin, the unit of thermodynamic temperature, in the International System of Units (SI). Furthermore, it is the most important fixed point of the International Temperature Scale of 1990 (ITS-90). Any uncertainty in the realization of the triple point of water contributes directly to the measurement uncertainty over the wide temperature range from 13.8033 K to 1234.93 K. The Consultative Committee for Thermometry (CCT) decided at its 21st meeting in 2001 to carry out a comparison of water triple point cells and charged the BIPM with its organization. Water triple point cells from 20 national metrology institutes were carried to the BIPM and were compared with highest accuracy with two reference cells. The small day-to-day changes of the reference cells were determined by a least-squares technique. Prior to the measurements at the BIPM, the transfer cells were compared with the corresponding national references and therefore also allow comparison of the national references of the water triple point. This report presents the results of this comparison and gives detailed information about the measurements made at the BIPM and in the participating laboratories. It was found that the transfer cells show a standard deviation of 50 µK the difference between the extremes is 160 µK. The same spread is observed between the national references. The most important result of this work is that a correlation between the isotopic composition of the cell water and the triple point temperature was observed. To reduce the spread between different realizations, it is therefore proposed that the definition of the kelvin should refer to water of a specified isotopic composition. The CCT recommended to the International Committee of Weights and Measures (CIPM) to clarify the definition of the kelvin in the SI brochure by explicitly referring to water with the isotopic composition of Vienna Standard Mean Ocean Water (VSMOW). The CIPM accepted this recommendation and the next edition of the SI brochure will include this specification. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCT, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

Reliability and validity of functional performance tests in dancers with hip dysfunction.

PubMed

Kivlan, Benjamin R; Carcia, Christopher R; Clemente, F Richard; Phelps, Amy L; Martin, Robroy L

2013-08-01

Quasi-experimental, repeated measures. Functional performance tests that identify hip joint impairments and assess the effect of intervention have not been adequately described for dancers. The purpose of this study was to examine the reliability and validity of hop and balance tests among a group of dancers with musculoskeletal pain in the hip region. NINETEEN FEMALE DANCERS (AGE: 18.90±1.11 years; height: 164.85±6.95 cm; weight: 60.37±8.29 kg) with unilateral hip pain were assessed utilizing the cross-over reach, medial triple hop, lateral triple hop, and cross-over hop tests on two occasions, 2 days apart. Test-retest reliability and comparisons between the involved and uninvolved side for each respective test were determined. Intra-class correlation coefficients for the functional performance tests ranged from 0.89-0.96. The cross-over reach test had a SEM of 2.79 cm and a MDC of 7.73 cm. The medial and lateral triple hop tests had SEM values of 7.51 cm and 8.17 cm, and MDC values of 20.81 cm and 22.62 cm, respectively. The SEM was 0.15 seconds and the MDC was 0.42 seconds for the cross-over hop test. Performance on the medial triple hop test was significantly less on the involved side (370.21±38.26 cm) compared to the uninvolved side (388.05±41.49 cm); t(18) = -4.33, p<0.01. The side-to-side comparisons of the cross-over reach test (involved mean=61.68±10.9 cm; uninvolved mean=61.69±8.63 cm); t(18) = -0.004, p=0.99, lateral triple hop test (involved mean=306.92±35.79 cm; uninvolved mean=310.68±24.49 cm); t(18) = -0.55, p=0.59, and cross-over hop test (involved mean=2.49±0.34 seconds; uninvolved mean= 2.61±0.42 seconds; t(18) = -1.84, p=0.08) were not statistically different between sides. The functional performance tests used in this study can be reliably performed on dancers with unilateral hip pain. The medial triple hop test was the only functional performance test with evidence of validity in side-to-side comparisons. These results suggest that the medial triple hop test may be a reliable and valid functional performance test to assess impairments related to hip pain among dancers. 3b. Non-consecutive cohort study.

RELIABILITY AND VALIDITY OF FUNCTIONAL PERFORMANCE TESTS IN DANCERS WITH HIP DYSFUNCTION

PubMed Central

Carcia, Christopher R.; Clemente, F. Richard; Phelps, Amy L.; Martin, RobRoy L.

2013-01-01

Study Design: Quasi-experimental, repeated measures. Purpose/Background: Functional performance tests that identify hip joint impairments and assess the effect of intervention have not been adequately described for dancers. The purpose of this study was to examine the reliability and validity of hop and balance tests among a group of dancers with musculoskeletal pain in the hip region. Methods: Nineteen female dancers (age: 18.90±1.11 years; height: 164.85±6.95 cm; weight: 60.37±8.29 kg) with unilateral hip pain were assessed utilizing the cross-over reach, medial triple hop, lateral triple hop, and cross-over hop tests on two occasions, 2 days apart. Test-retest reliability and comparisons between the involved and uninvolved side for each respective test were determined. Results: Intra-class correlation coefficients for the functional performance tests ranged from 0.89-0.96. The cross-over reach test had a SEM of 2.79 cm and a MDC of 7.73 cm. The medial and lateral triple hop tests had SEM values of 7.51 cm and 8.17 cm, and MDC values of 20.81 cm and 22.62 cm, respectively. The SEM was 0.15 seconds and the MDC was 0.42 seconds for the cross-over hop test. Performance on the medial triple hop test was significantly less on the involved side (370.21±38.26 cm) compared to the uninvolved side (388.05±41.49 cm); t(18) = −4.33, p<0.01. The side-to-side comparisons of the cross-over reach test (involved mean=61.68±10.9 cm; uninvolved mean=61.69±8.63 cm); t(18) = −0.004, p=0.99, lateral triple hop test (involved mean=306.92±35.79 cm; uninvolved mean=310.68±24.49 cm); t(18) = −0.55, p=0.59, and cross-over hop test (involved mean=2.49±0.34 seconds; uninvolved mean= 2.61±0.42 seconds; t(18) = −1.84, p=0.08) were not statistically different between sides. Conclusion: The functional performance tests used in this study can be reliably performed on dancers with unilateral hip pain. The medial triple hop test was the only functional performance test with evidence of validity in side-to-side comparisons. These results suggest that the medial triple hop test may be a reliable and valid functional performance test to assess impairments related to hip pain among dancers. Level of Evidence: 3b. Non-consecutive cohort study PMID:24175123

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.

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

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.

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.

The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrin

PubMed Central

Song, Yuanli; Pipalia, Nina H; Fung, L W-M

2009-01-01

The bundling of the N-terminal, partial domain helix (Helix C′) of human erythroid α-spectrin (αI) with the C-terminal, partial domain helices (Helices A′ and B′) of erythroid β-spectrin (βI) to give a spectrin pseudo structural domain (triple helical bundle A′B′C′) has long been recognized as a crucial step in forming functional spectrin tetramers in erythrocytes. We have used apparent polarity and Stern–Volmer quenching constants of Helix C′ of αI bound to Helices A′ and B′ of βI, along with previous NMR and EPR results, to propose a model for the triple helical bundle. This model was used as the input structure for molecular dynamics simulations for both wild type (WT) and αI mutant L49F. The simulation output structures show a stable helical bundle for WT, but not for L49F. In WT, four critical interactions were identified: two hydrophobic clusters and two salt bridges. However, in L49F, the region downstream of Helix C′ was unable to assume a helical conformation and one critical hydrophobic cluster was disrupted. Other molecular interactions critical to the WT helical bundle were also weakened in L49F, possibly leading to the lower tetramer levels observed in patients with this mutation-induced blood disorder. PMID:19593814

Multiplexed 3D FRET imaging in deep tissue of live embryos

PubMed Central

Zhao, Ming; Wan, Xiaoyang; Li, Yu; Zhou, Weibin; Peng, Leilei

2015-01-01

Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca2+ and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms. PMID:26387920

Unorthodox theoretical methods

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

Nedd, Sean

2012-01-01

The use of the ReaxFF force field to correlate with NMR mobilities of amine catalytic substituents on a mesoporous silica nanosphere surface is considered. The interfacing of the ReaxFF force field within the Surface Integrated Molecular Orbital/Molecular Mechanics (SIMOMM) method, in order to replicate earlier SIMOMM published data and to compare with the ReaxFF data, is discussed. The development of a new correlation consistent Composite Approach (ccCA) is presented, which incorporates the completely renormalized coupled cluster method with singles, doubles and non-iterative triples corrections towards the determination of heats of formations and reaction pathways which contain biradical species.

A dispersion relationship governing incompressible wall turbulence

NASA Technical Reports Server (NTRS)

Tsuge, S.

1978-01-01

The method of separation of variables is shown to make turbulent correlation equations of Karman-Howarth type tractable for shear turbulence as well under the condition of neglected triple correlation. The separated dependent variable obeys an Orr-Sommerfeld equation. A new analytical method is developed using a scaling law different from the classical one due to Heisenberg and Lin and more appropriate for wall turbulent profiles. A dispersion relationship between the wave number and the separation constant which has the dimension of a frequency is derived in support of experimental observations of wave or coherent structure of wall turbulence.

Pair and triple correlations in the A+B-->B diffusion-controlled reaction

NASA Astrophysics Data System (ADS)

Kuzovkov, Vladimir; Kotomin, Eugene

1994-03-01

An exact solution for the one-dimensional kinetics of the diffusion-controlled reaction A+B-->B is obtained by means of the three-particle correlation functions. Because of a lattice discreteness each site could be occupied by a single particle only which leads to the so-called ``bus effect'': Recombination of any particle A is defined by a spatial configuration of two nearest particles B only surrounding A from its left and right. This results in the unusual algebraic decay law, n(t)~t-1, which asymptotically (as t-->∞) does not depend on the trap B concentration.

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 ...

Insights into Alternanthera mosaic virus TGB3 functions: Interactions with Nicotiana benthamiana PsbO correlate with chloroplast vesiculation and veinal necrosis caused by TGB3 overexpression

USDA-ARS?s Scientific Manuscript database

Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculati...

Correlation consistent basis sets for lanthanides: The atoms La–Lu

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

Lu, Qing; Peterson, Kirk A., E-mail: kipeters@wsu.edu

Using the 3rd-order Douglas-Kroll-Hess (DKH3) Hamiltonian, all-electron correlation consistent basis sets of double-, triple-, and quadruple-zeta quality have been developed for the lanthanide elements La through Lu. Basis sets designed for the recovery of valence correlation (defined here as 4f5s5p5d6s), cc-pVnZ-DK3, and outer-core correlation (valence + 4s4p4d), cc-pwCVnZ-DK3, are reported (n = D, T, and Q). Systematic convergence of both Hartree-Fock and correlation energies towards their respective complete basis set (CBS) limits are observed. Benchmark calculations of the first three ionization potentials (IPs) of La through Lu are reported at the DKH3 coupled cluster singles and doubles with perturbative triples,more » CCSD(T), level of theory, including effects of correlation down through the 4s electrons. Spin-orbit coupling is treated at the 2-component HF level. After extrapolation to the CBS limit, the average errors with respect to experiment were just 0.52, 1.14, and 4.24 kcal/mol for the 1st, 2nd, and 3rd IPs, respectively, compared to the average experimental uncertainties of 0.03, 1.78, and 2.65 kcal/mol, respectively. The new basis sets are also used in CCSD(T) benchmark calculations of the equilibrium geometries, atomization energies, and heats of formation for Gd{sub 2}, GdF, and GdF{sub 3}. Except for the equilibrium geometry and harmonic frequency of GdF, which are accurately known from experiment, all other calculated quantities represent significant improvements compared to the existing experimental quantities. With estimated uncertainties of about ±3 kcal/mol, the 0 K atomization energies (298 K heats of formation) are calculated to be (all in kcal/mol): 33.2 (160.1) for Gd{sub 2}, 151.7 (−36.6) for GdF, and 447.1 (−295.2) for GdF{sub 3}.« less

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.

Tectonic history of the north portion of the San Andreas fault system, California, inferred from gravity and magnetic anomalies

USGS Publications Warehouse

Griscom, A.; Jachens, R.C.

1989-01-01

Geologic and geophysical data for the San Andreas fault system north of San Francisco suggest that the eastern boundary of the Pacific plate migrated eastward from its presumed original position at the base of the continental slope to its present position along the San Andreas transform fault by means of a series of eastward jumps of the Mendocino triple junction. These eastward jumps total a distance of about 150 km since 29 Ma. Correlation of right-laterally displaced gravity and magnetic anomalies that now have components at San Francisco and on the shelf north of Point Arena indicates that the presently active strand of the San Andreas fault north of the San Francisco peninsula formed recently at about 5 Ma when the triple junction jumped eastward a minimum of 100 km to its present location at the north end of the San Andreas fault. -from Authors

Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits

PubMed Central

Messa, Mirko; Fernández-Busnadiego, Rubén; Sun, Elizabeth Wen; Chen, Hong; Czapla, Heather; Wrasman, Kristie; Wu, Yumei; Ko, Genevieve; Ross, Theodora; Wendland, Beverly; De Camilli, Pietro

2014-01-01

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits. DOI: http://dx.doi.org/10.7554/eLife.03311.001 PMID:25122462

Collagenolytic Matrix Metalloproteinase Structure-Function Relationships: Insights From Molecular Dynamics Studies.

PubMed

Karabencheva-Christova, Tatyana G; Christov, Christo Z; Fields, Gregg B

2017-01-01

Several members of the zinc-dependent matrix metalloproteinase (MMP) family catalyze collagen degradation. Experimental data reveal a collaboration between different MMP domains in order to achieve efficient collagenolysis. Molecular dynamics (MD) simulations have been utilized to provide atomistic details of the collagenolytic process. The triple-helical structure of collagen exhibits local regions of flexibility, with modulation of interchain salt bridges and water bridges contributing to accessibility of individual chains by the enzyme. In turn, the hemopexin-like (HPX) domain of the MMP initially binds the triple helix and facilitates the presentation of individual strands to active site in the catalytic (CAT) domain. Extensive positive and negative correlated motions are observed between the CAT and HPX domains when collagen is bound. Ultimately, the MD simulation studies have complemented structural (NMR spectroscopy, X-ray crystallography) and kinetic analyses to provide a more detailed mechanistic view of MMP-catalyzed collagenolysis. © 2017 Elsevier Inc. All rights reserved.

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.

A New Kind of Blue Hybrid Electroluminescent Device.

PubMed

Wang, Junling; Li, Zhuan; Liu, Chunmei

2016-04-01

Bright blue Electroluminescence come from a ITO/BBOT doped silica (6 x 10(-3) M) made by a sol-gel method/Al driven by AC with 500 Hz at different voltages and Gaussian analysis under 55 V showed that blue emission coincidenced with typical triple emission from BBOT. This kind of device take advantage of organics (BBOT) and inorganics (silica). Electroluminescence from a single-layered sandwiched device consisting of blue fluorescent dye 2,5-bis (5-tert-butyl-2-benzoxazolyl) thiophene (BBOT) doped silica made by sol-gel method was investigated. A number of concentrations of hybrid devices were prepared and the maxium concentration was 6 x 10(-3) M. Blue electroluminescent (EL) always occurred above a threshold field 8.57 x 10(5) V/cm (30 V) at alternating voltage at 500 HZ. The luminance of the devices increased with the concentration of doped BBOT, but electroluminescence characteristics were different from a single molecule's photoluminescence properties of triple peaks. When analyzing in detail direct-current electroluminescence devices of pure BBOT, a single peak centered at 2.82 eV appeared with the driven voltage increase, which is similar to the hybrid devices. Comparing Gaussian decomposition date between two kinds of devices, the triple peak characteristic of BBOT was consistent. It is inferred that BBOT contributed EL of the hybrid devices mainly and silica may account for a very small part. Meanwhile the thermal stability of matrix silica was measured by Thermal Gravity-Mass Spectroscopy (TG-MS). There is 12 percent weight loss from room temperature to 1000 °C and silica has about 95% transmittance. So the matric silica played an important role in thermal stability and optical stability for BBOT. In addition, this kind of blue electroluminescence device can take advantages of organic materials BBOT and inorganic materials silica. This is a promising way to enrich EL devices, especially enriching inorganic EL color at a low cost.

Structure-function studies on hsp47: pH-dependent inhibition of collagen fibril formation in vitro.

PubMed Central

Thomson, C A; Ananthanarayanan, V S

2000-01-01

Hsp47, a 47 kDa heat shock protein whose expression level parallels that of collagen, has been regarded as a collagen-specific molecular chaperone. Studies from other laboratories have established the association of Hsp47 with the nascent as well as the triple-helical procollagen molecule in the endoplasmic reticulum and its dissociation from procollagen in the Golgi. One of several roles suggested for Hsp47 in collagen biosynthesis is the prevention of aggregation of procollagen in the endoplasmic reticulum. However, no experimental evidence has been available to verify this suggestion. In the present study we have followed the aggregation of mature triple-helical collagen molecules into fibrils by using turbidimetric measurements in the absence and presence of Hsp47. In the pH range 6-7, fibril formation of type I collagen, as monitored by turbidimetry, proceeds with a lag of approx. 10 min and levels off by approx. 60 min. The addition of Hsp47 at pH 7 effectively inhibits fibril formation at and above a 1:1 molar ratio of Hsp47 to triple-helical collagen. This inhibition is markedly pH-dependent, being significantly diminished at pH 6. CD and fluorescence spectral data of Hsp47 in the pH range 4.2-7.4 reveal a significant alteration in its structure at pH values below 6.2, with a decrease in alpha-helix and an increase in beta-structure. This conformational change is likely to be the basis of the decreased binding of Hsp47 to collagen in vitro at pH 6.3 as well as its inability to inhibit collagen fibril formation at this pH. Our results also provide a functional assay for Hsp47 that can be used in studies on collagen and Hsp47 interactions. PMID:10903151

Monitoring the development of xenograft triple-negative breast cancer models using diffusion-weighted magnetic resonance imaging.

PubMed

Stephen, Renu M; Pagel, Mark D; Brown, Kathy; Baker, Amanda F; Meuillet, Emmanuelle J; Gillies, Robert J

2012-11-01

Evaluations of tumor growth rates and molecular biomarkers are traditionally used to assess new mouse models of human breast cancers. This study investigated the utility of diffusion weighted (DW)-magnetic resonance imaging (MRI) for evaluating cellular proliferation of new tumor models of triple-negative breast cancer, which may augment traditional analysis methods. Eleven human breast cancer cell lines were used to develop xenograft tumors in severe combined immunodeficient mice, with two of these cell lines exhibiting sufficient growth to be serially passaged. DW-MRI was performed to measure the distributions of the apparent diffusion coefficient (ADC) in these two tumor xenograft models, which showed a correlation with tumor growth rates and doubling times during each passage. The distributions of the ADC values were also correlated with expression of Ki67, a biomarker of cell proliferation, and hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor receptor-2 (VEGFR2), which are essential proteins involved in regulating aerobic glycolysis and angiogenesis that support tumor cell proliferation. Although phosphatase and tensin homolog (PTEN) levels were different between the two xenograft models, AKT levels did not differ nor did they correlate with tumor growth. This last result demonstrates the complexity of signaling protein pathways and the difficulty in interpreting the effects of protein expression on tumor cell proliferation. In contrast, DW-MRI may be a more direct assessment of tumor growth and cancer cell proliferation.

An examination of triple jeopardy in rural youth physical activity participation.

PubMed

Shores, Kindal A; Moore, Justin B; Yin, Zenong

2010-01-01

Chances for a healthy life are not equally distributed across society. Instead, genetic, social, and environmental factors help determine the probability that a child will be healthy and active. We investigate the probability that youth will be physically active by examining 3 consistent correlates of physical activity. The individual and interaction effects of self-efficacy, social support, and access to physical activity areas are used to predict rural youth physical activity participation. We hypothesize that youth lacking all 3 activity supports will be in "triple jeopardy" for physical activity participation. Data were collected using a researcher-administered questionnaire with 147 youth in 2004. Youth ages 9-18 were enrolled in grades 4, 7, and 11 in 2 diverse rural counties in Georgia. Overall, a greater number of disadvantageous statuses were related to a lower probability of physical activity participation. Low self-efficacy, low social support, and no access to physical activity areas were related to lower levels of physical activity participation among rural youth. These variables exerted a stronger impact when factors were allowed to interact than when their isolated effects were summed. This study assessed correlates of physical activity among rural youth. These investigations, while scarce, can help identify subgroups of the population that may need to be targeted for intervention. Findings indicate that lived experience of youth (captured by the interaction of physical activity correlates) may be critical for understanding patterns of active and sedentary living. © 2010 National Rural Health Association.

Fluorescence, Absorption, and Excitation Spectra of Polycyclic Aromatic Hydrocarbons as a Tool for Quantitative Analysis

ERIC Educational Resources Information Center

Rivera-Figueroa, A. M.; Ramazan, K. A.; Finlayson-Pitts, B. J.

2004-01-01

A quantitative and qualitative study of the interplay between absorption, fluorescence, and excitation spectra of pollutants called polycyclic aromatic hydrocarbons (PAHs) is conducted. The study of five PAH displays the correlation of the above-mentioned properties along with the associated molecular changes.

Fluorescence hyperspectral imaging technique for the foreign substance detection on fresh-cut lettuce

USDA-ARS?s Scientific Manuscript database

Nondestructive methods based on fluorescence hyperspectral imaging (HSI) techniques were developed in order to detect worms on fresh-cut lettuce. The optimal wavebands for detecting worms on fresh-cut lettuce were investigated using the one-way ANOVA analysis and correlation analysis. The worm detec...

Optical fluorescence spectroscopy to detect hepatic necrosis after normothermic ischemia: animal model

NASA Astrophysics Data System (ADS)

Romano, Renan A.; Vollet-Filho, Jose D.; Pratavieira, Sebastião.; Fernandez, Jorge L.; Kurachi, Cristina; Bagnato, Vanderlei S.; Castro-e-Silva, Orlando; Sankarankutty, Ajith K.

2015-06-01

Liver transplantation is a well-established treatment for liver failure. However, the success of the transplantation procedure depends on liver graft conditions. The tissue function evaluation during the several transplantation stages is relevant, in particular during the organ harvesting, when a decision is made concerning the viability of the graft. Optical fluorescence spectroscopy is a good option because it is a noninvasive and fast technique. A partial normothermic hepatic ischemia was performed in rat livers, with a vascular occlusion of both median and left lateral lobes, allowing circulation only for the right lateral lobe and the caudate lobe. Fluorescence spectra under excitation at 532 nm (doubled frequency Nd:YAG laser) were collected using a portable spectrometer (USB2000, Ocean Optics, USA). The fluorescence emission was collected before vascular occlusion, after ischemia, and 24 hours after reperfusion. A morphometric histology analysis was performed as the gold standard evaluation - liver samples were analyzed, and the percentage of necrotic tissue was obtained. The results showed that changes in the fluorescence emission after ischemia can be correlated with the amount of necrosis evaluated by a morphometric analysis, the Pearson correlation coefficient of the generated model was 0.90 and the root mean square error was around 20%. In this context, the laser-induced fluorescence spectroscopy technique after normothermic ischemia showed to be a fast and efficient method to differentiate ischemic injury from viable tissues.

In vivo quantification of chromophore concentration using fluorescence differential path length spectroscopy

NASA Astrophysics Data System (ADS)

Kruijt, Bastiaan; Kascakova, Slavka; de Bruijn, Henriette S.; van der Ploeg-van den Heuvel, Angelique; Sterenborg, Henricus J. C. M.; Robinson, Dominic J.; Amelink, Arjen

2009-05-01

We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan®) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing. Concentration estimates of m-THPC measured by FDPS on the liver are compared with chemical extraction. Twenty-five rats were injected with 0.3 mg/kg m-THPC. In vivo optical concentration measurements were performed on tissue 3, 24, 48, and 96 h after m-THPC administration to yield a 10-fold variation in tissue concentration. After the optical measurements, the liver was harvested for chemical extraction. FDPS showed good correlation with chemical extraction. FDPS also showed a correlation between m-THPC fluorescence and blood volume fraction at the two shortest drug-light intervals. This suggests different compartmental localization of m-THPC for different drug-light intervals that can be resolved using fluorescence spectroscopy. Differences in measured m-THPC concentration between FDPS and chemical extraction are related to the interrogation volume of each technique; ~0.2 mm3 and ~102 mm3, respectively. This indicates intra-animal variation in m-THPC distribution in the liver on the scale of the FDPS sampling volume.

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice

NASA Astrophysics Data System (ADS)

Dobosz, Michael; Strobel, Steffen; Stubenrauch, Kay-Gunnar; Osl, Franz; Scheuer, Werner

2014-01-01

Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F( fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

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.

A Study of Neoadjuvant Paclitaxel in Combination With Bavituximab in Early- Stage Triple- Negative Breast Cancer

ClinicalTrials.gov

2017-03-08

Breast Cancer; Triple Negative Breast Neoplasms; Triple-Negative Breast Neoplasm; Triple-Negative Breast Cancer; Triple Negative Breast Cancer; ER-Negative PR-Negative HER2-Negative Breast Neoplasms; ER-Negative PR-Negative HER2-Negative Breast Cancer

Stream Dissolved Organic Matter Quantity and Quality Along a Wetland-Cropland Catchment Gradient

NASA Astrophysics Data System (ADS)

McDonough, O.; Hosen, J. D.; Lang, M. W.; Oesterling, R.; Palmer, M.

2012-12-01

Wetlands may be critical sources of dissolved organic matter (DOM) to stream networks. Yet, more than half of wetlands in the continental United States have been lost since European settlement, with the majority of loss attributed to agriculture. The degree to which agricultural loss of wetlands impacts stream DOM is largely unknown and may have important ecological implications. Using twenty headwater catchments on the Delmarva Peninsula (Maryland, USA), we investigated the seasonal influence of wetland and cropland coverage on downstream DOM quantity and quality. In addition to quantifying bulk downstream dissolved organic carbon (DOC) concentration, we used a suite of DOM UV-absorbance metrics and parallel factor analysis (PARAFAC) modeling of excitation-emission fluorescence spectra (EEMs) to characterize DOM composition. Percent bioavailable DOC (%BDOC) was measured during the Spring sampling using a 28-day incubation. Percent wetland coverage and % cropland within the watersheds were significantly negatively correlated (r = -0.93, p < 0.001). Results show that % wetland coverage was positively correlated with stream DOM concentration, molecular weight, aromaticity, humic-like fluorescence, and allochthonous origin. Conversely, increased wetland coverage was negatively correlated with stream DOM protein-like fluorescence. Percent BDOC decreased with DOM humic-like fluorescence and increased with protein-like fluorescence. We observed minimal seasonal interaction between % wetland coverage and DOM concentration and composition across Spring, Fall, and Winter sampling seasons. However, principal component analysis suggested more pronounced seasonal differences exist in stream DOM. This study highlights the influence of wetlands on downstream DOM in agriculturally impacted landscapes where loss of wetlands to cultivation may significantly alter stream DOM quantity and quality.

Determination of ethambutol by a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Wu, Wen-Ying; Yang, Ji-Yuan; Du, Li-Ming; Wu, Hao; Li, Chang-Feng

2011-08-01

The competitive reaction between ethambutol and two fluorescent probes (i.e., berberine and palmatine) for occupancy of the cucurbit[7]uril (CB[7]) cavity was studied by spectrofluorometry. The CB[7] reacts with these probes to form stable complexes, and the fluorescence intensity of the complexes is greatly enhanced. In addition, the excitation and emission wavelengths of their complexes moved to wavelengths of 343 nm and 495 nm, respectively. However, the addition of ethambutol dramatically quenches the fluorescence intensity of the two complexes. Accordingly, a couple of new fluorescence quenching methods for the determination of ethambutol were established. The methods can be applied for quantifying ethambutol. A linear relationship between the fluorescence quenching values (Δ F) and ethambutol concentration exists in the range of 5.0-1000.0 ng mL -1, with a correlation coefficient ( r) of 0.9997. The detection limit is 1.7 ng mL -1. The fluorescent probe of berberine has higher sensitivity than palmatine. This paper also discusses the mechanism of fluorescence indicator probes.

Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins.

PubMed

Ivanusic, Daniel; Denner, Joachim; Bannert, Norbert

2016-08-01

This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Correlations of trace elements in breast human tissues: Evaluation of spatial distribution using μ-XRF

NASA Astrophysics Data System (ADS)

Silva, Marina Piacenti da; Silva, Deisy Mara da; Ribeiro-Silva, Alfredo; Poletti, Martin Eduardo

2012-05-01

The aim of this work is to investigate microscopic correlations between trace elements in breast human tissues. A synchrotron X-ray fluorescence microprobe system (μ-XRF) was used to obtain two-dimensional distribution of trace element Ca, Fe, Cu and Zn in normal (6 samples) and malignant (14 samples) breast tissues. The experiment was performed in X-ray Fluorescence beam line at Laboratório Nacional de Luz Síncrotron (LNLS), Campinas, Brazil. The white microbeam was generated with a fine conical capillary with a 20 μm output diameter. The samples were supported on a XYZ table. An optical microscope with motorized zoom was used for sample positioning and choice the area to be scanned. Automatic two-dimensional scans were programmed and performed with steps of 30 μm in each direction (x, y) on the selected area. The fluorescence signals were recorded using a Si(Li) detector, positioned at 90 degrees with respect to the incident beam, with a collection time of 10 s per point. The elemental maps obtained from each sample were overlap to observe correlation between trace elements. Qualitative results showed that the pairs of elements Ca-Zn and Fe-Cu could to be correlated in malignant breast tissues. Quantitative results, achieved by Spearman correlation tests, indicate that there is a spatial correlation between these pairs of elements (p < 0.001) suggesting the importance of these elements in metabolic processes associated with the development of the tumor.