Removal of Chromophore-proximal Polar Atoms Decreases Water Content and Increases Fluorescence in a Near Infrared Phytofluor

Directory of Open Access Journals (Sweden)

Heli eLehtivuori

2015-11-01

Full Text Available Genetically encoded fluorescent markers have revolutionized cell and molecular biology due to their biological compatibility, controllable spatiotemporal expression, and photostability. To achieve in vivo imaging in whole animals, longer excitation wavelength probes are needed due to the superior ability of near infrared light to penetrate tissues unimpeded by absorbance from biomolecules or autofluorescence of water. Derived from near infrared-absorbing bacteriophytochromes, phytofluors are engineered to fluoresce in this region of the electromagnetic spectrum, although high quantum yield remains an elusive goal. An invariant aspartate residue is of utmost importance for photoconversion in native phytochromes, presumably due to the proximity of its backbone carbonyl to the pyrrole ring nitrogens of the biliverdin (BV chromophore as well as the size and charge of the side chain. We hypothesized that the polar interaction network formed by the charged side chain may contribute to the decay of the excited state via proton transfer. Thus, we chose to further probe the role of this amino acid by removing all possibility for polar interactions with its carboxylate side chain by incorporating leucine instead. The resultant fluorescent protein, WiPhy2, maintains BV binding, monomeric status, and long maximum excitation wavelength while minimizing undesirable protoporphyrin IXα binding in cells. A crystal structure and time-resolved fluorescence spectroscopy reveal that water near the BV chromophore is excluded and thus validate our hypothesis that removal of polar interactions leads to enhanced fluorescence by increasing the lifetime of the excited state. This new phytofluor maintains its fluorescent properties over a broad pH range and does not suffer from photobleaching. WiPhy2 achieves the best compromise to date between high fluorescence quantum yield and long illumination wavelength in this class of fluorescent proteins.

Photobleaching-induced changes in photosensitizing properties of dissolved organic matter

KAUST Repository

Niu, Xi-Zhi; Liu, Chao; Gutié rrez, Leonardo A.; Croue, Jean-Philippe

2014-01-01

Photosensitizing properties of different dissolved organic matter (DOM) were investigated according to their performance in singlet oxygen (1O2), triplet state of DOM (3DOM*), and hydroxyl radical (·OH) productions. The photobleaching of DOM solutions after irradiation was characterized by fluorescence excitation-emission matrix and UV-Vis spectroscopy. The photosensitizing properties of pre-irradiated DOM solutions were changed in a sunlight simulator. The performance of DOMs in photosensitized degradation of several contaminants was investigated. For a 20h exposure, the observed degradation rate constant (kobs) of some contaminants decreased as a function of exposure time, and highly depended on the properties of both DOM and contaminant. Degradation of contaminants with lower kobs was more susceptible to DOM photobleaching-induced decrease in kobs. Under the current experimental conditions, the photobleaching-induced decrease of DOM photo-reactivity in contaminant degradation was mainly attributed to indirect phototransformation of DOM caused by the interactions between photo-inductive DOM moieties and photochemically-produced reactive species. Reactive contaminants can inhibit DOM indirect photobleaching by scavenging reactive species, photosensitized degradation of these contaminants exhibited a stable kobs as a result. This is the first study to report DOM photobleaching-induced changes in the simultaneous DOM photosensitized degradation of contaminants and the inhibitory effect of reactive contaminants on DOM photobleaching.

Photobleaching-induced changes in photosensitizing properties of dissolved organic matter

KAUST Repository

Niu, Xi-Zhi

2014-12-01

Photosensitizing properties of different dissolved organic matter (DOM) were investigated according to their performance in singlet oxygen (1O2), triplet state of DOM (3DOM*), and hydroxyl radical (·OH) productions. The photobleaching of DOM solutions after irradiation was characterized by fluorescence excitation-emission matrix and UV-Vis spectroscopy. The photosensitizing properties of pre-irradiated DOM solutions were changed in a sunlight simulator. The performance of DOMs in photosensitized degradation of several contaminants was investigated. For a 20h exposure, the observed degradation rate constant (kobs) of some contaminants decreased as a function of exposure time, and highly depended on the properties of both DOM and contaminant. Degradation of contaminants with lower kobs was more susceptible to DOM photobleaching-induced decrease in kobs. Under the current experimental conditions, the photobleaching-induced decrease of DOM photo-reactivity in contaminant degradation was mainly attributed to indirect phototransformation of DOM caused by the interactions between photo-inductive DOM moieties and photochemically-produced reactive species. Reactive contaminants can inhibit DOM indirect photobleaching by scavenging reactive species, photosensitized degradation of these contaminants exhibited a stable kobs as a result. This is the first study to report DOM photobleaching-induced changes in the simultaneous DOM photosensitized degradation of contaminants and the inhibitory effect of reactive contaminants on DOM photobleaching.

Photobleaching response of different sources of chromophoric dissolved organic matter exposed to natural solar radiation using absorption and excitation-emission matrix spectra.

Science.gov (United States)

Zhang, Yunlin; Liu, Xiaohan; Osburn, Christopher L; Wang, Mingzhu; Qin, Boqiang; Zhou, Yongqiang

2013-01-01

CDOM biogeochemical cycle is driven by several physical and biological processes such as river input, biogeneration and photobleaching that act as primary sinks and sources of CDOM. Watershed-derived allochthonous (WDA) and phytoplankton-derived autochthonous (PDA) CDOM were exposed to 9 days of natural solar radiation to assess the photobleaching response of different CDOM sources, using absorption and fluorescence (excitation-emission matrix) spectroscopy. Our results showed a marked decrease in total dissolved nitrogen (TDN) concentration under natural sunlight exposure for both WDA and PDA CDOM, indicating photoproduction of ammonium from TDN. In contrast, photobleaching caused a marked increase in total dissolved phosphorus (TDP) concentration for both WDA and PDA CDOM. Thus TDN:TDP ratios decreased significantly both for WDA and PDA CDOM, which partially explained the seasonal dynamic of TDN:TDP ratio in Lake Taihu. Photobleaching rate of CDOM absorption a(254), was 0.032 m/MJ for WDA CDOM and 0.051 m/MJ for PDA CDOM from days 0-9, indicating that phototransformations were initially more rapid for the newly produced CDOM from phytoplankton than for the river CDOM. Extrapolation of these values to the field indicated that 3.9%-5.1% CDOM at the water surface was photobleached and mineralized every day in summer in Lake Taihu. Photobleaching caused the increase of spectral slope, spectral slope ratio and molecular size, indicating the CDOM mean molecular weight decrease which was favorable to further microbial degradation of mineralization. Three fluorescent components were validated in parallel factor analysis models calculated separately for WDA and PDA CDOM. Our study suggests that the humic-like fluorescence materials could be rapidly and easily photobleached for WDA and PDA CDOM, but the protein-like fluorescence materials was not photobleached and even increased from the transformation of the humic-like fluorescence substance to the protein

Photobleaching Response of Different Sources of Chromophoric Dissolved Organic Matter Exposed to Natural Solar Radiation Using Absorption and Excitation–Emission Matrix Spectra

Science.gov (United States)

Zhang, Yunlin; Liu, Xiaohan; Osburn, Christopher L.; Wang, Mingzhu; Qin, Boqiang; Zhou, Yongqiang

2013-01-01

CDOM biogeochemical cycle is driven by several physical and biological processes such as river input, biogeneration and photobleaching that act as primary sinks and sources of CDOM. Watershed-derived allochthonous (WDA) and phytoplankton-derived autochthonous (PDA) CDOM were exposed to 9 days of natural solar radiation to assess the photobleaching response of different CDOM sources, using absorption and fluorescence (excitation-emission matrix) spectroscopy. Our results showed a marked decrease in total dissolved nitrogen (TDN) concentration under natural sunlight exposure for both WDA and PDA CDOM, indicating photoproduction of ammonium from TDN. In contrast, photobleaching caused a marked increase in total dissolved phosphorus (TDP) concentration for both WDA and PDA CDOM. Thus TDN∶TDP ratios decreased significantly both for WDA and PDA CDOM, which partially explained the seasonal dynamic of TDN∶TDP ratio in Lake Taihu. Photobleaching rate of CDOM absorption a(254), was 0.032 m/MJ for WDA CDOM and 0.051 m/MJ for PDA CDOM from days 0–9, indicating that phototransformations were initially more rapid for the newly produced CDOM from phytoplankton than for the river CDOM. Extrapolation of these values to the field indicated that 3.9%–5.1% CDOM at the water surface was photobleached and mineralized every day in summer in Lake Taihu. Photobleaching caused the increase of spectral slope, spectral slope ratio and molecular size, indicating the CDOM mean molecular weight decrease which was favorable to further microbial degradation of mineralization. Three fluorescent components were validated in parallel factor analysis models calculated separately for WDA and PDA CDOM. Our study suggests that the humic-like fluorescence materials could be rapidly and easily photobleached for WDA and PDA CDOM, but the protein-like fluorescence materials was not photobleached and even increased from the transformation of the humic-like fluorescence substance to the protein

Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

Science.gov (United States)

Sarkar, Mitul; Koland, John G

2016-01-01

The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

OpenAIRE

Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

2014-01-01

Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows qu...

Protoporphyrin IX formation and photobleaching in different layers of normal human skin

DEFF Research Database (Denmark)

Togsverd-Bo, Katrine; Idorn, Luise W; Philipsen, Peter A

2012-01-01

human skin was tape-stripped and incubated with 20% methylaminolevulinate (MAL) or 20% hexylaminolevulinate (HAL) for 3 h. Fluorescence microscopy quantified PpIX accumulation in epidermis, superficial, mid and deep dermis, down to 2 mm. PpIX photobleaching by light-emitting diode (LED, 632 nm, 18......Topical photodynamic therapy (PDT) is used for various skin disorders, and selective targeting of specific skin structures is desirable. The objective was to assess accumulation of PpIX fluorescence and photobleaching within skin layers using different photosensitizers and light sources. Normal...... and 37 J/cm(2)), intense pulsed light (IPL, 500-650 nm, 36 and 72 J/cm(2)) and long-pulsed dye laser (LPDL, 595 nm, 7.5 and 15 J/cm(2)) was measured using fluorescence photography and microscopy. We found higher PpIX fluorescence intensities in epidermis and superficial dermis in HAL-incubated skin than...

Raman Enhancement and Photo-Bleaching of Organic Dyes in the Presence of Chemical Vapor Deposition-Grown Graphene

Directory of Open Access Journals (Sweden)

Jiaxin Weng

2017-10-01

Full Text Available Fluorescent organic dyes photobleach under intense light. Graphene has been shown to improve the photo-stability of organic dyes. In this paper, we investigated the Raman spectroscopy and photo-bleaching kinetics of dyes in the absence/presence of chemical vapor deposition (CVD-grown graphene. We show that graphene enhances the Raman signal of a wide range of dyes. The photo-bleaching of the dyes was reduced when the dyes were in contact with graphene. In contrast, monolayer hexagonal boron nitride (h-BN was much less effective in reducing the photo-bleaching rate of the dyes. We attribute the suppression of photo-bleaching to the energy or electron transfer from dye to graphene. The results highlight the potential of CVD graphene as a substrate for protecting and enhancing Raman response of organic dyes.

Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

Directory of Open Access Journals (Sweden)

Wüstner Daniel

2012-11-01

Full Text Available Abstract Background Fluorescence loss in photobleaching (FLIP is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp function is fitted to fluorescence loss (FL inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP, we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ disease proteins like mutant huntingtin (mtHtt can form large aggregates called inclusion bodies (IB’s. The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and

Spectral, stoichiometric ratio, physicochemical, polarity and photostability studies of newly synthesized chalcone dye in organized media

International Nuclear Information System (INIS)

Marwani, Hadi M.; Asiri, Abdullah M.; Khan, Salman A.

2013-01-01

The main focus of this study was to investigate spectroscopic properties, stoichiometric ratios, physicochemical parameters, polarity and photostability behaviors of newly synthesized chalcone dye in organized media. The chalcone dye, 1-(2,5-Dimethyl-thiophen-3-yl)-3-(9-etnyl-9H-carbazol-3-yl)-propenone (DTEP), was prepared by the reaction of carbazole aldehyde with 3-acetyl-2,5-dimethythiophene. Data obtained from FT-IR, 1 H-–NMR, 13 C-NMR and elemental analysis were consistent with chemical structure of newly prepared DTEP. Increases in fluorescence intensities of DTEP with cetyltrimethyl ammonium bromide (CTAB) were observed. In comparison of fluorescence intensities for DTEP with CTAB, reductions in fluorescence intensities for DTEP with sodium dodecyl sulfate (SDS) were noticed under the same experimental and instrumental conditions. Additionally, Benesi–Hildebrand method was applied to determine stoichiometric ratios and association constants of DTEP with CTAB and SDS. Stern–Volmer plot was used in order to further confirm the stoichiometric ratio and association constant of DTEP with SDS. Physicochemical parameters such as singlet absorption, molar absorptivity, oscillator strength, dipole moment and fluorescence quantum yield of DTEP were also determined. Fluorescence polarity study displayed that DTEP was sensitive to the polarity of the microenvironment provided by different solvents. Finally, fluorescence steady-state measurements revealed that DTEP has high photostability against photobleaching. -- Highlights: ► Mechanistic understanding of molecular structure of newly synthesized chalcone dye. ► Exploring spectral behaviors and physicochemical parameters of chalcone dye. ► Determination of stoichiometric ratios and association constants of chalcone dye. ► Determination of fluorescence quantum yield in different solvents. ► High photostability against photobleaching of chalcone dye was observed

Fluorescence Spectroscopy, Exciton Dynamics and Photochemistry of Single Allophycocyanin Trimers

International Nuclear Information System (INIS)

Ying, Liming; Xie, Xiaoliang

1998-01-01

We report a study of the spectroscopy and exciton dynamics of the allophycocyanin trimer (APC), a light harvesting protein complex from cyanobacteria, by room-temperature single-molecule measurements of fluorescence spectra, lifetimes, intensity trajectories and polarization modulation. Emission spectra of individual APC trimers are found to be homogeneous on the time scale of seconds. In contrast, their emission lifetimes are found to be widely distributed, because of generation of exciton traps during the course of measurements. The intensity trajectories and polarization modulation experiments indicate reversible ixciton trap formation within the three quasi-independent pairs of strong interacting a84 and B84 chromophores in APC, as well a photobleaching of individual chromophores. Comparison experiments under continuous wave and pulsed excitation reveal a two-photon mechanism for generating exciton traps and/or photobleaching, which involves exciton-exciton annihilation. These single-molecule experiments provide new insights into exciton dynamics and photochemistry of light-harvesting complexes

Polar plot representation of time-resolved fluorescence.

Science.gov (United States)

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

2014-01-01

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

EasyFRAP-web: a web-based tool for the analysis of fluorescence recovery after photobleaching data.

Science.gov (United States)

Koulouras, Grigorios; Panagopoulos, Andreas; Rapsomaniki, Maria A; Giakoumakis, Nickolaos N; Taraviras, Stavros; Lygerou, Zoi

2018-06-13

Understanding protein dynamics is crucial in order to elucidate protein function and interactions. Advances in modern microscopy facilitate the exploration of the mobility of fluorescently tagged proteins within living cells. Fluorescence recovery after photobleaching (FRAP) is an increasingly popular functional live-cell imaging technique which enables the study of the dynamic properties of proteins at a single-cell level. As an increasing number of labs generate FRAP datasets, there is a need for fast, interactive and user-friendly applications that analyze the resulting data. Here we present easyFRAP-web, a web application that simplifies the qualitative and quantitative analysis of FRAP datasets. EasyFRAP-web permits quick analysis of FRAP datasets through an intuitive web interface with interconnected analysis steps (experimental data assessment, different types of normalization and estimation of curve-derived quantitative parameters). In addition, easyFRAP-web provides dynamic and interactive data visualization and data and figure export for further analysis after every step. We test easyFRAP-web by analyzing FRAP datasets capturing the mobility of the cell cycle regulator Cdt2 in the presence and absence of DNA damage in cultured cells. We show that easyFRAP-web yields results consistent with previous studies and highlights cell-to-cell heterogeneity in the estimated kinetic parameters. EasyFRAP-web is platform-independent and is freely accessible at: https://easyfrap.vmnet.upatras.gr/.

A modified GFP facilitates counting membrane protein subunits by step-wise photobleaching in Arabidopsis.

Science.gov (United States)

Song, Kai; Xue, Yiqun; Wang, Xiaohua; Wan, Yinglang; Deng, Xin; Lin, Jinxing

2017-06-01

Membrane proteins exert functions by forming oligomers or molecular complexes. Currently, step-wise photobleaching has been applied to count the fluorescently labelled subunits in plant cells, for which an accurate and reliable control is required to distinguish individual subunits and define the basal fluorescence. However, the common procedure using immobilized GFP molecules is obviously not applicable for analysis in living plant cells. Using the spatial intensity distribution analysis (SpIDA), we found that the A206K mutation reduced the dimerization of GFP molecules. Further ectopic expression of Myristoyl-GFP A206K driven by the endogenous AtCLC2 promoter allowed imaging of individual molecules at a low expression level. As a result, the percentage of dimers in the transgenic pCLC2::Myristoyl-mGFP A206K line was significantly reduced in comparison to that of the pCLC2::Myristoyl-GFP line, confirming its application in defining the basal fluorescence intensity of GFP. Taken together, our results demonstrated that pCLC2::Myristoyl-mGFP A206K can be used as a standard control for monomer GFP, facilitating the analysis of the step-wise photobleaching of membrane proteins in Arabidopsis thaliana. Copyright © 2017 Elsevier GmbH. All rights reserved.

Photobleaching Response of Different Sources of Chromophoric Dissolved Organic Matter Exposed to Natural Solar Radiation Using Absorption and Excitation?Emission Matrix Spectra

OpenAIRE

Zhang, Yunlin; Liu, Xiaohan; Osburn, Christopher L.; Wang, Mingzhu; Qin, Boqiang; Zhou, Yongqiang

2013-01-01

CDOM biogeochemical cycle is driven by several physical and biological processes such as river input, biogeneration and photobleaching that act as primary sinks and sources of CDOM. Watershed-derived allochthonous (WDA) and phytoplankton-derived autochthonous (PDA) CDOM were exposed to 9 days of natural solar radiation to assess the photobleaching response of different CDOM sources, using absorption and fluorescence (excitation-emission matrix) spectroscopy. Our results showed a marked decrea...

Photobleaching effect in image fiber

International Nuclear Information System (INIS)

Hayashi, Shotaro; Wada, Yukio; Chigusa, Yoshiki; Fujiwara, Kunio; Hattori, Yasuji.

1985-01-01

The photobleaching effect in two types of image fibers is investigated using various light sources, light intensities, radiation dose rates and environmental temperatures. It is shown that the use of a xenon lamp, He-Cd laser or deuterium lamp can cause the photobleaching effect on the induced loss in an image fiber in the ultraviolet region. Of these light sources, a xenon lamp is found to have the greatest effect. This effectiveness appears to arise from the fact that this light source possesses a spectrum over the range from 0.3 to 0.36 μm. It is also revealed that when an image fiber is used for spectroscopic analysis under irradiation of gamma rays at a dose rate of 300 R/h, the available period of the fiber can be increase by 4 - 8 times with the aid of the photobleaching effect caused by a xenon lamp. In addition, the photobleaching effect is found to be dependent on temperature. It is inferred that this temperature dependence occur because electrons first excited to a level by the light energy tend to be further excited more easily at higher temperatures. (Nogami, K.)

Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching

International Nuclear Information System (INIS)

Vinnakota, Kalyan C; Wakatsuki, Tetsuro; Beard, Daniel A; Mitchell, David A; Deschenes, Robert J

2010-01-01

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane-localized fluorophore with the cytosol during fluorescence recovery after photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma membrane-localized wild-type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 µm × 1 µm bleach region-of-interest (ROI) and a 0.5 µm × 0.5 µm bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in the yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane-associated fluorophores using FRAP on commercial confocal laser scanning microscopes

High-contrast fluorescence imaging based on the polarization dependence of the fluorescence enhancement using an optical interference mirror slide.

Science.gov (United States)

Yasuda, Mitsuru; Akimoto, Takuo

2015-01-01

High-contrast fluorescence imaging using an optical interference mirror (OIM) slide that enhances the fluorescence from a fluorophore located on top of the OIM surface is reported. To enhance the fluorescence and reduce the background light of the OIM, transverse-electric-polarized excitation light was used as incident light, and the transverse-magnetic-polarized fluorescence signal was detected. As a result, an approximate 100-fold improvement in the signal-to-noise ratio was achieved through a 13-fold enhancement of the fluorescence signal and an 8-fold reduction of the background light.

Fluorescence confocal polarizing microscopy: Three-dimensional ...

Indian Academy of Sciences (India)

journal of. August 2003 physics pp. 373–384. Fluorescence confocal polarizing ... and focal conic domains in flat samples of lamellar LCs are practically indistinguishable. ... or less) LC layer confined between two transparent plates. ... in studies of electro-optic effects such as the Frederiks effect, defects, surface anchoring,.

Quantification of Lacunar-Canalicular Interstitial Fluid Flow Through Computational Modeling of Fluorescence Recovery After Photobleaching.

Science.gov (United States)

Kwon, Ronald Y; Frangos, John A

2010-09-01

Skeletal adaptation to mechanical loading has been widely hypothesized to involve the stimulation of osteocytes by interstitial fluid flow (IFF). However, direct investigation of this hypothesis has been difficult due in large part to the inability to directly measure IFF velocities within the lacunar-canalicular system. Measurements of fluorescence recovery after photobleaching (FRAP) within individual lacunae could be used to quantify lacunar-canalicular IFF when combined with mathematical modeling. In this study, we used a computational transport model to characterize the relationship between flow frequency (0.5-10 Hz), peak flow velocity (0-300 μm/s), tracer diffusion coefficient (100-300 μm(2)/s), and transport enhancement (i.e., (k/k(0)) - 1, where k and k(0) are the transport rates in the presence/absence of flow) during lacunar FRAP investigations. We show that this relationship is well described by a simple power law with frequency-dependent coefficients, and is relatively insensitive to variations in lacunar geometry. Using this power law relationship, we estimated peak IFF velocities in hindlimb mice subjected to intramedullary pressurization using values of k and k(0) previously obtained from ex vivo lacunar FRAP investigations. Together, our findings suggest that skeletal adaptation in hindlimb suspended mice subjected to dynamic intramedullary pressure occurred in the presence of IFF at levels associated with physiological loading.

Quantification of Lacunar–Canalicular Interstitial Fluid Flow Through Computational Modeling of Fluorescence Recovery After Photobleaching

Science.gov (United States)

Kwon, Ronald Y.; Frangos, John A.

2010-01-01

Skeletal adaptation to mechanical loading has been widely hypothesized to involve the stimulation of osteocytes by interstitial fluid flow (IFF). However, direct investigation of this hypothesis has been difficult due in large part to the inability to directly measure IFF velocities within the lacunar–canalicular system. Measurements of fluorescence recovery after photobleaching (FRAP) within individual lacunae could be used to quantify lacunar–canalicular IFF when combined with mathematical modeling. In this study, we used a computational transport model to characterize the relationship between flow frequency (0.5–10 Hz), peak flow velocity (0–300 μm/s), tracer diffusion coefficient (100–300 μm2/s), and transport enhancement (i.e., (k/k0) − 1, where k and k0 are the transport rates in the presence/absence of flow) during lacunar FRAP investigations. We show that this relationship is well described by a simple power law with frequency-dependent coefficients, and is relatively insensitive to variations in lacunar geometry. Using this power law relationship, we estimated peak IFF velocities in hindlimb mice subjected to intramedullary pressurization using values of k and k0 previously obtained from ex vivo lacunar FRAP investigations. Together, our findings suggest that skeletal adaptation in hindlimb suspended mice subjected to dynamic intramedullary pressure occurred in the presence of IFF at levels associated with physiological loading. PMID:21076644

Lateral mobility of plasma membrane lipids in a molluscan egg: Evidence for an animal/vegetal polarity

OpenAIRE

Laat, S.W. de; Speksnijder, J.E.; Dohmen, M.R.; Zoelen, E. van; Tertoolen, L.G.J.; Bluemink, J.G.

1984-01-01

The lateral diffusion of the lipid analog C₁₄-diI (3', 3'-dihexadecylindocarbocyanine iodide) was measured in the plasma membrane of early embryos of the mollusc Nassarius reticulatus using the FPR-(Fluorescence Photobleaching Recovery) method. At almost all stages measured (from fertilized egg up to 8-cell stage) the diffusion coefficient (D) of the mobile fraction (MF) of C₁₄-diI is significantly higher in the plasma membrane of the polar lobe as compared to the plasma membrane of the anima...

Mobility of adsorbed Cry1Aa insecticidal toxin from Bacillus thuringiensis (Bt) on montmorillonite measured by fluorescence recovery after photobleaching (FRAP)

Science.gov (United States)

Helassa, Nordine; Daudin, Gabrielle; Noinville, Sylvie; Janot, Jean-Marc; Déjardin, Philippe; Staunton, Siobhán; Quiquampoix, Hervé

2010-06-01

The insecticidal toxins produced by genetically modified Bt crops are introduced into soil through root exudates and tissue decomposition and adsorb readily on soil components, especially on clays. This immobilisation and the consequent concentration of the toxins in "hot spots" could increase the exposure of soil organisms. Whereas the effects on non-target organisms are well documented, few studies consider the migration of the toxin in soil. In this study, the residual mobility of Bt Cry1Aa insecticidal toxin adsorbed on montmorillonite was assessed using fluorescence recovery after photobleaching (FRAP). This technique, which is usually used to study dynamics of cytoplasmic and membrane molecules in live cells, was applied for the first time to a protein adsorbed on a finely divided swelling clay mineral, montmorillonite. No mobility of adsorbed toxin was observed at any pH and at different degrees of surface saturation.

Ratiometric fluorescence polarization as a cytometric functional parameter: theory and practice

International Nuclear Information System (INIS)

Yishai, Yitzhak; Fixler, Dror; Cohen-Kashi, Meir; Zurgil, Naomi; Deutsch, Mordechai

2003-01-01

The use of ratiometric fluorescence polarization (RFP) as a functional parameter in monitoring cellular activation is suggested, based on the physical phenomenon of fluorescence polarization dependency on emission wavelengths in multiple (at least binary) solutions. The theoretical basis of this dependency is thoroughly discussed and examined via simulation. For simulation, aimed to imitate a fluorophore-stained cell, real values of the fluorescence spectrum and polarization of different single fluorophore solutions were used. The simulation as well as the experimentally obtained values of RFP indicated the high sensitivity of this measure. Finally, the RFP parameter was utilized as a cytometric measure in three exemplary cellular bioassays. In the first, the apoptotic effect of oxLDL in a human Jurkat FDA-stained T cell line was monitored by RFP. In the second, the interaction between cell surface membrane receptors of human T lymphocyte cells was monitored by RFP measurements as a complementary means to the fluorescence resonance energy transfer (FRET) technique. In the third bioassay, cellular thiol level of FDA- and CMFDA-labelled Jurkat T cells was monitored via RFP

Polarization of fluorescence: a probe of molecular autoionization

International Nuclear Information System (INIS)

Leroi, G.E.; Dehmer, J.L.; Parr, A.C.; Poliakoff, E.D.

1983-01-01

The polarization of fluorescence from excited-state molecular photoions provides a direct probe of the photoionization dynamics and the symmetry signatures of autoionizing resonances. Measurements on CO 2 and CS 2 are presented as examples

Oral cancer detection based on fluorescence polarization of blood plasma at excitation wavelength 405 nm

Science.gov (United States)

Pachaiappan, Rekha; Prakasarao, Aruna; Manoharan, Yuvaraj; Dornadula, Koteeswaran; Singaravelu, Ganesan

2017-02-01

During metabolism the metabolites such as hormones, proteins and enzymes were released in to the blood stream by the cells. These metabolites reflect any change that occurs due to any disturbances in normal metabolic function of the human system. This was well observed with the altered spectral signatures observed with fluorescence spectroscopic technique. Previously many have reported on the significance of native fluorescence spectroscopic method in the diagnosis of cancer. As fluorescence spectroscopy is sensitive and simple, it has complementary techniques such as excitation-emission matrix, synchronous and polarization. The fluorescence polarization measurement provides details about any association or binding reactions and denaturing effects that occurs due to change in the micro environment of cells and tissues. In this study, we have made an attempt in the diagnosis of oral cancer at 405 nm excitation using fluorescence polarization measurement. The fluorescence anisotropic values calculated from polarized fluorescence spectral data of normal and oral cancer subjects yielded a good accuracy when analyzed with linear discriminant analysis based artificial neural network. The results will be discussed in detail.

Polarization Multiplexing of Fluorescent Emission Using Multiresonant Plasmonic Antennas.

Science.gov (United States)

De Leo, Eva; Cocina, Ario; Tiwari, Preksha; Poulikakos, Lisa V; Marqués-Gallego, Patricia; le Feber, Boris; Norris, David J; Prins, Ferry

2017-12-26

Combining the ability to localize electromagnetic fields at the nanoscale with a directional response, plasmonic antennas offer an effective strategy to shape the far-field pattern of coupled emitters. Here, we introduce a family of directional multiresonant antennas that allows for polarization-resolved spectral identification of fluorescent emission. The geometry consists of a central aperture surrounded by concentric polygonal corrugations. By varying the periodicity of each axis of the polygon individually, this structure can support multiple resonances that provide independent control over emission directionality for multiple wavelengths. Moreover, since each resonant wavelength is directly mapped to a specific polarization orientation, spectral information can be encoded in the polarization state of the out-scattered beam. To demonstrate the potential of such structures in enabling simplified detection schemes and additional functionalities in sensing and imaging applications, we use the central subwavelength aperture as a built-in nanocuvette and manipulate the fluorescent response of colloidal-quantum-dot emitters coupled to the multiresonant antenna.

Differential Polarization Nonlinear Optical Microscopy with Adaptive Optics Controlled Multiplexed Beams

Directory of Open Access Journals (Sweden)

Virginijus Barzda

2013-09-01

Full Text Available Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red, which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures.

Fluorescent nanodiamonds as non-photobleachable responsive probes

Czech Academy of Sciences Publication Activity Database

Cígler, Petr

2016-01-01

Roč. 14, č. 5 (2016), s. 232 ISSN 2336-7202. [Sjezd českých a slovenských chemických společností /68./. 04.09.2016-07.09.2016, Praha] R&D Projects: GA ČR(CZ) GA16-16336S Institutional support: RVO:61388963 Keywords : fluorescent nanodiamonds * FNDs * probes Subject RIV: CG - Electrochemistry

Spatiotemporal Distribution, Sources, and Photobleaching Imprint of Dissolved Organic Matter in the Yangtze Estuary and Its Adjacent Sea Using Fluorescence and Parallel Factor Analysis

Science.gov (United States)

Li, Penghui; Chen, Ling; Zhang, Wen; Huang, Qinghui

2015-01-01

To investigate the seasonal and interannual dynamics of dissolved organic matter (DOM) in the Yangtze Estuary, surface and bottom water samples in the Yangtze Estuary and its adjacent sea were collected and characterized using fluorescence excitation-emission matrices (EEMs) and parallel factor analysis (PARAFAC) in both dry and wet seasons in 2012 and 2013. Two protein-like components and three humic-like components were identified. Three humic-like components decreased linearly with increasing salinity (r>0.90, p<0.001), suggesting their distribution could primarily be controlled by physical mixing. By contrast, two protein-like components fell below the theoretical mixing line, largely due to microbial degradation and removal during mixing. Higher concentrations of humic-like components found in 2012 could be attributed to higher freshwater discharge relative to 2013. There was a lack of systematic patterns for three humic-like components between seasons and years, probably due to variations of other factors such as sources and characteristics. Highest concentrations of fluorescent components, observed in estuarine turbidity maximum (ETM) region, could be attributed to sediment resuspension and subsequent release of DOM, supported by higher concentrations of fluorescent components in bottom water than in surface water at two stations where sediments probably resuspended. Meanwhile, photobleaching could be reflected from the changes in the ratios between fluorescence intensity (Fmax) of humic-like components and chromophoric DOM (CDOM) absorption coefficient (a355) along the salinity gradient. This study demonstrates the abundance and composition of DOM in estuaries are controlled not only by hydrological conditions, but also by its sources, characteristics and related estuarine biogeochemical processes. PMID:26107640

Spatiotemporal Distribution, Sources, and Photobleaching Imprint of Dissolved Organic Matter in the Yangtze Estuary and Its Adjacent Sea Using Fluorescence and Parallel Factor Analysis.

Directory of Open Access Journals (Sweden)

Penghui Li

Full Text Available To investigate the seasonal and interannual dynamics of dissolved organic matter (DOM in the Yangtze Estuary, surface and bottom water samples in the Yangtze Estuary and its adjacent sea were collected and characterized using fluorescence excitation-emission matrices (EEMs and parallel factor analysis (PARAFAC in both dry and wet seasons in 2012 and 2013. Two protein-like components and three humic-like components were identified. Three humic-like components decreased linearly with increasing salinity (r>0.90, p<0.001, suggesting their distribution could primarily be controlled by physical mixing. By contrast, two protein-like components fell below the theoretical mixing line, largely due to microbial degradation and removal during mixing. Higher concentrations of humic-like components found in 2012 could be attributed to higher freshwater discharge relative to 2013. There was a lack of systematic patterns for three humic-like components between seasons and years, probably due to variations of other factors such as sources and characteristics. Highest concentrations of fluorescent components, observed in estuarine turbidity maximum (ETM region, could be attributed to sediment resuspension and subsequent release of DOM, supported by higher concentrations of fluorescent components in bottom water than in surface water at two stations where sediments probably resuspended. Meanwhile, photobleaching could be reflected from the changes in the ratios between fluorescence intensity (Fmax of humic-like components and chromophoric DOM (CDOM absorption coefficient (a355 along the salinity gradient. This study demonstrates the abundance and composition of DOM in estuaries are controlled not only by hydrological conditions, but also by its sources, characteristics and related estuarine biogeochemical processes.

The spectral properties of (--epigallocatechin 3-O-gallate (EGCG fluorescence in different solvents: dependence on solvent polarity.

Directory of Open Access Journals (Sweden)

Vladislav Snitsarev

Full Text Available (--Epigallocatechin 3-O-gallate (EGCG a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90, a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB at pH=7.0, acetonitrile (AN (a polar aprotic solvent, dimethylsulfoxide (DMSO (a polar aprotic solvent, and ethanol (EtOH (a polar protic solvent. We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.

Validation of photodynamic action via photobleaching of a new curcumin-based composite with enhanced water solubility.

Science.gov (United States)

Rego-Filho, Francisco G; de Araujo, Maria T; de Oliveira, Kleber T; Bagnato, Vanderlei S

2014-09-01

Motivated by the photochemical and photophysical properties of curcumin-based composites, the characteristics of a new curcumin-based water-soluble salt were investigated via absorption and fluorescence spectroscopy. Photobleaching was investigated using a set of LEDs in three different wavelengths (405 nm, 450 nm and 470 nm) to illuminate an aqueous solution of curcumin, evaluating its degradation for five different exposure times (0, 5, 15, 45 and 105 minutes). The results were compared with equivalent measurements of dark degradation and illumination in the presence of a singlet-oxygen quencher. Three solution concentrations (50, 100 and 150 μg/ml) were studied. To measure the fluorescence, it was used low power 405 nm excitation laser source. Time dependent photodegradation of curcumin was observed, as compared to the natural degradation of samples maintained on a dark environment. Two main absorption peaks were detected and their relation responded to both concentration and wavelength of the illumination source. A spectral correlation between absorption of curcumin and the emission bands of the sources showed an optimal spectral overlap for the 450 nm LED. For this source, photobleaching showed a less intense degradation on the presence of singlet oxygen quencher. This last result confirmed singlet oxygen production in vitro, indicating a strong potential of this composite to be used as a blue-light-activated photosensitizer.

Core-shell fluorescent silica nanoparticles for sensing near-neutral pH values

International Nuclear Information System (INIS)

Gao, F.; Chen, X.; Ye, Q.; Yao, Z.; Guo, X.; Wang, L.

2011-01-01

pH-responsive fluorescent core-shell silica nanoparticles (SiNPs) were prepared by encapsulating the pH-sensitive fluorophore 8-hydroxypyrene-1,3, 6-trisulfonate into their silica shell via a facile reverse microemulsion method. The resulting SiNPs were characterized by SEM, TEM, fluorescence lifetime spectroscopy, photobleaching experiments, and photoluminescence. The core-shell structure endows the SiNPs with reduced photobleaching, excellent photostability, minimized solvatachromic shift, and increased fluorescence efficiency compared to the free fluorophore in aqueous solution. The dynamic range for sensing pH ranges from 5. 5 to 9. 0. The nanosensors show excellent stability, are highly reproducible, and enable rapid detection of pH. The results obtained with the SiNPs are in good agreement with data obtained with a glass electrode. (author)

Characterization of microenvironment polarity and solvent accessibility of polysilsesquioxane xerogels by the fluorescent probe technique

Energy Technology Data Exchange (ETDEWEB)

Shea, K.J.; Zhu, H.D. [Univ., of California, Irvine, CA (United States). Dept. of Chemistry; Loy, D.A. [Sandia National Labs., Albuquerque, NM (United States)

1995-05-01

Poly (1, 4 bis(triethoxysilyl)benzene) (PTESB), a representative of a new type of organic-inorganic hybrid polysilsesquioxane material, was characterized by fluorescence spectroscopy for both microenvironmental polarity and solvent accessibility. A dansyl fluorescent molecule was incorporated into the bulk as well as onto the surface of both PTESB and silica materials. Information about the microenvironment polarity and accessibility of PTESB to various organic solvents was determined and compared to that of silica gel. This study found that both the bulk and surface of PTESB are less polar than that of the silica material. The silica material is accessible to polar solvents and water, while YMB is accessible to polar solvents but not to water. The hydrophobicity of PTESB differentiates these new materials from silica gel.

Room-temperature single-photon sources with definite circular and linear polarizations based on single-emitter fluorescence in liquid crystal hosts

International Nuclear Information System (INIS)

Winkler, Justin M; Lukishova, Svetlana G; Bissell, Luke J

2013-01-01

Definite circular and linear polarizations of room-temperature single-photon sources, which can serve as polarization bases for quantum key distribution, are produced by doping planar-aligned liquid crystal hosts with single fluorescence emitters. Chiral 1-D photonic bandgap microcavities for a single handedness of circularly polarized light were prepared from both monomeric and oligomeric cholesteric liquid crystals. Fluorescent emitters, such as nanocrystal quantum dots, nitrogen vacancy color centers in nanodiamonds, and rare-earth ions in nanocrystals, were doped into these microcavity structures and used to produce circularly polarized fluorescence of definite handedness. Additionally, we observed circularly polarized resonances in the spectrum of nanocrystal quantum dot fluorescence at the edge of the cholesteric microcavity's photonic stopband. For this polarization we obtained a ∼4.9 enhancement of intensity compared to the polarization of the opposite handedness that propagates without photonic bandgap microcavity effects. Such a resonance is indicative of coupling of quantum dot fluorescence to the cholesteric microcavity mode. We have also used planar-aligned nematic liquid crystal hosts to align DiI dye molecules doped into the host, thereby providing a single-photon source of linear polarization of definite direction. Antibunching is demonstrated for fluorescence of nanocrystal quantum dots, nitrogen vacancy color centers, and dye molecules in these liquid crystal structures.

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

Science.gov (United States)

Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki

2013-07-29

We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

Study on the fluorescence characteristics of carbon dots

Science.gov (United States)

Mao, Xiao-Jiao; Zheng, Hu-Zhi; Long, Yi-Juan; Du, Juan; Hao, Jian-Yu; Wang, Ling-Ling; Zhou, Dong-Bo

2010-02-01

Herein, we prepared water-soluble fluorescent carbon dots with diameter about 1.5 nm from cheap commercial lampblack. These fluorescent carbon nanoparticles are stable toward photobleaching and stable in water for more than half a year without fluorescence decrease. In order to improve its fluorescence properties, we passivated these nanoparticles with bisamino-terminated polyethylene glycol (PEG 1500N). Therefore, both fluorescence quantum yield and lifetime increased after this progress. In addition, the passivated carbon dots were more inert to solvent than the bare one and showed different responses to pH change.

APD detectors for biological fluorescence spectroscopy

International Nuclear Information System (INIS)

Mazeres, S.; Borrel, V.; Magenc, C.; Courrech, J.L.; Bazer-Bachi, R.

2006-01-01

Fluorescence spectroscopy is a very convenient and widely used method for studying the molecular background of biological processes [L. Salome, J.L. Cazeil, A. Lopez, J.F. Tocanne, Eur. Biophys. J. 27 (1998) 391-402]. Chromophores are included in the structure under study and a flash of laser light induces fluorescence (Fluorescence Recovery After Photo-bleaching), the decay of which yields information on the polarity, the speed of rotation, and the speed of diffusion as well as on the temporal and spatial evolution of interactions between molecular species. The method can even be used to study living cells [J.F. Tocanne, L. Cezanne, A. Lopez, Prog. Lipid Res. 33 (1994) 203-237, L. Cezanne, A. Lopez, F. Loste, G. Parnaud, O. Saurel, P. Demange, J.F. Tocanne, Biochemistry 38 (1999) 2779-2786]. This is classically performed with a PM-based system. For biological reasons a decrease of the excitation of the cells is highly desirable. Because the fluorescence response then becomes fainter a significant improvement in detector capability would be welcome. We present here results obtained with an Avalanche Photo Diode (APD)-based system. The small sensitive area of detection allows a very significant improvement in signal/noise ratio, improvement in gain, and the opening-up of a new parameter space. With these new detectors we can begin the study of information transmission between cells through morphine receptors. This work involves both electronics engineers and biophysicists, so results and techniques in both fields will be presented here

Regeneration of irradiated optical fibres by photo-bleaching?

International Nuclear Information System (INIS)

Henschel, H.; Koehn, O.

1999-01-01

It is known that a light power between 0,1 and 20 μW caused bleaching of colour centres, which implies a reduction of induced loss. Older fibres especially those with a core made of undoped, low OH silica, experience tremendous photo-bleaching. Light of shorter wavelengths has a higher bleaching efficiency than that of longer wavelengths and same light intensity. The investigations have demonstrated that the injection of photo-bleaching light of shorter wavelength and higher intensity can distinctly decrease the radiation-induced loss of Ge-doped fibres, especially at low temperatures. Another possibility to apply photo-bleaching by short wavelength is to regenerate fibres that are permanently installed in radiation environments. Modern undoped multi-mode (MM) step index (Si), Ge-doped MM graded index (Gi) and Ge-doped single-mode (SM) fibres that had been irradiated were submitted to bleaching light. In this article it is shown how loss reduction and necessary bleaching time depend on wavelength and intensity of the bleaching light, on fibre length (bleaching time) and on radiation dose. These results are promising for the regeneration of optical fibres in facilities where the fibres cannot be replaced easily by new ones. (A.C.)

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

Science.gov (United States)

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Polarized X-ray excitation for scatter reduction in X-ray fluorescence computed tomography.

Science.gov (United States)

Vernekohl, Don; Tzoumas, Stratis; Zhao, Wei; Xing, Lei

2018-05-25

X-ray fluorescence computer tomography (XFCT) is a new molecular imaging modality which uses X-ray excitation to stimulate the emission of fluorescent photons in high atomic number contrast agents. Scatter contamination is one of the main challenges in XFCT imaging which limits the molecular sensitivity. When polarized X-rays are used, it is possible to reduce the scatter contamination significantly by placing detectors perpendicular to the polarization direction. This study quantifies scatter contamination for polarized and unpolarized X-ray excitation and determines the advantages of scatter reduction. The amount of scatter in preclinical XFCT is quantified in Monte Carlo simulations. The fluorescent X-rays are emitted isotropically, while scattered X-rays propagate in polarization direction. The magnitude of scatter contamination is studied in XFCT simulations of a mouse phantom. In this study, the contrast agent gold is examined as an example but a scatter reduction from polarized excitation is also expected for other elements. The scatter reduction capability is examined for different polarization intensities with a monoenergetic X-ray excitation energy of 82 keV. The study evaluates two different geometrical shapes of CZT detectors which are modeled with an energy resolution of 1 keV FWHM at an X-ray energy of 80 keV. Benefits of a detector placement perpendicular to the polarization direction are shown in iterative and analytic image reconstruction including scatter correction. The contrast to noise ratio (CNR) and the normalized mean square error (NMSE) are analyzed and compared for the reconstructed images. A substantial scatter reduction for common detector sizes was achieved for 100% and 80% linear polarization while lower polarization intensities provide a decreased scatter reduction. By placing the detector perpendicular to the polarization direction, a scatter reduction by factor up to 5.5 can be achieved for common detector sizes. The image

Optimization of a polarized source for in vivo x-ray fluorescence analysis of platinum and other heavy metals

International Nuclear Information System (INIS)

Lewis, D.G.

1994-01-01

The Monte Carlo method was used to optimize a polarized photon source for the x-ray fluorescence analysis of platinum and other heavy metals in vivo. The source consisted of a 140 kVp, 25 mA x-ray tube with the photons plane-polarized by 90 o scattering. The use of plane-polarized photons results in a significant reduction in background when the fluorescent radiation is measured along the direction of polarization. A Monte Carlo computer programme was written to simulate the production and interaction of polarized photons in order to determine the optimal polarizing material and dimensions, together with beam width and geometrical arrangement of source, polarizer and beam collimators. Calculated photon energy distributions are compared with experimental data to test the validity of the model. (author)

Effects of photobleaching on selected advanced glycation end products in the human lens

DEFF Research Database (Denmark)

Holm, Thomas; Raghavan, Cibin T; Nahomi, Rooban

2015-01-01

at examining the optical and biochemical effects of the proposed treatment.MethodsHuman donor lenses were photobleaced using a 445 nm cw laser. Lens optical quality was assessed before and after photobleaching by light transmission and scattering. The concentration of the advanced glycation end products (AGEs...... of the photobleaching treatment on lens optical parameters but we could not associate the optical findings to a change in the concentration of the AGEs we measured. This finding suggests that other AGEs were responsible for the observed photobleaching of the human lens after laser treatment. The biochemical nature...

Nonclassical polarization effects in fluorescence emission spectra from microdroplets

Science.gov (United States)

Arnold, S.; Goddard, N. L.; Hill, S. C.

1999-12-01

We report a pronounced nonclassical polarization effect on the shape of fluorescence emission spectra from isolated microdroplets containing a dilute solution of soluble fluors or a dilute layer of surfactant fluors. We see different spectral shapes for 90° scattering when comparing between IVV, IVH, IHH, IHV. However, we measure the largest difference in spectral shape in the surfactant case, with the incident polarization directed toward the detector (IHV vs IHH). Imaging reveals that the emission in this case principally arises from two distinct regions near the surface of the droplet, which are diametrically opposed and along the axis of the incident laser beam. The effect appears to be the direct result of coupling between molecular emission moments and electromagnetic modes of the droplet. It is not the molecule which radiates but the molecule microvessel. Directional emission is sensitive to the polarization of the electromagnetic mode which is stimulated by the coupling.

Solvent polarity scale on the fluorescence spectra of a dansyl monomer copolymerizable in aqueous media

Science.gov (United States)

Ren, Biye; Gao, Feng; Tong, Zhen; Yan, Yu

1999-06-01

A copolymerizable fluorescent monomer N-[2-[[[5-(N,N-dimethylamino)-1-naphthalenyl]sulfonyl]-amino]ethyl]-2-propenamide (DANSAEP) was synthesized, which exhibits dual fluorescence due to the twisted intramolecular charge transfer in the excited state. The emission maximum λem shifts from 463.3 nm in n-hexane to 530.0 nm in water, showing solvent polarity dependence. The relations between λem and the conventional solvent polarity parameters ET(30) or Z are linear, dividing solvents into protic and aprotic groups. Kamlet's linear solvation energy relationship gives a good description for λem as a solvent polarity scale. The increment of dipole moment Δ μ at the excited state was estimated as 5.09 D with the solvatochromic analysis.

Intrinsic fluorescence for cervical precancer detection using polarized light based in-house fabricated portable device

Science.gov (United States)

Meena, Bharat Lal; Singh, Pankaj; Sah, Amar Nath; Pandey, Kiran; Agarwal, Asha; Pantola, Chayanika; Pradhan, Asima

2018-01-01

An in-house fabricated portable device has been tested to detect cervical precancer through the intrinsic fluorescence from human cervix of the whole uterus in a clinical setting. A previously validated technique based on simultaneously acquired polarized fluorescence and polarized elastic scattering spectra from a turbid medium is used to extract the intrinsic fluorescence. Using a diode laser at 405 nm, intrinsic fluorescence of flavin adenine dinucleotide, which is the dominant fluorophore and other contributing fluorophores in the epithelium of cervical tissue, has been extracted. Different grades of cervical precancer (cervical intraepithelial neoplasia; CIN) have been discriminated using principal component analysis-based Mahalanobis distance and linear discriminant analysis. Normal, CIN I and CIN II samples have been discriminated from one another with high sensitivity and specificity at 95% confidence level. This ex vivo study with cervix of whole uterus samples immediately after hysterectomy in a clinical environment indicates that the in-house fabricated portable device has the potential to be used as a screening tool for in vivo precancer detection using intrinsic fluorescence.

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

NARCIS (Netherlands)

Mastop, M.; Bindels, D.S.; Shaner, N.C.; Postma, M.; Gadella, T.W.J.; Goedhart, J.

2017-01-01

The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo

OpenAIRE

Speksnijder, J.E.; Dohmen, M.R.; Tertoolen, L.G.J.; Laat, S.W. de

1985-01-01

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1′-ditetradecyl 3,3,3′,3′-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lob...

MEMBRANE MOBILITY AND MICRODOMAIN LOCALIZATION OF THE DOPAMINE TRANSPORTER STUDIED BY CONFOCAL FLUORESCENCE CORRELATION SPECTROSCOPY (FCS) AND FRAP

DEFF Research Database (Denmark)

Adkins, Erica; (Vægter), Christian Bjerggaard; van Deurs, Bo

FCS measurements in transiently transfected N2A neuroblastoma cells were impaired by photobleachning suggesting immobilization of the transporter in the membrane. This was confirmed by the use of fluorescence recovery after photobleaching (FRAP), which showed clear recovery of YFP-DAT fluorescence...

Staphyloxanthin photobleaching sensitizes methicillin-resistant Staphylococcus aureus to reactive oxygen species attack

Science.gov (United States)

Dong, Pu-Ting; Mohammad, Haroon; Hui, Jie; Wang, Xiaoyu; Li, Junjie; Liang, Lijia; Seleem, Mohamed N.; Cheng, Ji-Xin

2018-02-01

Given that the dearth of new antibiotic development loads an existential burden on successful infectious disease therapy, health organizations are calling for alternative approaches to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report a drug-free photonic approach to eliminate MRSA through photobleaching of staphyloxanthin, an indispensable membrane-bound antioxidant of S. aureus. The photobleaching process, uncovered through a transient absorption imaging study and quantitated by absorption spectroscopy and mass spectrometry, decomposes staphyloxanthin, and sensitizes MRSA to reactive oxygen species attack. Consequently, staphyloxanthin bleaching by low-level blue light eradicates MRSA synergistically with external or internal reactive oxygen species. The effectiveness of this synergistic therapy is validated in MRSA culture, MRSAinfected macrophage cells. Collectively, these findings highlight broad applications of staphyloxanthin photobleaching for treatment of MRSA infections.

Monte Carlo modeling of in vivo protoporphyrin IX fluorescence and singlet oxygen production during photodynamic therapy for patients presenting with superficial basal cell carcinomas

Science.gov (United States)

Valentine, Ronan M.; Brown, C. Tom A.; Moseley, Harry; Ibbotson, Sally; Wood, Kenny

2011-04-01

We present protoporphyrin IX (PpIX) fluorescence measurements acquired from patients presenting with superficial basal cell carcinoma during photodynamic therapy (PDT) treatment, facilitating in vivo photobleaching to be monitored. Monte Carlo (MC) simulations, taking into account photobleaching, are performed on a three-dimensional cube grid, which represents the treatment geometry. Consequently, it is possible to determine the spatial and temporal changes to the origin of collected fluorescence and generated singlet oxygen. From our clinical results, an in vivo photobleaching dose constant, β of 5-aminolaevulinic acid-induced PpIX fluorescence is found to be 14 +/- 1 J/cm2. Results from our MC simulations suggest that an increase from our typical administered treatment light dose of 75-150 J/cm2 could increase the effective PDT treatment initially achieved at a depth of 2.7-3.3 mm in the tumor, respectively. Moreover, this increase reduces the surface PpIX fluorescence from 0.00012 to 0.000003 of the maximum value recorded before treatment. The recommendation of administrating a larger light dose, which advocates an increase in the treatment time after surface PpIX fluorescence has diminished, remains valid for different sets of optical properties and therefore should have a beneficial outcome on the total treatment effect.

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo

NARCIS (Netherlands)

Speksnijder, J.E.; Dohmen, M.R.; Tertoolen, L.G.J.; Laat, S.W. de

1985-01-01

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1′-ditetradecyl

Angular shaping of fluorescence from synthetic opal-based photonic crystal.

Science.gov (United States)

Boiko, Vitalii; Dovbeshko, Galyna; Dolgov, Leonid; Kiisk, Valter; Sildos, Ilmo; Loot, Ardi; Gorelik, Vladimir

2015-01-01

Spectral, angular, and temporal distributions of fluorescence as well as specular reflection were investigated for silica-based artificial opals. Periodic arrangement of nanosized silica globules in the opal causes a specific dip in the defect-related fluorescence spectra and a peak in the reflectance spectrum. The spectral position of the dip coincides with the photonic stop band. The latter is dependent on the size of silica globules and the angle of observation. The spectral shape and intensity of defect-related fluorescence can be controlled by variation of detection angle. Fluorescence intensity increases up to two times at the edges of the spectral dip. Partial photobleaching of fluorescence was observed. Photonic origin of the observed effects is discussed.

Waveguide evanescent field fluorescence microscopy & its application in cell biology

Science.gov (United States)

Hassanzadeh, Abdollah

are captured and collected during the experiment, permitting time lapse analysis. As a proof of concept, we have monitored the response of cells on the waveguide surface to an external lethal agent. Imaging analysis showed very low photobleaching. Therefore photobleaching can be neglected during the experiments. The effects of secondary patterns (inhomogeneities) in the grating and scratches and inhomogeneities in the wave guiding film on the fluorescence background, ultra-thin film and cell-substrate contact regions image were investigated. In conclusion, we developed and established WEFF microscopy and have visualized and quantified solid thin films thicknesses and cell-substrate contact regions. The achieved low scattering results in an improved signal-to-noise ratio and increased sensitivity. Photobleaching and phototoxicity are largely reduced compared to other microscopy techniques. Therefore imaging can be carried out over extended periods and having better temporal resolution without sample damage, such as effect of external agents on the cell-substrate contact regions. Keywords. Waveguide Evanescent Field Fluorescence Microscopy, Evanescent Field, Ion-exchanged Waveguides, TE modes, TM modes, Electromagnetic Field Distribution, Fluorescence, Microscopy, Optical Waveguides, Imaging, Interface, Triton X-100, Focal Contacts, Close Contacts, Cell-Substrate Contact Regions, Cell-Substrate Separation Distances, Photobleaching, Phototoxicity, Cell-Substrate Interaction, Langmuir-Blodgett Films, Phase Separated Lipid Films, SGG 11 Glass, Resolution, Total Internal Reflection, Total Internal Reflection Fluorescence Microscopy, Osteoblast Cells, Grating Coupling, Prism Coupling, Grating.

Fluorescence lifetime measurements to determine the core-shell nanostructure of FITC-doped silica nanoparticles: An optical approach to evaluate nanoparticle photostability

International Nuclear Information System (INIS)

Santra, Swadeshmukul; Liesenfeld, Bernd; Bertolino, Chiara; Dutta, Debamitra; Cao Zehui; Tan Weihong; Moudgil, Brij M.; Mericle, Robert A.

2006-01-01

In this paper, we described a novel fluorescence lifetime-based approach to determine the core-shell nanostructure of FITC-(fluorescein isothiocyanate, isomer I) doped fluorescent silica nanoparticles (FSNPs). Because of phase homogeneity between the core and the shell, electron microscopic technique could not be used to characterize such core-shell nanostructure. Our optical approach not only revealed the core-shell nanostructure of FSNPs but also evaluated photobleaching of FSNPs both in the solvated and non-solvated (dry) states. The FSNPs were produced via Stoeber's method by hydrolysis and co-condensation reaction of tetraethylorthosilicate (TEOS) and fluorescein linked (3-aminopropyl)triethoxysilane (FITC-APTS conjugate) in the presence of ammonium hydroxide catalyst. To obtain a pure silica surface coating, FSNPs were then post-coated with TEOS. The average particle size was 135 nm as determined by TEM (transmission electron microscope) measurements. Fluorescence excitation and emission spectral data demonstrated successful doping of FITC dye molecules in FSNPs. Fluorescence lifetime data revealed that approximately 62% of dye molecules remained in the solvated silica shell, while 38% of dye molecules remained in the non-solvated (dry) silica core. Photobleaching experiments of FSNPs were conducted both in DI water (solution state) and in air (dry state). Severe photobleaching of FSNPs was observed in air. However, FSNPs were moderately photostable in the solution state. Photostability of FSNPs in both solution and dry states was explained on the basis of fluorescence lifetime data

Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

Science.gov (United States)

Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

2017-06-01

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

High-sensitivity determination of Zn(II) and Cu(II) in vitro by fluorescence polarization

Science.gov (United States)

Thompson, Richard B.; Maliwal, Badri P.; Feliccia, Vincent; Fierke, Carol A.

1998-04-01

Recent work has suggested that free Cu(II) may play a role in syndromes such as Crohn's and Wilson's diseases, as well as being a pollutant toxic at low levels to shellfish and sheep. Similarly, Zn(II) has been implicated in some neural damage in the brain resulting from epilepsy and ischemia. Several high sensitivity methods exist for determining these ions in solution, including GFAAS, ICP-MS, ICP-ES, and electrochemical techniques. However, these techniques are generally slow and costly, require pretreatment of the sample, require complex instruments and skilled personnel, and are incapable of imaging at the cellular and subcellular level. To address these shortcomings we developed fluorescence polarization (anisotropy) biosensing methods for these ions which are very sensitivity, highly selective, require simple instrumentation and little pretreatment, and are inexpensive. Thus free Cu(II) or Zn(II) can be determined at picomolar levels by changes in fluorescence polarization, lifetime, or wavelength ratio using these methods; these techniques may be adapted to microscopy.

Investigation of β-lactam antibacterial drugs, β-lactamases, and penicillin-binding proteins with fluorescence polarization and anisotropy: a review

Science.gov (United States)

Shapiro, Adam B.

2016-06-01

This review covers the uses of fluorescence polarization and anisotropy for the investigation of bacterial penicillin binding proteins (PBPs), which are the targets of β-lactam antibacterial drugs (penicillins, cephalosporins, carbapenems, and monobactams), and of the β-lactamase enzymes that destroy these drugs and help to render bacterial pathogens resistant to them. Fluorescence polarization and anisotropy-based methods for quantitation of β-lactam drugs are also reviewed. A particular emphasis is on methods for quantitative measurement of the interactions of β-lactams and other inhibitors with PBPs and β-lactamases.

Synthesis and characterization of photoswitchable fluorescent silica nanoparticles.

Science.gov (United States)

Fölling, Jonas; Polyakova, Svetlana; Belov, Vladimir; van Blaaderen, Alfons; Bossi, Mariano L; Hell, Stefan W

2008-01-01

We have designed and synthesized a new functional (amino reactive) highly efficient fluorescent molecular switch (FMS) with a photochromic diarylethene and a rhodamine fluorescent dye. The reactive group in this FMS -N-hydroxysuccinimide ester- allows selective labeling of amino containing molecules or other materials. In ethanolic solutions, the compound displays a large fluorescent quantum yield of 52 % and a large fluorescence modulation ratio (94 %) between two states that may be interconverted with red and near-UV light. Silica nanoparticles incorporating the new FMS were prepared and characterized, and their spectroscopic and switching properties were also studied. The dye retained its properties after the incorporation into the silica, thereby allowing light-induced reversible high modulation of the fluorescence signal of a single particle for up to 60 cycles, before undergoing irreversible photobleaching. Some applications of these particles in fluorescence microscopy are also demonstrated. In particular, subdiffraction images of nanoparticles were obtained, in the focal plane of a confocal microscope.

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity

NARCIS (Netherlands)

Laat, S.W. de; Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van; Tetteroo, P.A.T.; Tertoolen, L.G.J.

1984-01-01

Regional differences in the lateral mobility properties of plasma membrane lipids were studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein- -1abelled fatty

Portable instrument that integrates irradiation with fluorescence and reflectance spectroscopies during clinical photodynamic therapy of cutaneous disease

Science.gov (United States)

Cottrell, W. J.; Oseroff, A. R.; Foster, T. H.

2006-06-01

We report a portable clinical instrument for delivering photodynamic therapy (PDT) while performing noninvasive spectroscopic monitoring in vivo. Using an off-surface probe, the instrument delivers the treatment beam to a user-defined field on the skin and performs reflectance and fluorescence spectroscopies at two regions within this field. The instrument is being used to monitor photosensitizer fluorescence photobleaching, fluorescent photoproduct kinetics, blood volume, and hemoglobin oxygen saturation during a pilot clinical trial of 5-aminolevulinic acid-PDT treatment of superficial basal cell carcinoma (BCC). Protoporphyrin IX and photoproduct fluorescence excited by the 633nm PDT treatment laser is collected between 655 and 800nm. During a series of brief treatment interruptions at programable time points, white light reflectance spectra between 475 and 800nm are acquired. Fluorescence spectra are corrected for the effects of absorption and scattering, informed by the reflectance measurements, and then decomposed into known fluorophore contributions in real time using a robust singular value decomposition fitting routine. Reflectance spectra additionally provide information on blood volume and hemoglobin oxygen saturation. Monitoring blood oxygenation and implicit dose metrics such as photosensitizer photobleaching during PDT allows the improved interpretation of clinical results and is helping to guide the treatment protocol for an anticipated low-irradiance PDT clinical trial of BCC.

A rate-equation model for polarized laser-induced fluorescence to measure electric field in glow discharge He plasmas

International Nuclear Information System (INIS)

Takiyama, K.; Watanabe, M.; Oda, T.

1998-01-01

Possibility of applying polarized laser-induced fluorescence (LIF) spectroscopy for measuring the electric field in a plasma with a large collisional depolarization has been investigated. A rate equation model including the depolarization process was employed to analyze the time evolution of LIF polarization components. The polarized LIF pulse shapes observed in the sheath of a He glow discharge plasma were successfully reproduced, and the electric field distribution was obtained with high accuracy. (author)

Fluorescent carbon dots and nanodiamonds for biological imaging: preparation, application, pharmacokinetics and toxicity.

Science.gov (United States)

Liu, Jia-Hui; Yang, Sheng-Tao; Chen, Xin-Xin; Wang, Haifang

2012-10-01

The rapid advancement of nanotechnology has brought us some new types of fluorescent probes, which are indispensable for bioimaging in life sciences. Because of their innate biocompatibility, good resistance against photobleaching, long fluorescence lifetime and wide fluorescence spectral region, fluorescent carbon quantum dots (C-Dots) and nanosized diamonds (nanodiamonds, NDs) are gradually evolving into promising reagents for bioimaging. In this review, we summarize the recent achievements in fluorescent C-Dots and NDs with emphases on their preparation, properties, imaging application, pharmacokinetics and toxicity. Perspectives on further investigations and opportunities to develop C-Dots and NDs into the safer and more sensitive imaging probes for both living cells and animal models are discussed.

Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy

Czech Academy of Sciences Publication Activity Database

Macháň, Radek; Hof, Martin

2010-01-01

Roč. 1798, č. 7 (2010), s. 1377-1391 ISSN 0005-2736 R&D Projects: GA ČR GA203/08/0114; GA AV ČR GEMEM/09/E006 Institutional research plan: CEZ:AV0Z40400503 Keywords : supported lipid bilayer * giant unilamellar vesicle * fluorescence recovery after photobleaching Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.647, year: 2010

Accessibility of nucleic acid-complexed biomolecules to hydroxyl radicals correlates with their conformation: a fluorescence polarization spectroscopy study

International Nuclear Information System (INIS)

Makrigiorgos, G.M.; Bump, E.; Huang, C.; Kassis, A.I.; Baranowska-Kortylewicz, J.

1994-01-01

A fluorescence methodology has been developed to examine the relationship between the conformational state of specific biomolecules in simple chromatin models and their accessibility to hydroxyl radicals ( . OH). Polylysine and histone H1 were labelled with SECCA, the succinimidyl ester of coumarin-3-carboxylic acid, which generates the fluorescent derivative 7-OH-SECCA following its interaction with radiation-induced . OH in aqueous solution. The fluorescence induced per unit γ-ray dose reflecting the accessibility of . OH to such SECCA-conjugated biomolecules was recorded. The biomolecules were also labelled with the fluorescent derivative 7-OH-SECCA in trace amounts to study their conformation under identical conditions via fluorescence polarization spectroscopy. (author)

Lateral mobility of plasma membrane lipids in a molluscan egg: Evidence for an animal/vegetal polarity

NARCIS (Netherlands)

Laat, S.W. de; Speksnijder, J.E.; Dohmen, M.R.; Zoelen, E. van; Tertoolen, L.G.J.; Bluemink, J.G.

1984-01-01

The lateral diffusion of the lipid analog C₁₄-diI (3', 3'-dihexadecylindocarbocyanine iodide) was measured in the plasma membrane of early embryos of the mollusc Nassarius reticulatus using the FPR-(Fluorescence Photobleaching Recovery) method. At almost all stages measured (from

Blueberry effects on dark vision and recovery after photobleaching: placebo-controlled crossover studies.

Science.gov (United States)

Kalt, Wilhelmina; McDonald, Jane E; Fillmore, Sherry A E; Tremblay, Francois

2014-11-19

Clinical evidence for anthocyanin benefits in night vision is controversial. This paper presents two human trials investigating blueberry anthocyanin effects on dark adaptation, functional night vision, and vision recovery after retinal photobleaching. One trial, S2 (n = 72), employed a 3 week intervention and a 3 week washout, two anthocyanin doses (271 and 7.11 mg cyanidin 3-glucoside equivalents (C3g eq)), and placebo. The other trial, L1 (n = 59), employed a 12 week intervention and an 8 week washout and tested one dose (346 mg C3g eq) and placebo. In both S2 and L1 neither dark adaptation nor night vision was improved by anthocyanin intake. However, in both trials anthocyanin consumption hastened the recovery of visual acuity after photobleaching. In S2 both anthocyanin doses were effective (P = 0.014), and in L1 recovery was improved at 8 weeks (P = 0.027) and 12 weeks (P = 0.030). Although photobleaching recovery was hastened by anthocyanins, it is not known whether this improvement would have an impact on everyday vision.

Rapid biochemical functionalization of technical surfaces by means of a photobleaching-based maskless projection lithography process

Science.gov (United States)

Waldbaur, Ansgar; Waterkotte, Björn; Leuthold, Juerg; Schmitz, Katja; Rapp, Bastian E.

2013-03-01

MEMS/MOEMS based systems are increasingly applied in the biological and biomedical context, e.g. in form of biosensors or substrates for monitoring biological responses such as cell migration. For such applications, technical surfaces have to be provided with suitable biochemical functionalization. Typical functionalization procedures include wet-chemical techniques based on self-assembled monolayers of thiols on gold or silanes on glass. These processes create binary patterns and are often of limited use if spatially constrained non-binary patterns like surface bound biochemical gradients have to be provided. In order to create gradients or patterns, methods such as direct spotting or dip pen nanolithography can be used. Here, gradients can be emulated by varying the spot density or the concentration of the solutions employed. However, these methods are serial in nature and are thus of limited use if large surface areas have to be patterned. We present a technique to generate gradients of biochemical function by a photobleaching-based process allowing fast large-scale patterning. The process is based on photobleaching resulting in light-induced coupling of a fluorescently tagged biomolecule to a technical surface by concerted bleaching of the fluorophore. We custom designed a maskless projection lithography system based on a digital mirror device that allows the rapid creation of 8-bit grayscale protein patterns on any technical surface from digital data (e.g. bitmap files). We demonstrate how this process can be used to obtain patterns of several cm2 lateral size at micrometer resolution within minutes.

A reaction–subdiffusion model of fluorescence recovery after photobleaching (FRAP)

International Nuclear Information System (INIS)

Yuste, S B; Abad, E; Lindenberg, K

2014-01-01

Anomalous diffusion, in particular subdiffusion, is frequently invoked as a mechanism of motion in dense biological media and may have a significant impact on the kinetics of binding/unbinding events at the cellular level. In this work we incorporate anomalous diffusion in a previously developed model for FRAP experiments. Our particular implementation of subdiffusive transport is based on a continuous time random walk (CTRW) description of the motion of fluorescent particles, as CTRWs lend themselves particularly well to the inclusion of binding/unbinding events. In order to model switching between bound and unbound states of fluorescent subdiffusive particles, we derive a fractional reaction–subdiffusion equation of rather general applicability. Using suitable initial and boundary conditions, this equation is then incorporated in the model describing 2D kinetics of FRAP experiments. We find that this model can be used to obtain excellent fits to experimental data. Moreover, recovery curves corresponding to different radii of the circular bleach spot can be fitted by a single set of parameters. While not enough evidence has been collected to claim with certainty that the underlying transport mechanism in FRAP experiments is one that leads to anomalous diffusion, the compatibility of our results with experimental data fuels the discussion as to whether normal diffusion or some form of anomalous diffusion is the appropriate model and as to whether anomalous diffusion effects are important to fully understand the outcomes of FRAP experiments. On a more technical side, we derive explicit analytic solutions of our model in certain limits. (paper)

Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

Directory of Open Access Journals (Sweden)

Yiyi Sun

Full Text Available Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (--arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.

Lateral mobility of plasma membrane lipids in Xenopus eggs: regional differences related to animal/vegetal polarity become extreme upon fertilization.

Science.gov (United States)

Dictus, W J; van Zoelen, E J; Tetteroo, P A; Tertoolen, L G; de Laat, S W; Bluemink, J G

1984-01-01

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-(N-hexadecanoyl)-aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching indicating the existence of lipidic microdomains. In the unfertilized egg the mobile fraction of plasma membrane lipids (approximately 50%) has a fivefold smaller lateral diffusion coefficient (D = 1.5 X 10(-8) cm2/sec) in the animal than in the vegetal plasma membrane (D = 7.6 X 10(-8) cm2/sec). This demonstrates the presence of an animal/vegetal polarity within the Xenopus egg plasma membrane. Upon fertilization this polarity is strongly (greater than 100X) enhanced leading to the formation of two distinct macrodomains within the plasma membrane. At the animal side of the egg lipids are completely immobilized on the time scale of FPR measurements (D less than 10(-10) cm2/sec), whereas at the vegetal side D is only slightly reduced (D = 4.4 X 10(-8) cm2/sec). The immobilization of animal plasma membrane lipids, which could play a role in the polyspermy block, probably arises by the fusion of cortical granules which are more numerous here. The transition between the animal and the vegetal domain is sharp and coincides with the boundary between the presumptive ecto- and endoderm. The role of regional differences in the plasma membrane is discussed in relation to cell diversification in early development.

Combined electromagnetic and photoreaction modeling of CLD-1 photobleaching in polymer microring resonators

Science.gov (United States)

Huang, Yanyi; Poon, Joyce K. S.; Liang, Wei; Yariv, Amnon; Zhang, Cheng; Dalton, Larry R.

2005-08-01

By combining a solid-state photoreaction model with the modal solutions of an optical waveguide, we simulate the refractive index change due to the photobleaching of CLD-1 chromophores in an amorphous polycarbonate microring resonator. The simulation agrees well with experimental results. The photobleaching quantum efficiency of the CLD-1 chromophores is determined to be 0.65%. The combined modeling of the electromagnetic wave propagation and photoreaction precisely illustrates the spatial and temporal evolution of the optical properties of the polymer material as manifested in the refractive index and their effects on the modal and physical properties of the optical devices.

Melanin fluorescence spectra by step-wise three photon excitation

Science.gov (United States)

Lai, Zhenhua; Kerimo, Josef; DiMarzio, Charles A.

2012-03-01

Melanin is the characteristic chromophore of human skin with various potential biological functions. Kerimo discovered enhanced melanin fluorescence by stepwise three-photon excitation in 2011. In this article, step-wise three-photon excited fluorescence (STPEF) spectrum between 450 nm -700 nm of melanin is reported. The melanin STPEF spectrum exhibited an exponential increase with wavelength. However, there was a probability of about 33% that another kind of step-wise multi-photon excited fluorescence (SMPEF) that peaks at 525 nm, shown by previous research, could also be generated using the same process. Using an excitation source at 920 nm as opposed to 830 nm increased the potential for generating SMPEF peaks at 525 nm. The SMPEF spectrum peaks at 525 nm photo-bleached faster than STPEF spectrum.

Alignment of Ar+ [3P]4p2P03/2 satellite state from the polarization analysis of fluorescent radiation after photoionization

International Nuclear Information System (INIS)

Yenen, O.; McLaughlin, K.W.; Jaecks, D.H.

1997-01-01

The measurement of the polarization of radiation from satellite states of Ar + formed after the photoionization of Ar provides detailed information about the nature of doubly excited states, magnetic sublevel cross sections and partial wave ratios of the photo-ejected electrons. Since the formation of these satellite states is a weak process, it is necessary to use a high flux beam of incoming photons. In addition, in order to resolve the many narrow doubly excited Ar resonances, the incoming photons must have a high resolution. The characteristics of the beam line 9.0.1 of the Advanced Light Source fulfill these requirements. The authors determined the polarization of 4765 Angstrom fluorescence from the Ar + [ 3 P] 4p 2 P 3/2 0 satellite state formed after photoionization of Ar by photons from the 9.0.1 beam line of ALS in the 35.620-38.261 eV energy range using a resolution of approximately 12,700. This is accomplished by measuring the intensities of the fluorescent light polarized parallel (I parallel) and perpendicular (I perpendicular) to the polarization axis of the incident synchrotron radiation using a Sterling Optics 105MB polarizing filter. The optical system placed at 90 degrees with respect to the polarization axis of the incident light had a narrow band interference filter (δλ=0.3 nm) to isolate the fluorescent radiation

Monitoring protein synthesis by fluorescence recovery after photobleaching (FRAP) in vivo

OpenAIRE

sprotocols

2015-01-01

Currently available methodologies for measuring protein synthesis rates rely on metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides. These approaches are hampered by several limitations and cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a novel method for monitoring protein synthesis in specific cells and tissues of live Caenorhabditis elegans animals. Fluorescent reporter proteins such as...

Photobleaching kinetics and time-integrated emission of fluorescent probes in cellular membranes

DEFF Research Database (Denmark)

Wüstner, Daniel; Christensen, Tanja; Solanko, Lukasz Michal

2014-01-01

Since the pioneering work of Hirschfeld, it is known that time-integrated emission (TiEm) of a fluorophore is independent of fluorescence quantum yield and illumination intensity. Practical implementation of this important result for determining exact probe distribution in living cells is often h...

DNA-functionalized gold nanoparticle-based fluorescence polarization for the sensitive detection of silver ions.

Science.gov (United States)

Wang, Gongke; Wang, Shuangli; Yan, Changling; Bai, Guangyue; Liu, Yufang

2018-04-05

Despite their practical applications, Ag + ions are environmental pollutants and affect human health. So the effective detection methods of Ag + ions are imperative. Herein, we developed a simple, sensitive, selective, and cost-effective fluorescence polarization sensor for Ag + detection in aqueous solution using thiol-DNA-functionalized gold nanoparticles (AuNPs). In this sensing strategy, Ag + ions can specifically interact with a cytosine-cytosine (CC) mismatch in DNA duplexes and form stable metal-mediated cytosine-Ag + -cytosine (C-Ag + -C) base pairs. The formation of the C-Ag + -C complex results in evident changes in the molecular volume and fluorescence polarization signal. To achieve our aims, we prepared two complementary DNA strands containing C-base mismatches (probe A: 5'-SH-A 10 -TACCACTCCTCAC-3' and probe B: 5'-TCCTCACCAGTCCTA-FAM-3'). The stable hybridization between probe A and probe B occurs with the formation of the C-Ag + -C complex in the presence of Ag + ions, leading to obvious fluorescence quenching in comparison to the system without AuNP enhancement. The assay can be used to identify nanomolar levels of Ag + within 6 min at room temperature, and has extremely high specificity for Ag + , even in the presence of higher concentrations of interfering metal ions. Furthermore, the sensor was successfully applied to the detection of Ag + ions in environmental water samples and showed excellent selectivity and high sensitivity, implying its promising application in the future. Copyright © 2018 Elsevier B.V. All rights reserved.

Photobleaching of chromophoric dissolved organic matter (CDOM) in the Yangtze River estuary: kinetics and effects of temperature, pH, and salinity.

Science.gov (United States)

Song, Guisheng; Li, Yijie; Hu, Suzheng; Li, Guiju; Zhao, Ruihua; Sun, Xin; Xie, Huixiang

2017-06-21

The kinetics and temperature-, pH- and salinity-dependences of photobleaching of chromophoric dissolved organic matter (CDOM) in the Yangtze River estuary (YRE) were evaluated using laboratory solar-simulated irradiation and compared to those of Suwannee River humic substances (SRHSs). Nearly all CDOM in water at the head of the estuary (headwater herein) was photobleachable in both summer and winter, while significant fractions of CDOM (13-29%) were resistant to photobleaching in saltier waters. The photobleaching rate constant in the headwater was 25% higher in summer than that in winter. The absorbed photon-based photobleaching efficiency (PE) increased with temperature following the linear Arrhenius equation. For a 20 °C increase in temperature, PE increased by ∼45% in the headwater and by 70-81% in the saltier waters. PE for YRE samples exhibited minima at pH from 6 to 7 and increased with both lower and higher pH values, contrasting the consistent increase in PE with pH shown by SRHSs. No consistent effect of salinity on PE was observed for both SRHSs and YRE samples. Photobleaching increased the spectral slope coefficient between 275 nm and 295 nm in summer, consistent with the behavior of SRHSs, but decreased it in winter, implying a difference in the molecular composition of chromophores between the two seasons. Temperature, salinity, and pH modified the photoalteration of the spectral shape but their effects varied spatially and seasonally. This study demonstrates that CDOM quality, temperature, and pH should be incorporated into models involving quantification of photobleaching.

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity become extreme upon fertilization

NARCIS (Netherlands)

Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van; Tetteroo, P.A.T.; Tertoolen, L.G.J.; Laat, S.W. de

1984-01-01

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilizedxaqpus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids

Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy

Directory of Open Access Journals (Sweden)

Molly McQuilken

2017-05-01

Full Text Available Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis.

mKikGR, a monomeric photoswitchable fluorescent protein.

Directory of Open Access Journals (Sweden)

Satoshi Habuchi

Full Text Available The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

Investigation of photobleaching and saturation of single molecules by fluorophore recrossing events

Energy Technology Data Exchange (ETDEWEB)

Burrows, Sean M.; Reif, Randall D. [Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061 (United States); Pappas, Dimitri [Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061 (United States)], E-mail: d.pappas@ttu.edu

2007-08-15

A method for investigation of photobleaching and saturation of single molecules by fluorophore recrossing events in a laser beam is described. The diffraction-limited probe volumes encountered in single-molecule detection (SMD) produce high excitation irradiance, which can decrease available signal. The single molecules of several dyes were detected and the data was used to extract interpeak times above a defined threshold value. The interpeak times revealed the number of fluorophore recrossing events. The number of molecules detected that were within 2 ms of each other represented a molecular recrossing for this work. Calcein, fluorescein and R-phycoerythrin were analyzed and the saturation irradiance and photobleaching effects were determined as a function of irradiance. This approach is simple and it serves as a method of optimizing experimental conditions for single-molecule detection.

Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

International Nuclear Information System (INIS)

Dietz, Marina S; Haße, Daniel; Ferraris, Davide M; Göhler, Antonia; Niemann, Hartmut H; Heilemann, Mike

2013-01-01

The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.

Molecular engineering of two-photon fluorescent probes for bioimaging applications

Science.gov (United States)

Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

2017-03-01

During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

A Light-Induced Reaction with Oxygen Leads to Chromophore Decomposition and Irreversible Photobleaching in GFP-Type Proteins.

Science.gov (United States)

Grigorenko, Bella L; Nemukhin, Alexander V; Polyakov, Igor V; Khrenova, Maria G; Krylov, Anna I

2015-04-30

Photobleaching and photostability of proteins of the green fluorescent protein (GFP) family are crucially important for practical applications of these widely used biomarkers. On the basis of simulations, we propose a mechanism for irreversible bleaching in GFP-type proteins under intense light illumination. The key feature of the mechanism is a photoinduced reaction of the chromophore with molecular oxygen (O2) inside the protein barrel leading to the chromophore's decomposition. Using quantum mechanics/molecular mechanics (QM/MM) modeling we show that a model system comprising the protein-bound Chro(-) and O2 can be excited to an electronic state of the intermolecular charge-transfer (CT) character (Chro(•)···O2(-•)). Once in the CT state, the system undergoes a series of chemical reactions with low activation barriers resulting in the cleavage of the bridging bond between the phenolic and imidazolinone rings and disintegration of the chromophore.

Fluorescence life-time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles.

Science.gov (United States)

Schwartz, Shmulik; Fixler, Dror; Popovtzer, Rachela; Shefi, Orit

2015-11-01

Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A sensitive fluorescent probe for the polar solvation dynamics at protein-surfactant interfaces.

Science.gov (United States)

Singh, Priya; Choudhury, Susobhan; Singha, Subhankar; Jun, Yongwoong; Chakraborty, Sandipan; Sengupta, Jhimli; Das, Ranjan; Ahn, Kyo-Han; Pal, Samir Kumar

2017-05-17

Relaxation dynamics at the surface of biologically important macromolecules is important taking into account their functionality in molecular recognition. Over the years it has been shown that the solvation dynamics of a fluorescent probe at biomolecular surfaces and interfaces account for the relaxation dynamics of polar residues and associated water molecules. However, the sensitivity of the dynamics depends largely on the localization and exposure of the probe. For noncovalent fluorescent probes, localization at the region of interest in addition to surface exposure is an added challenge compared to the covalently attached probes at the biological interfaces. Here we have used a synthesized donor-acceptor type dipolar fluorophore, 6-acetyl-(2-((4-hydroxycyclohexyl)(methyl)amino)naphthalene) (ACYMAN), for the investigation of the solvation dynamics of a model protein-surfactant interface. A significant structural rearrangement of a model histone protein (H1) upon interaction with anionic surfactant sodium dodecyl sulphate (SDS) as revealed from the circular dichroism (CD) studies is nicely corroborated in the solvation dynamics of the probe at the interface. The polarization gated fluorescence anisotropy of the probe compared to that at the SDS micellar surface clearly reveals the localization of the probe at the protein-surfactant interface. We have also compared the sensitivity of ACYMAN with other solvation probes including coumarin 500 (C500) and 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM). In comparison to ACYMAN, both C500 and DCM fail to probe the interfacial solvation dynamics of a model protein-surfactant interface. While C500 is found to be delocalized from the protein-surfactant interface, DCM becomes destabilized upon the formation of the interface (protein-surfactant complex). The timescales obtained from this novel probe have also been compared with other femtosecond resolved studies and molecular dynamics simulations.

Upconverting fluorescent nanoparticles for biodetection and photoactivation

Science.gov (United States)

Huang, Kai; Li, WenKai; Jayakumar, Muthu Kumara Gnanasammandhan; Zhang, Yong

2013-03-01

Fluorophores including fluorescent dyes/proteins and quantum dots (QDs) are used for fluorescence-based imaging and detection. These are based on `downconversion fluorescence' and have several drawbacks: photobleaching, autofluorescence, short tissue penetration depth and tissue photo-damage. Upconversion fluorescent nanoparticles (UCNs) emit detectable photons of higher energy in the short wavelength range upon irradiation with near-infrared (NIR) light based on a process termed `upconversion'. UCNs show absolute photostability, negligible autofluorescence, high penetration depth and minimum photodamage to biological tissues. Lanthanide doped nanocrystals with nearinfrared NIR-to-NIR and/or NIR-to-VIS and/or NIR-to-UV upconversion fluorescence emission have been synthesized. The nanocrystals with small size and tunable multi-color emission have been developed. The emission can be tuned by doping different upconverting lanthanide ions into the nanocrystals. The nanocrystals with core-shell structure have also been prepared to tune the emission color. The surfaces of these nanocrystals have been modified to render them water dispersible and biocompatible. They can be used for ultrasensitive interference-free biodetection because most biomolecules do not have upconversion properties. UCNs are also useful for light based therapy with enhanced efficiency, for example, photoactivation.

Fluorescence of the gamma, epsilon, and delta systems of nitric oxide - Polarization and use of calculated intensities for spectrometer calibration.

Science.gov (United States)

Poland, H. M.; Broida, H. P.

1971-01-01

Results of a study in which fluorescence of the gamma system of nitric oxide was obtained by excitation from both the 2144 A line of ionized cadmium and a continuum source. Individual rotational lines of the 2144 A excited fluorescence spectrum were found to be partially polarized and to have polarizations of differ ing sign. Measured relative vibrational band intensities from line and continuum excitation were compared to calculated Franck-Condon factors. Those Franck-Condon factors based on a single potential for the two spin states of the X super pi state agreed better with measured values than those based on separate potentials for the two spin states. Calculated intensities of the v prime = 3 progression were used to calibrate the instrument response in the wavelength region from 2000 to 2500 A and were checked with measured intensities of the v prime = 0.1, and 2 progressions. Fluorescence of the epsilon and delta bands obtained with continuum lamp excitation also were compared to calculated intensities.

A portable microscopy system for fluorescence, polarized, and brightfield imaging

Science.gov (United States)

Gordon, Paul; Wattinger, Rolla; Lewis, Cody; Venancio, Vinicius Paula; Mertens-Talcott, Susanne U.; Coté, Gerard

2018-02-01

The use of mobile phones to conduct diagnostic microscopy at the point-of-care presents intriguing possibilities for the advancement of high-quality medical care in remote settings. However, it is challenging to create a single device that can adapt to the ever-varying camera technologies in phones or that can image with the customization that multiple modalities require for applications such as malaria diagnosis. A portable multi-modal microscope system is presented that utilizes a Raspberry Pi to collect and transmit data wirelessly to a myriad of electronic devices for image analysis. The microscopy system is capable of providing to the user correlated brightfield, polarized, and fluorescent images of samples fixed on traditional microscopy slides. The multimodal diagnostic capabilities of the microscope were assessed by measuring parasitemia of Plasmodium falciparum-infected thin blood smears. The device is capable of detecting fluorescently-labeled DNA using FITC excitation (490 nm) and emission (525 nm), the birefringent P. falciparum byproduct hemozoin, and detecting brightfield absorption with a resolution of 0.78 micrometers (element 9-3 of a 1951 Air Force Target). This microscopy system is a novel portable imaging tool that may be a viable candidate for field implementation if challenges of system durability, cost considerations, and full automation can be overcome.

Microbubble embedded with upconversion nanoparticles as a bimodal contrast agent for fluorescence and ultrasound imaging

International Nuclear Information System (INIS)

Jin, Birui; Lin, Min; You, Minli; Xu, Feng; Lu, Tianjian; Zong, Yujin; Wan, Mingxi; Duan, Zhenfeng

2015-01-01

Bimodal imaging offers additional imaging signal thus finds wide spread application in clinical diagnostic imaging. Fluorescence/ultrasound bimodal imaging contrast agent using fluorescent dyes or quantum dots for fluorescence signal has emerged as a promising method, which however requires visible light or UV irradiation resulting in photobleaching, photoblinking, auto-fluorescence and limited tissue penetration depth. To surmount these problems, we developed a novel bimodal contrast agent using layer-by-layer assembly of upconversion nanoparticles onto the surface of microbubbles. The resulting microbubbles with average size of 2 μm provide enhanced ultrasound echo for ultrasound imaging and upconversion emission upon near infrared irradiation for fluorescence imaging. The developed bimodal contrast agent holds great potential to be applied in ultrasound target technique for targeted diseases diagnostics and therapy. (paper)

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

Science.gov (United States)

Blackman, S M; Cobb, C E; Beth, A H; Piston, D W

1996-01-01

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms. Images FIGURE 4 FIGURE 8 FIGURE 9 PMID:8804603

A fluorescence polarization based screening assay for identification of small molecule inhibitors of the PICK1 PDZ domain

DEFF Research Database (Denmark)

Thorsen, Thor S; Madsen, Kenneth L; Dyhring, Tino

2011-01-01

PDZ (PSD-95/Discs-large/ZO-1 homology) domains represent putative targets in several diseases including cancer, stroke, addiction and neuropathic pain. Here we describe the application of a simple and fast screening assay based on fluorescence polarization (FP) to identify inhibitors of the PDZ...

Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II

Czech Academy of Sciences Publication Activity Database

Alquicer, Glenda; Sedlák, David; Byun, Y.; Pavlíček, Jiří; Stathis, M.; Rojas, C.; Slusher, B.; Pomper, M.G.; Bartůněk, Petr; Bařinka, Cyril

2012-01-01

Roč. 17, č. 8 (2012), s. 1030-1040 ISSN 1087-0571 R&D Projects: GA MŠk(CZ) ME10031; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:68378050 Keywords : fluorescence polarization * glutamate carboxypeptidase II * high-throughput screening Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.207, year: 2012

Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging

Science.gov (United States)

Gravier, Julien; Navarro, Fabrice P.; Delmas, Thomas; Mittler, Frédérique; Couffin, Anne-Claude; Vinet, Françoise; Texier, Isabelle

2011-09-01

The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker®705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC50 > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.

Fluorescence and absorption properties of chromophoric dissolved organic matter (CDOM) in coastal surface waters of the Northwestern Mediterranean Sea (Bay of Marseilles, France)

Science.gov (United States)

Para, J.; Coble, P. G.; Charrière, B.; Tedetti, M.; Fontana, C.; Sempéré, R.

2010-07-01

Seawater samples were collected in surface waters (2 and 5 m depths) of the Bay of Marseilles (Northwestern Mediterranean Sea; 5°17'30'' E, 43°14'30'' N) during one year from November 2007 to December 2008 and studied for total organic carbon (TOC) as well as chromophoric dissolved organic matter (CDOM) optical properties (absorbance and fluorescence). The annual mean value of surface CDOM absorption coefficient at 350 nm [aCDOM(350)] was very low (0.10 ± 0.02 m-1) with in comparison to values usually found in coastal waters, and no significant seasonal trend in aCDOM(350) could be determined. By contrast, the spectral slope of CDOM absorption (SCDOM) was significantly higher (0.023 ± 0.003 nm-1) in summer than in fall and winter periods (0.017 ± 0.002 nm-1), reflecting either CDOM photobleaching or production in surface waters during stratified sunny periods. The CDOM fluorescence, assessed through excitation emission matrices (EEMs), was dominated by protein-like component (peak T; 1.30-21.94 QSU) and marine humic-like component (peak M; 0.55-5.82 QSU), while terrestrial humic-like fluorescence (peak C; 0.34-2.99 QSU) remained very low. This reflected a dominance of relatively fresh material from biological origin within the CDOM fluorescent pool. At the end of summer, surface CDOM fluorescence was very low and strongly blue shifted, reinforcing the hypothesis of CDOM photobleaching. Our results suggested that unusual Rhône River plume eastward intrusion events may reach Marseilles Bay within 2-3 days and induce local phytoplankton blooms and subsequent fluorescent CDOM production (peaks M and T) without adding terrestrial fluorescence signatures (peak C). Besides Rhône River plumes, mixing events of the entire water column injected humic (peaks C and M) CDOM from the bottom into the surface and thus appeared also as an important source of CDOM in surface waters of the Marseilles Bay. Therefore, the assessment of CDOM optical properties, within the

Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

Science.gov (United States)

Machida, Kazuya; Liu, Bernard

2017-01-01

Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

Use of astronomy filters in fluorescence microscopy.

Science.gov (United States)

Piper, Jörg

2012-02-01

Monochrome astronomy filters are well suited for use as excitation or suppression filters in fluorescence microscopy. Because of their particular optical design, such filters can be combined with standard halogen light sources for excitation in many fluorescent probes. In this "low energy excitation," photobleaching (fading) or other irritations of native specimens are avoided. Photomicrographs can be taken from living motile fluorescent specimens also with a flash so that fluorescence images can be created free from indistinctness caused by movement. Special filter cubes or dichroic mirrors are not needed for our method. By use of suitable astronomy filters, fluorescence microscopy can be carried out with standard laboratory microscopes equipped with condensers for bright-field (BF) and dark-field (DF) illumination in transmitted light. In BF excitation, the background brightness can be modulated in tiny steps up to dark or black. Moreover, standard industry microscopes fitted with a vertical illuminator for examinations of opaque probes in DF or BF illumination based on incident light (wafer inspections, for instance) can also be used for excitation in epi-illumination when adequate astronomy filters are inserted as excitatory and suppression filters in the illuminating and imaging light path. In all variants, transmission bands can be modulated by transmission shift.

Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds.

Science.gov (United States)

Wu, Tsai-Jung; Tzeng, Yan-Kai; Chang, Wei-Wei; Cheng, Chi-An; Kuo, Yung; Chien, Chin-Hsiang; Chang, Huan-Cheng; Yu, John

2013-09-01

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45(-)CD54(+)CD157(+) lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells' properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation.

Fluorescence quenching of 9-cyanoanthracene in presence of zinc tetraphenylporphyrin in a polar liquid medium

International Nuclear Information System (INIS)

Mandal, Paulami; Tiwari, Sanat Kumar; Ganguly, Tapan; Sinha, Subrata

2009-01-01

Steady-state and time-resolved techniques are used to study photoinduced electron and/or excitational energy transfer processes involved within a novel donor (zinc tetraphenylporphyrin)-acceptor (9-cyanoanthracene) system in a polar liquid medium (acetonitrile) at the ambient temperature (300 K). After photoexcitation of 9-cyanoanthracene, its fluorescence emission as well as lifetime are found to be quenched in presence of zinc tetraphenylporphyrin. The fluorescence quenching is ascribed to be due to the combined effect of electron transfer from zinc tetraphenylporphyrin to 9-cyanoanthracene and energy transfer (radiative as well as non-radiative) from 9-cyanoanthracene to zinc tetraphenylporphyrin. The highly exergonic values of Gibbs free energy change for both forward electron transfer reaction (-1.15 eV) and charge recombination reaction (-1.94 eV) indicate the possibilities of occurrences of these two processes in the Marcus inverted region. The fluorescence quenching rate due to photoinduced electron transfer reaction is found to be close to the diffusion-controlled limit within the present donor-acceptor system upon excitation of the acceptor molecules.

Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium

International Nuclear Information System (INIS)

Saloma, Caesar; Palmes-Saloma, Cynthia; Kondoh, Hisato

1998-01-01

Normally transparent biological structures in a turbid medium are imaged using a laser confocal microscope and multiwavelength site-specific fluorescence labelling. The spatial filtering capability of the detector pinhole in the confocal microscope limits the number of scattered fluorescent photons that reach the photodetector. Simultaneous application of different fluorescent markers on the same sample site minimizes photobleaching by reducing the excitation time for each marker. A high-contrast grey-level image is also produced by summing confocal images of the same site taken at different fluorescence wavelengths. Monte Carlo simulations are performed to obtain the quantitative behaviour of confocal fluorescence imaging in turbid media. Confocal images of the following samples were also obtained: (i) 15 μm diameter fluorescent spheres placed 1.16 mm deep beneath an aqueous suspension of 0.0823 μm diameter polystyrene latex spheres, and (ii) hindbrain of a whole-mount mouse embryo (age 10 days) that was stained to fluoresce at 515 nm and 580 nm peak wavelengths. Expression of RNA transcripts of a gene within the embryo hindbrain was detected by a fluorescence-based whole-mount in situ hybridization procedure that we recently tested. (author)

Correction of fluorescence for depth-specific optical and vascular properties using reflectance and differential path-length spectroscopy during PDT

Science.gov (United States)

van Zaane, F.; Middelburg, T. A.; de Bruijn, H. S.; van der Ploeg-van den Heuvel, A.; de Haas, E. R. M.; Sterenborg, H. J. C. M.; Neumann, H. A. M.; Robinson, D. J.

2009-06-01

Introduction: The rate of PpIX fluorescence photobleaching is routinely used as a dose metric for ALA-PDT. Diffuse reflection spectroscopy is often used to account for variations in tissue optical properties at the photosensitizer excitation and emission bands. It can be used to quantify changes in vascular parameters, such as blood volume fraction and saturation, and can aid understanding of tissue response to PDT. The volume and(/or) depth over which these signals are acquired are critical. The aim of this study is to use quantitative reflectance spectroscopy (DPS) to correct fluorescence for changes in tissue optical properties and monitor PDT. Materials & Methods: ALA was topically applied to hairless mice skin and the incubated spot was treated with PDT according to fractionated illumination schemes. DPS measurements of vascular parameters and optical properties were performed directly before and after illumination. Both the differential signal, delivery-and-collection-fiber signal and the collection fiber signal, which all probe different measurement volumes, are analyzed. Results & Conclusions: Analysis of DPS measurements shows that at the depth where most fluorescence originates, there is almost no blood present. During PDT vascular parameters at this depth stay constant. In more oxygenated layers of the tissue, the optical properties do change during PDT, suggesting that only a small part of PpIX fluorescence originates from the interesting depths where vascular response occurs. Correcting fluorescence emission spectra for optical changes at specific depths and not for the total of changes in a larger volume, as is usually done now, can improve PpIX photobleaching based treatment monitoring.

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

Czech Academy of Sciences Publication Activity Database

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

2016-01-01

Roč. 22, č. 2 (2016), s. 290-299 ISSN 1431-9276 R&D Projects: GA ČR(CZ) GAP305/11/2476; GA ČR(CZ) GPP501/12/P951 Institutional support: RVO:61389030 ; RVO:61388955 Keywords : raster image correlation spectroscopy * fluorescence recovery after photobleaching * auxin influx Subject RIV: EB - Genetics ; Molecular Biology; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 1.891, year: 2016

Abbott’s Fluorescence Polarization Immunoassay for Cyclosporine and Metabolites Compared with the Sandoz “Sandimmune” RIA

OpenAIRE

Sanghvi, Ajit; Diven, Warren; Seltman, Howard; Starzl, Thomas

1988-01-01

A new procedure for measuring cyclosporine in plasma has been introduced by Abbott Laboratories, involving their TDx instrumentation and fluorescence polarization immunoassay. Radioimmunoassay (RIA) and high-performance liquid chromatography are currently the conventional methods for measuring cyclosporine in plasma and whole blood. In an effort to find a method that will decrease the radioactive hazard, the reagent and supply cost, and the labor requirements associated with RIA procedures, w...

Fluorescence and absorption properties of chromophoric dissolved organic matter (CDOM) in coastal surface waters of the northwestern Mediterranean Sea, influence of the Rhône River

Science.gov (United States)

Para, J.; Coble, P. G.; Charrière, B.; Tedetti, M.; Fontana, C.; Sempéré, R.

2010-12-01

Seawater samples were collected monthly in surface waters (2 and 5 m depths) of the Bay of Marseilles (northwestern Mediterranean Sea; 5°17'30" E, 43°14'30" N) during one year from November 2007 to December 2008 and studied for total organic carbon (TOC) as well as chromophoric dissolved organic matter (CDOM) optical properties (absorbance and fluorescence). The annual mean value of surface CDOM absorption coefficient at 350 nm [aCDOM(350)] was very low (0.10 ± 0.02 m-1) in comparison to values usually found in coastal waters, and no significant seasonal trend in aCDOM(350) could be determined. By contrast, the spectral slope of CDOM absorption (SCDOM) was significantly higher (0.023 ± 0.003 nm-1) in summer than in fall and winter periods (0.017 ± 0.002 nm-1), reflecting either CDOM photobleaching or production in surface waters during stratified sunny periods. The CDOM fluorescence, assessed through excitation emission matrices (EEMs), was dominated by protein-like component (peak T; 1.30-21.94 QSU) and marine humic-like component (peak M; 0.55-5.82 QSU), while terrestrial humic-like fluorescence (peak C; 0.34-2.99 QSU) remained very low. This reflected a dominance of relatively fresh material from biological origin within the CDOM fluorescent pool. At the end of summer, surface CDOM fluorescence was very low and strongly blue shifted, reinforcing the hypothesis of CDOM photobleaching. Our results suggested that unusual Rhône River plume eastward intrusion events might reach Marseilles Bay within 2-3 days and induce local phytoplankton blooms and subsequent fluorescent CDOM production (peaks M and T) without adding terrestrial fluorescence signatures (peaks C and A). Besides Rhône River plumes, mixing events of the entire water column injected relative aged (peaks C and M) CDOM from the bottom into the surface and thus appeared also as an important source of CDOM in surface waters of the Marseilles Bay. Therefore, the assessment of CDOM optical properties

Hybrid fluorescent layer emitting polarized light

Directory of Open Access Journals (Sweden)

Mohammad Mohammadimasoudi

2017-07-01

Full Text Available Semiconductor nanorods have anisotropic absorption and emission properties. In this work a hybrid luminescent layer is produced based on a mixture of CdSe/CdS nanorods dispersed in a liquid crystal that is aligned by an electric field and polymerized by UV illumination. The film emits light with polarization ratio 0.6 (polarization contrast 4:1. Clusters of nanorods in liquid crystal can be avoided by applying an AC electric field with sufficient amplitude. This method can be made compatible with large-scale processing on flexible transparent substrates. Thin polarized light emitters can be used in LCD backlights or solar concentrators to increase the efficiency.

Picocyanobacteria and deep-ocean fluorescent dissolved organic matter share similar optical properties

Science.gov (United States)

Zhao, Zhao; Gonsior, Michael; Luek, Jenna; Timko, Stephen; Ianiri, Hope; Hertkorn, Norbert; Schmitt-Kopplin, Philippe; Fang, Xiaoting; Zeng, Qinglu; Jiao, Nianzhi; Chen, Feng

2017-05-01

Marine chromophoric dissolved organic matter (CDOM) and its related fluorescent components (FDOM), which are widely distributed but highly photobleached in the surface ocean, are critical in regulating light attenuation in the ocean. However, the origins of marine FDOM are still under investigation. Here we show that cultured picocyanobacteria, Synechococcus and Prochlorococcus, release FDOM that closely match the typical fluorescent signals found in oceanic environments. Picocyanobacterial FDOM also shows comparable apparent fluorescent quantum yields and undergoes similar photo-degradation behaviour when compared with deep-ocean FDOM, further strengthening the similarity between them. Ultrahigh-resolution mass spectrometry (MS) and nuclear magnetic resonance spectroscopy reveal abundant nitrogen-containing compounds in Synechococcus DOM, which may originate from degradation products of the fluorescent phycobilin pigments. Given the importance of picocyanobacteria in the global carbon cycle, our results indicate that picocyanobacteria are likely to be important sources of marine autochthonous FDOM, which may accumulate in the deep ocean.

Fluorescence confocal polarizing microscopy

Indian Academy of Sciences (India)

Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by ...

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity

OpenAIRE

Laat, S.W. de; Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van; Tetteroo, P.A.T.; Tertoolen, L.G.J.

1984-01-01

Regional differences in the lateral mobility properties of plasma membrane lipids were studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein- -1abelled fatty acids HEDAF (5-(N-hexadecanoyl)- aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to distribute itself in the plasma membrane. Under all experimental conditions used these molecules s...

Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

Directory of Open Access Journals (Sweden)

Blachutzik Jörg O

2012-08-01

Full Text Available Abstract Background Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.

Alignment of Ar{sup +} [{sup 3}P]4p{sup 2}P{sup 0}{sub 3/2} satellite state from the polarization analysis of fluorescent radiation after photoionization

Energy Technology Data Exchange (ETDEWEB)

Yenen, O.; McLaughlin, K.W.; Jaecks, D.H. [Univ. of Nebraska, Lincoln, NE (United States)] [and others

1997-04-01

The measurement of the polarization of radiation from satellite states of Ar{sup +} formed after the photoionization of Ar provides detailed information about the nature of doubly excited states, magnetic sublevel cross sections and partial wave ratios of the photo-ejected electrons. Since the formation of these satellite states is a weak process, it is necessary to use a high flux beam of incoming photons. In addition, in order to resolve the many narrow doubly excited Ar resonances, the incoming photons must have a high resolution. The characteristics of the beam line 9.0.1 of the Advanced Light Source fulfill these requirements. The authors determined the polarization of 4765 {Angstrom} fluorescence from the Ar{sup +} [{sup 3}P] 4p {sup 2}P{sub 3/2}{sup 0} satellite state formed after photoionization of Ar by photons from the 9.0.1 beam line of ALS in the 35.620-38.261 eV energy range using a resolution of approximately 12,700. This is accomplished by measuring the intensities of the fluorescent light polarized parallel (I{parallel}) and perpendicular (I{perpendicular}) to the polarization axis of the incident synchrotron radiation using a Sterling Optics 105MB polarizing filter. The optical system placed at 90{degrees} with respect to the polarization axis of the incident light had a narrow band interference filter ({delta}{lambda}=0.3 nm) to isolate the fluorescent radiation.

Color-tunable electrophosphorescent device fabricated by a photo-bleaching method

International Nuclear Information System (INIS)

Kim, Tae-Ho; Park, Jong Hyeok; Park, O Ok

2011-01-01

We demonstrated an efficient color-tunable electrophosphorescent device fabricated by a photo-bleaching method. Electroluminescence studies indicate that excellent device performance can be achieved through efficient Foerster energy transfer from the conjugated polymer to the iridium complexes by improving their miscibility. The use of a very low concentration of red phosphorescent dye and the easy degradation characteristics of conjugated structure of the red dopant enable color-tuning from red to green emission by a simple UV-irradiation process without a sacrifice of luminescent properties.tp

A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

Science.gov (United States)

Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

2012-06-01

N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. Copyright © 2012 Elsevier Inc. All rights reserved.

Quantitative Light Fluorescence (QLF and Polarized White Light (PWL assessments of dental fluorosis in an epidemiological setting

Directory of Open Access Journals (Sweden)

Pretty Iain A

2012-05-01

Full Text Available Abstract Background To determine if a novel dual camera imaging system employing both polarized white light (PWL and quantitative light induced fluorescence imaging (QLF is appropriate for measuring enamel fluorosis in an epidemiological setting. The use of remote and objective scoring systems is of importance in fluorosis assessments due to the potential risk of examiner bias using clinical methods. Methods Subjects were recruited from a panel previously characterized for fluorosis and caries to ensure a range of fluorosis presentation. A total of 164 children, aged 11 years (±1.3 participated following consent. Each child was examined using the novel imaging system, a traditional digital SLR camera, and clinically using the Dean’s and Thylstrup and Fejerskov (TF Indices on the upper central and lateral incisors. Polarized white light and SLR images were scored for both Dean’s and TF indices by raters and fluorescence images were automatically scored using software. Results Data from 164 children were available with a good distribution of fluorosis severity. The automated software analysis of QLF images demonstrated significant correlations with the clinical examinations for both Dean’s and TF index. Agreement (measured by weighted Kappa’s between examiners scoring clinically, from polarized photographs and from SLR images ranged from 0.56 to 0.92. Conclusions The study suggests that the use of a digital imaging system to capture images for either automated software analysis, or remote assessment by raters is suitable for epidemiological work. The use of recorded images enables study archiving, assessment by multiple examiners, remote assessment and objectivity due to the blinding of subject status.

Fluorescence and absorption properties of chromophoric dissolved organic matter (CDOM in coastal surface waters of the northwestern Mediterranean Sea, influence of the Rhône River

Directory of Open Access Journals (Sweden)

J. Para

2010-12-01

Full Text Available Seawater samples were collected monthly in surface waters (2 and 5 m depths of the Bay of Marseilles (northwestern Mediterranean Sea; 5°17'30" E, 43°14'30" N during one year from November 2007 to December 2008 and studied for total organic carbon (TOC as well as chromophoric dissolved organic matter (CDOM optical properties (absorbance and fluorescence. The annual mean value of surface CDOM absorption coefficient at 350 nm [a_CDOM(350] was very low (0.10 ± 0.02 m⁻¹ in comparison to values usually found in coastal waters, and no significant seasonal trend in a_CDOM(350 could be determined. By contrast, the spectral slope of CDOM absorption (S_CDOM was significantly higher (0.023 ± 0.003 nm⁻¹ in summer than in fall and winter periods (0.017 ± 0.002 nm⁻¹, reflecting either CDOM photobleaching or production in surface waters during stratified sunny periods. The CDOM fluorescence, assessed through excitation emission matrices (EEMs, was dominated by protein-like component (peak T; 1.30–21.94 QSU and marine humic-like component (peak M; 0.55–5.82 QSU, while terrestrial humic-like fluorescence (peak C; 0.34–2.99 QSU remained very low. This reflected a dominance of relatively fresh material from biological origin within the CDOM fluorescent pool. At the end of summer, surface CDOM fluorescence was very low and strongly blue shifted, reinforcing the hypothesis of CDOM photobleaching. Our results suggested that unusual Rhône River plume eastward intrusion events might reach Marseilles Bay within 2–3 days and induce local phytoplankton blooms and subsequent fluorescent CDOM production (peaks M and T without adding terrestrial fluorescence signatures (peaks C and A. Besides Rhône River plumes, mixing events of the entire water column injected relative aged (peaks C and M CDOM from the bottom into the surface and thus appeared also as an important source

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity become extreme upon fertilization

OpenAIRE

Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van; Tetteroo, P.A.T.; Tertoolen, L.G.J.; Laat, S.W. de

1984-01-01

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilizedxaqpus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-(N-hexadecanoyl)-aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show par...

THE IMPACT OF CDOM PHOTOBLEACHING ON UV ATTENUATION NEAR CORAL REEFS IN THE FLORIDA KEYS

Science.gov (United States)

We have investigated how the loss of chromophoric dissolved organic matter (CDOM) in the water column due to photobleaching allows for increased penetration of UV radiation near coral reefs in the Florida Keys. Extended exposure to UV may contribute to coral bleaching episodes. C...

Silica encapsulation of fluorescent nanodiamonds for colloidal stability and facile surface functionalization.

Science.gov (United States)

Bumb, Ambika; Sarkar, Susanta K; Billington, Neil; Brechbiel, Martin W; Neuman, Keir C

2013-05-29

Fluorescent nanodiamonds (FNDs) emit in the near-IR and do not photobleach or photoblink. These properties make FNDs better suited for numerous imaging applications compared with commonly used fluorescence agents such as organic dyes and quantum dots. However, nanodiamonds do not form stable suspensions in aqueous buffer, are prone to aggregation, and are difficult to functionalize. Here we present a method for encapsulating nanodiamonds with silica using an innovative liposome-based encapsulation process that renders the particle surface biocompatible, stable, and readily functionalized through routine linking chemistries. Furthermore, the method selects for a desired particle size and produces a monodisperse agent. We attached biotin to the silica-coated FNDs and tracked the three-dimensional motion of a biotinylated FND tethered by a single DNA molecule with high spatial and temporal resolution.

Pancreatic tumor detection using hypericin-based fluorescence spectroscopy and cytology

Science.gov (United States)

Lavu, Harish; Geary, Kevin; Fetterman, Harold R.; Saxton, Romaine E.

2005-04-01

Hypericin is a novel, highly fluorescent photosensitizer that exhibits selective tumor cell uptake properties and is particularly resistant to photobleaching. In this study, we have characterized hypericin uptake in human pancreatic tumor cells with relation to incubation time, cell number, and drug concentration. Ex vivo hypericin based fluorescence spectroscopy was performed to detect the presence of MIA PaCa-2 pancreatic tumor cells in the peritoneal cavity of BALB/c nude mice, as well as to quantify gross tumor burden. Hypericin based cytology of peritoneal lavage samples, using both one and two photon laser confocal microscopy, demonstrated more than a two-fold increase in fluorescence emission of pancreatic tumor cells as compared to control samples. In vitro treatment of pancreatic cancer cells with hypericin based photodynamic therapy showed tumor cell cytotoxicity in a drug dose, incident laser power, and time dependent manner. For these experiments, a continuous wavelength solid-state laser source (532 nm) was operated at power levels in the range of 100-400 mW. Potential applications of hypericin in tumor diagnosis, staging, and therapy will be presented.

Potential applications of near infrared auto-fluorescence spectral polarized imaging for assessment of food quality

Science.gov (United States)

Zhou, Kenneth J.; Chen, Jun

2016-03-01

The current growing of food industry for low production costs and high efficiency needs for maintenance of high-quality standards and assurance of food safety while avoiding liability issues. Quality and safety of food depend on physical (texture, color, tenderness etc.), chemical (fat content, moisture, protein content, pH, etc.), and biological (total bacterial count etc.) features. There is a need for a rapid (less than a few minutes) and accurate detection system in order to optimize quality and assure safety of food. However, the fluorescence ranges for known fluorophores are limited to ultraviolet emission bands, which are not in the tissue near infrared (NIR) "optical window". Biological tissues excited by far-red or NIR light would exhibit strong emission in spectral range of 650-1,100 nm although no characteristic peaks show the emission from which known fluorophores. The characteristics of the auto-fluorescence emission of different types of tissues were found to be different between different tissue components such as fat, high quality muscle food. In this paper, NIR auto-fluorescence emission from different types of muscle food and fat was measured. The differences of fluorescence intensities of the different types of muscle food and fat emissions were observed. These can be explained by the change of the microscopic structure of physical, chemical, and biological features in meat. The difference of emission intensities of fat and lean meat tissues was applied to monitor food quality and safety using spectral polarized imaging, which can be detect deep depth fat under the muscle food up to several centimeter.

Solvent-dependent fluorescence enhancement and piezochromism of a carbazole-substituted naphthopyran

Energy Technology Data Exchange (ETDEWEB)

Liu, Lihui; Wang, Aixia [Faculty of Chemistry, Northeast Normal University, Changchun 130024 (China); Wang, Guang, E-mail: wangg923@nenu.edu.cn [Faculty of Chemistry, Northeast Normal University, Changchun 130024 (China); Munyentwari, Alexis [Faculty of Chemistry, Northeast Normal University, Changchun 130024 (China); Zhou, Yihan, E-mail: yhzhou@ciac.ac.cn [National Analytical Research Center of Electrochemistry and Spectroscopy, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China)

2015-09-15

A novel carbazole-substituted naphthopyran, 3,3-bis-(4-carbazolylphenyl)-[3H]-naphtho[2,1-b]pyran (CzNP) was designed and synthesized. The new compound exhibited normal photochromism in dichloromethane solution and the UV irradiation did not influence its fluorescence. On the contrary, the fluorescence of CzNP in N,N-dimethylformamide (DMF) was intensively enhanced to 29 times after 60 min of the UV irradiation and this enhanced fluorescence can be quenched by addition of triethylamine (TEA). The study of enhanced extent of fluorescence of CzNP in solvents with different polarities and in mixed solvents demonstrated that the enhanced fluorescence is dependent on the polarity of solvents. The larger the polarity of solvent was, the stronger was the fluorescence of CzNP. CzNP also exhibited piezochromic performance and the pressure led to the cleavage of the C–O bond of pyran ring. - Highlights: • A carbazole-substituted photochromic naphthopyran was designed and synthesized. • The fluorescence was enhanced under the existence of DMF and UV irradiation. • The polarity of solvent was the dominating factor to affect the fluorescence. • The new compound also displayed piezochromic performance.

A new optical method improves fluorescence guided diagnosis of bladder tumor in the outpatient department and reveals significant photo bleaching problems in established inpatients PDD techniques

Science.gov (United States)

Lindvold, Lars R.; Hermann, Gregers G.

2013-03-01

Photo dynamic diagnosis (PDD) is a convenient and well-documented procedure for diagnosis of bladder cancer and tumours using endoscopic techniques. At present, this procedure is available only for routine use in an operating room (OR) and often with substantial photobleaching effects of the photosensitizer. We present a novel optical design of the endoscopic PDD procedure that allows the procedure to be performed in the outpatient department (OPD) and not only in the OR. Thereby, inpatient procedures lasting 1-2 days may be replaced by a few hours lasting procedure in the OPD. Urine blurs the fluorescence during PDD used in the OPD. Urine contains fluorescent metabolites that are excited by blue light giving an opaque green fluorescence confounding the desired red fluorescence (PDD) from the tumour tissue. Measurements from the clinical situation has shown that some systems for PPD based on blue light illumination (PDD mode) and white light illumination used for bladder tumour diagnosis and surgery suffers some inherent disadvantages, i.e., photo bleaching in white light that impairs the possibility for PDD as white light usually is used before the blue light for PDD. Based on spectroscopic observations of urine and the photoactive dye Protoporphyrin IX used in PDD a novel optical system for use with the cystoscope has been devised that solves the problem of green fluorescence from urine. This and the knowledge of photo-bleaching pitfalls in established systems make it possible to perform PDD of bladder tumours in the OPD and to improve PDD in the OR.

Exciplex fluorescence emission from simple organic intramolecular constructs in non-polar and highly polar media as model systems for DNA-assembled exciplex detectors.

Science.gov (United States)

Bichenkova, Elena V; Sardarian, Ali R; Wilton, Amanda N; Bonnet, Pascal; Bryce, Richard A; Douglas, Kenneth T

2006-01-21

Organic intramolecular exciplexes, N-(4-dimethylaminobenzyl)-N-(1-pyrenemethyl)amine (1) and N'-4-dimethylaminonaphthyl-N-(1-pyrenemethyl)amine (2), were used as model systems to reveal major factors affecting their exciplex fluorescence, and thus lay the basis for developing emissive target-assembled exciplexes for DNA-mounted systems in solution. These models with an aromatic pyrenyl hydrocarbon moiety as an electron acceptor appropriately connected to an aromatic dimethylamino electron donor component (N,N-dimethylaminophenyl or N,N-dimethylaminonaphthyl) showed strong intramolecular exciplex emission in both non-polar and highly polar solvents. The effect of dielectric constant on the maximum wavelength for exciplex emission was studied, and emission was observed for 1 and 2 over the full range of solvent from non-polar hydrocarbons up to N-methylformamide with a dielectric constant of 182. Quantum yields were determined for these intramolecular exciplexes in a range of solvents relative to that for Hoechst 33,258. Conformational analysis of 1 was performed both computationally and via qualitative 2D NMR using (1)H-NOESY experiments. The results obtained indicated the contribution of pre-folded conformation(s) to the ground state of 1 conducive to exciplex emission. This research provides the initial background for design of self-assembled, DNA-mounted exciplexes and underpins further development of exciplex-based hybridisation bioassays.

NANODIAMONDS FOR FLUORESCENT CELL AND SENSOR NANOTECHNOLOGIES

Directory of Open Access Journals (Sweden)

V. I. Nazarenko

2013-10-01

Full Text Available This review addresses the analysis of properties and applications of fluorescent nanodiamonds. They are carbon nanostructures with atomic arrangement of a diamond and carry all its properties, including record — high density, rigidity and refraction index. They are of almost spherical shape, and their small size (~4–10 nm creates substantial surface area that can be used for absorption of different compounds including drugs. Their surface is formed by different chemical groups (hydroxyls, carboxyls, etc. exhibits also chemical reactivity that allows different types of modifications. This opens innumerable possibilities for constructing different functional nanomaterials. The technologies have been developed for making these nanodiamonds fluorescent. Particularly, these properties are achieved by radioactive treatment with the formation of N–V impurities. These particles absorb and emit light in convenient for observation visible range of spectrum. They do not photobleach, which is very attractive for fluorescent microscopy of the cell. And, finally, these nanoparticles do not display toxicity on the cellular or whole — body level, and because of their biocompatibility they can be used in vivo as contrast agents and drug carriers. It is expected that future biotechnological applications of these nanoparticles will be connected with the creation of nanocomposites that combine multiple useful functions.

Fluorescent probe studies of polarity and solvation within room temperature ionic liquids: a review.

Science.gov (United States)

Pandey, Shubha; Baker, Sheila N; Pandey, Siddharth; Baker, Gary A

2012-09-01

Ionic liquids display an array of useful and sometimes unconventional, solvent features and have attracted considerable interest in the field of green chemistry for the potential they hold to significantly reduce environmental emissions. Some of these points have a bearing on the chemical reactivity of these systems and have also generated interest in the physical and theoretical aspects of solvation in ionic liquids. This review presents an introduction to the field of ionic liquids, followed by discussion of investigations into the solvation properties of neat ionic liquids or mixed systems including ionic liquids as a major or minor component. The ionic liquid based multicomponent systems discussed are composed of other solvents, other ionic liquids, carbon dioxide, surfactants or surfactant solutions. Although we clearly focus on fluorescence spectroscopy as a tool to illuminate ionic liquid systems, the issues discussed herein are of general relevance to discussions of polarity and solvent effects in ionic liquids. Transient solvation measurements carried out by means of time-resolved fluorescence measurements are particularly powerful for their ability to parameterize the kinetics of the solvation process in ionic liquids and are discussed as well.

Energy dispersive X-ray fluorescence analysis with Bragg polarized Mo radiation. Energiedispersive Roentgenfluoreszenzanalyse mit Bragg-polarisierter Mo Strahlung

Energy Technology Data Exchange (ETDEWEB)

Gloeckl, H

1983-01-01

The aim of introducing energy dispersive analysis into X-ray fluorescence analysis is to suppress background from the Bremsstrahlung spectrum and the characteristic radiation without an undue reduction of the signal. The variant under consideration uses linearly polarization radiation obtained after a Bragg reflection,under delta = 90/sup 0/. In an introductory part, Bragg reflection, fluorescence and strong radiation are considered quantitatively with respect to counting statistics and detection limits. In the experimental part two combinations are describe, of a Ta crystal with a Cr tube and of a Mo crystal with a Mo tube. Details of adjustment, sample preparation and calibration and detection limits are given. The pros and cons of the Ta/Cr and the Mo/Mo are contrasted and proposals for further improvements are given.

Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging.

Science.gov (United States)

Sreejith, Sivaramapanicker; Joseph, James; Lin, Manjing; Menon, Nishanth Venugopal; Borah, Parijat; Ng, Hao Jun; Loong, Yun Xian; Kang, Yuejun; Yu, Sidney Wing-Kwong; Zhao, Yanli

2015-06-23

Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1(micelle)) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1(micelle) exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1(micelle) in the NIR window of 800-900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1(micelle), as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1(micelle) proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging.

Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs

DEFF Research Database (Denmark)

Eriksen, Jacob; Rasmussen, Søren G F; Jørgensen, Trine Nygaard

2009-01-01

(fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction less than approximately 30%). DAT was constitutively internalized into vesicular...... and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular...

Reducing the photo-bleaching effect of a new europium complex embedded in styrene butadiene copolymer

Science.gov (United States)

Jiménez, G. Lesly; Reyes-Rodríguez, J. L.; Padilla, Isela; Alarcón-Flores, G.; Falcony, C.

2018-02-01

A highly luminescent europium complex obtained with two different ligands, succinimide (SI) and 2-thenoyltrifluoroacetone (TTA) , was synthetized with different TTA concentrations. The photoluminescence (PL) emission from these materials corresponds to the characteristic inter-electronic energy level transitions of the Eu3+ ions. However, the excitation spectrum is strongly dependent on the presence of TTA, having an optimum response when 0.75 mmol of this compound is added to the EuL3(H2O)3 complex. The quantum yield obtained by these powders were around 72 % ± 1.7 % indicating an optimum sensitization of these complex. The EuL3 TTA complex with the best PL properties was embedded in a styrene butadiene copolymer (SBC) film, produced by the drop casting method, obtaining similar PL behavior at different concentrations, the highest intensity was observed at 1.2% (w/v) of EuL3 TTA complex and the quantum yield of these composite films was 60.5 % ± 2 % . These films were exposed to continuous UV irradiation and after 141 h no photo-bleaching effect was observed in contrast with the EuL3 TTA complex that exhibited a noticeable photoluminescence intensity degradation at much shorter exposure times. Both the Eu-complexes and the composite films were characterized by FT-IR, XRD, SEM and fluorescence spectroscopy.

Preparation and Characterization of Highly Fluorescent, Glutathione-coated Near Infrared Quantum Dots for in Vivo Fluorescence Imaging

Directory of Open Access Journals (Sweden)

Yoshichika Yoshioka

2008-10-01

Full Text Available Fluorescent probes that emit in the near-infrared (NIR, 700-1,300 nm region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4. The GSH-QDs (800 nm emission were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer, and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM, the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIRfluorescence imaging of a lymph node in a mouse is presented.

Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

Science.gov (United States)

Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

2015-02-01

A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

Fluorescence Intrinsic Characterization of Excitation-Emission Matrix Using Multi-Dimensional Ensemble Empirical Mode Decomposition

Directory of Open Access Journals (Sweden)

Tzu-Chien Hsiao

2013-11-01

Full Text Available Excitation-emission matrix (EEM fluorescence spectroscopy is a noninvasive method for tissue diagnosis and has become important in clinical use. However, the intrinsic characterization of EEM fluorescence remains unclear. Photobleaching and the complexity of the chemical compounds make it difficult to distinguish individual compounds due to overlapping features. Conventional studies use principal component analysis (PCA for EEM fluorescence analysis, and the relationship between the EEM features extracted by PCA and diseases has been examined. The spectral features of different tissue constituents are not fully separable or clearly defined. Recently, a non-stationary method called multi-dimensional ensemble empirical mode decomposition (MEEMD was introduced; this method can extract the intrinsic oscillations on multiple spatial scales without loss of information. The aim of this study was to propose a fluorescence spectroscopy system for EEM measurements and to describe a method for extracting the intrinsic characteristics of EEM by MEEMD. The results indicate that, although PCA provides the principal factor for the spectral features associated with chemical compounds, MEEMD can provide additional intrinsic features with more reliable mapping of the chemical compounds. MEEMD has the potential to extract intrinsic fluorescence features and improve the detection of biochemical changes.

Effect of light polarization on the efficiency of photodynamic therapy of basal cell carcinomas: an in vitro cellular study.

Science.gov (United States)

JalalKamali, M; Nematollahi-Mahani, S N; Shojaei, M; Shamsoddini, A; Arabpour, N

2018-02-01

In an in vitro study, the effect of light polarization on the efficiency of 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) of basal cell carcinoma (BCC) was investigated. Three states of light polarization (non-polarized, linearly polarized, and circularly polarized) were considered. Cells were exposed to green (532 pm 20 nm) irradiation from light emitting diodes. Cell survival was measured by the colorimetric assay (WST-1) and Trypan blue staining. The colorimetric assay showed a pronounced decrease in the cell viability (up to 30%) using polarized light compared to the non-polarized one in the wavelength region used. Similar results were obtained by the cell counting method (20-30% increase in cell death). The observed effect was dependent on the concentration of photosensitizer. The effect is more expressed in the case of linearly polarized light compared to the circularly polarized one. Results show that the use of polarized light increases the efficiency of in vitro ALA-PDT of BCC. Utilizing polarized light, it is possible to obtain the same effect from PDT by lower concentrations of photosensitizer. Additionally, the concentration dependency of PDT response and photo-bleaching is also reduced.

Relative Roles of Gap Junction Channels and Cytoplasm in Cell-to-Cell Diffusion of Fluorescent Tracers

Science.gov (United States)

Safranyos, Richard G. A.; Caveney, Stanley; Miller, James G.; Petersen, Nils O.

1987-04-01

Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic diffusion, which may limit intercellular diffusion.

Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy

Science.gov (United States)

Glazachev, Yu I.; Orlova, D. Y.; Řezníčková, P.; Bártová, E.

2018-05-01

We proposed an effective kinetics scheme of photolysis of green fluorescent protein (GFP) observed in live cells with a commercial confocal fluorescence microscope. We investigated the photolysis of GFP-tagged heterochromatin protein, HP1β-GFP, in live nucleus with the pulse position modulation approach, which has several advantages over the classical pump-and-probe method. At the basis of the proposed scheme lies a process of photoswitching from the native fluorescence state to the intermediate fluorescence state, which has a lower fluorescence yield and recovers back to native state in the dark. This kinetics scheme includes four effective parameters (photoswitching, reverse switching, photodegradation rate constants, and relative brightness of the intermediate state) and covers the time scale from dozens of milliseconds to minutes of the experimental fluorescence kinetics. Additionally, the applicability of the scheme was demonstrated in the cases of continuous irradiation and the classical pump-and-probe approach using numerical calculations and analytical solutions. An interesting finding of experimental data analysis was that the overall photodegradation of GFP proceeds dominantly from the intermediate state, and demonstrated approximately the second-order reaction versus irradiation power. As a practical example, the proposed scheme elucidates the artifacts of fluorescence recovery after the photobleaching method, and allows us to propose some suggestions on how to diminish them.

Subsurface femtosecond tissue alteration: selectively photobleaching macular degeneration pigments in near retinal contact.

Science.gov (United States)

Manevitch, Zakhariya; Lewis, Aaron; Levy, Carol; Zeira, Evelyne; Banin, Eyal; Manevitch, Alexandra; Khatchatouriants, Artium; Pe'er, Jacob; Galun, Eithan; Hemo, Itzhak

2012-06-14

This paper uses advances in the ultrafast manipulation of light to address a general need in medicine for a clinical approach that can provide a solution to a variety of disorders requiring subsurface tissue manipulation with ultralow collateral damage. Examples are age-related macular degeneration (AMD), fungal infections, tumors surrounded by overlying tissue, cataracts, etc. Although lasers have revolutionized the use of light in clinical settings, most lasers employed in medicine cannot address such problems of depth-selective tissue manipulation. This arises from the fact that they are mostly based on one photon based laser tissue interactions that provide a cone of excitation where the energy density is sufficiently high to excite heat or fluorescence in the entire cone. Thus, it is difficult to excite a specific depth of a tissue without affecting the overlying surface. However, the advent of femtosecond (fs) lasers has caused a revolution in multiphoton microscopy (Zipfel et al. Nat. Biotechnol. 2003, 21, 1369-1377; Denk et al. Science 1990, 248, 73-76) and fabrication (Kawata et al. Nature 2001, 412, 697-698). With such lasers, the photon energy density is only high enough for multiphoton processes in the focal volume, and this opens a new direction to address subsurface tissue manipulation. Here we show in an AMD animal model, Ccr2 KO knockout mutant mice, noninvasive, selective fs two-photon photobleaching of pigments associated with AMD that accumulate under and in ultraclose proximity to the overlying retina. Pathological evidence is presented that indicates the lack of collateral damage to the overlying retina or other surrounding tissue.

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

Science.gov (United States)

Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

2013-08-09

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in

New method for preparing a liquid crystal polymer that exhibits linearly polarized white fluorescence

International Nuclear Information System (INIS)

Zheng Shijun; Kun, Wang; Kobayashi, Takaomi

2011-01-01

With the aim of developing a single-chain white-light-emitting polymer, liquid crystal (LC) polymers with a shish-kebab-type moiety on their cross-conjugated (p-phenylene)s-poly(p-phenylenevinylene)s main chain were synthesized by Gilch polymerization. They were characterized by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and polarizing optical microscopy (POM). 1 H-NMR indicated that the polymers had a shish-kebab structure, which strongly suppressed the formation of structural defects in the polymers. DSC revealed that the polymers had thermotropic LC properties, indicating that the LC polymers were enantiotropic. XRD showed that the polymers had a mesophase, which implies that they were in a smectic LC phase. A polymer with 'kebabs' of 2,5-bis(4'-alkoxyphenyl)benzene was combined with an aligned polyimide film with orientated microgrooves. The polymer main chain was aligned due to the orientation of the 'kebabs' of the uniform cross-conjugated structure. It lay between the kebabs and the 'shish' of the polymer main chains. The aligned polymer main chain emitted yellow light while and the oriented LC side chains emitted blue light emission. These two emissions resulted in linearly polarized white fluorescence.

Detection of volatile and soluble general anesthetics using a fluorescence-based fiber optic sensor: recent progress in chemical sensitivity and noise sources

Science.gov (United States)

Yager, Paul; Abrams, Susan B.

1992-04-01

A fiber optic sensor for general anesthetics based on the phase transition of immobilized phospholipid vesicles is under development. Current work centers on evaluating the sensor response to different anesthetics and instrumentation design. The fluorescence of laurdan- doped liposomes is found to respond linearly to the infusible anesthetics thiopental sodium and Propofol. Preliminary experiments have been performed to determine sources of noise in the optical and electronic components of the sensor as it is now configured. One potential noise source is the liposome sample at the fiber tip; photobleaching and thermal fluctuations due to heating by the illuminating 360 nm radiation can affect measurement of the anesthetic level. Heating of the sample is a factor at high illumination levels, but photobleaching, which reduces the signal intensity, does not alter the intensity ratio upon which the anesthetic concentration measurement is based. Optical microscopy of fiber tips embedded in liposomes allows direct observation of the light intensity near the tip of the fiber despite the extreme turbidity of the suspension. Light intensity drops to less than 10% of its maximum intensity at the fiber tip within 300 micrometers . Further use of this technique should allow monitoring the effects of photobleaching on the spatial distribution of the liposomes responsible for the measured optical signal.

Heidelberg polarized alkali source

International Nuclear Information System (INIS)

Kraemer, D.; Steffens, E.; Jaensch, H.; Philipps Universitaet, Marburg, Germany)

1984-01-01

A new atomic beam type polarized alkali ion source has been installed at Heidelberg. In order to improve the beam polarization considerably optical pumping is applied in combination with an adiabatic medium field transition which results in beams in single hyperfine sublevels. The m state population is determined by laser-induced fluorescence spectroscopy. Highly polarized beams (P/sub s/ > 0.9, s = z, zz) with intensities of 30 to 130 μA can be extracted for Li + and Na + , respectively

Stepwise multiphoton activation fluorescence reveals a new method of melanin detection

Science.gov (United States)

Lai, Zhenhua; Kerimo, Josef; Mega, Yair; DiMarzio, Charles A.

2013-06-01

The stepwise multiphoton activated fluorescence (SMPAF) of melanin, activated by a continuous-wave mode near infrared (NIR) laser, reveals a broad spectrum extending from the visible spectra to the NIR and has potential application for a low-cost, reliable method of detecting melanin. SMPAF images of melanin in mouse hair and skin are compared with conventional multiphoton fluorescence microscopy and confocal reflectance microscopy (CRM). By combining CRM with SMPAF, we can locate melanin reliably. However, we have the added benefit of eliminating background interference from other components inside mouse hair and skin. The melanin SMPAF signal from the mouse hair is a mixture of a two-photon process and a third-order process. The melanin SMPAF emission spectrum is activated by a 1505.9-nm laser light, and the resulting spectrum has a peak at 960 nm. The discovery of the emission peak may lead to a more energy-efficient method of background-free melanin detection with less photo-bleaching.

Enhanced localized fluorescence in plasmonic nanoantennae

DEFF Research Database (Denmark)

Bakker, R.M.; Yuan, H.-K.; Liu, Z.

2008-01-01

in fluorescence that reaches 100 times enhancement. Near-field excitation shows enhanced fluorescence from a single nanoantenna localized in a subwavelength area of similar to 0.15 mu m(2). The polarization of enhanced emission is along the main antenna axis. These observed experimental results are important...

Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells

DEFF Research Database (Denmark)

Wüstner, Daniel; Lund, Frederik Wendelboe; Röhrl, Clemens

2016-01-01

and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after...... photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we...

Fluorescence brightness and photostability of individual copper (I) oxide nanocubes.

Science.gov (United States)

Zohora, Nafisa; Kandjani, Ahmad Esmaielzadeh; Orth, Antony; Brown, Hannah M; Hutchinson, Mark R; Gibson, Brant C

2017-12-04

Conventional organic fluorophores lose their ability to fluoresce after repeated exposure to excitation light due to photobleaching. Therefore, research into emerging bright and photostable nanomaterials has become of great interest for a range of applications such as bio-imaging and tracking. Among these emerging fluorophores, metal oxide-based nanomaterials have attracted significant attention as a potential multifunctional material with photocatalytic and angeogenisis abilities in addition to fluorescnce applications. However, most of these applications are highly dependent on size, morphology, and chemo-physical properties of individual particles. In this manuscript, we present a method to study the intrinsic optical characteristics of individual copper (I) oxide (Cu 2 O) nanocubes. When excited at 520 nm using only 11 µW excitation power (1.7 W/cm2), individual nanocubes were observed to emit light with peak wavelengths ~760 nm which is conveniently within the near-infrared 1 (NIR1) biological window where tissue autofluorescence is minimal. Bright and photostable fluorescence was observed with intensities up to 487 K counts/s under constant illumination for at least 2 minutes with a brightness approximately four times higher than the autofluorescence from a fixed cumulus-oocyte complex. With near-IR emission, high fluorescence brightness, and outstanding photostability, Cu 2 O nanocubes are attractive candidates for long-term fluorescent bioimaging applications.

Polarization Sensitive Coherent Raman Measurements of DCVJ

Science.gov (United States)

Anderson, Josiah; Cooper, Nathan; Lawhead, Carlos; Shiver, Tegan; Ujj, Laszlo

2014-03-01

Coherent Raman spectroscopy which recently developed into coherent Raman microscopy has been used to produce label free imaging of thin layers of material and find the spatial distributions of certain chemicals within samples, e.g. cancer cells.(1) Not all aspects of coherent scattering have been used for imaging. Among those for example are special polarization sensitive measurements. Therefore we have investigated the properties of polarization sensitive CARS spectra of a highly fluorescent molecule, DCVJ.(2) Spectra has been recorded by using parallel polarized and perpendicular polarized excitations. A special polarization arrangement was developed to suppress the non-resonant background scattering from the sample. These results can be used to improve the imaging properties of a coherent Raman microscope in the future. This is the first time coherent Raman polarization sensitive measurements have been used to characterize the vibrational modes of DCVJ. 1: K. I. Gutkowski, et al., ``Fluorescence of dicyanovinyl julolidine in a room temperature ionic liquid '' Chemical Physics Letters 426 (2006) 329 - 333 2: Fouad El-Diasty, ``Coherent anti-Stokes Raman scattering: Spectroscopy and microscopy'' Vibrational Spectroscopy 55 (2011) 1-37

Broadband polarized emission from P(NDI2OD-T2) polymer.

Science.gov (United States)

Ulrich, Steve; Sutch, Tabitha; Szulczewski, Greg; Schweizer, Matthias; Barbosa, Newton; Araujo, Paulo

2018-05-18

We investigate the P(NDI2OD-T2) photophysical properties via absorbance and fluorescence spectroscopy, in association with the experimental approach baptized Stokes Spectroscopy, which provides valuable material information through the acquisition and analysis of the fluorescence polarization degree. By changing solvents and using different samples such as solutions, thick, and thin films, it is possible to control the polarization degree spectrum associated to the fluorescence emitted by the polymer's isolated chains and aggregates. We show that the polarization degree could become a powerful tool to obtain information related to the samples morphology, which is connected to their microscopic structure. Moreover, the polarization degree spectra suggest that depolarization effects linked to energy and charge transfer mechanisms are likely taking place. Our findings indicate that P(NDI2OD-T2) polymers are excellent candidates for the advancement of organic technologies that rely on the emission and detection of polarized lights. © 2018 IOP Publishing Ltd.

d-PET-controlled “off-on” Polarity-sensitive Probes for Reporting Local Hydrophilicity within Lysosomes

Science.gov (United States)

Zhu, Hao; Fan, Jiangli; Mu, Huiying; Zhu, Tao; Zhang, Zhen; Du, Jianjun; Peng, Xiaojun

2016-10-01

Polarity-sensitive fluorescent probes are powerful chemical tools for studying biomolecular structures and activities both in vitro and in vivo. However, the lack of “off-on” polarity-sensing probes has limited the accurate monitoring of biological processes that involve an increase in local hydrophilicity. Here, we design and synthesize a series of “off-on” polarity-sensitive fluorescent probes BP series consisting of the difluoroboron dippyomethene (BODIPY) fluorophore connected to a quaternary ammonium moiety via different carbon linkers. All these probes showed low fluorescence quantum yields in nonpolar solution but became highly fluorescent in polar media. BP-2, which contains a two-carbon linker and a trimethyl quaternary ammonium, displayed a fluorescence intensity and quantum yield that were both linearly correlated with solvent polarity. In addition, BP-2 exhibited high sensitivity and selectivity for polarity over other environmental factors and a variety of biologically relevant species. BP-2 can be synthesized readily via an unusual Mannich reaction followed by methylation. Using electrochemistry combined with theoretical calculations, we demonstrated that the “off-on” sensing behavior of BP-2 is primarily due to the polarity-dependent donor-excited photoinduced electron transfer (d-PET) effect. Live-cell imaging established that BP-2 enables the detection of local hydrophilicity within lysosomes under conditions of lysosomal dysfunction.

Fluorescence confocal polarizing microscopy: Three-dimensional ...

Indian Academy of Sciences (India)

Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by ...

Demonstration of Single-Barium-Ion Sensitivity for Neutrinoless Double-Beta Decay Using Single-Molecule Fluorescence Imaging

Energy Technology Data Exchange (ETDEWEB)

McDonald, A. D.; Jones, B. J. P.; Nygren, D. R.; Adams, C.; Álvarez, V.; Azevedo, C. D. R.; Benlloch-Rodríguez, J. M.; Borges, F. I. G. M.; Botas, A.; Cárcel, S.; Carrión, J. V.; Cebrián, S.; Conde, C. A. N.; Díaz, J.; Diesburg, M.; Escada, J.; Esteve, R.; Felkai, R.; Fernandes, L. M. P.; Ferrario, P.; Ferreira, A. L.; Freitas, E. D. C.; Goldschmidt, A.; Gómez-Cadenas, J. J.; González-Díaz, D.; Gutiérrez, R. M.; Guenette, R.; Hafidi, K.; Hauptman, J.; Henriques, C. A. O.; Hernandez, A. I.; Hernando Morata, J. A.; Herrero, V.; Johnston, S.; Labarga, L.; Laing, A.; Lebrun, P.; Liubarsky, I.; López-March, N.; Losada, M.; Martín-Albo, J.; Martínez-Lema, G.; Martínez, A.; Monrabal, F.; Monteiro, C. M. B.; Mora, F. J.; Moutinho, L. M.; Muñoz Vidal, J.; Musti, M.; Nebot-Guinot, M.; Novella, P.; Palmeiro, B.; Para, A.; Pérez, J.; Querol, M.; Repond, J.; Renner, J.; Riordan, S.; Ripoll, L.; Rodríguez, J.; Rogers, L.; Santos, F. P.; dos Santos, J. M. F.; Simón, A.; Sofka, C.; Sorel, M.; Stiegler, T.; Toledo, J. F.; Torrent, J.; Tsamalaidze, Z.; Veloso, J. F. C. A.; Webb, R.; White, J. T.; Yahlali, N.

2018-03-01

A new method to tag the barium daughter in the double beta decay of $^{136}$Xe is reported. Using the technique of single molecule fluorescent imaging (SMFI), individual barium dication (Ba$^{++}$) resolution at a transparent scanning surface has been demonstrated. A single-step photo-bleach confirms the single ion interpretation. Individual ions are localized with super-resolution ($\\sim$2~nm), and detected with a statistical significance of 12.9~$\\sigma$ over backgrounds. This lays the foundation for a new and potentially background-free neutrinoless double beta decay technology, based on SMFI coupled to high pressure xenon gas time projection chambers.

Demonstration of Single-Barium-Ion Sensitivity for Neutrinoless Double-Beta Decay Using Single-Molecule Fluorescence Imaging

Science.gov (United States)

McDonald, A. D.; Jones, B. J. P.; Nygren, D. R.; Adams, C.; Álvarez, V.; Azevedo, C. D. R.; Benlloch-Rodríguez, J. M.; Borges, F. I. G. M.; Botas, A.; Cárcel, S.; Carrión, J. V.; Cebrián, S.; Conde, C. A. N.; Díaz, J.; Diesburg, M.; Escada, J.; Esteve, R.; Felkai, R.; Fernandes, L. M. P.; Ferrario, P.; Ferreira, A. L.; Freitas, E. D. C.; Goldschmidt, A.; Gómez-Cadenas, J. J.; González-Díaz, D.; Gutiérrez, R. M.; Guenette, R.; Hafidi, K.; Hauptman, J.; Henriques, C. A. O.; Hernandez, A. I.; Hernando Morata, J. A.; Herrero, V.; Johnston, S.; Labarga, L.; Laing, A.; Lebrun, P.; Liubarsky, I.; López-March, N.; Losada, M.; Martín-Albo, J.; Martínez-Lema, G.; Martínez, A.; Monrabal, F.; Monteiro, C. M. B.; Mora, F. J.; Moutinho, L. M.; Muñoz Vidal, J.; Musti, M.; Nebot-Guinot, M.; Novella, P.; Palmeiro, B.; Para, A.; Pérez, J.; Querol, M.; Repond, J.; Renner, J.; Riordan, S.; Ripoll, L.; Rodríguez, J.; Rogers, L.; Santos, F. P.; dos Santos, J. M. F.; Simón, A.; Sofka, C.; Sorel, M.; Stiegler, T.; Toledo, J. F.; Torrent, J.; Tsamalaidze, Z.; Veloso, J. F. C. A.; Webb, R.; White, J. T.; Yahlali, N.; NEXT Collaboration

2018-03-01

A new method to tag the barium daughter in the double-beta decay of Xe 136 is reported. Using the technique of single molecule fluorescent imaging (SMFI), individual barium dication (Ba++ ) resolution at a transparent scanning surface is demonstrated. A single-step photobleach confirms the single ion interpretation. Individual ions are localized with superresolution (˜2 nm ), and detected with a statistical significance of 12.9 σ over backgrounds. This lays the foundation for a new and potentially background-free neutrinoless double-beta decay technology, based on SMFI coupled to high pressure xenon gas time projection chambers.

Three-ring filters increase the effective NA up to 1.46 in optical sectioning fluorescence microscopy

International Nuclear Information System (INIS)

Martinez-Corral, M; Ibanez-Lopez, C; Caballero, M T; Munoz-Escriva, L; Saavedra, G

2003-01-01

Single-photon fluorescence confocal microscopy techniques can be combined with the use of specific binary filters in order to increase their optical sectioning capability. We present a novel class of axially super-resolving binary pupil filters specially designed to reach this aim. These filters let us to obtain a relevant compression of the z-response together with the reduction of the photo-bleaching effect typically inherent to apodization techniques. The fact of joining both the three-ring filters we propose in the illumination path, and the confocal detection gives rise to an important effective increase of lenses of effective numerical aperture

Calibration of a DG–model for fluorescence microscopy

DEFF Research Database (Denmark)

Hansen, Christian Valdemar

It is well known that diseases like Alzheimer, Parkinson, Corea Huntington and Arteriosclerosis are caused by a jam in intracellular membrane traffic [2]. Hence to improve treatment, a quantitative analysis of intracellular transport is essential. Fluorescence loss in photobleaching (FLIP......) is an impor- tant and widely used microscopy method for visualization of molecular transport processes in living cells. Thus, the motivation for making an automated reliable analysis of the image data is high. In this contribution, we present and comment on the calibration of a Discontinuous......–Galerkin simulator [3, 4] on segmented cell images. The cell geometry is extracted from FLIP images using the Chan– Vese active contours algorithm [1] while the DG simulator is implemented in FEniCS [5]. Simulated FLIP sequences based on optimal parameters from the PDE model are presented, with an overall goal...

Sources of linear polarized x-rays

International Nuclear Information System (INIS)

Aiginger, H.; Wobrauschek, P.

1989-01-01

Linear polarized X-rays are used in X-ray fluorescence analysis to decrease the background caused by scattered photons. Various experiments, calculations and constructions have demonstrated the possibility to produce polarized radiation in an analytical laboratory with an X-ray tube and polarizer-analyzer facilities as auxiliary equipment. The results obtained with Bragg-polarizers of flat and curved focussing geometry and of Barkla-polarizers are presented. The advantages and disadvantages of the method are discussed and compared with the respective quality of synchrotron radiation. Polarization by scattering reduces the intensity of the primary radiation. Recently much effort is devoted to the construction of integrated high power X-ray tube polarizer-analyzer arrangements. The detailed design, geometry and performance of such a facility is described. (author)

Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic vesicles.

Science.gov (United States)

Pierzyńska-Mach, Agnieszka; Janowski, Paweł A; Dobrucki, Jurek W

2014-08-01

Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis. © 2014 International Society for Advancement of Cytometry.

Improved Immunoassay Sensitivity in Serum as a Result of Polymer-Entrapped Quantum Dots: 'Papaya Particles'

NARCIS (Netherlands)

Ranzoni, A.; den Hamer, A.; Karoli, T.; Buechler, J.; Cooper, M.A.

2015-01-01

Fluorescent labels are widely employed in biomarker quantification and diagnostics, however they possess narrow Stokes shifts and can photobleach, limiting multiplexed detection applications and compromising sensitivity. In contrast, quantum dots do not photobleach and have much wider Stokes shifts,

Screening for Antifibrotic Compounds Using High Throughput System Based on Fluorescence Polarization

Directory of Open Access Journals (Sweden)

Branko Stefanovic

2014-04-01

Full Text Available Fibroproliferative diseases are one of the leading causes of death worldwide. They are characterized by reactive fibrosis caused by uncontrolled synthesis of type I collagen. There is no cure for fibrosis and development of therapeutics that can inhibit collagen synthesis is urgently needed. Collagen α1(I mRNA and α2(I mRNA encode for type I collagen and they have a unique 5' stem-loop structure in their 5' untranslated regions (5'SL. Collagen 5'SL binds protein LARP6 with high affinity and specificity. The interaction between LARP6 and the 5'SL is critical for biosynthesis of type I collagen and development of fibrosis in vivo. Therefore, this interaction represents is an ideal target to develop antifibrotic drugs. A high throughput system to screen for chemical compounds that can dissociate LARP6 from 5'SL has been developed. It is based on fluorescence polarization and can be adapted to screen for inhibitors of other protein-RNA interactions. Screening of 50,000 chemical compounds yielded a lead compound that can inhibit type I collagen synthesis at nanomolar concentrations. The development, characteristics, and critical appraisal of this assay are presented.

Some fluorescence properties of dimethylaminochalcone and its novel cyclic analogues

Science.gov (United States)

Tomečková, Vladimíra; Poškrobová, Martina; Štefanišinová, Miroslava; Perjési, Pál

2009-12-01

This paper demonstrates the basic character (polarity, solubility, colour, absorption and fluorescence quantum yield) of synthetic dimethylaminochalcone ( 1) and its cyclic analogues measured in toluene, chloroform, dimethylsulfoxide and ethanol, which have been studied by absorption and fluorescence spectroscopy. The biologically active dye 4'-dimethylaminochalcone ( 1b) and its less flexible analogues 4-dimethylaminoindanone ( 2b), -tetralone ( 3b), and -benzosuberone ( 4b) are lipophilic molecules that displayed the best solubility in toluene and chloroform. The highest fluorescence and quantum yields of compounds 1 and 2 have been obtained in DMSO and chloroform. Quenching effect of fluorescence compounds ( 1- 4) has been studied in the mixture of the most polar organic solvents DMSO and water. In the presence of water, fluorescence of compound 1 has been quenched the best from all studied chalcones and emission maxima of molecules 1- 4 have been shifted to the longer wavelengths. Quenching effect of fluorescence by water was in order 1 > 2 > 3 > 4.

Power-Law-Distributed Dark States are the Main Pathway for Photobleaching of Single Organic Molecules

OpenAIRE

Hoogenboom, J.P.; Hoogenboom, Jacob; van Dijk, E.M.H.P.; Hernando Campos, J.; van Hulst, N.F.; Garcia Parajo, M.F.

2005-01-01

We exploit the strong excitonic coupling in a superradiant trimer molecule to distinguish between long-lived collective dark states and photobleaching events. The population and depopulation kinetics of the dark states in a single molecule follow power-law statistics over 5 orders of magnitude in time. This result is consistent with the formation of a radical unit via electron tunneling to a time-varying distribution of trapping sites in the surrounding polymer matrix. We furthermore demonstr...

Polar and low polar solvents media effect on dipole moments of some diazo Sudan dyes

Science.gov (United States)

Zakerhamidi, M. S.; Golghasemi Sorkhabi, Sh.; Shamkhali, A. N.

2014-06-01

Absorption and fluorescence spectra of three Sudan dyes (SudanIII, SudanIV and Sudan black B) were recorded in various solvents with different polarity in the range of 300-800 nm, at room temperature. The solvatochromic method was used to investigate dipole moments of these dyes in ground and excited states, in different media. The solvatochromic behavior of these substances and their solvent-solute interactions were analyzed via solvent polarity parameters. Obtained results express the effects of solvation on tautomerism and molecular configuration (geometry) of Sudan dyes in solvent media with different polarity. Furthermore, analyze of solvent-solute interactions and value of ground and excited states dipole moments suggests different forms of resonance structures for Sudan dyes in polar and low-polar solvents.

Ultrafast polarized fluorescence measurements on monomeric and self-associated melittin

NARCIS (Netherlands)

Pandit, A.; Larsen, O.F.A.; Stokkum, van I.H.M.; Grondelle, van R.; Kraayenhof, R.; Amerongen, van H.

2003-01-01

The anisotropic and magic-angle fluorescence decay of the single tryptophan (Trp) residue of melittin, a bee venom peptide, was investigated by time-resolved fluorescence anisotropy using a streak camera setup. The peptide was dissolved either in distilled water or in Hepes/NaOH buffer containing

Simple apparatus for polarization sensing of analytes

Science.gov (United States)

Gryczynski, Zygmunt; Gryczynski, Ignacy; Lakowicz, Joseph R.

2000-09-01

We describe a simple device for fluorescence sensing based on an unexpansive light source, a dual photocell and a Watson bridge. The emission is detected from two fluorescent samples, one of which changes intensity in response to the analyte. The emission from these two samples is observed through two orthogonally oriented polarizers and an analyzer polarizer. The latter polarizer is rotated to yield equal intensities from both sides of the dual photocell, as determined by a zero voltage from the Watson bridge. Using this device, we are able to measure fluorescein concentration to an accuracy near 2% at 1 (mu) M fluorescein, and pH values accurate to +/- 0.02 pH units. We also use this approach with a UV hand lamp and a glucose-sensitive protein to measure glucose concentrations near 2 (mu) M to an accuracy of +/- 0.1 (mu) M. This approach requires only simple electronics, which can be battery powered. Additionally, the method is generic, and can be applied with any fluorescent sample that displays a change in intensity. One can imagine this approach being used to develop portable point-of-care clinical devices.

Wide-range tuning of polymer microring resonators by the photobleaching of CLD-1 chromophores

Science.gov (United States)

Poon, Joyce K. S.; Huang, Yanyi; Paloczi, George T.; Yariv, Amnon; Zhang, Cheng; Dalton, Larry R.

2004-11-01

We present a simple and effective method for the postfabrication trimming of optical microresonators. We photobleach CLD-1 chromophores to tune the resonance wavelengths of polymer microring resonator optical notch filters. A maximum wavelength shift of -8.73 nm is observed. The resonators are fabricated with a soft-lithography molding technique and have an intrinsic Q value of 2.6×10^4 and a finesse of 9.3. The maximum extinction ratio of the resonator filters is -34 dB, indicating that the critical coupling condition has been satisfied.

Polarized spectral features of human breast tissues through wavelet ...

Indian Academy of Sciences (India)

Abstract. Fluorescence characteristics of human breast tissues are investigated through wavelet transform and principal component analysis (PCA). Wavelet transform of polar- ized fluorescence spectra of human breast tissues is found to localize spectral features that can reliably differentiate different tissue types.

Fluorescence and Cytotoxicity of Cadmium Sulfide Quantum Dots Stabilized on Clay Nanotubes

Directory of Open Access Journals (Sweden)

Anna V. Stavitskaya

2018-05-01

Full Text Available Quantum dots (QD are widely used for cellular labeling due to enhanced brightness, resistance to photobleaching, and multicolor light emissions. CdS and CdxZn1−xS nanoparticles with sizes of 6–8 nm were synthesized via a ligand assisted technique inside and outside of 50 nm diameter halloysite clay nanotubes (QD were immobilized on the tube’s surface. The halloysite–QD composites were tested by labeling human skin fibroblasts and prostate cancer cells. In human cell cultures, halloysite–QD systems were internalized by living cells, and demonstrated intense and stable fluorescence combined with pronounced nanotube light scattering. The best signal stability was observed for QD that were synthesized externally on the amino-grafted halloysite. The best cell viability was observed for CdxZn1−xS QD immobilized onto the azine-grafted halloysite. The possibility to use QD clay nanotube core-shell nanoarchitectures for the intracellular labeling was demonstrated. A pronounced scattering and fluorescence by halloysite–QD systems allows for their promising usage as markers for biomedical applications.

Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer

Directory of Open Access Journals (Sweden)

Cohen Sarit

2012-08-01

Full Text Available Abstract Background The use of near-infrared (NIR fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. Methods The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR. Tumor-targeting ligands such as peanut agglutinin (PNA, anti-carcinoembryonic antigen antibodies (anti-CEA and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72 were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. Results and discussion Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. Conclusions These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA

Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer.

Science.gov (United States)

Cohen, Sarit; Margel, Shlomo

2012-08-14

The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon

Synthesis of Fluorescent Carbon Dots by Gastrointestinal Fluid Treatment of Mongolia Har Gabur

Directory of Open Access Journals (Sweden)

Tegexibaiyin Wang

2017-01-01

Full Text Available Har Gabur is the carbide obtained from pig manure by burning. The fluorescent carbon dots (CDs of Har Gabur were successfully synthesized through simulating the digestion process of human gastrointestinal tract. Transmission Electron Microscope (TEM analysis showed that the average size of the prepared Har Gabur CDs was 4 nm, with good solubility in water and strong fluorescence under UV irradiation. The X-ray and Raman results showed that the Har Gabur CDs were mainly composed of oxygen “O” and carbon “C” elements, in the forms of “C=O” and “C-O.” The bond energy results showed that the nitrogen “N” atom presented as “C-N” form, which indicated that Har Gabur CDs also contain “N.” In photobleaching assay, Har Gabur CDs showed excellent light stability compared with ordinary organic dye, fluorescein, and Rhodamine B. The fluorescence intensity of Har Gabur CDs was fairly stable within a wide pH range of 3–10. When L-lysine and L-cysteine were applied for the passivation stage, the relative quantum yields were improved by 1.53 and 3.68 times, respectively. Finally, the fluorescence properties of Har Gabur CDs were tested in cells and zebrafish, illustrating that Har Gabur CD has potential in the application of biological labeling and imaging.

Correlative FRET: new method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture

Science.gov (United States)

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.

A disordered polaron model for polarized fluorescence excitation spectra of LH1 and LH2 bacteriochlorophyll antenna aggregates

International Nuclear Information System (INIS)

Trinkunas, Gediminas; Freiberg, Arvi

2006-01-01

Excitonic polarons in antenna complexes are subject to static lattice disorder. A model has been developed to analyze polarized fluorescence excitation spectra of circular light-harvesting complexes from purple photosynthetic bacteria containing bacteriochlorophyll as the main photoactive pigment that includes both diagonal (energetic) and off-diagonal (structural) disorders. Essential differences of disorder realizations seem to exist between the core LH1 and peripheral LH2 complexes from the bacterium Rhodobacter sphaeroides. The disorder in LH1 appears to be dominated by the structural disorder, while that in LH2, by energetic one. These differences may be due to relatively bigger size of the LH1 complex and, consequently, with its enhanced structural flexibility

Crystallography and Molecular Arrangement of Polymorphic Monolayer J-Aggregates of a Cyanine Dye: Multiangle Polarized Light Fluorescence Optical Microscopy Study.

Science.gov (United States)

Prokhorov, Valery V; Pozin, Sergey I; Perelygina, Olga M; Mal'tsev, Eugene I

2018-04-24

The molecular orientation in monolayer J-aggregates of 3,3-di(γ-sulfopropyl)-5,5-dichlorotiamonomethinecyanine dye has been precisely estimated using improved linear polarization measurements in the fluorescence microscope in which a multiangle set of polarization data is obtained using sample rotation. The estimated molecular orientation supplemented with the previously established crystallographic constraints based on the analysis of the well-developed two-dimensional J-aggregate shapes unambiguously indicate the staircase type of molecular arrangement for striplike J-aggregates with the staircases oriented along strips. The molecular transition dipoles are inclined at an angle of ∼25° to the strip direction, whereas the characteristic strip vertex angle ∼45° is formed by the [100] and [1-10] directions of the monoclinic unit cell. Measurements of the geometry of partially unwound tubes and their polarization properties support the model of tube formation by close-packed helical winding of flexible monolayer strips. In the tubes, the long molecular axes are oriented at a small angle in the range of 5-15° to the normal to the tube axis providing low bending energy. At a nanoscale, high-resolution atomic force microscopy imaging of J-aggregate monolayers reveals a complex quasi-one-dimensional organization.

Rapid screening of aflatoxin B1 in beer by fluorescence polarization immunoassay.

Science.gov (United States)

Beloglazova, N V; Eremin, S A

2015-09-01

This manuscript describes the development of a sensitive, fast and easily-performed fluorescence polarization immunoassay (FPIA) for the mycotoxin aflatoxin B1 (AFB1) in various beer samples, both lager and dark. The highest sensitivity was determined for six poly- and monoclonal antibodies selective towards aflatoxins. The sample pretreatment design was emphasized since beer samples are characterized by extremely diverse matrices. Herein, the choice of sorbent for effective removal of matrix interferences prior to analysis was crucial. The samples were diluted with a borate buffer solution containing 1% PEG 6000 and passed through the clean-up column packed with NH2-derivated silica. This sample pretreatment technique was perfectly suitable for the FPIA of lager beer samples, but for dark beer and ale it did not suffice. An artificial matrix was constructed to plot a calibration curve and quantify the results of the latter samples. The developed immunoassay was characterized by a limit of detection of 1 ng mL(-1). Apparent recovery values of 89-114% for lager and 80-125% for dark beer were established. The FPIA data for AFB1 was characterized by elevated linear regression coefficients, 0.9953 for spiked lager and 0.9895 for dark beer samples respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantification of Material Fluorescence and Light Scattering Cross Sections Using Ratiometric Bandwidth-Varied Polarized Resonance Synchronous Spectroscopy.

Science.gov (United States)

Xu, Joanna Xiuzhu; Hu, Juan; Zhang, Dongmao

2018-05-25

Presented herein is the ratiometric bandwidth-varied polarized resonance synchronous spectroscopy (BVPRS2) method for quantification of material optical activity spectra. These include the sample light absorption and scattering cross-section spectrum, the scattering depolarization spectrum, and the fluorescence emission cross-section and depolarization spectrum in the wavelength region where the sample both absorbs and emits. This ratiometric BVPRS2 spectroscopic method is a self-contained technique capable of quantitatively decoupling material fluorescence and light scattering signal contribution to its ratiometric BVPRS2 spectra through the linear curve-fitting of the ratiometric BVPRS2 signal as a function of the wavelength bandwidth used in the PRS2 measurements. Example applications of this new spectroscopic method are demonstrated with materials that can be approximated as pure scatterers, simultaneous photon absorbers/emitters, simultaneous photon absorbers/scatterers, and finally simultaneous photon absorbers/scatterers/emitters. Because the only instruments needed for this ratiometric BVPRS2 technique are the conventional UV-vis spectrophotometer and spectrofluorometer, this work should open doors for routine decomposition of material UV-vis extinction spectrum into its absorption and scattering component spectra. The methodology and insights provided in this work should be of broad significance to all chemical research that involves photon/matter interactions.

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

Science.gov (United States)

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.

Synchronous fluorescence spectroscopic study of solvatochromic curcumin dye

Science.gov (United States)

Patra, Digambara; Barakat, Christelle

2011-09-01

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

Selective nonspecific solvation under dielectric saturation and fluorescence spectra of dye solutions in binary solvents.

Science.gov (United States)

Bakhshiev, N G; Kiselev, M B

1991-09-01

The influence of selective nonspecific solvation on the fluorescence spectra of three substitutedN-methylphthalimides in a binary solvent system consisting of a nonpolar (n-heptane) and a polar (pyridine) component has been studied under conditions close to dielectric saturation. The substantially nonlinearity of the effect is confirmation that the spectral shifts of fluorescence bands depend on the number of polar solvent molecules involved in solvating the dye molecule. The measured fluorescence spectral shifts determined by substituting one nonpolar solvent molecula with a polar one in the proximity of the dye molecule agree quantitatively with the forecasts of the previously proposed semiempirical theory which describes this nonlinear solvation phenomenon.

Monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in Streptococcus pneumoniae using fluorescent dyes.

Science.gov (United States)

Clementi, Emily A; Marks, Laura R; Roche-Håkansson, Hazeline; Håkansson, Anders P

2014-02-17

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial

DART: Recent Advances in Remote Sensing Data Modeling With Atmosphere, Polarization, and Chlorophyll Fluorescence

Science.gov (United States)

Gastellu-Etchegorry, Jean-Phil; Lauret, Nicolas; Yin, Tiangang; Landier, Lucas; Kallel, Abdelaziz; Malenovsky, Zbynek; Bitar, Ahmad Al; Aval, Josselin; Benhmida, Sahar; Qi, Jianbo;

Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening

International Nuclear Information System (INIS)

Johansson, Daniel X.; Brismar, Hjalmar; Persson, Mats A.A.

2007-01-01

The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5 ± 1.5% (mean ± SEM) and 8.0 ± 0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8 ± 2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1 ± 1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals

Computational Modeling of Fluorescence Loss in Photobleaching

DEFF Research Database (Denmark)

Hansen, Christian Valdemar; Schroll, Achim; Wüstner, Daniel

2015-01-01

sequences as reaction– diffusion systems on segmented cell images. The cell geometry is extracted from microscopy images using the Chan–Vese active contours algorithm [8]. The PDE model is subsequently solved by the automated Finite Element software package FEniCS [20]. The flexibility of FEniCS allows...

Fluorescence Decay Dynamics of Ethidium Bromide in Polymers

International Nuclear Information System (INIS)

Jee, Ah Young; Min Yung

2010-01-01

The fluorescence lifetimes of EB in five polymers covering LDPE, HDPE, PC, PS, and PAA were measured by picosecond time-correlated single photon counting. The lifetime change of EB has been previously described by hydrogen bonding ability. In this work, we have observed that the lifetime of EB depends strongly on the Young's modulus of medium. Thus, it is possible that the fluorescence decay dynamics of EB could be influenced by medium rigidity rather than hydrogen bonding ability in polymer. The medium influence on the fluorescence decay dynamics of ethidium bromide (EB) has been investigated in various environments. For example, Ohmstead and Kearns related the fluorescence lifetime of EB to the excited-state proton transfer process. In addition, they reported that the solvent viscosity plays a minor role in the excited state decay process of EB. Chirico et al. measured the fluorescence decay of EB as 1.7 ns in water and 6.5 ns in ethanol and concluded that hydrogen bonding ability is a key factor for the nonradiative relaxation. Pal et al. measured the fluorescence decay time of EB in acetone, acetonitrile, and their mixtures. They observed that the fluorescence decay processes were independent on the solvent polarity. These results show that the EB lifetime does not depend much on polarity or viscosity, but is mainly influenced by hydrogen bonding. Overall, EB is one of most widely used dyes for probing DNA. When EB is intercalated into the helical structure of DNA, a large increase in the fluorescence lifetime has been observed in comparison with water environment, and the fluorescence enhancement was attributed to the blocking of the excited-state proton transfer

Comparison of capillary electrophoresis-based immunoassay with fluorescence polarization immunoassay for the immunodetermination of methamphetamine using various methamphetamine antibodies.

Science.gov (United States)

Choi, J; Kim, C; Choi, M J

1998-11-01

An accurate and simple immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was performed for the detection of methamphetamine (MA) in urine. The CE-LIF was conducted with an untreated fused-silica column using antiserum and a tracer of fluorescein isothiocyanate (FITC)-labeled MA. This CE-LIF system was compared with fluorescence polarization immunoassay (FPIA) in a TDx analyzer in the photo-check mode using the same FITC-labeled tracer and the same antiserum. Various antibodies, not only those prepared by our own immunogens but also those from commercial sources, were screened and characterized in both assay systems with regard to sensitivity, precision, and cross-reactivity. Both systems satisfied analytical precision and gave similar cross-reactivity patterns. However, the CE-LIF-based immunoassay was approximately one order superior to FPIA in sensitivity, requiring less volume of sample, antiserum, and tracer for the assay. Considering that the FPIA system is well known to be a useful tool for screening antibodies and detecting drugs, the CE-LIF-based immunoassay system, which is seemingly more advantageous than the FPIA system, appears to have great power for the characterization of antibodies and for the detection of MA in urine.

Solvent effects on the photochemistry of dimethyl sulfoxide-Cl complexes studied by combined pulse radiolysis and laser flash photolysis

International Nuclear Information System (INIS)

Sumiyoshi, Takashi; Minegishi, Hideki; Fujiyoshi, Ryoko; Sawamura, Sadashi

2006-01-01

Photolysis of complexes of dimethyl sulfoxide (DMSO) with chlorine atoms results in rapid and permanent photobleaching which may be due to intramolecular hydrogen abstraction. The effects of solvent polarity were examined in a wide variety of DMSO-carbon tetrachloride mixed solvents. The quantum yields of photobleaching decreased from 0.27 to 0.08 as the solvent polarity increased, while significant changes were observed in the low DMSO concentration range ( -3 ). This cannot be accounted for by simple solvent polarity effects. The effects of polar and nonpolar additives were also examined and it is concluded that the specific solvation effect of DMSO was the main cause of the significant change in quantum yields in the low concentration range of DMSO

Propagation of ionization waves during ignition of fluorescent lamps

International Nuclear Information System (INIS)

Langer, R; Tidecks, R; Horn, S; Garner, R; Hilscher, A

2008-01-01

The propagation of the first ionization wave in a compact fluorescent lamp (T4 tube with standard electrodes) during ignition was investigated for various initial dc-voltages (both polarities measured against ground) and gas compositions (with and without mercury). In addition the effect of the presence of a fluorescent powder coating was studied. The propagation velocity of the initial wave was measured by an assembly of photomultipliers installed along the tube, which detected the light emitted by the wave head. The propagation was found to be faster for positive than for negative polarity. This effect is explained involving processes in the electrode region as well as in the wave head. Waves propagate faster in the presence of a fluorescent powder coating than without it and gases of lighter mass show a faster propagation than gases with higher mass

Proceedings of the Japan-US workshop on plasma polarization spectroscopy and the international seminar on plasma polarization spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Fujimoto, Takashi; Beiersdorfer, Peter [eds.

1998-06-01

The international meeting on Plasma Polarization Spectroscopy (PPS) was held in Kyoto during January 26-28, 1998. This Proceedings book includes the papers of the talks given at the meeting. These include: overviews of PPS from the aspects of atomic physics, and of plasma physics; several PPS and MSE (motional Stark effect) experiments on magnetically confined plasmas and a laser-produced plasma; polarized laser-induced fluorescence spectroscopy, several experiments on EBITs (electron beam ion trap) and their theoretical interpretations; polarized profiles of spectral lines, basic formulation of PPS; inelastic and elastic electron collisions leading to polarized atomic states; polarization in recombining plasma; relationship between the collisional polarization relaxation and the line broadening; and characteristics of the plasma produced by very short pulse and high power laser irradiation. The 19 of the presented papers are indexed individually. (J.P.N.)

Portable ultrahigh-vacuum sample storage system for polarization-dependent total-reflection fluorescence x-ray absorption fine structure spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Watanabe, Yoshihide, E-mail: e0827@mosk.tytlabs.co.jp; Nishimura, Yusaku F.; Suzuki, Ryo; Beniya, Atsushi; Isomura, Noritake [Toyota Central R& D Labs., Inc., Yokomichi 41-1, Nagakute, Aichi 480-1192 (Japan); Uehara, Hiromitsu; Asakura, Kiyotaka; Takakusagi, Satoru [Catalysis Research Center, Hokkaido University, Kita 21-10, Sapporo, Hokkaido 001-0021 (Japan); Nimura, Tomoyuki [AVC Co., Ltd., Inada 1450-6, Hitachinaka, Ibaraki 312-0061 (Japan)

2016-03-15

A portable ultrahigh-vacuum sample storage system was designed and built to investigate the detailed geometric structures of mass-selected metal clusters on oxide substrates by polarization-dependent total-reflection fluorescence x-ray absorption fine structure spectroscopy (PTRF-XAFS). This ultrahigh-vacuum (UHV) sample storage system provides the handover of samples between two different sample manipulating systems. The sample storage system is adaptable for public transportation, facilitating experiments using air-sensitive samples in synchrotron radiation or other quantum beam facilities. The samples were transferred by the developed portable UHV transfer system via a public transportation at a distance over 400 km. The performance of the transfer system was demonstrated by a successful PTRF-XAFS study of Pt{sub 4} clusters deposited on a TiO{sub 2}(110) surface.

State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation.

Science.gov (United States)

Kitamura, Akira; Kinjo, Masataka

2018-03-23

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and Huntington's disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In the current review, we present an overview of the principles and application of several methodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N&B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases.

Spectroscopic Analysis of Red Fluorescent Proteins and Development of a Microfluidic Cell Sorter for the Generation of Improved Variants

Science.gov (United States)

Lubbeck, Jennifer L.

The discovery of the green fluorescent protein (GFP) launched the development of a wide variety of fluorescent protein (FP) mutants whose spectral and photophysical diversity revolutionized in vivo imaging. The excitation and emission spectra of red fluorescent proteins (RFPs), in particular, have been ideally tuned to a window optically favorable for in vivo work. However, their quantum yields, photostabilities and fluorescence intermittency properties require improvement if they are to be broadly employed for low-copy or single-molecule measurements. Attempts to engineer improved RFPs often result in optimization of one photophysical property at the expense of others. We developed a microfluidic-based cytometer for screening HeLa cell-based genetic RFP-libraries simultaneously on the basis of fluorescence lifetime (a proxy for quantum yield), photostability, and brightness. Ten 532 nm excitation beams interrogate each cell in flow. The first is electro-optically modulated (30 MHz) to enable lifetime measurement with phase fluorimetry. The remaining beams act as a pulse sequence for isolating the irreversible photobleaching time constant. Optical-force switching is employed to sort cells based on any combination of the photophysical parameters. Screening with this instrument enables identification of regions of the structure that synergistically affect quantum yield and photostability and the sorting capability provides a new tool for accelerating the development of next generation RFPs.

Fluorenyl benzothiadiazole and benzoselenadiazole near-IR fluorescent probes for two-photon fluorescence imaging (Conference Presentation)

Science.gov (United States)

Belfield, Kevin D.; Yao, Sheng; Kim, Bosung; Yue, Xiling

2016-03-01

Imaging biological samples with two-photon fluorescence (2PF) microscopy has the unique advantage of resulting high contrast 3D resolution subcellular image that can reach up to several millimeters depth. 2PF probes that absorb and emit at near IR region need to be developed. Two-photon excitation (2PE) wavelengths are less concerned as 2PE uses wavelengths doubles the absorption wavelength of the probe, which means 2PE wavelengths for probes even with absorption at visible wavelength will fall into NIR region. Therefore, probes that fluoresce at near IR region with high quantum yields are needed. A series of dyes based on 5-thienyl-2, 1, 3-benzothiadiazole and 5-thienyl-2, 1, 3-benzoselenadiazole core were synthesized as near infrared two-photon fluorophores. Fluorescence maxima wavelengths as long as 714 nm and fluorescence quantum yields as high as 0.67 were achieved. The fluorescence quantum yields of the dyes were nearly constant, regardless of solvents polarity. These diazoles exhibited large Stokes shift (GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.

Concentration Effect on Quenching of Chlorophyll a Fluorescence by All-Trans-β-Carotene in Photosynthesis

Directory of Open Access Journals (Sweden)

Chen Chen

2017-09-01

Full Text Available Absorption, fluorescence spectra of chlorophyll a (Chl-a and all-trans-β-carotene (β-Car mixing solution are investigated in different polarity and polarizability solvents. The carotenoids regulate the energy flow in photosynthesis by interaction with chlorophyll, leading to an observable reduction of Chl-a fluorescence. The fluorescence red shifts with the increasing solvent polarizability. The energy transfer in the Chl-a and β-Car system is proposed. The electron transfer should be dominant in quenching Chl-a fluorescence rather than the energy transfer in this system. Polar solvent with large polarizability shows high quenching efficiency. When dissolved in carbon tetrachloride, Chl-a presents red shift of absorption and blue shift of fluorescence spectra with increasing β-Car concentration, which implies a Chl-a conformational change.

Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis.

Science.gov (United States)

Erdel, Fabian; Rippe, Karsten

2012-11-20

Interactions between nuclear proteins and chromatin frequently occur on the time scale of seconds and below. These transient binding events are important for the fast identification of target sites as concluded from our previous analysis of the human chromatin remodelers Snf2H and Snf2L from the imitation switch (ISWI) family. Both ATP-driven molecular motor proteins are able to translocate nucleosomes along the DNA and appear to exert this activity only on a small number of nucleosomes to which they bind more tightly. For mechanistic studies, one needs to distinguish such translocation reactions or other long-lived interactions associated with conformational changes and/or ATP hydrolysis from nonproductive chromatin sampling during target search. These processes can be separated by measuring the duration of nucleosome binding with subsecond time resolution. To reach this goal, we have developed a fluorescence bleaching technique termed pixel-wise photobleaching profile evolution analysis (3PEA). It exploits the inherent time structure of confocal microscopy images and yields millisecond resolution. 3PEA represents a generally applicable approach to quantitate transient chromatin interactions in the 2- to 500-ms time regime within only ∼1 s needed for a measurement. The green autofluorescent protein (GFP)-tagged Snf2H and Snf2L and the inactive Snf2L+13 splice variant were studied by 3PEA in comparison to the isolated GFP or red autofluorescent protein and a GFP pentamer. Our results reveal that the residence time for transient chromatin binding of Snf2H and Snf2L is <2 ms, and strongly support the view that ISWI-type remodelers are only rarely active in unperturbed cells during G1 phase.

Intradermal indocyanine green for in vivo fluorescence laser scanning microscopy of human skin: a pilot study.

Directory of Open Access Journals (Sweden)

Constanze Jonak

Full Text Available BACKGROUND: In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. METHODOLOGY/PRINCIPAL FINDINGS: Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. CONCLUSIONS/SIGNIFICANCE: Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of

Microscopic oxygen imaging based on fluorescein bleaching efficiency measurements

DEFF Research Database (Denmark)

Beutler, Martin; Heisterkamp, Ines M.; Piltz, Bastian

2014-01-01

by a charge-coupled-device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen-consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen......Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time-dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi-fluorescence microscopy. The molecular...... states of the fluorophore can be expressed by a three-state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide-field fluorescence microscope, the fluorescence...

Modelling the competition between photo-darkening and photo-bleaching effects in high-power ytterbium-doped fibre amplifiers

Science.gov (United States)

Jolly, A.; Vinçont, C.; Pierre, Ch.; Boullet, J.

2017-08-01

We propose an innovative, fully space-time model to take into account the seed-dependent nature of ageing penalties in high-power ytterbium-doped fibre amplifiers. Ageing is shown to be based on the on-going competition between photo-darkening and photo-bleaching phenomena. Our approach is based on the natural interplay between the excited states of co-existing ytterbium pairs and colour centres in highly doped fibres, in the presence of thermal coupling between the closely spaced excited states. As initiated from IR photons, the excitation of colour centres up to the UV band is supposed to be governed by multi-photon absorption. The interactions of interest in the kinetics of photo-bleaching then take the form of highly efficient charge transfers, which imply the reduction of some fraction of the basically trivalent ions to their divalent state. Due to the activation of ytterbium pairs by means of energy transfer up-conversion, these interactions get more and more effective at elevated operating powers. Computational results using these principles actually help to fit our experimental data regarding seeding effects, as well as fully generic trends already evidenced in the literature. This gives a fine demonstration for the need to discriminate co-active pump and signal contributions. Our self-consistent, still simplified model then consists of a valuable tool to help for a deeper understanding of the ageing issues. Furthermore, considering higher-order ytterbium aggregates, this should open new routes towards more comprehensive models.

Nanoantenna array-induced fluorescence enhancement and reduced lifetimes

DEFF Research Database (Denmark)

Bakker, R. M.; Drachev, V. P.; Liu, Z.

2008-01-01

Enhanced fluorescence is observed from dye molecules interacting with optical nanoantenna arrays. Elliptical gold dimers form individual nanoantennae with tunable plasmon resonances depending upon the geometry of the two particles and the size of the gap between them. A fluorescent dye, Rhodamine...... 800, is uniformly embedded in a dielectric host that coats the nanoantennae. The nanoantennae act to enhance the dye absorption. In turn, emission from the dye drives the plasmon resonance of the antennae; the nanoantennae act to enhance the fluorescence signal and change the angular distribution...... of emission. These effects depend upon the overlap of the plasmon resonance with the excitation wavelength and the fluorescence emission band. A decreased fluorescence lifetime is observed along with highly polarized emission that displays the characteristics of the nanoantenna's dipole mode. Being able...

Dual fluorescence and laser emissions from fluorescein-Na and eosin-B

International Nuclear Information System (INIS)

Math, N.N.; Naik, L.R.; Suresh, H.M.; Inamdar, S.R.

2006-01-01

Dual laser emissions were observed from fluorescein-Na and eosin-B in ethanolic solutions individually in the concentration range from 10 -2 to 10 -3 mol dm -3 under N 2 laser excitation. The first compound was found to lase at two distinct regions with wavelength maxima around 540, 550 nm, while the second one around 558, 574 nm. Steady-state absorption, fluorescence excitation, fluorescence polarization, fluorescence emission and decays of the dyes in various solvents under varying conditions of excitation and detection systems were carried out to identify the nature of the emitting species responsible for laser emissions in two distinct regions. Both the dyes exhibited concentration and excitation wavelength dependence of fluorescence and the effects were found to be more pronounced in binary solution. The fluorescence decays of dyes were monoexponential in ethanol, while in some other solvents used, the decays showed biexponential behavior. The absorption and excitation studies using thin layers of solutions revealed the formation of dimers with the dye concentration around 1x10 -3 mol dm -3 . Fluorescence polarization and decay studies confirmed the presence of dimers. The two laser bands observed in the shorter and longer wavelengths were respectively ascribed to monomeric and dimeric species

Dual fluorescence and laser emissions from fluorescein-Na and eosin-B

Energy Technology Data Exchange (ETDEWEB)

Math, N.N. [Laser Spectroscopy (DRDO/KU) Programme, Department of Physics, Karnatak University, Dharwad 580 003 (India)]. E-mail: nnm31@rediffmail.com; Naik, L.R. [Laser Spectroscopy (DRDO/KU) Programme, Department of Physics, Karnatak University, Dharwad 580 003 (India); Suresh, H.M. [Laser Spectroscopy (DRDO/KU) Programme, Department of Physics, Karnatak University, Dharwad 580 003 (India); Inamdar, S.R. [Laser Spectroscopy (DRDO/KU) Programme, Department of Physics, Karnatak University, Dharwad 580 003 (India)

2006-12-15

Dual laser emissions were observed from fluorescein-Na and eosin-B in ethanolic solutions individually in the concentration range from 10{sup -2} to 10{sup -3} mol dm{sup -3} under N{sub 2} laser excitation. The first compound was found to lase at two distinct regions with wavelength maxima around 540, 550 nm, while the second one around 558, 574 nm. Steady-state absorption, fluorescence excitation, fluorescence polarization, fluorescence emission and decays of the dyes in various solvents under varying conditions of excitation and detection systems were carried out to identify the nature of the emitting species responsible for laser emissions in two distinct regions. Both the dyes exhibited concentration and excitation wavelength dependence of fluorescence and the effects were found to be more pronounced in binary solution. The fluorescence decays of dyes were monoexponential in ethanol, while in some other solvents used, the decays showed biexponential behavior. The absorption and excitation studies using thin layers of solutions revealed the formation of dimers with the dye concentration around 1x10{sup -3} mol dm{sup -3}. Fluorescence polarization and decay studies confirmed the presence of dimers. The two laser bands observed in the shorter and longer wavelengths were respectively ascribed to monomeric and dimeric species.

A Brief Introduction to Single-Molecule Fluorescence Methods.

Science.gov (United States)

van den Wildenberg, Siet M J L; Prevo, Bram; Peterman, Erwin J G

2018-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

Studying atomic-resolution by X-ray fluorescence holography

International Nuclear Information System (INIS)

Gao Hongyi; Chen Jianwen; Xie Honglan; Zhu Huafeng; Li Ruxin; Xu Zhizhan

2005-01-01

In this work, the results of numerical simulations of X-ray fluorescence holograms and the reconstructed atomic images for Fe single crystal are given. The influences of the recording angles ranges and the polarization effect on the reconstruction of the atomic images are discussed. The process for removing twin images by multiple energy fluorescence holography and expanding the energy range of the incident X-rays to improve the resolution of the reconstructed images is presented

Nanodiamonds as multi-purpose labels for microscopy

NARCIS (Netherlands)

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.

2017-01-01

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

Proceedings of the Japan-US workshop on plasma polarization spectroscopy and the fourth international symposium on plasma polarization spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Fujimoto, Takashi; Beiersdorfer, Peter [eds.

2004-07-01

The international meeting on Plasma Polarization Spectroscopy (PPS) was held at Kyoto University during February 4-6, 2004. This Proceedings book includes the summaries of the talks given in that meeting. Starting with the Overview talk by Csanak, the subjects cover: x-ray polarization experiments on z-pinches (plasma foci), and an x-pinch, a laser-produced plasma in a gas atmosphere, an interpretation of the polarized 1<- 0 x-ray laser line, polarization observation from various laser-produced plasmas including a recombining phase plasma, a report on the on-going project of a laser facility, several polarization observations on magnetically confined plasmas including the Large Helical Device and an ECR plasma, a new laser-induced fluorescence diagnostic method. On atomic physics side given are: various polarization measurements on EBIT, precision spectroscopy on the TEXTOR, user-friendly atomic codes. Instrumentation is also a subject of this book. The 18 of the presented papers are indexed individually. (J.P.N.)

Design of near-infrared fluorescent bioactive conjugated functional iron oxide nanoparticles for optical detection of colon cancer

Directory of Open Access Journals (Sweden)

Corem-Salkmon E

2012-10-01

Full Text Available Enav Corem-Salkmon, Benny Perlstein, Shlomo MargelThe Institute of Nanotechnology and Advanced Materials, Department of Chemistry, Bar-Ilan University, Ramat-Gan, IsraelBackground: Colon cancer is one of the major causes of death in the Western world. Early detection significantly improves long-term survival for patients with the disease. Near-infrared (NIR fluorescent nanoparticles hold great promise as contrast agents for tumor detection. NIR offers several advantages for bioimaging compared with fluorescence in the visible spectrum, ie, lower autofluorescence of biological tissues, lower absorbance, and consequently deeper penetration into biomatrices.Methods and results: NIR fluorescent iron oxide nanoparticles with a narrow size distribution were prepared by nucleation, followed by controlled growth of thin iron oxide films onto cyanine NIR dye conjugated gelatin-iron oxide nuclei. For functionalization, and in order to increase the NIR fluorescence intensity, the NIR fluorescent iron oxide nanoparticles obtained were coated with human serum albumin containing cyanine NIR dye. Leakage of the NIR dye from these nanoparticles into phosphate-buffered saline solution containing 4% albumin was not detected. The work presented here is a feasibility study to test the suitability of iron oxide-human serum albumin NIR fluorescent nanoparticles for optical detection of colon cancer. It demonstrates that encapsulation of NIR fluorescent dye within these nanoparticles significantly reduces photobleaching of the dye. Tumor-targeting ligands, peanut agglutinin and anticarcinoembryonic antigen antibodies (αCEA, were covalently conjugated with the NIR fluorescent iron oxide-human serum albumin nanoparticles via a poly(ethylene glycol spacer. Specific colon tumor detection was demonstrated in chicken embryo and mouse models for both nonconjugated and the peanut agglutinin-conjugated or αCEA-conjugated NIR fluorescent iron oxide-human serum albumin

Nanodiamonds as multi-purpose labels for microscopy

NARCIS (Netherlands)

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

2017-01-01

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 Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Science.gov (United States)

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Resonance fluorescence from an atom in a squeezed vacuum

Science.gov (United States)

Carmichael, H. J.; Lane, A. S.; Walls, D. F.

1987-06-01

The fluorescent spectrum for a two-level atom which is damped by a squeezed vacuum shows striking differences from the spectrum for ordinary resonance fluorescence. For strong coherent driving fields the Mollow triplet depends on the relative phase of the driving field and the squeezed vacuum field. The central peak may have either subnatural linewidth or supernatural linewidth depending on this phase. The mean atomic polarization also shows a phase sensitivity.

Curcumin as fluorescent probe for directly monitoring in vitro uptake of curcumin combined paclitaxel loaded PLA-TPGS nanoparticles

Science.gov (United States)

Nguyen, Hoai Nam; Thu Ha, Phuong; Sao Nguyen, Anh; Nguyen, Dac Tu; Doan Do, Hai; Nguyen Thi, Quy; Nhung Hoang Thi, My

2016-06-01

Theranostics, which is the combination of both therapeutic and diagnostic capacities in one dose, is a promising tool for both clinical application and research. Although there are many chromophores available for optical imaging, their applications are limited due to the photobleaching property or intrinsic toxicity. Curcumin, a natural compound extracted from the rhizome of curcuma longa, is well known thanks to its bio-pharmaceutical activities and strong fluorescence as biocompatible probe for bio-imaging. In this study, we aimed to fabricate a system with dual functions: diagnostic and therapeutic, based on poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) micelles co-loaded curcumin (Cur) and paclitaxel (PTX). Two kinds of curcumin nanoparticle (NP) were fabricated and characterized by Fourier transform infrared spectroscopy, field emission scanning electron microscopy and dynamic light scattering methods. The cellular uptake and fluorescent activities of curcumin in these systems were also tested by bioassay studies, and were compared with paclitaxe-oregon. The results showed that (Cur + PTX)-PLA-TPGS NPs is a potential system for cancer theranostics.

Curcumin as fluorescent probe for directly monitoring in vitro uptake of curcumin combined paclitaxel loaded PLA-TPGS nanoparticles

International Nuclear Information System (INIS)

Nguyen, Hoai Nam; Ha, Phuong Thu; Do, Hai Doan; Nguyen, Anh Sao; Nguyen, Dac Tu; Thi, Quy Nguyen; Thi, My Nhung Hoang

2016-01-01

Theranostics, which is the combination of both therapeutic and diagnostic capacities in one dose, is a promising tool for both clinical application and research. Although there are many chromophores available for optical imaging, their applications are limited due to the photobleaching property or intrinsic toxicity. Curcumin, a natural compound extracted from the rhizome of curcuma longa, is well known thanks to its bio-pharmaceutical activities and strong fluorescence as biocompatible probe for bio-imaging. In this study, we aimed to fabricate a system with dual functions: diagnostic and therapeutic, based on poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) micelles co-loaded curcumin (Cur) and paclitaxel (PTX). Two kinds of curcumin nanoparticle (NP) were fabricated and characterized by Fourier transform infrared spectroscopy, field emission scanning electron microscopy and dynamic light scattering methods. The cellular uptake and fluorescent activities of curcumin in these systems were also tested by bioassay studies, and were compared with paclitaxe-oregon. The results showed that (Cur + PTX)-PLA-TPGS NPs is a potential system for cancer theranostics. (paper)

Advances in polarization sensitive multiphoton nano-bio-imaging

Directory of Open Access Journals (Sweden)

Zyss J.

2010-06-01

Full Text Available In this talk, we shall shortly review four main directions of ongoing research in our laboratories, directed at the conception and demonstration of a variety of innovative configurations in nanoscale multiphoton imaging. A common feature to all of these directions appears to be the central role played by the involvement of polarization features, both in- and outgoing, moreover so in view of the tensorial aspects inherent to nonlinear schemes such second-harmonic generation, electro-optic modulation or two-photon fluorescence which will ne emphasized. These advances relate to the new domain of nonlinear ellipsometry in multiphoton imaging [1], of high relevance to fundamental aspects of nanophotonics and nanomaterial engineering as well as towards basic life science issues. The four domains to be shortly reported are: a polarization resolved second-harmonic generation in semiconductor QD’s with record small sizes in the 10-12 nm range [2] b original use of two-photon confocal polarization resolved microscopy in DNA stained by two photon fluorescent dyes in different LC phases arrangements so as to characterize these as well as ascertain the respective DNA-dye orientation (intercalant or groves [3] c elaboration and demonstration of an electrooptic confocal microscope in a highly sensitive interferometric and homodyne detection configuration allowing to map weak electric potentials such as in artificial functionalized membranes, the dynamical investigation of firing and propagation aspects of action potentials in neurones being currently the next step [4] d original plasmon based enhanced nanoscale confocal imaging involving a dual detection scheme (fluorescence imaging and ATR plasmon coupling in reflection whereby adequate preparation and switching of the incoming polarization state between radial, linear and azimuthal configurations, entail different images and plasmon enhancement levels [5].

Tungsten disulfide nanosheet and exonuclease III co-assisted amplification strategy for highly sensitive fluorescence polarization detection of DNA glycosylase activity

International Nuclear Information System (INIS)

Zhao, Jingjin; Ma, Yefei; Kong, Rongmei; Zhang, Liangliang; Yang, Wen; Zhao, Shulin

2015-01-01

Herein, we introduced a tungsten disulfide (WS 2 ) nanosheet and exonuclease III (Exo III) co-assisted signal amplification strategy for highly sensitive fluorescent polarization (FP) assay of DNA glycosylase activity. Two DNA glycosylases, uracil-DNA glycosylase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1), were tested. A hairpin-structured probe (HP) which contained damaged bases in the stem was used as the substrate. The removal of damaged bases from substrate by DNA glycosylase would lower the melting temperature of HP. The HP was then opened and hybridized with a FAM dye-labeled single strand DNA (DP), generating a duplex with a recessed 3′-terminal of DP. This design facilitated the Exo III-assisted amplification by repeating the hybridization and digestion of DP, liberating numerous FAM fluorophores which could not be adsorbed on WS 2 nanosheet. Thus, the final system exhibited a small FP signal. However, in the absence of DNA glycosylases, no hybridization between DP and HP was occurred, hampering the hydrolysis of DP by Exo III. The intact DP was then adsorbed on the surface of WS 2 nanosheet that greatly amplified the mass of the labeled-FAM fluorophore, resulting in a large FP value. With the co-assisted amplification strategy, the sensitivity was substantially improved. In addition, this method was applied to detect UDG activity in cell extracts. The study of the inhibition of UDG was also performed. Furthermore, this method is simple in design, easy in implementation, and selective, which holds potential applications in the DNA glycosylase related mechanism research and molecular diagnostics. - Highlights: • A fluorescence polarization strategy for DNA glycosylase activity detection was developed. • The present method was based on WS 2 nanosheet and exonuclease III co-assisted signal amplification. • A high sensitivity and desirable selectivity were achieved. • This method provides a promising universal platform for DNA glycosylase

Tungsten disulfide nanosheet and exonuclease III co-assisted amplification strategy for highly sensitive fluorescence polarization detection of DNA glycosylase activity

Energy Technology Data Exchange (ETDEWEB)

Zhao, Jingjin; Ma, Yefei [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004 (China); Kong, Rongmei [The Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165 (China); Zhang, Liangliang, E-mail: liangzhang319@163.com [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004 (China); Yang, Wen; Zhao, Shulin [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004 (China)

2015-08-05

Herein, we introduced a tungsten disulfide (WS{sub 2}) nanosheet and exonuclease III (Exo III) co-assisted signal amplification strategy for highly sensitive fluorescent polarization (FP) assay of DNA glycosylase activity. Two DNA glycosylases, uracil-DNA glycosylase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1), were tested. A hairpin-structured probe (HP) which contained damaged bases in the stem was used as the substrate. The removal of damaged bases from substrate by DNA glycosylase would lower the melting temperature of HP. The HP was then opened and hybridized with a FAM dye-labeled single strand DNA (DP), generating a duplex with a recessed 3′-terminal of DP. This design facilitated the Exo III-assisted amplification by repeating the hybridization and digestion of DP, liberating numerous FAM fluorophores which could not be adsorbed on WS{sub 2} nanosheet. Thus, the final system exhibited a small FP signal. However, in the absence of DNA glycosylases, no hybridization between DP and HP was occurred, hampering the hydrolysis of DP by Exo III. The intact DP was then adsorbed on the surface of WS{sub 2} nanosheet that greatly amplified the mass of the labeled-FAM fluorophore, resulting in a large FP value. With the co-assisted amplification strategy, the sensitivity was substantially improved. In addition, this method was applied to detect UDG activity in cell extracts. The study of the inhibition of UDG was also performed. Furthermore, this method is simple in design, easy in implementation, and selective, which holds potential applications in the DNA glycosylase related mechanism research and molecular diagnostics. - Highlights: • A fluorescence polarization strategy for DNA glycosylase activity detection was developed. • The present method was based on WS{sub 2} nanosheet and exonuclease III co-assisted signal amplification. • A high sensitivity and desirable selectivity were achieved. • This method provides a promising universal platform for DNA

Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

Science.gov (United States)

Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

2009-02-01

Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

Directory of Open Access Journals (Sweden)

Heidi J Chial

2010-08-01

Full Text Available Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR domain, a central pleckstrin homology (PH domain, and a C-terminal phosphotyrosine binding (PTB domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2 and heterotypic (i.e., APPL1-APPL2 manner on curved cell membranes. Furthermore, the results of our experiments

Solvent effects on the fluorescence and effective three-photon absorption of a Zn(II)-[meso-tetrakis(4-octyloxyphenyl)porphyrin

Science.gov (United States)

Wan, Yong; Xue, Yuxiong; Sheng, Ning; Rui, Guanghao; Lv, Changgui; He, Jun; Gu, Bing; Cui, Yiping

2018-06-01

The fluorescence and effective three-photon absorption (3PA) properties of Zn(II)-[meso-tetrakis(4-octyloxyphenyl)porphyrin] (labeled Zn(II)-porphyrin) dissolved in three different polar solvents were systematically investigated. The electrochemical and photophysical properties of Zn(II)-porphyrin were investigated by 1H NMR spectra, IR spectra, mass spectroscopy, and electronic absorption spectra. The fluorescence emission of Zn(II)-porphyrin in three different solvents excited at the wavelengths of 420 nm (Soret band) and 550 nm (Q-band) were analyzed. By performing Z-scan experiments with femtosecond laser pulses at a wavelength of 800 nm, the effective 3PA process of Zn(II)-porphyrin in three different solvents was observed and the underlying mechanism was discussed in detail. It is found that the fluorescence spectra slightly depend on the polarity of the solvent. Interestingly, the effective 3PA properties of Zn(II)-porphyrin strongly depend on the solvent polarity. The lower the solvent polarity is, the larger effective 3PA cross-section is. Low polar solvents are beneficial to applications of Zn(II)-porphyrin in optical limiting, photodynamic therapy, etc.

Evolution of group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer.

Science.gov (United States)

Koide, Yuichiro; Urano, Yasuteru; Hanaoka, Kenjiro; Terai, Takuya; Nagano, Tetsuo

2011-06-17

The absorption and emission wavelengths of group 14 pyronines and rhodamines, which contain silicon, germanium, or tin at the 10 position of the xanthene chromophore, showed large bathochromic shifts compared to the original rhodamines, owing to stabilization of the LUMO energy levels by σ*-π* conjugation between group 14 atom-C (methyl) σ* orbitals and a π* orbital of the fluorophore. These group 14 pyronines and rhodamines retain the advantages of the original rhodamines, including high quantum efficiency in aqueous media (Φ(fl) = 0.3-0.45), tolerance to photobleaching, and high water solubility. Group 14 rhodamines have higher values of reduction potential than other NIR light-emitting original rhodamines, and therefore, we speculated their NIR fluorescence could be controlled through the photoinduced electron transfer (PeT) mechanism. Indeed, we found that the fluorescence quantum yield (Φ(fl)) of Si-rhodamine (SiR) and Ge-rhodamine (GeR) could be made nearly equal to zero, and the threshold level for fluorescence on/off switching lies at around 1.3-1.5 V for the SiRs. This is about 0.1 V lower than in the case of TokyoGreens, in which the fluorophore is well established to be effective for PeT-based probes. That is to say, the fluorescence of SiR and GeR can be drastically activated by more than 100-fold through a PeT strategy. To confirm the validity of this strategy for developing NIR fluorescence probes, we employed this approach to design two kinds of novel fluorescence probes emitting in the far-red to NIR region, i.e., a series of pH-sensors for use in acidic environments and a Zn(2+) sensor. We synthesized these probes and confirmed that they work well.

Kinetics of bacterial fluorescence staining with 3,3'-diethylthiacyanine.

Science.gov (United States)

Thomas, Marlon S; Nuñez, Vicente; Upadhyayula, Srigokul; Zielins, Elizabeth R; Bao, Duoduo; Vasquez, Jacob M; Bahmani, Baharak; Vullev, Valentine I

2010-06-15

For more than a century, colorimetric and fluorescence staining have been the foundation of a broad range of key bioanalytical techniques. The dynamics of such staining processes, however, still remains largely unexplored. We investigated the kinetics of fluorescence staining of two gram-negative and two gram-positive species with 3,3'-diethylthiacyanine (THIA) iodide. An increase in the THIA fluorescence quantum yield, induced by the bacterial dye uptake, was the principal reason for the observed emission enhancement. The fluorescence quantum yield of THIA depended on the media viscosity and not on the media polarity, which suggested that the microenvironment of the dye molecules taken up by the cells was restrictive. The kinetics of fluorescence staining did not manifest a statistically significant dependence neither on the dye concentration, nor on the cell count. In the presence of surfactant additives, however, the fluorescence-enhancement kinetic patterns manifested species specificity with statistically significant discernibility.

Characterization of nanostructures in the live cell plasma membrane utilizing advanced single molecule fluorescence techniques

International Nuclear Information System (INIS)

Brameshuber, M.

2009-01-01

Unrevealing the detailed structure of the cellular plasma membrane at a nanoscopic length scale is the key for understanding the regulation of various signaling pathways or interaction mechanism. Hypotheses postulate the existence of nanoscopic lipid platforms in the cell membrane which are termed lipid- or membrane rafts. Based on biochemical studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. In this thesis I present an ultra-sensitive fluorescence based method which allows for the first time the direct imaging of single mobile rafts in the live cell plasma membrane. The method senses rafts by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. A special photobleaching protocol was developed and used to reduce the surface density of labeled mobile rafts down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per raft was determined by single molecule brightness analysis. For demonstration, I used the consensus markers Bodipy-GM1, a fluorescent lipid analogue, and glycosylphosphatidyl-inositol-anchored monomeric GFP. For both markers I found cholesterol-dependent association in the plasma membrane of living CHO and Jurkat T cells in the resting state, indicating the presence of mobile, stable rafts hosting these probes. I further characterized these structures by taking cell-to-cell variations under consideration. By comparing Bodipy-GM1 with mGFP-GPI homo-association upon temperature variation, two different states - a non-equilibrated and an equilibrated state - could be identified. I conclude that rafts are loaded non-randomly; the characteristic load is maintained during its lifetime in the plasma membrane of a non-activated cell. Beside these

Angle-resolved polarimetry of antenna-mediated fluorescence

NARCIS (Netherlands)

Mohtashami, A.; Osorio, C.I.; Koenderink, A.F.

2015-01-01

Optical phase-array antennas can be used to control not only the angular distribution but also the polarization of fluorescence from quantum emitters. The emission pattern of the resulting system is determined by the properties of the antenna, the properties of the emitters, and the strength of the

A cutin fluorescence pattern in developing embryos of some angiosperms

Directory of Open Access Journals (Sweden)

Ewa Szczuka

2014-01-01

Full Text Available A cuticle visualized by auramine O fluorescence appears on the developing embryos of 9 species belonging to Cruciferae, Caryophyllaceae, Plantaginaceae, Linaceae and Papilionaceae. In the investigated species the formation and extent of fluorescing and non-fluorescing embryonic areas follow a similar pattern. At first the cutin fluorescing layer is formed on the apical part of the proembryo without delimited protoderm. This layer extends and at the late globular stage envelops the embryo proper, except for a cell adjoining the suspensor. Fluorescing cutin persists during the heart stage but disappears from the torpedo embryo. During these stages there is no cutine fluorescence on suspensorial cells. Continuous cutin fluorescence appears again on the surface of the whole embryo by the late torpedo stage. Then fluorescence disappears from the radicular part of U-shaped embryos, but persists on the shoot apex, cotyledons and at least on the upper part of hypocotyl. It is assumed that polarization and nutrition of the embryo may be influenced by cuticular changes.

Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

Science.gov (United States)

Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

2007-08-17

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

Variety of Polarized Line Profiles in Interacting Supernovae

Science.gov (United States)

Hoffman, Jennifer L.; Huk, L. N.; Peters, C. L.

2013-01-01

The dense circumstellar material that creates strong emission lines in the spectra of interacting supernovae also gives rise to complex line polarization behavior. Viewed in polarized light, the emission line profiles of these supernovae encode information about the geometrical and optical characteristics of their surrounding circumstellar material (CSM) that is inaccessible by other observational techniques. To facilitate quantitative interpretation of these spectropolarimetric signatures, we have created a large grid of model polarized line profiles using a three-dimensional radiative transfer code that simulates polarization via electron and resonant/fluorescent line scattering. The simulated polarized lines take on an array of profile shapes that vary with viewing angle and CSM properties. We present the major results from the grid and investigate the dependence of polarized line profiles on CSM characteristics including temperature, optical depth, and geometry. These results will allow more straightforward interpretation of polarized line profiles in interacting supernovae than has previously been possible. This research is supported by the National Science Foundation through the AAG program and the XSEDE collaboration, and uses the resources of the Texas Advanced Computing Center.

Double-gated spectral snapshots for biomolecular fluorescence

International Nuclear Information System (INIS)

Nakamura, Ryosuke; Hamada, Norio; Ichida, Hideki; Tokunaga, Fumio; Kanematsu, Yasuo

2007-01-01

A versatile method to take femtosecond spectral snapshots of fluorescence has been developed based on a double gating technique in the combination of an optical Kerr gate and an image intensifier as an electrically driven gate set in front of a charge-coupled device detector. The application of a conventional optical-Kerr-gate method is limited to molecules with the short fluorescence lifetime up to a few hundred picoseconds, because long-lifetime fluorescence itself behaves as a source of the background signal due to insufficiency of the extinction ratio of polarizers employed for the Kerr gate. By using the image intensifier with the gate time of 200 ps, we have successfully suppressed the background signal and overcome the application limit of optical-Kerr-gate method. The system performance has been demonstrated by measuring time-resolved fluorescence spectra for laser dye solution and the riboflavin solution as a typical sample of biomolecule

MR image enhancement as a function of tissue gadolinium concentration, measured with polarized X-ray fluorescence analysis

International Nuclear Information System (INIS)

Wang, S.C.; Morita, Y.; White, D.L.; Kaufman, L.; Brasch, R.C.

1988-01-01

MR imaging contrast agents alter intensities nonlinearly relative to their tissue concentrations. To extract Gd concentrations from image intensity data, a 13-tube phantom (Gd-DTPA dilutions, 0-10/sup -2/M) was imaged (2 T, 3 mm, spin echo, 300 = msec repetition time, 15 = msec echo time, 128 X 256, four excitations). Also, 18 rats were studied with Gd-DTPA or albumin-(Gd-DTPA)/sub 19/ (nine each, three doses). Liver and renal cortex were imaged before and 10 minutes after contrast material administration, with immediate killing and harvesting, and enhancement was calculated. These samples were assayed by x-ray fluorescent excitation analysis (150-kVp beam, B/sub 4/C ceramic polarizer, Mo-Cu-Ni filter, Si[Li] detector). Gd levels as low as 0.5 ppm (--3.18 x 10/sup -6/M) could be detected in liquid or solid samples. Enhancement increased with a nonlinear relationship to Gd in the range measured. This assay for Gd permits empiric assessment of the relationship between pulse variables, intensity, and paramagnet concentration, allowing Gd values to be estimated from image intensities

Development of a novel ozone- and photo-stable HyPer5 red fluorescent dye for array CGH and microarray gene expression analysis with consistent performance irrespective of environmental conditions

Directory of Open Access Journals (Sweden)

Kille Peter

2008-11-01

Full Text Available Abstract Background Array-based comparative genomic hybridization (CGH and gene expression profiling have become vital techniques for identifying molecular defects underlying genetic diseases. Regardless of the microarray platform, cyanine dyes (Cy3 and Cy5 are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. However, combining both dyes on arrays can become problematic during summer months when ozone levels rise to near 25 parts per billion (ppb. Under such conditions, Cy5 is known to rapidly degrade leading to loss of signal from either "homebrew" or commercial arrays. Cy5 can also suffer disproportionately from dye photobleaching resulting in distortion of (Cy5/Cy3 ratios used in copy number analysis. Our laboratory has been active in fluorescent dye research to find a suitable alternative to Cy5 that is stable to ozone and resistant to photo-bleaching. Here, we report on the development of such a dye, called HyPer5, and describe its' exceptional ozone and photostable properties on microarrays. Results Our results show HyPer5 signal to be stable to high ozone levels. Repeated exposure of mouse arrays hybridized with HyPer5-labeled cDNA to 300 ppb ozone at 5, 10 and 15 minute intervals resulted in no signal loss from the dye. In comparison, Cy5 arrays showed a dramatic 80% decrease in total signal during the same interval. Photobleaching experiments show HyPer5 to be resistant to light induced damage with 3- fold improvement in dye stability over Cy5. In high resolution array CGH experiments, HyPer5 is demonstrated to detect chromosomal aberrations at loci 2p21-16.3 and 15q26.3-26.2 from three patient sample using bacterial artificial chromosome (BAC arrays. The photostability of HyPer5 is further documented by repeat array scanning without loss of detection. Additionally, HyPer5 arrays are shown to preserve sensitivity and

Excited state hydrogen bonding fluorescent probe: Role of structure and environment

Energy Technology Data Exchange (ETDEWEB)

Dey, Debarati, E-mail: debaratidey07@gmail.com [Department of Chemistry, Vidyasagar College, 39 Sankar Ghosh Lane, Kolkata 700006 (India); Sarangi, Manas Kumar [Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064 (India); Ray, Angana; Bhattacharyya, Dhananjay [Computational Science Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064 (India); Maity, Dilip Kumar [Department of Chemistry, University College of Science and Technology, 92 A.P.C. Road, Kolkata 700009 (India)

2016-05-15

An environment sensitive fluorescent probe, 11-benzoyl-dibenzo[a,c]phenazine (BDBPZ), has been synthesized and characterized that acts via excited state hydrogen bonding (ESHB). On interaction with hydrogen bond donating solvents the fluorescence intensity of BDBPZ increases abruptly with a concomitant bathochromic shift. The extent of fluorescence increment and the red-shift of λ{sub max} depend on hydrogen bond donating ability of the solvent associated. ESHB restricts the free rotation of the benzoyl group and hence blocks the non-radiative deactivation pathway. BDBPZ forms an exciplex with organic amine in nonpolar medium that readily disappears on increasing the polarity of the solvent. In polar environment the fluorescence of both the free molecule and excited state hydrogen bonded species are quenched on addition of amine unlike its parent dibenzo[a,c]phenazine (DBPZ), that remains very much inaccessible towards the solvent as well as quencher molecules due to its structure. This newly synthesized derivative BDBPZ is much more interactive due to the benzoyl group that is flanked outside the skeletal aromatic rings of DBPZ, which helps to sense the environment properly and thus shows better ESHB capacity than DBPZ.

Using fluorescent dissolved organic matter to trace and distinguish the origin of Arctic surface waters

DEFF Research Database (Denmark)

Goncalves-Araujo, Rafael; Granskog, Mats A.; Bracher, Astrid

2016-01-01

were performed in the Fram and Davis Straits, and on the east Greenland Shelf (EGS), in late summer 2012/2013. Meteoric (f(mw)), sea-ice melt, Atlantic and Pacific water fractions were determined and the fluorescence properties of dissolved organic matter (FDOM) were characterized. In Fram Strait...... and EGS, a robust correlation between visible wavelength fluorescence and f(mw) was apparent, suggesting it as a reliable tracer of polar waters. However, a pattern was observed which linked the organic matter characteristics to the origin of polar waters. At depth in Davis Strait, visible wavelength FDOM...

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay.

Science.gov (United States)

Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen

2017-01-01

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

In vivo x-ray fluorescence of lead and other toxic trace elements

International Nuclear Information System (INIS)

Chettle, D.R.

1995-01-01

The first in vivo x-ray fluorescence measurements of lead in bone used y-rays from a 57 Co source to excite Pb K x-rays. Later systems used γ-rays from 109 Cd to excite Pb K x-rays or polarized x-rays to excite Ph L x-rays. All three approaches involve an extremely low effective dose to the subject. Of the two K x-ray techniques, 109 Cd is more precise and more flexible in choice of measurement site. Pb L x-ray fluorescence (L-XRF) effectively samples lead at bone surfaces, whereas Ph K x-ray fluorescence (K-XRF) samples through the bulk of a bone. Both the polarized L-XRF and 109 Cd K-XRF achieve similar precision. Renal mercury has recently been determined using a polarized x-ray source, Both renal and hepatic cadmium can be measured using polarized x-rays in conjunction with a Si(Li) detector. Platinum and gold have been measured both by radioisotopic source excitation and by using polarized x-rays, but the latter is to be preferred. Applications of Pb K-XRF have shown that measured bone lead relates strongly to cumulative lead exposure. Secondly, biological half lives of lead in different bone types have been estimated from limited longitudinal data sets and from some cross sectional surveys. Thirdly, the effect of bone lead as an endogenous source of lead has been demonstrated and it has been shown that a majority of circulating blood lead can be mobilized from bone, rather than deriving from new exposure, in some retired lead workers. 35 refs., 5 tabs

Time-resolved fluorescence analysis of the mobile flavin cofactor

Indian Academy of Sciences (India)

Conformational heterogeneity of the FAD cofactor in -hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations ...

Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

Directory of Open Access Journals (Sweden)

Markiv Anatoliy

2011-11-01

Full Text Available Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell

Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)

NARCIS (Netherlands)

Lidke, D.S.; Nagy, P.; Barisas, B.G.; Heintzmann, R.; Post, Janine Nicole; Lidke, K.A.; Clayton, A.H.A.; Arndt-jovin, D.J.; Jovin, T.M.

2003-01-01

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow

Nuclear resonance fluorescence of {sup 203,205}Tl

Energy Technology Data Exchange (ETDEWEB)

Pfeifer, Fabian; Fritzsche, Matthias; Pietralla, Norbert; Savran, Deniz; Weller, Henry; Zweidinger, Markus [Institut fuer Kernphysik, Technische Universitaet, Darmstadt (Germany); Rusev, Gencho; Tonchev, Anton P.; Tornow, Werner [Triangle Universities Nuclear Laboratory, Duke University, Durham (United States); Zilges, Andreas [Institut fuer Kernphysik, Universitaet Koeln (Germany)

2009-07-01

In order to investigate the dipole strength distribution in Thalium isotopes we have studied Nuclear Resonance Fluorescence of a sample composed of natural Thallium (consisting of 30% {sup 203}Tl and 70% {sup 205}Tl). Unpolarized bremsstrahlung with photo energies up to 7.5 MeV was used at the High Intensity Photon Setup (HIPS) at S-DALINAC at the IKP Darmstadt. 24 fluorescent {gamma}-ray transitions were observed, 19 of them for the first time. For the assignment of the polarity of two prominent {gamma}-ray transitions, one at 4.7 MeV and one at 4.9 MeV, the polarized photon beam of the High Intensity {gamma}-ray Source (HI{gamma}S) at Duke University was used. The experiment at HI{gamma}S revealed the existence of a photo-excited state of {sup 205}Tl at an excitation energy of 4.971 MeV that exhibits a transition to the first excited state at 203 keV.

Attomolar detection of proteins via cascade strand-displacement amplification and polystyrene nanoparticle enhancement in fluorescence polarization aptasensors.

Science.gov (United States)

Huang, Yong; Liu, Xiaoqian; Huang, Huakui; Qin, Jian; Zhang, Liangliang; Zhao, Shulin; Chen, Zhen-Feng; Liang, Hong

2015-08-18

Extremely sensitive and accurate measurements of protein markers for early detection and monitoring of diseases pose a formidable challenge. Herein, we develop a new type of amplified fluorescence polarization (FP) aptasensor based on allostery-triggered cascade strand-displacement amplification (CSDA) and polystyrene nanoparticle (PS NP) enhancement for ultrasensitive detection of proteins. The assay system consists of a fluorescent dye-labeled aptamer hairpin probe and a PS NP-modified DNA duplex (assistant DNA/trigger DNA duplex) probe with a single-stranded part and DNA polymerase. Two probes coexist stably in the absence of target, and the dye exhibits relatively low FP background. Upon recognition and binding with a target protein, the stem of the aptamer hairpin probe is opened, after which the opened hairpin probe hybridizes with the single-stranded part in the PS NP-modified DNA duplex probe and triggers the CSDA reaction through the polymerase-catalyzed recycling of both target protein and trigger DNA. Throughout this CSDA process, numerous massive dyes are assembled onto PS NPs, which results in a substantial FP increase that provides a readout signal for the amplified sensing process. Our newly proposed amplified FP aptasensor enables the quantitative measurement of proteins with the detection limit in attomolar range, which is about 6 orders of magnitude lower than that of traditional homogeneous aptasensors. Moreover, this sensing method also exhibits high specificity for target proteins and can be performed in homogeneous solutions. In addition, the suitability of this method for the quantification of target protein in biological samples has also been shown. Considering these distinct advantages, the proposed sensing method can be expected to provide an ultrasensitive platform for the analysis of various types of target molecules.

Studies of radiation induced membrane damage in lymphocytes using fluorescent probes

International Nuclear Information System (INIS)

Nikesch, W.

1974-01-01

The fluorescent probes perylene (PER), 1-anilino-8-naphthalene sulfonic acid (ANS), and fluorescein diacetate (FDA) were used to investigate membrane changes caused by ionizing radiation. Probe response to various other perturbations (variation of pH, temperature, and salt concentration, and treatment with phythohemagglutinin (PHA) and saponins) was also investigated to better understand membrane-probe interactions. ANS was used to probe the membrane surface, PER to probe the membrane interior, and FDA to investigate membrane integrity. Polarization of fluorescent light from ANS and PER was used to investigate the microviscosity and order of the membrane surface and interior respectively. Irradiated cells (600 R) were shown to have a decreased rate of hydrolysis of FDA probably due to cytoplasmic changes effecting the enzymatic reaction. Also evident was an increase in loss of intracellular fluorescein and a decrease in PER polarization indicating that the cells have a decreased membrane integrity, possibly the result of an increased disorganization of the phospholipid hydrocarbon chains in the membrane interior. Experiments with PHA link the decreased membrane integrity with the eventual interphase death of the cells. In general it is shown that the fluorescent probes ANS, PER, and FDA provide useful ways to investigate order and microviscosity in the cell membrane surface and interior, membrane surface charges, internal membrane polarity changes, and membrane integrity. (U.S.)

Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Takashi Jin

2009-11-01

Full Text Available The early detection of HER2 (human epidermal growth factor receptor 2 status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC. As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs with fluorescence quantum yields of 0.23~0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission on the fluorescence image of KPL-4 cells.

Plasmonics Enhanced Smartphone Fluorescence Microscopy

KAUST Repository

Wei, Qingshan

2017-05-12

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

Plasmonics Enhanced Smartphone Fluorescence Microscopy

KAUST Repository

Wei, Qingshan; Acuna, Guillermo; Kim, Seungkyeum; Vietz, Carolin; Tseng, Derek; Chae, Jongjae; Shir, Daniel; Luo, Wei; Tinnefeld, Philip; Ozcan, Aydogan

2017-01-01

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

Near-infrared fluorescence glucose sensing based on glucose/galactose-binding protein coupled to 651-Blue Oxazine

Energy Technology Data Exchange (ETDEWEB)

Khan, Faaizah; Pickup, John C., E-mail: john.pickup@kcl.ac.uk

2013-08-30

Highlights: •We showed that the NIR fluorophore, 651-Blue Oxazine, is solvatochromic (polarity sensitive). •Blue Oxazine was covalently attached to mutants of glucose/galactose-binding protein (GBP). •Fluorescence intensity of GBP-Blue Oxazine increased with addition of glucose. •Fluorescence from bead-immobilised GBP-Blue Oxazine was detectable through skin in vitro. •This shows proof-of-concept for non-invasive glucose sensing using GBP-Blue Oxazine. -- Abstract: Near-infrared (NIR) fluorescent dyes that are environmentally sensitive or solvatochromic are useful tools for protein labelling in in vivo biosensor applications such as glucose monitoring in diabetes since their spectral properties are mostly independent of tissue autofluorescence and light scattering, and they offer potential for non-invasive analyte sensing. We showed that the fluorophore 651-Blue Oxazine is polarity-sensitive, with a marked reduction in NIR fluorescence on increasing solvent polarity. Mutants of glucose/galactose-binding protein (GBP) used as the glucose receptor were site-specifically and covalently labelled with Blue Oxazine using click chemistry. Mutants H152C/A213R and H152C/A213R/L238S showed fluorescence increases of 15% and 21% on addition of saturating glucose concentrations and binding constants of 6 and 25 mM respectively. Fluorescence responses to glucose were preserved when GBP-Blue Oxazine was immobilised to agarose beads, and the beads were excited by NIR light through a mouse skin preparation studied in vitro. We conclude GBP-Blue Oxazine shows proof-of-concept as a non-invasive continuous glucose sensing system.

PsbS-specific zeaxanthin-independent changes in fluorescence emission spectrum as a signature of energy-dependent non-photochemical quenching in higher plants.

Science.gov (United States)

Zulfugarov, Ismayil S; Tovuu, Altanzaya; Dogsom, Bolormaa; Lee, Chung Yeol; Lee, Choon-Hwan

2010-05-01

The PsbS protein of photosystem II is necessary for the development of energy-dependent quenching of chlorophyll (Chl) fluorescence (qE), and PsbS-deficient Arabidopsis plant leaves failed to show qE-specific changes in the steady-state 77 K fluorescence emission spectra observed in wild-type leaves. The difference spectrum between the quenched and un-quenched states showed a negative peak at 682 nm. Although the level of qE development in the zeaxanthin-less npq1-2 mutant plants, which lacked violaxanthin de-epoxidase enzyme, was only half that of wild type, there were no noticeable changes in this qE-dependent difference spectrum. This zeaxanthin-independent DeltaF682 signal was not dependent on state transition, and the signal was not due to photobleaching of pigments either. These results suggest that DeltaF682 signal is formed due to PsbS-specific conformational changes in the quenching site of qE and is a new signature of qE generation in higher plants.

Exchange of rotor components in functioning bacterial flagellar motor

International Nuclear Information System (INIS)

Fukuoka, Hajime; Inoue, Yuichi; Terasawa, Shun; Takahashi, Hiroto; Ishijima, Akihiko

2010-01-01

The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP, and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007 s -1 , meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.

Spitzenkorper, exocyst, and polarisome components in Candida albicans hyphae show different patterns of localization and have distinct dynamic properties.

Science.gov (United States)

Jones, Laura A; Sudbery, Peter E

2010-10-01

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.

Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis.

Science.gov (United States)

Schulze, Philipp; Ludwig, Martin; Belder, Detlev

2008-12-01

A high intensity 266 nm continuous wave (cw-) laser developed for material processing was utilised as an excitation source for sensitive native fluorescence detection of unlabelled compounds in MCE. This 120 mW laser was attached via an optical fibre into a commercial epifluorescence microscope. With this MCE set-up we evaluated the impact of laser power on the S/N of aromatic compounds as well as of proteins. Compared with a previous work which used a 4 mW pulsed laser for excitation, improved S/N for small aromatics and to a lesser extent for proteins could be attained. The LOD of the system was determined down to 24 ng/mL for serotonin (113 nM), 24 ng/mL for propranolol (81 nM), 80 ng/mL for tryptophan (392 nM) and 80 ng/mL for an aromatic diol (475 nM). Sensitive protein detection was obtained at concentrations of 5 microg/mL for lysocyme, trypsinogen and chymotrypsinogen (340, 208 and 195 nM, respectively). Finally, a comparison of the cw- with a pulsed 266 nm laser, operating at the same average power, showed a higher attainable sensitivity of the cw-laser. This can be attributed to fluorescence saturation and photobleaching effects of the pulsed laser at high pulse energies.

From Dark to Light to Fluorescence Resonance Energy Transfer (FRET): Polarity-Sensitive Aggregation-Induced Emission (AIE)-Active Tetraphenylethene-Fused BODIPY Dyes with a Very Large Pseudo-Stokes Shift.

Science.gov (United States)

Şen, Esra; Meral, Kadem; Atılgan, Serdar

2016-01-11

The work presented herein is devoted to the fabrication of large Stokes shift dyes in both organic and aqueous media by combining dark resonance energy transfer (DRET) and fluorescence resonance energy transfer (FRET) in one donor-acceptor system. In this respect, a series of donor-acceptor architectures of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes substituted by one, two, or three tetraphenylethene (TPE) luminogens were designed and synthesised. The photophysical properties of these three chromophore systems were studied to provide insight into the nature of donor-acceptor interactions in both THF and aqueous media. Because the generation of emissive TPE donor(s) is strongly polarity dependent, due to its aggregation-induced emission (AIE) feature, one might expect the formation of appreciable fluorescence emission intensity with a very large pseudo-Stokes shift in aqueous media when considering FRET process. Interestingly, similar results were also recorded in THF for the chromophore systems, although the TPE fragment(s) of the dyes are non-emissive. The explanation for this photophysical behaviour lies in the DRET. This is the first report on combining two energy-transfer processes, namely, FRET and DRET, in one polarity-sensitive donor-acceptor pair system. The accuracy of the dark-emissive donor property of the TPE luminogen is also presented for the first time as a new feature for AIE phenomena. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CDOM Sources and Photobleaching Control Quantum Yields for Oceanic DMS Photolysis

KAUST Repository

Galí, Martí

2016-11-14

Photolysis is a major removal pathway for the biogenic gas dimethylsulfide (DMS) in the surface ocean. Here we tested the hypothesis that apparent quantum yields (AQY) for DMS photolysis varied according to the quantity and quality of its photosensitizers, chiefly chromophoric dissolved organic matter (CDOM) and nitrate. AQY compiled from the literature and unpublished studies ranged across 3 orders of magnitude at the 330 nm reference wavelength. The smallest AQY(330) were observed in coastal waters receiving major riverine inputs of terrestrial CDOM (0.06-0.5 m3 (mol quanta)-1). In open-ocean waters, AQY(330) generally ranged between 1 and 10 m3 (mol quanta)-1. The largest AQY(330), up to 34 m3 (mol quanta)-1), were seen in the Southern Ocean potentially associated with upwelling. Despite the large AQY variability, daily photolysis rate constants at the sea surface spanned a smaller range (0.04-3.7 d-1), mainly because of the inverse relationship between CDOM absorption and AQY. Comparison of AQY(330) with CDOM spectral signatures suggests there is an interplay between CDOM origin (terrestrial versus marine) and photobleaching that controls variations in AQYs, with a secondary role for nitrate. Our results can be used for regional or large-scale assessment of DMS photolysis rates in future studies.

CDOM Sources and Photobleaching Control Quantum Yields for Oceanic DMS Photolysis.

Science.gov (United States)

Galí, Martí; Kieber, David J; Romera-Castillo, Cristina; Kinsey, Joanna D; Devred, Emmanuel; Pérez, Gonzalo L; Westby, George R; Marrasé, Cèlia; Babin, Marcel; Levasseur, Maurice; Duarte, Carlos M; Agustí, Susana; Simó, Rafel

2016-12-20

Photolysis is a major removal pathway for the biogenic gas dimethylsulfide (DMS) in the surface ocean. Here we tested the hypothesis that apparent quantum yields (AQY) for DMS photolysis varied according to the quantity and quality of its photosensitizers, chiefly chromophoric dissolved organic matter (CDOM) and nitrate. AQY compiled from the literature and unpublished studies ranged across 3 orders of magnitude at the 330 nm reference wavelength. The smallest AQY(330) were observed in coastal waters receiving major riverine inputs of terrestrial CDOM (0.06-0.5 m 3 (mol quanta) -1 ). In open-ocean waters, AQY(330) generally ranged between 1 and 10 m 3 (mol quanta) -1 . The largest AQY(330), up to 34 m 3 (mol quanta) -1 ), were seen in the Southern Ocean potentially associated with upwelling. Despite the large AQY variability, daily photolysis rate constants at the sea surface spanned a smaller range (0.04-3.7 d -1 ), mainly because of the inverse relationship between CDOM absorption and AQY. Comparison of AQY(330) with CDOM spectral signatures suggests there is an interplay between CDOM origin (terrestrial versus marine) and photobleaching that controls variations in AQYs, with a secondary role for nitrate. Our results can be used for regional or large-scale assessment of DMS photolysis rates in future studies.

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Energy Technology Data Exchange (ETDEWEB)

Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen (UMASS, MED); (Pfizer)

2017-09-21

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (K_D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

Study of ciclosporine blood levels in patients after kidney or bone-marrow transplantation. Comparison between the two methods, fluorescence polarization immunoassay and radioimmunoassay

International Nuclear Information System (INIS)

Sadeg, N.; Pham Huy, C.; Claude, J.R.; Postaire, M.; Lebrec, H.; Hamon, M.; Broyer, M.; Gagnadoux, M.F.; Fischer, A.

1989-01-01

The apparition of ciclosporine, immunodepressive drug, has largely improved the organ transplantations. However, the range of blood concentrations must be defined to allow the efficacity of ciclosporine therapy and to avoid toxic reactions, because there are very important variations for a same dosage according to the individuals and the diseases. Relative to the low concentrations to be determined (about one hundred ng/ml), the most useful methods for ciclosporine measurement are based on immunochemical assays. This work compares the two methods: radioimmunoassay (RIA) and fluorescence polarization immunoassay (FPIA) simultaneously performed on several hundred samples. A very significant correlation exists between the two techniques (r = 0.80). The advantages of immunofluorescent assay consists in rapidity, sensibility and facility to realize emergency analysis [fr

Fluorescent glycosidase inhibiting 1,5-dideoxy-1,5-iminoalditols

DEFF Research Database (Denmark)

Greimel, Peter; Häusler, Herwig; Lundt, Inge

2006-01-01

1,5-Dideoxy-1,5-iminoalditols of various configurations as well as isofagomine were N-alkylated with non-polar straight chain spacer-arms by a set of simple standard procedures. The spacer-arms’ terminal functional groups, primary amines, were employed to introduce fluorescent tags such as dansyl...

Polarized spectral properties of Sm3+:LiYF4 crystal

International Nuclear Information System (INIS)

Wang, G.Q.; Lin, Y.F.; Gong, X.H.; Chen, Y.J.; Huang, J.H.; Luo, Z.D.; Huang, Y.D.

2014-01-01

A trivalent samarium-doped LiYF 4 single crystal was grown by the vertical Bridgman technique. Its polarized absorption and fluorescence spectra and fluorescence decay curves were recorded at room temperature. On the basis of the Judd–Ofelt theory, the spectral parameters of the Sm 3+ :LiYF 4 crystal were calculated. The emission cross sections for the 4 G 5/2 → 6 H J (J=5/2, 7/2. 9/2, and 11/2) transitions of special interest for visible laser application were obtained by the Fuchtbauer–Ladenburg formula. -- Highlights: • Polarized spectral properties of Sm 3+ :LiYF 4 crystal at room temperature were analyzed in detail. • The emission cross sections for the transitions of special interest for visible laser application are calculated. • Sm 3+ :LiYF 4 is a promising laser material for 401 nm GaN LD pumped 605 nm visible laser

Nine New Fluorescent Probes

Science.gov (United States)

Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

1989-06-01

Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

Polarized spectroscopic properties of Nd3+-doped KGd(WO4)2 single crystal

International Nuclear Information System (INIS)

Chen Yujin; Lin Yanfu; Gong Xinghong; Tan Qiguang; Zhuang Jian; Luo Zundu; Huang Yidong

2007-01-01

The polarized absorption spectra, infrared fluorescence spectra, upconversion visible fluorescence spectra, and fluorescence decay curve of orientated Nd 3+ :KGd(WO 4 ) 2 crystal were measured at room-temperature. Some important spectroscopic parameters were investigated in detail in the framework of the Judd-Ofelt theory and the Fuchtbauer-Ladenburg formula. The effect of the crystal structure on the spectroscopic properties of the Nd 3+ ions was analyzed. The relation among the spectroscopic parameters and the laser performances of the Nd 3+ :KGd(WO 4 ) 2 crystal was discussed

Viability of exploiting L-shell fluorescence for X-ray polarimetry

Energy Technology Data Exchange (ETDEWEB)

Weisskopf, M C; Elsner, R F; Ramsey, B D [National Aeronautics and Space Administration, Huntsville, AL (USA). Space Sciences Lab.; Sutherland, P G [McMaster Univ., Hamilton, Ontario (Canada). Dept. of Physics

1985-05-15

It has been suggested that one may build an X-ray polarimeter by exploiting the polarization dependence of the angular distribution of L-shell fluorescence photons. In this paper we examine, theoretically, the sensitivity of this approach to polarimetry. We apply our calculations to several detection schemes using imaging proportional counters that would have direct application in X-ray astronomy. We find, however, that the sensitivity of this method for measuring X-ray polarization is too low to be of use for other than laboratory applications.

Fluorescence properties of 6-aryl-2‧-deoxy-furanouridine and -pyrrolocytidine and their derivatives

Science.gov (United States)

Ro, Jong Jin; Go, Gui Han; Wilhelmsson, L. Marcus; Hyean Kim, Byeang

2018-01-01

2‧-deoxyfuranouridine derivatives presenting various aryl groups have been synthesized through Cu(I)-catalyzed intramolecular cyclizations. Moreover, corresponding pyrrolo-dC derivatives have been synthesized and both families of compounds thoroughly characterized using UV/vis and fluorescence spectroscopy as well as time-dependent density functional theory calculations. The photophysical characterization, show that our newly synthesized derivatives of the important pyrrolo-dC family have high fluorescence quantum yields (QYs) and brightness values. Pyrrolo-dC derivative, 3a, shows an environment sensitive QY of up to >60% and brightness of almost 3000, in low polarity solvents and excitation and emission maxima between 365-381 nm and 479-510 nm, respectively, in solvents of different polarities. Two other derivatives, 3b and 3c, show high QYs and brightness values of up to 3300 that are fairly insensitive to their microenvironment. These promising photophysical features suggest future applicability as fluorescent nucleobase analogs.

Molecular Level Understanding of Sodium Dodecyl Sulfate (SDS) Induced Sol-Gel Transition of Pluronic F127 Using Fisetin as a Fluorescent Molecular Probe.

Science.gov (United States)

Mishra, Jhili; Swain, Jitendriya; Mishra, Ashok Kumar

2018-01-11

The thermoreversible sol-gel transition of pluronic F127 is markedly altered even with addition of submicellar concentration of sodium dodecyl sulfate (SDS) surfactant. Multiple fluorescence parameters like fluorescence intensity, fluorescence anisotropy and fluorescence lifetime of both the prototropic forms (anion (A - *) and phototautomer FT*) of the photoprototropic fluorescent probe fisetin has been efficiently used to understand the molecular level properties like polarity and microviscosity of the PF127-SDS system as a function of temperature. The SDS-induced increase in the interfacial hydrophobicity level is seen to affect the sol-gel phase transition of PF127 (21-18 °C). The E T (30) polarity parameter value of anionic emission of fisetin suggests that there is a considerable decrease in the polarity of the PF127 medium with increase in temperature and with the addition of SDS. The microviscosity progressively increases from ∼5 mPa s (sol state, 10 °C) to ∼22.01 mPa s (gel state 35 °C) in aqueous solution of PF127. The variation in microviscosity with addition of SDS in PF127-SDS mixed system is significant in sol phase whereas in gel phase this variation is significantly less. Temperature dependent fluorescence lifetime of FT* indicates that there is heterogeneity in distribution of fisetin molecules at different domains of PF127. This work also show-cases the sensitivity of fisetin toward change in polarity and change in sol-gel transition temperature of copolymer PF127 with variation in temperature (both forward and reverse directions) and SDS.

Analysis of signal to background ratio in synchrotron radiation X-ray fluorescence

International Nuclear Information System (INIS)

Sakurai, Kenji; Gohshi, Yohichi; Iida, Atsuo.

1988-01-01

The signal to background (S/B) ratio in energy dispersive X-ray fluorescence using synchrotron radiation (SR) was quantitatively analyzed. The S/B ratio, which has been significantly improved by taking advantage of the polarized nature of SR, was found to be strongly dependent on geometrical factors of the measurement system. From the analysis on the origin of the scattered background, the dependence of the S/B ratio on the geometry was quantitatively explained, mainly by the polarization properties of SR. Experimental conditions could be optimized by adjusting the degree of polarization of the incident beam and the detector solid angle. (author)

Development of a screening fluorescence polarization immunoassay for the simultaneous detection of fumonisins B₁ and B₂ in maize.

Science.gov (United States)

Li, Chenglong; Mi, Tiejun; Conti, Gea Oliveri; Yu, Qing; Wen, Kai; Shen, Jianzhong; Ferrante, Margherita; Wang, Zhanhui

2015-05-27

This paper reports the development of a screening fluorescence polarization immunoassay (FPIA) for the simultaneous detection of fumonisins B1 (FB1) and B2 (FB2) in maize. Three FB1 tracers including FB1-fluorescein isothiocyanate isomer I (FB1-FITC), FB1-5-([4,6-dichlorotriazine-2-yl]amino)-fluorescein (FB1-5-DTAF), and FB1-Texas Red-X succinimidyl ester (FB1-TRX) were synthesized and studied to select appropriate tracer-antibody pairs using seven previously produced monoclonal antibodies (mAbs). An FPIA employing the pair of FB1-FITC and mAb 4B9 showing 98.9% cross-reactivity (CR) toward FB2 was used to simultaneously detect FB1 and FB2. Maize flour samples were extracted with methanol/water (2:3, v/v). After optimization, the FPIA revealed a limit of detection (LOD) of 157.4 μg/kg for FB1 and an LOD of 290.6 μg/kg for FB2, respectively. Recoveries were measured for spiked samples of FB1 or FB2 separately, ranging from 84.7 to 93.6%, with a coefficient of variation (CV) of fumonisins in maize.

Development of a novel combined fluorescence and reflectance spectroscopy system for guiding high-grade glioma resections: confirmation of capability in lab experiments

Science.gov (United States)

Mousavi, Monirehalsadat; Xie, Haiyan; Xie, Zhiyuan; Brydegaard, Mikkel; Axelsson, Johan; Andersson-Engels, Stefan

2013-11-01

Total resection of glioblastoma multiform (GBM), the most common and aggressive malignant brain tumor, is challenging among other things due to difficulty in intraoperative discrimination between normal and residual tumor cells. This project demonstrates the potential of a system based on a combination of autofluorescence and diffuse reflectance spectroscopy to be useful as an intraoperative guiding tool. In this context, a system based on 5 LEDs coupled to optical fibers was employed to deliver UV/visible light to the sample sequentially. Remitted light from the tissue; including diffuse reflected and fluorescence of endogenous and exogenous fluorophores, as well as its photobleaching product, is transmitted to one photodiode and four avalanche photodiodes. This instrument has been evaluated with very promising results by performing various tissue-equivalent phantom laboratory and clinical studies on skin lesions.

Mapping the Local Organization of Cell Membranes Using Excitation-Polarization-Resolved Confocal Fluorescence Microscopy

OpenAIRE

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-01-01

International audience; Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive t...

Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy.

Science.gov (United States)

Goudsmits, Joris M H; van Oijen, Antoine M; Slotboom, Dirk J

2017-01-01

Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data. © 2017 Elsevier Inc. All rights reserved.

A novel fluorescent array for mercury (II) ion in aqueous solution with functionalized cadmium selenide nanoclusters

International Nuclear Information System (INIS)

Chen Jinlong; Gao Yingchun; Xu, ZhiBing; Wu, GenHua; Chen, YouCun; Zhu, ChangQing

2006-01-01

Mono-disperse CdSe nanoclusters have been prepared facilely and functionalized with L-cysteine through two steps by using safe and low cost substances. They are water-soluble and biocompatible. Especially these functionalized quantum dots can be stably soluble in water more than for 30 days, and the intensity of fluorescence and absorbance was decreased less than 15% of fresh prepared CdSe colloids. These functionalized CdSe QDs exhibited strong specific affinity for mercury (II) through QDs interface functional groups. Based on the quenching of fluorescence signals of functionalized CdSe QDs at 530 nm and no obvious wavelength shift or no new emission band in present of Hg (II) at pH 7.75 of phosphate buffer solution, a simple, rapid and specific array for Hg (II) was proposed. In comparison with conventional organic fluorophores, these nanoparticles are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimum conditions, the response of linearly proportional to the concentration of Hg (II) between 0 and 2.0 x 10 -6 mol L -1 , and the limit of detection is 6.0 x 10 -9 mol L -1 . The relative standard deviation of six replicate measurements is 1.8% for 1.0 x 10 -7 mol L -1 Hg (II). The mechanism of reaction is also discussed. The proposed method was successfully applied for Hg (II) detection in four real samples with a satisfactory result that was obtained by cold vapor atomic fluorescence spectrometry (CV-AFS)

Quantitative measurement of brightness from living cells in the presence of photodepletion.

Directory of Open Access Journals (Sweden)

Kwang-Ho Hur

Full Text Available The brightness of fluorescently labeled proteins provides an excellent marker for identifying protein interactions in living cells. Quantitative interpretation of brightness, however, hinges on a detailed understanding of the processes that affect the signal fluctuation of the fluorescent label. Here, we focus on the cumulative influence of photobleaching on brightness measurements in cells. Photobleaching within the finite volume of the cell leads to a depletion of the population of fluorescently labeled proteins with time. The process of photodepletion reduces the fluorescence signal which biases the analysis of brightness data. Our data show that even small reductions in the signal can introduce significant bias into the analysis of the data. We develop a model that quantifies the bias and introduce an analysis method that accurately determines brightness in the presence of photodepletion as verified by experiments with mammalian and yeast cells. In addition, photodepletion experiments with the fluorescent protein EGFP reveal the presence of a photoconversion process, which leads to a marked decrease in the brightness of the EGFP protein. We also identify conditions where the effect of EGFP's photoconversion on brightness experiments can be safely ignored.

Preparation and application of new fluorescein-labeled fumonisins B1 in fluorescence polarization analysis technique

Science.gov (United States)

Objective: To prepare a new fluorescent tracer against common mycotoxins such as fumonisin B1 in order to replace 6-(4,6-Dichlorotriazinyl) aminofluorescein (6-DTAF), an expensive marker, and to develop a technique for quick detection of fumonisin B1 based on the principle of fluorescence polarizati...

Photodegradation and polarization properties of vertical external surface-emitting organic laser

International Nuclear Information System (INIS)

Leang, Tatiana

2014-01-01

Although organic solid-state dye lasers can provide wavelength tunability in the whole visible spectrum and offers perspectives of low-cost compact lasers, they are still limited by several drawbacks, especially photodegradation. The geometry of a Vertical External Cavity Surface-emitting Organic Laser (VECSOL) enables organic lasers to reach high energies, excellent conversion efficiencies and good beam quality, it also enables an external control on many parameters, a feature that we have used here to study the photodegradation phenomenon as well as some polarization properties of organic solid-state lasers. In the first part of this thesis, we studied the lifetime of the laser upon varying several parameters (pump pulse-width, repetition rate, output coupling,...) and we found that the intracavity laser intensity, independently of the pump intensity, had a major on photodegradation rate. Moreover, we observed that the profile of the laser beam was also degrading with time: while it is Gaussian in the beginning it gradually shifts to an annular shape. In the second part, we investigated the polarization properties of VECSOLs, with a special emphasis on fluorescence properties of some typical dyes used in lasers. The crucial role played by resonant non-radiative energy transfers between dye molecules (HOMO-FRET) is evidenced and enables explaining the observed fluorescence depolarization, compared to the expected limiting fluorescence anisotropy. Energy transfers happen to play a negligible role above laser threshold, as the organic laser beam is shown to be linearly polarized in a wide range of experimental conditions when excitation occurs in the first singlet state. (author) [fr

Modelling spatio-temporal interactions within the cell

Indian Academy of Sciences (India)

Prakash

Cell signalling pathways make up the regulatory systems of mammalian cells. ... This organization makes it possible to study the signalling networks in a modelling ..... energy transfer (FRET) and fluorescence recovery after photobleaching ...

Status report of the S-DALINAC polarized electron injector SPIN at Darmstadt

Energy Technology Data Exchange (ETDEWEB)

Eckardt, Christian; Bahlo, Thore; Bangert, Phillip; Barday, Roman; Bonnes, Uwe; Brunken, Marco; Eichhorn, Ralf; Enders, Joachim; Platz, Markus; Poltoratska, Yuliya; Roth, Markus; Schneider, Fabian; Wagner, Markus; Weber, Antje; Zwicker, Benjamin [Institut fuer Kernphysik, Technische Universitaet Darmstadt (Germany); Ackermann, Wolfgang; Mueller, Wolfgang F.O.; Weiland, Thomas [Institut fuer Theorie Elektromagnetischer Felder, Technische Universitaet Darmstadt (Germany)

2010-07-01

At the superconducting 130 MeV Darmstadt electron linac S-DALINAC a source of polarized electrons is being installed. Polarized electrons are produced by photoemission from a negative electron affinity strained superlattice GaAs cathode and preaccelerated to 100 keV. With a Wien filter and Mott polarimeter in the beam line the polarization is manipulated and measured. For beam diagnostics wire scanners, fluorescent screens and a coaxial Faraday cup are included. To measure the beam polarization at higher energies, a 5-10 MeV Mott polarimeter and a 50-130 MeV Moeller polarimeter as well as a Compton transmission polarimeter will be installed. We report on the status of the implementation and show plans for future development and experiments.

Evidence for the TICT mediated nonradiative deexcitation process for the excited coumarin-1 dye in high polarity protic solvents

Energy Technology Data Exchange (ETDEWEB)

Barik, Atanu [Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Center, Trombay, Mumbai 400 085 (India); Kumbhakar, Manoj [Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Center, Trombay, Mumbai 400 085 (India); Nath, Sukhendu [Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Center, Trombay, Mumbai 400 085 (India); Pal, Haridas [Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Center, Trombay, Mumbai 400 085 (India)

2005-08-29

Photophysical properties of coumarin-1 (C1) dye in different protic solvents have been investigated using steady-state and time-resolved fluorescence measurements. Correlation of the Stokes' shifts ({delta}{nu}-bar ) with the solvent polarity ({delta}f) suggests the intramolecular charge transfer (ICT) character for the dye fluorescent state. Fluorescence quantum yields ({phi}{sub f}) and lifetimes ({tau}{sub f}) of the dye show an abrupt reduction in high polarity solvents having {delta}f >{approx}0.28. In these solvents {tau}{sub f} is seen to be strongly temperature dependent, though it is temperature independent in solvents with {delta}f <{approx}0.28. It is inferred that in high polarity protic solvents there is a participation of an additional nonradiative decay process via the involvement of twisted intramolecular charge transfer (TICT) state. Unlike present results, no involvement of TICT state was observed even in strongly polar aprotic solvent like acetonitrile. It is indicated that the intermolecular hydrogen bonding of the dye with protic solvents in addition with the solvent polarity helps in the stabilization of the TICT state for C1 dye. Unlike most TICT molecules, the activation barrier ({delta}E{sub a}) for the TICT mediated nonradiative process for C1 dye is seen to increase with solvent polarity. This is rationalized on the basis of the assumption that the TICT to ground state conversion is the activation-controlled rate-determining step for the present system than the usual ICT to TICT conversion as encountered for most other TICT molecules.

Optical characterization and polarization calibration for rigid endoscopes

Science.gov (United States)

Garcia, Missael; Gruev, Viktor

2017-02-01

Polarization measurements give orthogonal information to spectral images making them a great tool in the characterization of environmental parameters in nature. Thus, polarization imagery has proven to be remarkably useful in a vast range of biomedical applications. One such application is the early diagnosis of flat cancerous lesions in murine colorectal tumor models, where polarization data complements NIR fluorescence analysis. Advances in nanotechnology have led to compact and precise bio-inspired imaging sensors capable of accurately co-registering multidimensional spectral and polarization information. As more applications emerge for these imagers, the optics used in these instruments get very complex and can potentially compromise the original polarization state of the incident light. Here we present a complete optical and polarization characterization of three rigid endoscopes of size 1.9mm x 10cm (Karl Storz, Germany), 5mm x 30cm, and 10mm x 33cm (Olympus, Germany), used in colonoscopy for the prevention of colitis-associated cancer. Characterization results show that the telescope optics act as retarders and effectively depolarize the linear component. These incorrect readings can cause false-positives or false-negatives leading to an improper diagnosis. In this paper, we offer a polarization calibration scheme for these endoscopes based on Mueller calculus. By modeling the optical properties from training data as real-valued Mueller matrices, we are able to successfully reconstruct the initial polarization state acquired by the imaging system.

MitoGen: A Framework for Generating 3D Synthetic Time-Lapse Sequences of Cell Populations in Fluorescence Microscopy.

Science.gov (United States)

Svoboda, David; Ulman, Vladimir

2017-01-01

The proper analysis of biological microscopy images is an important and complex task. Therefore, it requires verification of all steps involved in the process, including image segmentation and tracking algorithms. It is generally better to verify algorithms with computer-generated ground truth datasets, which, compared to manually annotated data, nowadays have reached high quality and can be produced in large quantities even for 3D time-lapse image sequences. Here, we propose a novel framework, called MitoGen, which is capable of generating ground truth datasets with fully 3D time-lapse sequences of synthetic fluorescence-stained cell populations. MitoGen shows biologically justified cell motility, shape and texture changes as well as cell divisions. Standard fluorescence microscopy phenomena such as photobleaching, blur with real point spread function (PSF), and several types of noise, are simulated to obtain realistic images. The MitoGen framework is scalable in both space and time. MitoGen generates visually plausible data that shows good agreement with real data in terms of image descriptors and mean square displacement (MSD) trajectory analysis. Additionally, it is also shown in this paper that four publicly available segmentation and tracking algorithms exhibit similar performance on both real and MitoGen-generated data. The implementation of MitoGen is freely available.

Synthesis and spectroscopic characterization of a fluorescent pyrrole derivative containing electron acceptor and donor groups

Science.gov (United States)

Almeida, A. K. A.; Monteiro, M. P.; Dias, J. M. M.; Omena, L.; da Silva, A. J. C.; Tonholo, J.; Mortimer, R. J.; Navarro, M.; Jacinto, C.; Ribeiro, A. S.; de Oliveira, I. N.

2014-07-01

The synthesis and fluorescence characterization of a new pyrrole derivative (PyPDG) containing the electron donor-acceptor dansyl substituent is reported. The effects of temperature and solvent polarity on the steady-state fluorescence of this compound are investigated. Our results show that PyPDG exhibits desirable fluorescent properties which makes it a promising candidate to be used as the photoactive material in optical thermometry and thermography applications. Further, the electrochemical and emission properties of polymeric films obtained from the oxidation polymerization of PyPDG are also analyzed.

Combining polar organic chemical integrative samplers (POCIS) with toxicity testing to evaluate pesticide mixture effects on natural phototrophic biofilms

International Nuclear Information System (INIS)

Pesce, Stephane; Morin, Soizic; Lissalde, Sophie; Montuelle, Bernard; Mazzella, Nicolas

2011-01-01

Polar organic chemical integrative samplers (POCIS) are valuable tools in passive sampling methods for monitoring polar organic pesticides in freshwaters. Pesticides extracted from the environment using such methods can be used to toxicity tests. This study evaluated the acute effects of POCIS extracts on natural phototrophic biofilm communities. Our results demonstrate an effect of POCIS pesticide mixtures on chlorophyll a fluorescence, photosynthetic efficiency and community structure. Nevertheless, the range of biofilm responses differs according to origin of the biofilms tested, revealing spatial variations in the sensitivity of natural communities in the studied stream. Combining passive sampler extracts with community-level toxicity tests offers promising perspectives for ecological risk assessment. - Research highlights: → Polar organic chemical integrative samplers (POCIS) were used for monitoring polar organic pesticides in a contaminated river. → The acute effects of POCIS extracts were tested on natural phototrophic biofilm communities. → POCIS pesticide mixtures affected chlorophyll a fluorescence, photosynthetic efficiency and community structure. → Biofilm responses differed according to origin of the biofilms tested, revealing variations in the sensitivity of natural communities. → Combining passive sampler extracts with community-level toxicity tests offers promising perspectives for ecological risk assessment. - Pesticide mixtures extracted from POCIS can affect chl a fluorescence, photosynthetic efficiency and community structure of natural biofilms.

Fluorescent nanodiamond and lanthanide labelled in situ hybridization for the identification of RNA transcripts in fixed and CLARITY-cleared central nervous system tissues (Conference Presentation)

Science.gov (United States)

Parker, Lindsay M.; Staikopoulos, Vicky; Cordina, Nicole M.; Sayyadi, Nima; Hutchinson, Mark R.; Packer, Nicolle H.

2016-03-01

Despite significant advancement in the methodology used to conjugate, incorporate and visualize fluorescent molecules at the cellular and tissue levels, biomedical imaging predominantly relies on the limitations of established fluorescent molecules such as fluorescein, cyanine and AlexaFluor dyes or genetic incorporation of fluorescent proteins by viral or other means. These fluorescent dyes and conjugates are highly susceptible to photobleaching and compete with cellular autofluorescence, making biomedical imaging unreliable, difficult and time consuming in many cases. In addition, some proteins have low copy numbers and/or poor antibody recognition, further making detection and imaging difficult. We are developing better methods for imaging central nervous system neuroinflammatory markers using targeted mRNA transcripts labelled with fluorescent nanodiamonds or lanthanide chelates. These tags have increased signal and photostability and can also discriminate against tissue/cell autofluorescence. Brains and spinal cords from BALB/c mice with a chronic constriction model of neuropathic pain (neuroinflammation group) or that have undergone sham surgeries (control group) were collected. A subset of brains and spinal cords were perfused and fixed with paraformaldehyde (n=3 sham and n=3 pain groups) prior to sectioning and in situ hybridization using nanodiamond or lanthanide chelate conjugated complementary RNA probes. Another subset of brains and spinal cords from the same cohort of animals were perfused and processed for CLARITY hydrogel based clearing prior to in situ hybridization with the same probes. We will present our findings on the photostability, sensitivity and discrimination from background tissue autofluorescence of our novel RNA probes, compared to traditional fluorophore tags.

Spatio-seasonal variability of chromophoric dissolved organic matter absorption and responses to photobleaching in a large shallow temperate lake

Science.gov (United States)

Encina Aulló-Maestro, María; Hunter, Peter; Spyrakos, Evangelos; Mercatoris, Pierre; Kovács, Attila; Horváth, Hajnalka; Preston, Tom; Présing, Mátyás; Torres Palenzuela, Jesús; Tyler, Andrew

2017-03-01

The development and validation of remote-sensing-based approaches for the retrieval of chromophoric dissolved organic matter (CDOM) concentrations requires a comprehensive understanding of the sources and magnitude of variability in the optical properties of dissolved material within lakes. In this study, spatial and seasonal variability in concentration and composition of CDOM and the origin of its variation was studied in Lake Balaton (Hungary), a large temperate shallow lake in central Europe. In addition, we investigated the effect of photobleaching on the optical properties of CDOM through in-lake incubation experiments. There was marked variability throughout the year in CDOM absorption in Lake Balaton (aCDOM(440) = 0. 06-9.01 m-1). The highest values were consistently observed at the mouth of the main inflow (Zala River), which drains humic-rich material from the adjoining Kis-Balaton wetland, but CDOM absorption decreased rapidly towards the east where it was consistently lower and less variable than in the westernmost lake basins. The spectral slope parameter for the interval of 350-500 nm (SCDOM(350-500)) was more variable with increasing distance from the inflow (observed range 0.0161-0.0181 nm-1 for the mouth of the main inflow and 0.0158-0.0300 nm-1 for waters closer to the outflow). However, spatial variation in SCDOM was more constant exhibiting a negative correlation with aCDOM(440). Dissolved organic carbon (DOC) was strongly positively correlated with aCDOM(440) and followed a similar seasonal trend but it demonstrated more variability than either aCDOM or SCDOM with distance through the system. Photobleaching resulting from a 7-day exposure to natural solar UV radiation resulted in a marked decrease in allochthonous CDOM absorption (7.04 to 3.36 m-1, 42 % decrease). Photodegradation also resulted in an increase in the spectral slope coefficient of dissolved material.

Effects of hyperthermia on intracellular CA/sup 2+/ monitored by digitized video image fluorescence microscopy

International Nuclear Information System (INIS)

Asher, C.R.; Mikkelsen, R.B.

1987-01-01

With digitized video image fluorescence microscopy and the fluorescent Ca/sup 2+/ dye, fuca-2, the authors examined heat effects on intracellular free Ca/sup 2+/, [Ca/sup 2/]/sub f/. HT-29 human colon cancer cells grown on coverslip were equilibrated with 2.0 μM fura-2 in RPMI 1540 (20 0 , 15 min), washed three times and incubated at 20 0 for 1 h. Coverslips were mounted in a Dvorok perfusion chamber sitting within a temperature controlled microscope stage. Fluorescence was monitored at 500 nm by epi-illumination at 385 nm, excitation maximum for free dye, and 340 nm, maximum for Ca/sup 2+/ complexed dye, with a computer controlled filter wheel. The emission intensity ratio, I/sub 340//I/sub 385/, which corrects for dye leakage, photo-bleaching and cell thickness was used to calculate [Ca/sup 2+/]/sub f/. Measurements of 200 cells at 37 0 using a bit pad and mouse to select 0.6 x 0.6 μ cytoplasmi areas indicated 3 populations of cells in terms of [Ca/sup 2+/]/sub f/ (70%, 40-60nM; 15% 70-110nM; 15%, 120-200 nM). Heating to 43 0 for 1 h resulted in an overall decrease in [Ca/sup 2+/]/sub f/ with greater than 90% cells within 30-50 nM. Not all cells responded to heat. Post-incubation for 3 h at 37 0 showed the identical cell distribution; at 24 h, cell distribution was that of non-heated cells. The relationship of these results to cell killing and thermotolerance are not understood, but these results indicated the importance of cell heterogeneity in response to heat

Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

Science.gov (United States)

Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

2010-07-01

Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

8-Anilino-1-naphthalenesulfonate/Layered Double Hydroxide Ultrathin Films: Small Anion Assembly and Its Potential Application as a Fluorescent Biosensor.

Science.gov (United States)

Zhang, Ping; Li, Ling; Zhao, Yun; Tian, Zeyun; Qin, Yumei; Lu, Jun

2016-09-06

The fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is a widely used fluorescent probe molecule for biochemistry analysis. This paper reported the fabrication of ANS/layered double hydroxide nanosheets (ANS/LDH)n ultrathin films (UTFs) via the layer-by-layer small anion assembly technique based on electrostatic interaction and two possible weak interactions: hydrogen-bond and induced electrostatic interactions between ANS and positive-charged LDH nanosheets. The obtained UTFs show a long-range-ordered periodic layered stacking structure and weak fluorescence in dry air or water, but it split into three narrow strong peaks in a weak polarity environment induced by the two-dimensional (2D) confinement effect of the LDH laminate; the fluorescence intensity increases with decreasing the solvent polarity, concomitant with the blue shift of the emission peaks, which show good sensoring reversibility. Meanwhile, the UTFs exhibit selective fluorescence enhancement to the bovine serum albumin (BSA)-like protein biomolecules, and the rate of fluorescence enhancement with the protein concentration is significantly different with the different protein aggregate states. The (ANS/LDH)n UTF has the potential to be a novel type of biological flourescence sensor material.

Quantum Dot-based Immunohistochemistry for Pathological Applications

Directory of Open Access Journals (Sweden)

Li Zhou

2016-01-01

Full Text Available Quantum dots (QDs are novel light emitting semiconductor nanocrystals with diameter ranging from 2 to 20 nm. In comparison with traditional organic dyes and fluorescent proteins, QDs possess unique optical properties including extremely high fluorescence efficiency and minimal photobleaching which make them emerge as a new class of fluorescent labels for molecular imaging and biomedical analysis. Herein, recent advances in fundamental mechanisms and pathological applications of QD were reviewed.

Fluorescence ON–OFF switching using micelle of stimuli-responsive double hydrophilic block copolymers: Nile Red fluorescence in micelles of poly(acrylic acid-b-N-isopropylacrylamide)

Energy Technology Data Exchange (ETDEWEB)

Yee, Min Min; Tsubone, Miyabi; Morita, Takuya [Department of Chemistry, Graduate School of Science & Engineering, Saga University, 1 Honjo, Saga 840-8502 (Japan); Yusa, Shin-ichi [Department of Materials Science and Chemistry, University of Hyogo, 2167 Shosha, Himeji 671-2280 (Japan); Nakashima, Kenichi, E-mail: nakashik@cc.saga-u.ac.jp [Department of Chemistry, Graduate School of Science & Engineering, Saga University, 1 Honjo, Saga 840-8502 (Japan)

2016-08-15

The dual-mode fluorescence ON–OFF switching of Nile Red (NR) by using stimuli-responsive polymeric micelle of poly(acrylic acid-b-N-isopropylacrylamide) (PAA-b-PNIPAM) has been studied. PAA-b-PNIPAM, one of double hydrophilic block copolymers, is known to form PNIPAM-core/PAA-corona micelles in aqueous solutions when the temperature of the solution is elevated up to the lower critical solution temperature (LCST) of PNIPAM block. It also forms PAA-core/PNIPAM-corona micelles when the anionic PAA block is charge-neutralized with cationic cetyltrimethylammonium ion. Fluorescence properties of NR in the micelles are elucidated by observing various fluorescence parameters such as intensity, polarization, and quantum yield. It is found that the fluorescence intensity is negligibly low (OFF-state) when PAA-b-PNIPAM exists as a form of unimer, whereas it is remarkably enhanced (ON-state) when the PNIPAM-core or PAA-core micelles are formed. These results demonstrate that a novel fluorescence ON–OFF switching system can be constructed by using PAA-b-PNIPAM micelles and NR.

Enhancing molecular logic through modulation of temporal and spatial constraints with quantum dot-based systems that use fluorescent (Förster) resonance energy transfer

Science.gov (United States)

Claussen, Jonathan C.; Algar, W. Russ; Hildebrandt, Niko; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

2013-10-01

Luminescent semiconductor nanocrystals or quantum dots (QDs) contain favorable photonic properties (e.g., resistance to photobleaching, size-tunable PL, and large effective Stokes shifts) that make them well-suited for fluorescence (Förster) resonance energy transfer (FRET) based applications including monitoring proteolytic activity, elucidating the effects of nanoparticles-mediated drug delivery, and analyzing the spatial and temporal dynamics of cellular biochemical processes. Herein, we demonstrate how unique considerations of temporal and spatial constraints can be used in conjunction with QD-FRET systems to open up new avenues of scientific discovery in information processing and molecular logic circuitry. For example, by conjugating both long lifetime luminescent terbium(III) complexes (Tb) and fluorescent dyes (A647) to a single QD, we can create multiple FRET lanes that change temporally as the QD acts as both an acceptor and donor at distinct time intervals. Such temporal FRET modulation creates multi-step FRET cascades that produce a wealth of unique photoluminescence (PL) spectra that are well-suited for the construction of a photonic alphabet and photonic logic circuits. These research advances in bio-based molecular logic open the door to future applications including multiplexed biosensing and drug delivery for disease diagnostics and treatment.

Development of UV-curable liquid for in-liquid fluorescence alignment in ultraviolet nanoimprint lithography

Science.gov (United States)

Ochiai, Kento; Kikuchi, Eri; Ishito, Yota; Kumagai, Mari; Nakamura, Takahiro; Nakagawa, Masaru

2018-06-01

We studied a fluorescent UV-curable resin suitable for fluorescence alignment in UV nanoimprinting. The addition of a cationic fluorescent dye caused radical photopolymerization of a UV-curable resin by exposure to visible excitation light for fluorescence microscope observation. The microscope observation of a resin film prepared by pressing resin droplets on a silica substrate with a fluorinated silica superstrate revealed that the cationic dye molecules were preferably adsorbed onto the silica surface. It was indicated that the dye molecules concentrated on the silica surface may cause the photocuring. A nonionic fluorescent dye was selected owing to its low polar symmetrical structure and its solubility parameter close to monomers. The fluorescent UV-curable resin with the nonionic dye showed uncured stability to exposure to visible excitation light for 30 min with a light intensity of 8.5 mW cm‑2 detected at 530 nm.

Trials and Tribulations of Fluorescent Dissolved Organic Matter Chemical Interpretations: A case study of polar ice cores

Science.gov (United States)

D'Andrilli, J.

2017-12-01

Excitation emission matrix fluorescence spectroscopy is widely applied for rapid dissolved organic matter (DOM) characterization in aquatic systems. Fluorescent DOM surveys are booming, not only as a central focus in aquatic environments, but also as an important addition to interdisciplinary research (e.g., DOM analysis in concert with ice core paleoclimate reconstructions, stream metabolism, hydrologic regimes, agricultural developments, and biological activity), opening new doors, not just for novelty, but also for more challenges with chemical interpretations. Recently, the commonly used protein- versus humic-like classifications of DOM have been ineffective at describing DOM chemistry in various systems (e.g., ice cores, wastewaters, incubations/engineered). Moreover, the oversimplification of such classifications used to describe fluorescing components, without further scrutiny, has become commonplace, ultimately producing vague reporting. For example, West Antarctic ice core DOM was shown to contain fluorescence in the low excitation/emission wavelength region, however resolved fluorophores depicting tyrosine- and tryptophan-like DOM were not observed. At first, as literature suggested, we reported this result as protein-like, and concluded that microbial contributions were dominant in deep ice. That initial interpretation would disintegrate the conservation paradigm of atmospheric composition during deposition, the crux of ice core research, and contradict other lines of evidence. This begged the question, "How can we describe DOM chemistry without distinct fluorophores?" Antarctic ice core DOM was dominated by neither tyrosine- nor tryptophan-like fluorescence, causing "unusual" looking fluorescent components. After further examination, deep ice DOM was reported to contain fluorescent species most similar to monolignols and tannin-like phenols, describing the precursors of lignin from low carbon producing environments, consistent with marine sediment

Fluorescent ampicillin analogues as multifunctional disguising agents against opsonization

Science.gov (United States)

Kotagiri, Nalinikanth; Sakon, Joshua; Han, Haewook; Zharov, Vladimir P.; Kim, Jin-Woo

2016-06-01

Cancer nanomedicines are opening new paradigms in cancer management and recent research points to how they can vastly improve imaging and therapy through multimodality and multifunctionality. However, challenges to achieving optimal efficacy are manifold starting from processing materials and evaluating their intended effectiveness on biological tissue, to developing new strategies aimed at improving transport of these materials through the biological milieu to the target tissue. Here, we report a fluorescent derivative of a beta-lactam antibiotic, ampicillin (termed iAmp) and its multifunctional physicobiochemical characteristics and potential as a biocompatible shielding agent and an effective dispersant. Carbon nanotubes (CNTs) were chosen to demonstrate the efficacy of iAmp. CNTs are known for their versatility and have been used extensively for cancer theranostics as photothermal and photoacoustic agents, but have limited solubility in water and biocompatibility. Traditional dispersants are associated with imaging artifacts and are not fully biocompatible. The chemical structure of iAmp is consistent with a deamination product of ampicillin. Although the four-membered lactam ring is intact, it does not retain the antibiotic properties. The iAmp is an effective dispersant and simultaneously serves as a fluorescent label for single-walled CNTs (SWNTs) with minimal photobleaching. The iAmp also enables bioconjugation of SWNTs to bio-ligands such as antibodies through functional carboxyl groups. Viability tests show that iAmp-coated SWNTs have minimal toxicity. Bio-stability tests under physiological conditions reveal that iAmp coating not only remains stable in a biologically relevant environment with high protein and salt concentrations, but also renders SWNTs transparent against nonspecific protein adsorption, also known as protein corona. Mammalian tissue culture studies with macrophages and opsonins validate that iAmp coating affords immunological resistance

On the viability of exploiting L-shell fluorescence for X-ray polarimetry

Energy Technology Data Exchange (ETDEWEB)

Weisskopf, M C; Elsner, R F; Ramsey, B D; Sutherland, P G

1985-05-15

It has been suggested that one may build an X-ray polarimeter by exploiting the polarization dependence of the angular distribution of L-shell fluorescence photons. In this paper we examine, theoretically, the sensitivity of this approach to polarimetry. We apply our calculations to several detection schemes using imaging proportional counters that would have direct application in X-ray astronomy. We find, however, that the sensitivity of this method for measuring X-ray polarization is too low to be of use for other than laboratory applications. (orig.).

Resonance Fluorescence of a Trapped Four-Level Atom with Bichromatic Driving

International Nuclear Information System (INIS)

Bergou, J.; Jakob, M.; Abranyos, Y.

1999-01-01

The resonance fluorescence spectrum of a bichromatically driven four-level atom is polarization dependent. Very narrow lines occur in the incoherent parts of the spectrum for polarization directions which are different from that of the driving fields. The degree of squeezing has a maximum of 56% which should make it easily observable. The second-order correlation function exhibits anti bunching for zero time delay and strong super bunching for certain values of the interaction parameter and time delay. For these parameters resonant two-photon emission takes place in the form of polarization entangled photon pairs. The system can be a novel source of photons in the EPR and/or Bell states. Some experiments will be proposed which make use of this unique source. (Authors)

Surface recognition and fluorescence sensing of histone by dansyl-appended cyclophane-based resorcinarene trimer.

Science.gov (United States)

Hayashida, Osamu; Ogawa, Naoyuki; Uchiyama, Masaki

2007-11-07

A cyclophane-based resorcinarene trimer (3) bearing a dansyl moiety as an environmentally sensitive fluorophore was prepared by stepwise condensation of a tetraaza[6.1.6.1]paracyclophane skeleton with a dansyl moiety and three resorcinarene derivatives having heptacarboxylic acid residues in this sequence. The dansyl-appended cyclophane exhibited the following fluorescence properties regarding solvent polarity dependency and histone surface recognition: With increasing dioxane contents in dioxane/water solvents, the fluorescence intensity originating from the dansyl moiety of 3 increased along with a concomitant blue shift of the fluorescence maximum (lambdaem). The microenvironmentally sensitive fluorescence properties of dansyl fluorophore were maintained, even when the dansyl moiety was covalently attached to a cyclophane. Most interestingly, the cyclophane-based resorcinarene trimer exhibited recognition and fluorescence sensing capabilities toward histone, a small basic protein of eukaryotic chromatins. The fluorescence intensity originating from 3 increased along with a concomitant blue shift of lambdaem upon the addition of histone, reflecting the formation of 3-histone complexes. A relatively large fluorescence polarization (P) value was obtained for the 3-histone complexes (0.15), reflecting highly restricted conformations of 3, and the obtained P value was much larger than that of 3 alone in aqueous medium (0.07). The binding constant (K) of 3 with histone (unit basis) was estimated to be 2.1 x 106 M-1. On the other hand, upon the addition of acetylated histone (Ac-histone) to an aqueous solution containing 3, the extent of change in fluorescence intensity originating from the dansyl group of 3 was almost negligible, indicating that the electrostatic interactions between 3 and Ac-histone were weak. In addition, the fluorescence spectral changes were also small or negligible upon the addition of other proteins such as albumin, ovalbumin, peanut agglutinin

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

Science.gov (United States)

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

Directory of Open Access Journals (Sweden)

Prerna Grover

Full Text Available The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery

Fluorescence resonance energy transfer (FRET-based subcellular visualization of pathogen-induced host receptor signaling

Directory of Open Access Journals (Sweden)

Zimmermann Timo

2009-11-01

Full Text Available Abstract Background Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET to visualize pathogen-initiated signaling events in human cells. Results Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3. In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2 domain of Hck (Hck-SH2 was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet-Hck-SH2 and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact. Conclusion These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.

Analysis of trace element compositions in adhesive cloth tapes using high-energy x-ray fluorescence spectrometer with three-dimensional polarization optics for forensic discrimination

International Nuclear Information System (INIS)

Goto, Akiko; Hokura, Akiko; Nakai, Izumi

2008-01-01

The forensic discrimination of adhesive cloth tapes often used in crimes was developed using a high-energy energy-dispersive X-ray fluorescence spectrometer with 3-dimensional polarization optics. The best measurement condition for discrimination of the tape was as follows: secondary targets, Rh and Al 2 O 3 ; measurement time, 300 s for Rh and 600 s for Al 2 O 3 ; 14 elements (Ca, Ti, Cr, Mn, Fe, Ni, Zn, Sr, Zr, Nb, Mo, Sb, Ba and Pb) were used for discrimination. It is found that the combined information of yarn density and the XRF peak intensity of the 14 elements successfully discriminated 29 out of 31 samples, of which 2 probably had the same origin. This technique is useful for forensic analysis, because it is nondestructive, rapid and easy. Therefore, it can be applied to actual forensic identification. (author)

Structural and dynamical aspects of skin studied by multiphoton excitation fluorescence microscopy-based methods

DEFF Research Database (Denmark)

Bloksgaard, Maria; Brewer, Jonathan R.; Bagatolli, Luis

2013-01-01

' parameters. Specifically, by applying these methods, spatially resolved maps of water dipolar relaxation (generalized polarization function using the 6-lauroyl-2-(N,N-dimethylamino)naphthale probe), activity of protons (fluorescence lifetime imaging using a proton sensitive fluorescence probe--2,7-bis-(2......-carboxyethyl)-5-(and-6)-carboxyfluorescein) and diffusion coefficients of distinct fluorescence probes (raster imaging correlation spectroscopy) can be obtained from different regions of the tissue. Comparative studies of different tissue strata, but also between equivalent regions of normal and abnormal......This mini-review reports on applications of particular multiphoton excitation microscopy-based methodologies employed in our laboratory to study skin. These approaches allow in-depth optical sectioning of the tissue, providing spatially resolved information on specific fluorescence probes...

Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli.

Directory of Open Access Journals (Sweden)

Kwang-Ho Hur

Full Text Available The brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined it with the analysis of axial scans through the E. coli cell. The MSQ method recovers brightness, concentration, and diffusion time of soluble proteins in E. coli. We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements. We further used MSQ analysis to determine the oligomeric state of nuclear transport factor 2 labeled with EGFP expressed in E. coli cells. The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

Differential Cotton leaf crumple virus-VIGS-mediated gene silencing and viral genome localization in different Gossypium hirsutum genetic backgrounds

KAUST Repository

Idris, Ali; Tuttle, John Richard; Robertson, Dominique Niki; Haigler, Candace H.; Brown, Judith K.

2010-01-01

inoculation resulted in systemic and persistent photo-bleaching of the leaves and bolls of the seven cultivars tested, however, the intensity of silencing was variable. CLCrV-VIGS-mediated expression of green fluorescent protein was used to monitor

Imaging of polarity during zygotic and somatic embryogenesis of carrot (Daucus carota L.)

NARCIS (Netherlands)

Timmers, A.C.J.

1993-01-01

In this thesis a study of the regulation of coordinated growth and the development of polarity during embryogenesis of carrot, Daucus carota L., is described. To this end, several microscopical techniques were used, such as light microscopy, fluorescence microscopy,

Resonance Polarization and Phase-Mismatched CARS of Pheophytin b Excited in the Qy Band

NARCIS (Netherlands)

de Boeij, W.P.; Lucassen, G.W.; Lucassen, Gerald; Otto, Cornelis; Greve, Jan

1993-01-01

Resonance polarization and phase-mismatched coherent anti-Stokes Raman scattering (CARS) measurements were performed on pheophytin b dissolved in acetone excited in the Qy absorption band, where strong broad fluorescence makes spontaneous Raman spectroscopy impossible. The phase-mismatching

Nanodiamonds and silicon quantum dots: ultrastable and biocompatible luminescent nanoprobes for long-term bioimaging.

Science.gov (United States)

Montalti, M; Cantelli, A; Battistelli, G

2015-07-21

Fluorescence bioimaging is a powerful, versatile, method for investigating, both in vivo and in vitro, the complex structures and functions of living organisms in real time and space, also using super-resolution techniques. Being poorly invasive, fluorescence bioimaging is suitable for long-term observation of biological processes. Long-term detection is partially prevented by photobleaching of organic fluorescent probes. Semiconductor quantum dots, in contrast, are ultrastable, fluorescent contrast agents detectable even at the single nanoparticle level. Emission color of quantum dots is size dependent and nanoprobes emitting in the near infrared (NIR) region are ideal for low back-ground in vivo imaging. Biocompatibility of nanoparticles, containing toxic elements, is debated. Recent safety concerns enforced the search for alternative ultrastable luminescent nanoprobes. Most recent results demonstrated that optimized silicon quantum dots (Si QDs) and fluorescent nanodiamonds (FNDs) show almost no photobleaching in a physiological environment. Moreover in vitro and in vivo toxicity studies demonstrated their unique biocompatibility. Si QDs and FNDs are hence ideal diagnostic tools and promising non-toxic vectors for the delivery of therapeutic cargos. Most relevant examples of applications of Si QDs and FNDs to long-term bioimaging are discussed in this review comparing the toxicity and the stability of different nanoprobes.

Using fluorescent dissolved organic matter to trace and distinguish the origin of Arctic surface waters

Science.gov (United States)

Gonçalves-Araujo, Rafael; Granskog, Mats A.; Bracher, Astrid; Azetsu-Scott, Kumiko; Dodd, Paul A.; Stedmon, Colin A.

2016-01-01

Climate change affects the Arctic with regards to permafrost thaw, sea-ice melt, alterations to the freshwater budget and increased export of terrestrial material to the Arctic Ocean. The Fram and Davis Straits represent the major gateways connecting the Arctic and Atlantic. Oceanographic surveys were performed in the Fram and Davis Straits, and on the east Greenland Shelf (EGS), in late summer 2012/2013. Meteoric (fmw), sea-ice melt, Atlantic and Pacific water fractions were determined and the fluorescence properties of dissolved organic matter (FDOM) were characterized. In Fram Strait and EGS, a robust correlation between visible wavelength fluorescence and fmw was apparent, suggesting it as a reliable tracer of polar waters. However, a pattern was observed which linked the organic matter characteristics to the origin of polar waters. At depth in Davis Strait, visible wavelength FDOM was correlated to apparent oxygen utilization (AOU) and traced deep-water DOM turnover. In surface waters FDOM characteristics could distinguish between surface waters from eastern (Atlantic + modified polar waters) and western (Canada-basin polar waters) Arctic sectors. The findings highlight the potential of designing in situ multi-channel DOM fluorometers to trace the freshwater origins and decipher water mass mixing dynamics in the region without laborious samples analyses. PMID:27667721

Detection and Interpretation of Fluorescence Signals Generated by Excitable Cells and Tissues

Science.gov (United States)

Costantino, Anthony J.

Part 1: High-Sensitivity Amplifiers for Detecting Fluorescence . Monitoring electrical activity and Cai 2+ transients in biological tissues and individual cells increasingly utilizes optical sensors based on voltage-dependent and Cai 2+-dependent fluorescent dyes. However, achieving satisfactory signal-to-noise ratios (SNR) often requires increased illumination intensities and/or dye concentrations, which results in photo-toxicity, photo-bleaching and other adverse effects limiting the utility of optical recordings. The most challenging are the recordings from individual cardiac myocytes and neurons. Here we demonstrate that by optimizing a conventional transimpedance topology one can achieve a 10-20 fold increase of sensitivity with photodiode-based recording systems (dependent on application). We provide a detailed comparative analysis of the dynamic and noise characteristics of different transimpedance amplifier topologies as well as the example(s) of their practical implementation. Part 2: Light-Scattering Models for Interpretation of Fluorescence Data. Current interest in understanding light transport in cardiac tissue has been motivated in part by increased use of voltage-sensitive and Ca i2+-sensitive fluorescent probes to map electrical impulse propagation and Cai2+-transients in the heart. The fluorescent signals are recorded using such probes represent contributions from different layers of myocardial tissue and are greatly affected by light scattering. The interpretation of these signals thus requires deconvolution which would not be possible without detailed models of light transport in the respective tissue. Which involves the experimental measurements of the absorption, scattering, and anisotropy coefficients, mua, mu s, and g respectively. The aim of the second part of our thesis was to derive a new method for deriving these parameters from high spatial resolution measurements of forward-directed flux (FDF). To this end, we carried out high spatial

A KIND OF FLUORESCENCE PROBE TO STUDY THE KINETICS OF POLYMERIZATION PROCESS

Institute of Scientific and Technical Information of China (English)

YANG Guoqiang; WU Shikang

1994-01-01

Fluorescence properties of 1-phenyl-3-(4'-nitrophenyl) pyrazoline (PNP) were studied in bulk polymerization process of methylmethacrylate (MMA). The fluorescence intensity of PNP was enhanced and the emission maximum was blue shifted with the polymerization progress. In the period of auto-acceleration of the polymerization the enhancement of fluorescence intensity and blue shift of peak wavelength in spectra could be observed evidently. This means that the solvatochromic properties of PNP are influenced not only by the solvent polarity but also by the viscosity of the medium(especially by the phase transition). In solid state PNP emits from the charge transfer excited state without solvent relaxation. The transient emission spectra and the results from Bakhshiev model of solvent relaxation coincide with that from the polymerization experiment.

Evidence of excited state localization and static disorder in LH2 investigated by 2D-polarization single-molecule imaging at room temperature.

Science.gov (United States)

Tubasum, Sumera; Camacho, Rafael; Meyer, Matthias; Yadav, Dheerendra; Cogdell, Richard J; Pullerits, Tõnu; Scheblykin, Ivan G

2013-12-07

Two-dimensional polarization fluorescence imaging of single light harvesting complexes 2 (LH2) of Rps. acidophila was carried out to investigate the polarization properties of excitation and fluorescence emission simultaneously, at room temperature. In two separate experiments we excited LH2 with a spectrally narrow laser line matched to the absorption bands of the two chromophore rings, B800 and B850, thereby indirectly and directly triggering fluorescence of the B850 exciton state. A correlation analysis of the polarization modulation depths in excitation and emission for a large number of single complexes was performed. Our results show, in comparison to B800, that the B850 ring is a more isotropic absorber due to the excitonic nature of its excited states. At the same time, we observed a strong tendency for LH2 to emit with dipolar character, from which preferential localization of the emissive exciton, stable for minutes, is inferred. We argue that the observed effects can consistently be explained by static energetic disorder and/or deformation of the complex, with possible involvement of exciton self-trapping.

Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

OpenAIRE

Wargocki, Piotr; Deng, Wei; Anwer, Ayad G.; Goldys, Ewa M.

2015-01-01

Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera) and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate s...

Single Molecule Study of Photoconversion and Spectral Heterogeneities of Fluorophores

DEFF Research Database (Denmark)

Liao, Zhiyu

of conformational changes and dynamics. The photophysical properties of organic dyes directly determine the quality of the experiments. So the better understanding of the photophysical properties of organic dyes, the better we are able to design the experiments and interpret the data, especially in single molecule...... important criteria for a good fluorophore. Improving the photostability of organic dyes by designing the structure is always a difficult task for organic chemists. It requires a comprehensive understanding of the mechanism of the photobleaching behavior of fluorophores. It is the aim of this work...... to understand the mechanisms of photobleaching behaviors of organic dyes, terrylene diimide (TDI) and amino-trioxatriangulenium dye (A3-TOTA+). Photobleaching is usually seen as permanent loss of fluorescence. In this work, we show that organic fluorophores can be converted into another chemical compound after...

Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

DEFF Research Database (Denmark)

Lund, F. W.; Lomholt, M. A.; Solanko, L. M.

2012-01-01

to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter...

78 FR 64916 - Application(s) for Duty-Free Entry of Scientific Instruments

Science.gov (United States)

2013-10-30

... Minnesota--Twin Cities, 421 Washington Avenue SE., Minneapolis, MN 55455. Instrument: Diode-Pumped Solid... imaging of cellular organelles and calcium flux, photo-activation and photo-bleaching fluorescent proteins to study cellular organelles (mitochondria) and intracellular ion flux. The unique characteristics of...

New insight in the template decomposition process of large zeolite ZSM-5 crystals: an in situ UV-Vis/fluorescence micro-spectroscopy study

NARCIS (Netherlands)

Karwacki, L.|info:eu-repo/dai/nl/304824283; Weckhuysen, B.M.|info:eu-repo/dai/nl/285484397

2011-01-01

A combination of in situ UV-Vis and confocal fluorescence micro-spectroscopy was used to study the template decomposition process in large zeolite ZSM-5 crystals. Correlation of polarized light dependent UV-Vis absorption spectra with confocal fluorescence emission spectra in the 400–750 nm region

Polarized two-photon photoselection in EGFP: Theory and experiment.

Science.gov (United States)

Masters, T A; Marsh, R J; Blacker, T S; Armoogum, D A; Larijani, B; Bain, A J

2018-04-07

In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the "hidden" degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S 0 → S 1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S 0 → S 1 transition.

Polarized two-photon photoselection in EGFP: Theory and experiment

Science.gov (United States)

Masters, T. A.; Marsh, R. J.; Blacker, T. S.; Armoogum, D. A.; Larijani, B.; Bain, A. J.

2018-04-01

In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the "hidden" degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S0 → S1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S0 → S1 transition.

A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility

DEFF Research Database (Denmark)

Lund, F. W.; Wustner, D.

2013-01-01

Single particle tracking (SPT) is becoming a standard method to extract transport parameters from time-lapse image sequences of fluorescent vesicles in living cells. Another method to obtain these data is temporal image correlation spectroscopy (TICS), but this method is less often used for measu......Single particle tracking (SPT) is becoming a standard method to extract transport parameters from time-lapse image sequences of fluorescent vesicles in living cells. Another method to obtain these data is temporal image correlation spectroscopy (TICS), but this method is less often used...... for measurement of intracellular vesicle transport. Here, we present an extensive comparison of SPT and TICS. First we examine the effect of photobleaching, shading and noise on SPT and TICS analysis using simulated image sequences. To this end, we developed a simple photophysical model, which relates spatially...... varying illumination intensity to the bleaching propensity and fluorescence intensity of the moving particles. We found that neither SPT nor TICS are affected by photobleaching per se, but the transport parameters obtained by both methods are sensitive to the signal-to-noise ratio. In addition, the number...

Steady State and Time-Resolved Fluorescence Dynamics of Triphenylamine Based Oligomers with Phenylene/Thiophene/Furan in Solvents

International Nuclear Information System (INIS)

Qi, Zeng; Ying-Liang, Liu; Kang, Meng; Xiang-Jie, Zhao; Shu-Feng, Wang; Qi-Huang, Gong

2009-01-01

We investigate the photo-physical properties of a series of triphenylamine-based oligomers by steady-state and picosecond transient fluorescence measurements in solvents. The oligomers are composed alternatively with triphenylamine and phenylene/thiophene/furan group, bridged by vinyl group (PNB/PNT/PNF). Their fluorescence spectra show bathochromic phenomenon with solvent polarity and viscosity increasing. The fluorescence decays are bi-exponential for PNB and PNT, and tri-exponential for PNF in THF and aniline. The strong viscosity dependence suggests conformational relaxation along the PNF chain after photo excitation. (condensed matter: electronicstructure, electrical, magnetic, and opticalproperties)

First attempt of the measurement of the beam polarization at an accelerator with the optical electron polarimeter POLO

CERN Document Server

Collin, B; Essabaa, S; Frascaria, R; Gacougnolle, R; Kunne, Ronald Alexander; Aulenbacher, K; Tioukine, V

2004-01-01

The conventional methods for measuring the polarization of electron beams are either time consuming, invasive or accurate only to a few percent. We developped a method to measure electron beam polarization by observing the light emitted by argon atoms following their excitation by the impact of polarized electrons. The degree of circular polarization of the emitted fluorescence is directly related to the electron polarization. We tested the polarimeter on a test GaAs source available at the MAMI electron accelerator in Mainz, Germany. The polarimeter determines the polarization of a 50 keV electron beam decelerated to a few eV and interacting with an effusive argon gas jet. The resulting decay of the excited states produces the emission of a circularly polarized radiation line at 811.5 nm which is observed and analyzed.

Crystal growth, polarized spectroscopy and Judd-Ofelt analysis of Tb:YAlO3.

Science.gov (United States)

Liu, Bin; Shi, Jiaojiao; Wang, Qingguo; Tang, Huili; Liu, Junfang; Zhao, Hengyu; Li, Dongzhen; Liu, Jian; Xu, Xiaodong; Wang, Zhanshan; Xu, Jun

2018-07-05

Tb 3+ -doped YAlO 3 (YAP) single crystal was grown by Czochralski (Cz) method. Based on the polarized absorption spectra, the spectroscopic parameters were calculated to be Ω 2 =3.49×10 -20 cm 2 , Ω 4 =5.87×10 -20 cm 2 and Ω 6 =2.55×10 -20 cm 2 , and then the spontaneous transition rate, fluorescent branching ratio and radiative lifetime of 5 D 4 multiplet were obtained. The yellow emission cross sections of 5 D 4 → 7 F 4 transition were calculated to be 1.72×10 -22 cm 2 , 2.73×10 -22 cm 2 and 2.65×10 -22 cm 2 for a, b and c polarization, respectively. The fluorescence lifetime of the 5 D 4 multiplet was fitted to be 1.72ms. All the data indicate that Tb:YAP crystal is a promising candidate for yellow laser operation. Copyright © 2018. Published by Elsevier B.V.

Polarization control of intermediate state absorption in resonance-mediated multi-photon absorption process

International Nuclear Information System (INIS)

Xu, Shuwu; Yao, Yunhua; Jia, Tianqing; Ding, Jingxin; Zhang, Shian; Sun, Zhenrong; Huang, Yunxia

2015-01-01

We theoretically and experimentally demonstrate the control of the intermediate state absorption in an (n + m) resonance-mediated multi-photon absorption process by the polarization-modulated femtosecond laser pulse. An analytical solution of the intermediate state absorption in a resonance-mediated multi-photon absorption process is obtained based on the time-dependent perturbation theory. Our theoretical results show that the control efficiency of the intermediate state absorption by the polarization modulation is independent of the laser intensity when the transition from the intermediate state to the final state is coupled by the single-photon absorption, but will be affected by the laser intensity when this transition is coupled by the non-resonant multi-photon absorption. These theoretical results are experimentally confirmed via a two-photon fluorescence control in (2 + 1) resonance-mediated three-photon absorption of Coumarin 480 dye and a single-photon fluorescence control in (1 + 2) resonance-mediated three-photon absorption of IR 125 dye. (paper)

Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

Science.gov (United States)

Ciaccio, Mark F.; Chuu, Chih-pin; Jones, Richard B.

2012-01-01

First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This

Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional diversification of ErbB receptors.

Directory of Open Access Journals (Sweden)

Ronald J Hause

Full Text Available First-generation interaction maps of Src homology 2 (SH2 domains with receptor tyrosine kinase (RTK phosphosites have previously been generated using protein microarray (PM technologies. Here, we developed a large-scale fluorescence polarization (FP methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry

High resolution x-ray fluorescence spectroscopy - a new technique for site- and spin-selectivity

International Nuclear Information System (INIS)

Wang, Xin

1996-12-01

X-ray spectroscopy has long been used to elucidate electronic and structural information of molecules. One of the weaknesses of x-ray absorption is its sensitivity to all of the atoms of a particular element in a sample. Through out this thesis, a new technique for enhancing the site- and spin-selectivity of the x-ray absorption has been developed. By high resolution fluorescence detection, the chemical sensitivity of K emission spectra can be used to identify oxidation and spin states; it can also be used to facilitate site-selective X-ray Absorption Near Edge Structure (XANES) and site-selective Extended X-ray Absorption Fine Structure (EXAFS). The spin polarization in K fluorescence could be used to generate spin selective XANES or spin-polarized EXAFS, which provides a new measure of the spin density, or the nature of magnetic neighboring atoms. Finally, dramatic line-sharpening effects by the combination of absorption and emission processes allow observation of structure that is normally unobservable. All these unique characters can enormously simplify a complex x-ray spectrum. Applications of this novel technique have generated information from various transition-metal model compounds to metalloproteins. The absorption and emission spectra by high resolution fluorescence detection are interdependent. The ligand field multiplet model has been used for the analysis of Kα and Kβ emission spectra. First demonstration on different chemical states of Fe compounds has shown the applicability of site selectivity and spin polarization. Different interatomic distances of the same element in different chemical forms have been detected using site-selective EXAFS

Mapping microscopic order in plant and mammalian cells and tissues: novel differential polarization attachment for new generation confocal microscopes (DP-LSM)

Science.gov (United States)

Steinbach, G.; Pawlak, K.; Pomozi, I.; Tóth, E. A.; Molnár, A.; Matkó, J.; Garab, G.

2014-03-01

Elucidation of the molecular architecture of complex, highly organized molecular macro-assemblies is an important, basic task for biology. Differential polarization (DP) measurements, such as linear (LD) and circular dichroism (CD) or the anisotropy of the fluorescence emission (r), which can be carried out in a dichrograph or spectrofluorimeter, respectively, carry unique, spatially averaged information about the molecular organization of the sample. For inhomogeneous samples—e.g. cells and tissues—measurements on macroscopic scale are not satisfactory, and in some cases not feasible, thus microscopic techniques must be applied. The microscopic DP-imaging technique, when based on confocal laser scanning microscope (LSM), allows the pixel by pixel mapping of anisotropy of a sample in 2D and 3D. The first DP-LSM configuration, which, in fluorescence mode, allowed confocal imaging of different DP quantities in real-time, without interfering with the ‘conventional’ imaging, was built on a Zeiss LSM410. It was demonstrated to be capable of determining non-confocally the linear birefringence (LB) or LD of a sample and, confocally, its FDLD (fluorescence detected LD), the degree of polarization (P) and the anisotropy of the fluorescence emission (r), following polarized and non-polarized excitation, respectively (Steinbach et al 2009 Acta Histochem.111 316-25). This DP-LSM configuration, however, cannot simply be adopted to new generation microscopes with considerably more compact structures. As shown here, for an Olympus FV500, we designed an easy-to-install DP attachment to determine LB, LD, FDLD and r, in new-generation confocal microscopes, which, in principle, can be complemented with a P-imaging unit, but specifically to the brand and type of LSM.

Mapping microscopic order in plant and mammalian cells and tissues: novel differential polarization attachment for new generation confocal microscopes (DP-LSM)

International Nuclear Information System (INIS)

Steinbach, G; Pawlak, K; Garab, G; Pomozi, I; Tóth, E A; Molnár, A; Matkó, J

2014-01-01

Elucidation of the molecular architecture of complex, highly organized molecular macro-assemblies is an important, basic task for biology. Differential polarization (DP) measurements, such as linear (LD) and circular dichroism (CD) or the anisotropy of the fluorescence emission (r), which can be carried out in a dichrograph or spectrofluorimeter, respectively, carry unique, spatially averaged information about the molecular organization of the sample. For inhomogeneous samples—e.g. cells and tissues—measurements on macroscopic scale are not satisfactory, and in some cases not feasible, thus microscopic techniques must be applied. The microscopic DP-imaging technique, when based on confocal laser scanning microscope (LSM), allows the pixel by pixel mapping of anisotropy of a sample in 2D and 3D. The first DP-LSM configuration, which, in fluorescence mode, allowed confocal imaging of different DP quantities in real-time, without interfering with the ‘conventional’ imaging, was built on a Zeiss LSM410. It was demonstrated to be capable of determining non-confocally the linear birefringence (LB) or LD of a sample and, confocally, its FDLD (fluorescence detected LD), the degree of polarization (P) and the anisotropy of the fluorescence emission (r), following polarized and non-polarized excitation, respectively (Steinbach et al 2009 Acta Histochem.111 316–25). This DP-LSM configuration, however, cannot simply be adopted to new generation microscopes with considerably more compact structures. As shown here, for an Olympus FV500, we designed an easy-to-install DP attachment to determine LB, LD, FDLD and r, in new-generation confocal microscopes, which, in principle, can be complemented with a P-imaging unit, but specifically to the brand and type of LSM. (paper)

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

NARCIS (Netherlands)

Laat, S.W. de; Tetteroo, P.A.T.; Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van

1984-01-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xaopus Levis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein (“HEDAF”) and 5-(N-tetradecanoyl)aminofluorescein (“TEDAF”) as probes. The

Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

Science.gov (United States)

Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

2010-09-01

The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

Studies of the laser-induced fluorescence of explosives and explosive compositions.

Energy Technology Data Exchange (ETDEWEB)

Hargis, Philip Joseph, Jr. (,; .); Thorne, Lawrence R.; Phifer, Carol Celeste; Parmeter, John Ethan; Schmitt, Randal L.

2006-10-01

Continuing use of explosives by terrorists throughout the world has led to great interest in explosives detection technology, especially in technologies that have potential for standoff detection. This LDRD was undertaken in order to investigate the possible detection of explosive particulates at safe standoff distances in an attempt to identify vehicles that might contain large vehicle bombs (LVBs). The explosives investigated have included the common homogeneous or molecular explosives, 2,4,6-trinitrotoluene (TNT), pentaerythritol tetranitrate (PETN), cyclonite or hexogen (RDX), octogen (HMX), and the heterogeneous explosive, ammonium nitrate/fuel oil (ANFO), and its components. We have investigated standard excited/dispersed fluorescence, laser-excited prompt and delayed dispersed fluorescence using excitation wavelengths of 266 and 355 nm, the effects of polarization of the laser excitation light, and fluorescence imaging microscopy using 365- and 470-nm excitation. The four nitro-based, homogeneous explosives (TNT, PETN, RDX, and HMX) exhibit virtually no native fluorescence, but do exhibit quenching effects of varying magnitude when adsorbed on fluorescing surfaces. Ammonium nitrate and fuel oil mixtures fluoresce primarily due to the fuel oil, and, in some cases, due to the presence of hydrophobic coatings on ammonium nitrate prill or impurities in the ammonium nitrate itself. Pure ammonium nitrate shows no detectable fluorescence. These results are of scientific interest, but they provide little hope for the use of UV-excited fluorescence as a technique to perform safe standoff detection of adsorbed explosive particulates under real-world conditions with a useful degree of reliability.

Pulsed Laser Deposition of Polymers Doped with Fluorescent Probes. Application to Environmental Sensors

International Nuclear Information System (INIS)

Rebollar, E; Villavieja, Mm; Gaspard, S; Oujja, M; Corrales, T; Georgiou, S; Domingo, C; Bosch, P; Castillejo, M

2007-01-01

Pulsed laser deposition (PLD) has been used to obtain thin films of poly(methyl methacrylate) and polystyrene doped with fluorescent probes, amino aromatic compounds S5 and S6, that could be used to sense the presence of contaminating environmental agents. These dopants both in solution and inserted in polymeric films are sensitive to changes in pH, viscosity and polarity, increasing their fluorescence emission and/or modifying the position of their emission band. Films deposits on quartz substrates, obtained by irradiating targets with a Ti:Sapphire laser (800 nm, 120 fs pulse) were analyzed by optical and Environmental Scanning Electron Microscopy, Fluorescence Microscopy, Laser-Induced Fluorescence, Micro Raman Spectroscopy and Flow Injection Analysis-Mass Spectrometry. The transfer of the polymer and the probe to the substrate is observed to be strongly dependent on the optical absorption coefficient of the polymeric component of the target at the irradiation wavelength

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non-polarized and polarized cells.

Science.gov (United States)

Mennesson, Eric; Erbacher, Patrick; Piller, Véronique; Kieda, Claudine; Midoux, Patrick; Pichon, Chantal

2005-06-01

Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively. PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency. We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes. Copyright (c) 2005 John Wiley & Sons, Ltd.

Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

Science.gov (United States)

Ghosh, Shirsendu; Nandi, Somen; Ghosh, Catherine; Bhattacharyya, Kankan

2016-09-19

Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time () of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nuclear spin polarized alkali beams (Li and Na): Production and acceleration

International Nuclear Information System (INIS)

Jaensch, H.; Becker, K.; Blatt, K.; Leucker, H.; Fick, D.

1987-01-01

Recent improvements of the Heidelberg source for polarized heavy ions (PSI) are described. By means of optical pumping in combination with the existing multipole separation magnet the beam figure of merit (polarization 2 x intensity) was doubled. 7 Li and 23 Na atomic beams can now be produced in pure hyperfine magnetic substates. Fast switching of the polarization is achieved by an adiabatic medium field transition. The hyperfine magnetic substate population is determined by laser-induced fluorescence spectroscopy. In routine operation atomic beams with nuclear polarization p α ≥0.85 (α=z, zz) are obtained. The acceleration of polarized 23 Na - ions by a 12 MV tandem accelerator introduces a new problem: the energy at the terminal stripper foil is not sufficient to produce a usable yield of naked ions. For partially stripped ions hyperfine interaction of the remaining electrons with the nuclear spin reduces the nuclear polarization. Using in addition the Heidelberg postaccelerator 23 Na 9+ beams of energies between 49 and 184 MeV were obtained with an alignment on target of P zz ≅0.45. 7 Li beams have also been accelerated up to 45 MeV with an alignment of P zz =0.69. (orig.)

Light sheet-based fluorescence microscopy (LSFM) reduces phototoxic effects and provides new means for the modern life sciences

Science.gov (United States)

Pampaloni, Francesco; Ansari, Nari; Girard, Philippe; Stelzer, Ernst H. K.

2011-07-01

Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack). The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed

Novel dansyl-appended calix[4]arene frameworks: fluorescence properties and mercury sensing.

Science.gov (United States)

Pandey, Shubha; Azam, Amir; Pandey, Siddharth; Chawla, H M

2009-01-21

Covalently-attached fluorophores may impart enhanced chemosensing capabilities to calixarene frameworks. Synthesis and characterization of six novel dansyl-appended calix[4]arenes, namely, H/Dan4, NO2/Dan4, H/(OH)2Dan2, H/(Ester)2(Dan)2, t-Bu/(OH)2Dan2, and t-Bu/(Ester)2Dan2, containing two or four dansyl moieties are reported. Among these, fluorescence intensity of NO2/Dan4 is observed to decrease significantly in the presence Hg2+ in the solution. Based on the decrease in fluorescence, a limit of detection for Hg2+ of 20 ppb is obtained. NO2/Dan4 as a chemosensing agent for Hg2+ shows excellent selectivity and adequate reversibility. Complexation of NO2/Dan4 with Hg2+ is investigated using fluorescence spectroscopy and is observed to be 2:1. The formation constant of (NO2/Dan4)2Hg2+ is estimated to be 5.2(+/- 0.8) x 10(10) M(-2) at ambient conditions. These observations are traced to the fact that while all other dansyl-appended calix[4]arenes show cone conformation in the solution, NO2/Dan4 is in the 1,3-alternate conformation. Stokes shift versus solvent orientational polarizability for NO2/Dan4 also indicates the difference in the ground- to excited-state dipole moment of this compound to be the maximum among all six, rendering it most sensitive to its environment. Fluorescence emission of NO2/Dan4 in nonpolar chloroform, polar-aprotic acetonitrile, and polar-protic ethanol is observed to be different than that of the rest of the dansyl-appended compounds as well.

An organic dye with very large Stokes-shift and broad tunability of fluorescence: Potential two-photon probe for bioimaging and ultra-sensitive solid-state gas sensor

Energy Technology Data Exchange (ETDEWEB)

He, Tingchao; Tian, Xiaoqing; Lin, Xiaodong, E-mail: linxd@szu.edu.cn, E-mail: hdsun@ntu.edu.sg [College of Physics Science and Technology, Shenzhen University, Shenzhen 518060 (China); Wang, Yue; Zhao, Xin; Sun, Handong, E-mail: linxd@szu.edu.cn, E-mail: hdsun@ntu.edu.sg [Division of Physics and Applied Physics, and Centre for Disruptive Photonic Technologies (CDPT), School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371 (Singapore); Gao, Yang; Grimsdale, Andrew C. [School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798 (Singapore)

2016-01-04

Light-emitting nonlinear optical molecules, especially those with large Stokes shifts and broad tunability of their emission wavelength, have attracted considerable attention for various applications including biomedical imaging and fluorescent sensors. However, most fluorescent chromophores have only limited potential for such applications due to small Stokes shifts, narrow tunability of fluorescence emissions, and small optical nonlinearity in highly polar solvents. In this work, we demonstrate that a two-photon absorbing stilbene chromophore exhibits a large two-photon absorption action cross-section (ηδ = 320 GM) in dimethylsulfoxide (DMSO) and shows broad fluorescence tunability (125 nm) by manipulating the polarity of the surrounding medium. Importantly, a very large Stokes shift of up to 227 nm is achieved in DMSO. Thanks to these features, this chromophore can be utilized as a two-photon probe for bioimaging applications and in an ultrasensitive solid-state gas detector.

Critical aggregates concentration of fatty esters present in biodiesel determined by turbidity and fluorescence.

Science.gov (United States)

Froehner, Sandro; Sánez, Juan; Dombroski, Luiz Fernando; Gracioto, Maria Paula

2017-09-01

Biodiesel for combustible engine is available as mixture of fossil diesel and fatty esters obtained by transesterification of vegetable oils. The use of biodiesel reduces the amount of SO x , mainly. However, it was already observed that biodiesel has a different behavior in environment in cases of accidental spill and groundwater contamination. It was noticed that the biodegradation of hydrocarbons (cyclic and aliphatic) in the presence of biodiesel are speeded, although the mechanism is still unclear. Considering the chemical structure of fatty esters, it was investigated the formation of aggregates in water solution by fatty esters present in commercial biodiesel. In Brazil, biodiesel is composed by 95% of fossil diesel and 5% of fatty esters mixture. In this work, fatty esters were treated as neutral surfactant, i.e., it was treated as a molecule with polar and non-polar part. Turbidity and fluorescence were used to determine the critical aggregates concentration (CAC). Water solutions containing fatty esters were examined exploiting changes in turbidity and fluorescence intensity of pyrene. Abrupt changes were attributed to aggregates formation, following the same behavior of traditional amphiphilic compounds. It was determined the CAC for ethyl palmitate, ethyl stearate, ethyl oleate, and ethyl linoleate. The values of CAC for fatty esters varied from 1.91 to 4.27 μmol/L, while CAC for the mixture of esters (biodiesel) was 2.01 for methyl esters and 1.19 for ethyl esters, both prepared using soybean oil. The aggregates formation was also determined by fluorescence measurements considering the changes in intensity of peaks I and III of pyrene. Pyrene senses the changes in environment polarity. The values found of CAC by fluorescence for individual ethyl esters varied from 1.85 to 3.21 μmol/L, while mixtures of ethyl esters was 2.23 and 2.07 μmol/L for mixture of methyl esters. The results clearly showed that fatty esters form aggregates and might be

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

Science.gov (United States)

Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe; Zurzolo, Chiara

2017-12-01

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. © 2017 The Author(s).

Single-Photon Source for Quantum Information Based on Single Dye Molecule Fluorescence in Liquid Crystal Host

International Nuclear Information System (INIS)

Lukishova, S.G.; Knox, R.P.; Freivald, P.; McNamara, A.; Boyd, R.W.; Stroud, Jr. C.R.; Schmid, A.W.; Marshall, K.L.

2006-01-01

This paper describes a new application for liquid crystals: quantum information technology. A deterministically polarized single-photon source that efficiently produces photons exhibiting antibunching is a pivotal hardware element in absolutely secure quantum communication. Planar-aligned nematic liquid crystal hosts deterministically align the single dye molecules which produce deterministically polarized single (antibunched) photons. In addition, 1-D photonic bandgap cholesteric liquid crystals will increase single-photon source efficiency. The experiments and challenges in the observation of deterministically polarized fluorescence from single dye molecules in planar-aligned glassy nematic-liquid-crystal oligomer as well as photon antibunching in glassy cholesteric oligomer are described for the first time

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

Science.gov (United States)

Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

2016-04-01

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

OpenAIRE

Laat, S.W. de; Tetteroo, P.A.T.; Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van

1984-01-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xaopus Levis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein (“HEDAF”) and 5-(N-tetradecanoyl)aminofluorescein (“TEDAF”) as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FP...

Electric Dipole Transition Moments and Solvent-Dependent Interactions of Fluorescent Boron-Nitrogen Substituted Indole Derivatives.

Science.gov (United States)

Saif, Mari; Widom, Julia R; Xu, Senmiao; Abbey, Eric R; Liu, Shih-Yuan; Marcus, Andrew H

2015-06-25

Fluorescent analogues of the indole side chain of tryptophan can be useful spectroscopic probes of protein-protein and protein-DNA interactions. Here we present linear dichroism and solvent-dependent spectroscopic studies of two fluorescent analogues of indole, in which the organic C═C unit is substituted with the isosteric inorganic B-N unit. We studied the so-called "external" BN indole, which has C2v symmetry, and the "fused" BN indole with Cs symmetry. We performed a combination of absorption and fluorescence spectroscopy, ultraviolet linear dichroism (UV-LD) in stretched poly(ethylene) (PE) films, and quantum chemical calculations on both BN indole compounds. Our measurements allowed us to characterize the degree of alignment for both molecules in stretched PE films. We thus determined the orientations and magnitudes of the two lowest energy electric dipole transition moments (EDTMs) for external BN indole, and the two lowest energy EDTMs for fused BN indole within the 30 000-45 000 cm(-1) spectral range. We compared our experimental results to those of quantum chemical calculations using standard density functional theory (DFT). Our theoretical predictions for the low-energy EDTMs are in good agreement with our experimental data. The absorption and fluorescence spectra of the external and the fused BN indoles are sensitive to solvent polarity. Our results indicate that the fused BN indole experiences much greater solvation interactions with polar solvents than does the external BN indole.

Strong plasmonic enhancement of single molecule photostability in silver dimer optical antennas

Directory of Open Access Journals (Sweden)

Kaminska Izabela

2018-02-01

Full Text Available Photobleaching is an effect terminating the photon output of fluorophores, limiting the duration of fluorescence-based experiments. Plasmonic nanoparticles (NPs can increase the overall fluorophore photostability through an enhancement of the radiative rate. In this work, we use the DNA origami technique to arrange a single fluorophore in the 12-nm gap of a silver NP dimer and study the number of emitted photons at the single molecule level. Our findings yielded a 30× enhancement in the average number of photons emitted before photobleaching. Numerical simulations are employed to rationalize our results. They reveal the effect of silver oxidation on decreasing the radiative rate enhancement.

Real-Time Dosimetry and Optimization of Prostate Photodynamic Therapy

Science.gov (United States)

2005-05-01

85cm’ 0:k -" l)=O5.0O25,=0.10cnf’ Ps4 ൱ crm’ 0 cik)=5.012p 5,4=5.45c,1’. G~cff’* - 0~n) .012,P. 0.49cff’,0:=3.56c-0 cink) = - 1,00 c ’", V-= 1.75 ccml...tissue to make a measurement. This limits the resolution of absorption spectroscopy, and makes it more time- consuming than fluorescence spectroscopy...photobleaching versus those that do not. Further research into the photobleaching behavior and in vivo vascular effects of MLu is needed to resolve the

Environment-sensitive quinolone demonstrating long-lived fluorescence and unusually slow excited-state intramolecular proton transfer kinetics

Czech Academy of Sciences Publication Activity Database

Zamotaiev, O. M.; Shvadchak, Volodymyr; Sych, T. P.; Melnychuk, N. A.; Yushchenko, Dmytro A.; Mely, Y.; Pivovarenko, V. G.

2016-01-01

Roč. 4, č. 3 (2016), č. článku 034004. ISSN 2050-6120 Institutional support: RVO:61388963 Keywords : quinolone * fluorescent probes * local polarity * hydration * excited-state intramolecular proton transfer * kinetics Subject RIV: CC - Organic Chemistry Impact factor: 2.656, year: 2016

Photoinduced intramolecular charge transfer (ICT) reaction in trans-methyl p-(dimethylamino) cinnamate: A combined fluorescence measurement and quantum chemical calculations

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, Amrita [Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata 700009 (India); Kar, Samiran [Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032 (India); Guchhait, Nikhil [Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata 700009 (India)], E-mail: nikhilg@postmark.net

2006-01-05

The photophysical behaviour of trans-methyl p-(dimethylamino) cinnamate (t-MDMAC) donor-acceptor system has been investigated by steady-state absorption and emission spectroscopy and quantum chemical calculations. The molecule t-MDMAC shows an emission from the locally excited state in non-polar solvents. In addition to weak local emission, a strong solvent dependent red shifted fluorescence in polar aprotic solvents is attributed to highly polar intramolecular charge transfer state. However, the formation of hydrogen-bonded clusters with polar protic solvents has been suggested from a linear correlation between the observed red shifted fluorescence band maxima with hydrogen bonding parameters ({alpha}). Calculations by ab initio and density functional theory show that the lone pair electron at nitrogen center is out of plane of the benzene ring in the global minimum ground state structure. In the gas phase, a potential energy surface along the twist coordinate at the donor (-NMe{sub 2}) and acceptor (-CH = CHCOOMe) sites shows stabilization of S{sub 1} state and destabilization S{sub 2} and S{sub 0} states. A similar potential energy calculation along the twist coordinate in acetonitrile solvent using non-equilibrium polarized continuum model also shows more stabilization of S{sub 1} state relative to other states and supports solvent dependent red shifted emission properties. In all types of calculations it is found that the nitrogen lone pair is delocalized over the benzene ring in the global minimum ground state and is localized on the nitrogen centre at the 90 deg. twisted configuration. The S{sub 1} energy state stabilization along the twist coordinate at the donor site and localized nitrogen lone pair at the perpendicular configuration support well the observed dual fluorescence in terms of proposed twisted intramolecular charge transfer (TICT) model.

A customized light sheet microscope to measure spatio-temporal protein dynamics in small model organisms.

Directory of Open Access Journals (Sweden)

Matthias Rieckher

Full Text Available We describe a customizable and cost-effective light sheet microscopy (LSM platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP, which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.

Functionality and stability data of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology

Directory of Open Access Journals (Sweden)

Luis F. Padilla-Morales

2016-03-01

Full Text Available The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor (nAChR rich membrane solubilized with long chain (16 saturated carbons lysophospholipid with glycerol headgroup (LFG-16. The assessment of stability and functionality of solubilized membrane protein is a critical step prior to further crystallization trails. One of the key factors for this task is the appropriate choice of a detergent that can support nAChR activity and stability comparable to the crude membranes. The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique. Keywords: Detergents, Fluorescence recovery after photobleaching, Lipidic Cubic Phase, nAChR, Planar lipid bilayer, Two-electrode voltage clamp

Insights into bird wing evolution and digit specification from polarizing region fate maps.

Science.gov (United States)

Towers, Matthew; Signolet, Jason; Sherman, Adrian; Sang, Helen; Tickle, Cheryll

2011-08-09

The proposal that birds descended from theropod dinosaurs with digits 2, 3 and 4 was recently given support by short-term fate maps, suggesting that the chick wing polarizing region-a group that Sonic hedgehog-expressing cells-gives rise to digit 4. Here we show using long-term fate maps that Green fluorescent protein-expressing chick wing polarizing region grafts contribute only to soft tissues along the posterior margin of digit 4, supporting fossil data that birds descended from theropods that had digits 1, 2 and 3. In contrast, digit IV of the chick leg with four digits (I-IV) arises from the polarizing region. To determine how digit identity is specified over time, we inhibited Sonic hedgehog signalling. Fate maps show that polarizing region and adjacent cells are specified in parallel through a series of anterior to posterior digit fates-a process of digit specification that we suggest is involved in patterning all vertebrate limbs with more than three digits.

Production and degradation of fluorescent dissolved organic matter in surface waters of the eastern north Atlantic ocean

Science.gov (United States)

Lønborg, Christian; Yokokawa, Taichi; Herndl, Gerhard J.; Antón Álvarez-Salgado, Xosé

2015-02-01

The distribution and fate of coloured dissolved organic matter (CDOM) in the epipelagic Eastern North Atlantic was investigated during a cruise in the summer 2009 by combining field observations and culture experiments. Dissolved organic carbon (DOC) and nitrogen (DON), the absorption spectra of CDOM and the fluorescence intensity of proteins (Ex/Em 280/320 nm; F(280/320)) and marine humic-like substances (F(320/410)) were measured in the upper 200 m. DOC and DON showed higher concentrations in the top 20 m than below, and DOC increased southwards, while DON decreased. F(280/320) and F(320/410) showed maxima near the deep chlorophyll maximum (at about 50 m), suggesting that these fluorophores were linked to phytoplankton production and the metabolism of the associated microbial community. The coloured and fluorescent fractions of DOM showed low levels south of the Azores Front, at about 35 °N, likely due to the accumulated photobleaching of the waters transported eastwards by the Azores current into the study area (at 20°W). Twelve culture experiments were also conducted with surface water (5 m) to assess the impact of microbial degradation processes on the bulk, coloured and fluorescent fractions of DOM. After 72 h of incubation in the darkness, 14±9% (average±SD) of the initial DON was consumed at an average rate of 0.24±0.14 μmol l-1 d-1 and the protein-like fluorescence decayed by 29±9% at a net rate of 0.06±0.03 QSU d-1. These rates were significantly lower south of the Azores front, suggesting that DOM in this region was of a more recalcitrant nature. Conversely, the marine humic-like fluorescence increased at a net rate of 0.013±0.003 QSU d-1. The close linear relationship of DON uptake with F(280/320) consumption (R2= 0.91, p <0.0001, n=12) and F(320/410) production (R2= 0.52, p <0.008, n=12) that we found during these incubation experiments suggest that the protein-like fluorescence can be used as a proxy for the dynamics of the labile DON pool

Unusual case of a polar copper(II) uranyl phosphonate that fluoresces

Energy Technology Data Exchange (ETDEWEB)

Nelson, A.G.D.; Albrecht-Schmitt, Th.E. [Department of Civil Engineering and Geological Sciences and Department of Chemistry and Biochemistry, 156 Fitzpatrick Hall, University of Notre Dame, Notre Dame, Indiana (United States)

2010-06-15

A polar Cu(II) uranyl diphosphonate, Cu(H{sub 2}O){sub 4}(UO{sub 2}){sub 3}(H{sub 2}O){sub 2}[CH{sub 2}(PO{sub 3}){sub 2}]{sub 2}.5H{sub 2}O, has been prepared under mild hydrothermal conditions. This compound has direct linkages between the oxo atoms of the uranyl moieties and the Cu(II) centers. Despite the presence of Cu(II) in the structure, vibronically-coupled emission is still observed, most likely because there are two crystallographically unique uranyl moieties, only one of which bonds to Cu(II). (authors)

Vertical profiles of atmospheric fluorescent aerosols observed by a mutil-channel lidar spectrometer system

Science.gov (United States)

Huang, Z.; Huang, J.; Zhou, T.; Sugimoto, N.; Bi, J.

2015-12-01

Zhongwei Huang1*, Jianping Huang1, Tian Zhou1, Nobuo Sugimoto2, Jianrong Bi1 and Jinsen Shi11Key Laboratory for Semi-Arid Climate Change of the Ministry of Education, College of Atmospheric Sciences, Lanzhou University, Lanzhou, China. 2Atmospheric Environment Division, National Institutes for Environmental Studies, Tsukuba, Japan Email: huangzhongwei@lzu.edu.cn Abstract Atmospheric aerosols have a significant impact on regional and globe climate. The challenge in quantifying aerosol direct radiative forcing and aerosol-cloud interactions arises from large spatial and temporal heterogeneity of aerosol concentrations, compositions, sizes, shape and optical properties (IPCC, 2007). Lidar offers some remarkable advantages for determining the vertical structure of atmospheric aerosols and their related optical properties. To investigate the characterization of atmospheric aerosols (especially bioaerosols) with high spatial and temporal resolution, we developed a Raman/fluorescence/polarization lidar system employed a multi-channel spectrometer, with capabilities of providing measurements of Raman scattering and laser-induced fluorescence excitation at 355 nm from atmospheric aerosols. Meanwhile, the lidar system operated polarization measurements both at 355nm and 532nm wavelengths, aiming to obtain more information of aerosols. It employs a high power pulsed laser and a received telescope with 350mm diameter. The receiver could simultaneously detect a wide fluorescent spectrum about 178 nm with spectral resolution 5.7 nm, mainly including an F/3.7 Crossed Czerny-Turner spectrograph, a grating (1200 gr/mm) and a PMT array with 32 photocathode elements. Vertical structure of fluorescent aerosols in the atmosphere was observed by the developed lidar system at four sites across northwest China, during 2014 spring field observation that conducted by Lanzhou University. It has been proved that the developed lidar could detect the fluorescent aerosols with high temporal and

Chlorophyll bleaching by UV-irradiation in vitro and in situ: Absorption and fluorescence studies

International Nuclear Information System (INIS)

Zvezdanovic, Jelena; Cvetic, Tijana; Veljovic-Jovanovic, Sonja; Markovic, Dejan

2009-01-01

Chlorophyll bleaching by UV-irradiation has been studied by absorbance and fluorescence spectroscopy in extracts containing mixtures of photosynthetic pigments, in acetone and n-hexane solutions, and in aqueous thylakoid suspensions. Chlorophyll undergoes destruction (bleaching) accompanied by fluorescent transient formation obeying first-order kinetics. The bleaching is governed by UV-photon energy input, as well as by different chlorophyll molecular organizations in solvents of different polarities (in vitro), and in thylakoids (in situ). UV-C-induced bleaching of chlorophylls in thylakoids is probably caused by different mechanisms compared to UV-A- and UV-B-induced bleaching

Polarization measurement for internal polarized gaseous targets

International Nuclear Information System (INIS)

Ye Zhenyu; Ye Yunxiu; Lv Haijiang; Mao Yajun

2004-01-01

The authors present an introduction to internal polarized gaseous targets, polarization method, polarization measurement method and procedure. To get the total nuclear polarization of hydrogen atoms (including the polarization of the recombined hydrogen molecules) in the target cell, authors have measured the parameters relating to atomic polarization and polarized hydrogen atoms and molecules. The total polarization of the target during our measurement is P T =0.853 ± 0.036. (authors)

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

Science.gov (United States)

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-03-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

Differential dynamics of splicing factor SC35 during the cell cycle

Indian Academy of Sciences (India)

Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of ...

[Fluorescence polarization used to investigate the cell membrane fluidity of Saccharomyces cerevisiae treated by pulsed electric field].

Science.gov (United States)

Zhang, Ying; Zeng, Xin-An; Wen, Qi-Biao; Li, Lin

2008-01-01

To know the lethal mechanism of microorganisms under pulsed electric field treatment, the relationship between the inactivation of Saccharomyces cerevisiae (CICC1308) cell and the permeability and fluidity changes of its cell membrane treated by pulsed electric field (0-25 kV x cm(-1), 0-266 ms) was investigated. With 1,6-diphenyl-1,3,5-hexatriene (DPH) used as a probe, the cell membrane fluidity of Saccharomyces cerevisiae treated by pulsed electric field was expressed by fluorescence polarization. Results showed that the cell membrane fluidity decreases when the electric flied strength is up to 5 kV x cm(-1), and decreases with the increase in electric field strength and treatment time. The plate counting method and ultraviolet spectrophotometer were used to determine the cell viability and to investigate the cell membrane permeability, respectively, treated by pulsed electric field. Results showed that the lethal ratio and the content of protein and nucleic acid leaked from intracellular plasma increased with the increase in the electric field strength and the extension of treatment time. Even in a quite lower electric field of 5 kV x cm(-1) with a tiny microorganism lethal level, the increase in UV absorption value and the decrease in fluidity were significant. It was demonstrated that the cell membrane fluidity decreases with the increase in lethal ratio and cell membrane permeability. The viscosity of cell membrane increases with the decrease in fluidity. These phenomena indicated that cell membrane is one of the most key sites during the pulsed electric field treatment, and the increased membrane permeability and the decreased cell membrane fluidity contribute to the cell death.

Myosin II dynamics are regulated by tension in intercalating cells.

Science.gov (United States)

Fernandez-Gonzalez, Rodrigo; Simoes, Sérgio de Matos; Röper, Jens-Christian; Eaton, Suzanne; Zallen, Jennifer A

2009-11-01

Axis elongation in Drosophila occurs through polarized cell rearrangements driven by actomyosin contractility. Myosin II promotes neighbor exchange through the contraction of single cell boundaries, while the contraction of myosin II structures spanning multiple pairs of cells leads to rosette formation. Here we show that multicellular actomyosin cables form at a higher frequency than expected by chance, indicating that cable assembly is an active process. Multicellular cables are sites of increased mechanical tension as measured by laser ablation. Fluorescence recovery after photobleaching experiments show that myosin II is stabilized at the cortex in regions of increased tension. Myosin II is recruited in response to an ectopic force and relieving tension leads to a rapid loss of myosin, indicating that tension is necessary and sufficient for cortical myosin localization. These results demonstrate that myosin II dynamics are regulated by tension in a positive feedback loop that leads to multicellular actomyosin cable formation and efficient tissue elongation.

Self diffusion and spectral modifications of a membrane protein, the Rubrivivax gelatinosus LH2 complex, incorporated into a monoolein cubic phase.

OpenAIRE

Tsapis, N; Reiss-Husson, F; Ober, R; Genest, M; Hodges, R S; Urbach, W

2001-01-01

The light-harvesting complex LH2 from a purple bacterium, Rubrivivax gelatinosus, has been incorporated into the Q230 cubic phase of monoolein. We measured the self-diffusion of LH2 in detergent solution and in the cubic phase by fluorescence recovery after photobleaching. We investigated also the absorption and fluorescence properties of this oligomeric membrane protein in the cubic phase, in comparison with its beta-octyl glucoside solution. In these experiments, native LH2 and LH2 labeled ...

New insights into the dual fluorescence of methyl salicylate: effects of intermolecular hydrogen bonding and solvation.

Science.gov (United States)

Zhou, Panwang; Hoffmann, Mark R; Han, Keli; He, Guozhong

2015-02-12

In this paper, we propose a new and complete mechanism for dual fluorescence of methyl salicylate (MS) under different conditions using a combined experimental (i.e., steady-state absorption and emission spectra and time-resolved fluorescence spectra) and theoretical (i.e., time-dependent density function theory) study. First, our theoretical study indicates that the barrier height for excited state intramolecular proton transfer (ESIPT) reaction of ketoB depends on the solvent polarity. In nonpolar solvents, the ESIPT reaction of ketoB is barrierless; the barrier height will increase with increasing solvent polarity. Second, we found that, in alcoholic solvents, intermolecular hydrogen bonding plays a more important role. The ketoB form of MS can form two hydrogen bonds with alcoholic solvents; one will facilitate ESIPT and produce the emission band in the blue region; the other one precludes ESIPT and produces the emission band in the near-UV region. Our proposed new mechanism can well explain previous results as well as our new experimental results.

Microtubule dynamics of the centrosome-like polar organizers from the basal land plant Marchantia polymorpha.

Science.gov (United States)

Buschmann, Henrik; Holtmannspötter, Michael; Borchers, Agnes; O'Donoghue, Martin-Timothy; Zachgo, Sabine

2016-02-01

The liverwort Marchantia employs both modern and ancestral devices during cell division: it forms preprophase bands and in addition it shows centrosome-like polar organizers. We investigated whether polar organizers and preprophase bands cooperate to set up the division plane. To this end, two novel green fluorescent protein-based microtubule markers for dividing cells of Marchantia were developed. Cells of the apical notch formed polar organizers first and subsequently assembled preprophase bands. Polar organizers were formed de novo from multiple mobile microtubule foci localizing to the nuclear envelope. The foci then became concentrated by bipolar aggregation. We determined the comet production rate of polar organizers and show that microtubule plus ends of astral microtubules polymerize faster than those found on cortical microtubules. Importantly, it was observed that conditions increasing polar organizer numbers interfere with preprophase band formation. The data show that polar organizers have much in common with centrosomes, but that they also have specialized features. The results suggest that polar organizers contribute to preprophase band formation and in this way are involved in controlling the division plane. Our analyses of the basal land plant Marchantia shed new light on the evolution of plant cell division. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Stress tolerance and stress-induced injury in crop plants measured by chlorophyll fluorescence in vivo: chilling, freezing, ice cover, heat, and high light.

Science.gov (United States)

Smillie, R M; Hetherington, S E

1983-08-01

The proposition is examined that measurements of chlorophyll fluorescence in vivo can be used to monitor cellular injury caused by environmental stresses rapidly and nondestructively and to determine the relative stress tolerances of different species. Stress responses of leaf tissue were measured by F(R), the maximal rate of the induced rise in chlorophyll fluorescence. The time taken for F(R) to decrease by 50% in leaves at 0 degrees C was used as a measure of chilling tolerance. This value was 4.3 hours for chilling-sensitive cucumber. In contrast, F(R) decreased very slowly in cucumber leaves at 10 degrees C or in chilling-tolerant cabbage leaves at 0 degrees C. Long-term changes in F(R) of barley, wheat, and rye leaves kept at 0 degrees C were different in frost-hardened and unhardened material and in the latter appeared to be correlated to plant frost tolerance. To simulate damage caused by a thick ice cover, wheat leaves were placed at 0 degrees C under N(2). Kharkov wheat, a variety tolerant of ice encapsulation, showed a slower decrease in F(R) than Gatcher, a spring wheat. Relative heat tolerance was also indicated by the decrease in F(R) in heated leaves while changes in vivo resulting from photoinhibition, ultraviolet radiation, and photobleaching can also be measured.

Quantitative comparison of X-ray fluorescence microtomography setups: Standard and confocal collimator apparatus

Energy Technology Data Exchange (ETDEWEB)

Chukalina, M. [Institute of Microelectronics Technology RAS, 142432, Chernogolovka, Moscow District (Russian Federation)], E-mail: marina@ipmt-hpm.ac.ru; Simionovici, A. [Laboratoire de Geophysique Interne et Tectonophysique, University of Grenoble, BP 53, 38041, Grenoble (France)], E-mail: alexandre.simionovici@ujf-grenoble.fr; Zaitsev, S. [Institute of Microelectronics Technology RAS, 142432, Chernogolovka, Moscow District (Russian Federation)], E-mail: zaitsev@ipmt-hpm.ac.ru; Vanegas, C.J. [Institute of Microelectronics Technology RAS, 142432, Chernogolovka, Moscow District (Russian Federation)], E-mail: vanegas@ipmt-hpm.ac.ru

2007-07-15

Recently, there has been a renewed interest for fluorescence spectroscopy, as provided by modern setups which allow 2D and 3D imaging of elemental distributions. Two directions are currently under development: the SR-based fluorescence tomography in polar scanning geometry, provided by the new generation of X-ray microprobes and the confocal scanning geometry, which can be fielded in both SR and laboratory environments. The new probes bring forth a new age in fluorescence spectrometry: high resolution, high intensity and high sensitivity which allow 3D elemental mapping of volumes. The major task now is the development of these complex tools into fully quantitative probes, reproducible and straightforward for general use. In this work we analyze two X-ray fluorescence microtomography techniques: an apparatus tomography using a confocal collimator for the data collection and a standard first generation Computed Tomography (CT) in the parallel scanning scheme. We calculate the deposited dose (amount of energy deposited and distributed in the sample during the data collection time) and find the conditions for the choice of the tomography scheme.

A polarized view on DNA under tension

Science.gov (United States)

van Mameren, Joost; Vermeulen, Karen; Wuite, Gijs J. L.; Peterman, Erwin J. G.

2018-03-01

In the past decades, sensitive fluorescence microscopy techniques have contributed significantly to our understanding of the dynamics of DNA. The specific labeling of DNA using intercalating dyes has allowed for quantitative measurement of the thermal fluctuations the polymers undergo. On the other hand, recent advances in single-molecule manipulation techniques have unraveled the mechanical and elastic properties of this intricate polymer. Here, we have combined these two approaches to study the conformational dynamics of DNA under a wide range of tensions. Using polarized fluorescence microscopy in conjunction with optical-tweezers-based manipulation of YOYO-intercalated DNA, we controllably align the YOYO dyes using DNA tension, enabling us to disentangle the rapid dynamics of the dyes from that of the DNA itself. With unprecedented control of the DNA alignment, we resolve an inconsistency in reports about the tilted orientation of intercalated dyes. We find that intercalated dyes are on average oriented perpendicular to the long axis of the DNA, yet undergo fast dynamics on the time scale of absorption and fluorescence emission. In the overstretching transition of double-stranded DNA, we do not observe changes in orientation or orientational dynamics of the dyes. Only beyond the overstretching transition, a considerable depolarization is observed, presumably caused by an average tilting of the DNA base pairs. Our combined approach thus contributes to the elucidation of unique features of the molecular dynamics of DNA.

Preparation of Plasmonic Platforms of Silver Wires on Gold Mirrors and Their Application to Surface Enhanced Fluorescence

Science.gov (United States)

2015-01-01

In this report we describe a preparation of silver wires (SWs) on gold mirrors and its application to surface enhanced fluorescence (SEF) using a new methodology. Silica protected gold mirrors were drop-coated with a solution of silver triangular nanoprisms. The triangular nanoprisms were slowly air-dried to get silver wires that self-assembled on the gold mirrors. Fluorescence enhancement was studied using methyl azadioxatriangulenium chloride (Me-ADOTA·Cl) dye in PVA spin-coated on a clean glass coverslip. New Plasmonic Platforms (PPs) were assembled by placing a mirror with SWs in contact with a glass coverslip spin-coated with a uniform Me-ADOTA·Cl film. It was shown that surface enhanced fluorescence is a real phenomenon, not just an enhancement of the fluorescence signal due to an accumulation of the fluorophore on rough nanostructure surfaces. The average fluorescence enhancement was found to be about 15-fold. The lifetime of Me-ADOTA·Cl dye was significantly reduced (∼4 times) in the presence of SWs. Moreover, fluorescence enhancement and lifetime did not show any dependence on the excitation light polarization. PMID:25296293

Capillary Ion Concentration Polarization for Power-Free Salt Purification

Science.gov (United States)

Park, Sungmin; Jung, Yeonsu; Cho, Inhee; Kim, Ho-Young; Kim, Sung Jae

2014-11-01

In this presentation, we experimentally and theoretically demonstrated the capillary based ion concentration polarization for power-free salt purification system. Traditional ion concentration polarization phenomenon has been studied for a decade for both fundamental nanoscale fluid dynamics and novel engineering applications such as desalination, preconcentration and energy harvesting devices. While the conventional system utilizes an external power source, the system based on capillary ion concentration polarization is capable of perm-selective ion transportation only by capillarity so that the same ion depletion zone can be formed without any external power sources. An ion concentration profile near the nanostructure was tracked using fluorescent probes and analyzed by solving the modified Nernst-Planck equation. As a result, the concentration in the vicinity of the nanostructure was at least 10 times lower than that of bulk electrolyte and thus, the liquid absorbed into the nanostructure had the low concentration. This mechanism can be used for the power free salt purification system which would be significantly useful in underdeveloped and remote area. This work was supported by Samsung Research Funding Center of Samsung Electronics under Project Number SRFC-MA1301-02.

EPR and Fluorescence Spectroscopy in the Photodegradation Study of Arabian and Colombian Crude Oils

Directory of Open Access Journals (Sweden)

Carmen L. B. Guedes

2006-01-01

W/m2. The reduction in the linewidth of the free radical of 9.8% in Arabian oil and 18.5% in Colombian oil, as well as the decrease in radical numbers, indicated photochemical degradation, especially in Colombian oil. The linewidth narrowing corresponding to free radicals in the irradiated oils occurred due to the rearrangement among radicals and aromatic carbon consumption. The irradiated oils showed a reduction in the relative intensity of fluorescence of the aromatics with high molecular mass, polar aromatics, and asphaltene. The fluorescent fraction was reduced by 61% in Arabian oil and 72% in Colombian oil, corresponding to photochemical degradation of crude oil aromatic compounds.

Enriching PMMA nanospheres with adjustable charges as novel templates for multicolored dye-PMMA nanocomposites

International Nuclear Information System (INIS)

Wang Xumei; Xu Shuping; Xu Weiqing; Liang Chongyang; Li Hongrui; Sun Fei

2011-01-01

Multicolored fluorescent dye loaded PMMA nanospheres were synthesized by the electrostatic adsorption of dye molecules on the charged PMMA nanospheres, whose charges were adjusted by choosing different initiators. The charged PMMA nanospheres have a wider capacity and advantage for combining the charged dyes. The fluorescent dye-PMMA composite nanospheres possess the advantages of higher brightness, longer lifetime and stronger resistance to photobleaching relative to dye molecules. Dye leakage remained lower than 5% over one week. These fluorescent nanospheres have been used in biological labels in cell imaging. They can easily stain blood cancer cells without further surface modification.

M2 polarization enhances silica nanoparticle uptake by macrophages

Directory of Open Access Journals (Sweden)

Jessica eHoppstädter

2015-03-01

Full Text Available While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth.We employed different models of M1 and M2 polarization: GM-CSF/LPS/IFN-gamma was used to generate primary human M1 cells and M-CSF/IL-10 to differentiate M2 monocyte-derived macrophages. PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-gamma and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø 26 and 41 nm and microparticles (Ø 1.75 µm was quantified. At the concentration used (50 µg/ml, silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human monocyte-derived macrophages compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages (TAM obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue.In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but might also open up therapeutic perspectives allowing to specifically target M2

Fluorescence line-narrowing studies of rare earths in disordered solids

International Nuclear Information System (INIS)

Hall, D.W.

1982-01-01

This dissertation is made up of two experimental studies dealing with apparently diverse topics within the subject of rare earths (RE) in solids. The first study, described in Part II, concerns the vibrations of a disordered host material about an optically active rare-earth ion as manifested by vibrationally-assisted-electronic, or vibronic transitions. Part III of the dissertation describes an investigation of the influence of site anisotropy on the purely electronic, laser transition of Nd 3+ in glass. These two studies are bound together by the common experimental technique of laser-induced fluorescence line narrowing (FLN). By exciting fluorescence with monochromatic light of well-characterized polarization, one may select and observe the response of a single subset of the optically active ions and obtain information that is usually masked by the inhomogeneous nature of disordered solids

Energy transfer between surface-immobilized light-harvesting chlorophyll a/b complex (LHCII) studied by surface plasmon field-enhanced fluorescence spectroscopy (SPFS).

Science.gov (United States)

Lauterbach, Rolf; Liu, Jing; Knoll, Wolfgang; Paulsen, Harald

2010-11-16

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that combines rapid and efficient excitation energy transfer with effective protection of its pigments from photobleaching. These properties make LHCII potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Most of such applications would require the LHCII to be immobilized on a solid surface. In a previous study we showed the immobilization of recombinant LHCII on functionalized gold surfaces via a 6-histidine tag (His tag) in the protein moiety. In this work the occurrence and efficiency of Förster energy transfer between immobilized LHCII on a functionalized surface have been analyzed by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). A near-infrared dye was attached to some but not all of the LHC complexes, serving as an energy acceptor to chlorophylls. Analysis of the energy transfer from chlorophylls to this acceptor dye yielded information about the extent of intercomplex energy transfer between immobilized LHCII.

Response of biomembrane domains to external stimuli

Science.gov (United States)

Urbancic, Iztok

To enrich our knowledge about membrane domains, new measurement techniques with extended spatial and temporal windows are being vigorously developed by combining various approaches. Following such efforts of the scientific community, we set up fluorescence microspectroscopy (FMS), bridging two well established methods: fluorescence microscopy, which enables imaging of the samples with spatial resolution down to 200 nm, and fluorescence spectroscopy that provides molecular information of the environment at nanometer and nanosecond scale. The combined method therefore allows us to localize this type of information with the precision suitable for studying various cellular structures. Faced with weak available fluorescence signals, we have put considerable efforts into optimization of measurement processes and analysis of the data. By introducing a novel acquisition scheme and by fitting the data with a mathematical model, we preserved the spectral resolution, characteristic for spectroscopic measurements of bulk samples, also at microscopic level. We have at the same time overcome the effects of photobleaching, which had previously considerably distorted the measured spectral lineshape of photosensitive dyes and consequently hindered the reliability of FMS. Our new approach has therefore greatly extended the range of applicable environmentally sensitive probes, which can now be designed to better accommodate the needs of each particular experiment. Moreover, photobleaching of fluorescence signal can now even be exploited to obtain new valuable information about molecular environment of the probes, as bleaching rates of certain probes also depend on physical and chemical properties of the local surroundings. In this manner we increased the number of available spatially localized spectral parameters, which becomes invaluable when investigating complex biological systems that can only be adequately characterized by several independent variables. Applying the developed

Mn(II)-coordinated Fluorescent Carbon Dots: Preparation and Discrimination of Organic Solvents

Science.gov (United States)

Wang, Yuru; Wang, Tianren; Chen, Xi; Xu, Yang; Li, Huanrong

2018-04-01

Herein, we prepared a Mn(II)-coordinated carbon dots (CDs) with fluorescence and MRI (magnetic resonance imaging) bimodal properties by a one-pot solvothermal method and separated via silica column chromatography. The quantum yield of the CDs increased greatly from 2.27% to 6.75% with increase of Mn(II) doping, meanwhile the CDs exhibited a higher MR activity (7.28 mM-1s-1) than that of commercial Gd-DTPA (4.63 mM-1s-1). In addition, white light emitting CDs were obtained by mixing the different types of CDs. Notably, these CDs exhibited different fluorescence emissions in different organic solvents and could be used to discriminate organic solvents based on the polarity and protonation of the solvents.

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

DEFF Research Database (Denmark)

Börner, Richard; Ehrlich, Nicky; Hohlbein, Johannes

2016-01-01

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels...... interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination...

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo.

Science.gov (United States)

Speksnijder, J E; Dohmen, M R; Tertoolen, L G; de Laat, S W

1985-07-01

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1'-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lobe area as compared to the animal plasma membrane area (on average 30%), demonstrating the existence of an animal-vegetal polarity in plasma membrane properties. At third cleavage, the differences between animal and vegetal plasma membrane region become even more pronounced; in the four animal micromeres the diffusion coefficient (D) and mobile fraction (MF) are 2.9 +/- 0.2 X 10(-9) cm2/sec and 51 +/- 2%, respectively, while in the four vegetal macromeres D = 5.0 +/- 0.3 X 10(-9) cm2/sec and MF = 78 +/- 2%. Superimposed upon the observed animal-vegetal polarity, the lateral diffusion in the polar lobe membrane area shows a cell-cycle-dependent modulation. The highest mean values for D are reached during the S phase (ranging from 7.0 to 7.8 X 10(-9) cm2/sec in the three cycles measured), while at the end of G2 phase and during early mitosis mean values for D have decreased significantly (ranging from 5.0 to 5.9 X 10(-9) cm2/sec). Diffusion rates in the animal membranes of the embryo are constant during the three successive cell cycles (D = 4.3-5.0 X 10(-9) cm2/sec), except for a peak at the S phase of the first cell cycle (D = 6.0 X 10(-9) cm2/sec). These results are discussed in relation with previously observed ultrastructural heterogeneities in the Nassarius egg plasma membrane. It is speculated that the observed animal-vegetal polarity in the organization of the egg membrane might play an important role in the process of cell diversification during early development.

Widefield and total internal reflection fluorescent structured illumination microscopy with scanning galvo mirrors

Science.gov (United States)

Chen, Youhua; Cao, Ruizhi; Liu, Wenjie; Zhu, Dazhao; Zhang, Zhiming; Kuang, Cuifang; Liu, Xu

2018-04-01

We present an alternative approach to realize structured illumination microscopy (SIM), which is capable for live cell imaging. The prototype utilizes two sets of scanning galvo mirrors, a polarization converter and a piezo-platform to generate a fast shifted, s-polarization interfered and periodic variable illumination patterns. By changing the angle of the scanning galvanometer, we can change the position of the spots at the pupil plane of the objective lens arbitrarily, making it easy to switch between widefield and total internal reflection fluorescent-SIM mode and adapting the penetration depth in the sample. Also, a twofold resolution improvement is achieved in our experiments. The prototype offers more flexibility of pattern period and illumination orientation changing than previous systems.

Characterization of a Biomimetic Mesophase Composed of Nonionic Surfactants and an Aqueous Solvent.

Science.gov (United States)

Adrien, V; Rayan, G; Reffay, M; Porcar, L; Maldonado, A; Ducruix, A; Urbach, W; Taulier, N

2016-10-11

We have investigated the physical and biomimetic properties of a sponge (L 3 ) phase composed of pentaethylene glycol monododecyl ether (C 12 E 5 ), a nonionic surfactant, an aqueous solvent, and a cosurfactant. The following cosurfactants, commonly used for solubilizing membrane proteins, were incorporated: n-octyl-β-d-glucopyranoside (β-OG), n-dodecyl-β-d-maltopyranoside (DDM), 4-cyclohexyl-1-butyl-β-d-maltoside (CYMAL-4), and 5-cyclohexyl-1-pentyl-β-d-maltoside (CYMAL-5). Partial phase diagrams of these systems were created. The L 3 phase was characterized using crossed polarizers, diffusion of a fluorescent probe by fluorescence recovery after pattern photobleaching (FRAPP), and freeze fracture electron microscopy (FFEM). By varying the hydration of the phase, we were able to tune the distance between adjacent bilayers. The characteristic distance (d b ) of the phase was obtained from small angle scattering (SAXS/SANS) as well as from FFEM, which yielded complementary d b values. These d b values were neither affected by the nature of the cosurfactant nor by the addition of membrane proteins. These findings illustrate that a biomimetic surfactant sponge phase can be created in the presence of several common membrane protein-solubilizing detergents, thus making it a versatile medium for membrane protein studies.

Fluorescence and NMR spectroscopy together with molecular simulations reveal amphiphilic characteristics of a Burkholderia biofilm exopolysaccharide.

Science.gov (United States)

Kuttel, Michelle M; Cescutti, Paola; Distefano, Marco; Rizzo, Roberto

2017-06-30

Biofilms are a collective mode of bacterial life in which a self-produced matrix confines cells in close proximity to each other. Biofilms confer many advantages, including protection from chemicals (including antibiotics), entrapment of useful extracellular enzymes and nutrients, as well as opportunities for efficient recycling of molecules from dead cells. Biofilm matrices are aqueous gel-like structures composed of polysaccharides, proteins, and DNA stabilized by intermolecular interactions that may include non-polar connections. Recently, polysaccharides extracted from biofilms produced by species of the Burkholderia cepacia complex were shown to possess clusters of rhamnose, a 6-deoxy sugar with non-polar characteristics. Molecular dynamics simulations are well suited to characterizing the structure and dynamics of polysaccharides, but only relatively few such studies exist of their interaction with non-polar molecules. Here we report an investigation into the hydrophobic properties of the exopolysaccharide produced by Burkholderia multivorans strain C1576. Fluorescence experiments with two hydrophobic fluorescent probes established that this polysaccharide complexes hydrophobic species, and NMR experiments confirmed these interactions. Molecular simulations to model the hydrodynamics of the polysaccharide and the interaction with guest species revealed a very flexible, amphiphilic carbohydrate chain that has frequent dynamic interactions with apolar molecules; both hexane and a long-chain fatty acid belonging to the quorum-sensing system of B. multivorans were tested. A possible role of the non-polar domains of the exopolysaccharide in facilitating the diffusion of aliphatic species toward specific targets within the biofilm aqueous matrix is proposed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A low cost multi-purpose experimental arrangement for variants in energy dispersive X-ray fluorescence analysis

International Nuclear Information System (INIS)

Nascimento Filho, V.F.; Silva, R.M.C.; Moraes, L.M.B.; Parreira, P.S.; Appoloni, R.C.; Silva, R.M.C.

2005-01-01

Based in an X-ray tower with four exits (two line and two point beams) experimental conditions were arranged to carry out variants in energy dispersive X-ray fluorescence analysis: (1) the conventional one (EDXRF), with excitation/detection of thin and thick samples, under vacuum and air atmosphere, (2) the X-ray energy dispersive micro- fluorescence analysis(μ-EDXRF), with 2D mapping, using a quartz capillar, (3) the total reflection X-ray fluorescence (TXRF), under He and air atmosphere, and (4) secondary target/polarized X-ray fluorescence (P-EDXRF). It was possible to use a Cu, Mo or W target on the X-ray tube, with or without filter (V, Fe, Ni and Zr), and Si(Li) or Si-PIN semicondutor detectors coupled to a multichannel analyzer. In addition, it was possible to use the point beam to carry out experiments on (5) X-ray radiography and (6) X-ray absorption, and the line beam on (7) X-ray diffractometry studies.

Spectroscopic imaging studies of nanoscale polarity and mass transport phenomena in self-assembled organic nanotubes.

Science.gov (United States)

Xu, Hao; Nagasaka, Shinobu; Kameta, Naohiro; Masuda, Mitsutoshi; Ito, Takashi; Higgins, Daniel A

2017-08-02

Synthetic organic nanotubes self-assembled from bolaamphiphile surfactants are now being explored for use as drug delivery vehicles. In this work, several factors important to their implementation in drug delivery are explored. All experiments are performed with the nanotubes immersed in ethanol. First, Nile Red (NR) and a hydroxylated Nile Red derivative (NR-OH) are loaded into the nanotubes and spectroscopic fluorescence imaging methods are used to determine the apparent dielectric constant of their local environment. Both are found in relatively nonpolar environments, with the NR-OH molecules preferring regions of relatively higher dielectric constant compared to NR. Unique two-color imaging fluorescence correlation spectroscopy (imaging FCS) measurements are then used along with the spectroscopic imaging results to deduce the dielectric properties of the environments sensed by mobile and immobile populations of probe molecules. The results reveal that mobile NR molecules pass through less polar regions, likely within the nanotube walls, while immobile NR molecules are found in more polar regions, possibly near the nanotube surfaces. In contrast, mobile and immobile NR-OH molecules are found to locate in environments of similar polarity. The imaging FCS results also provide quantitative data on the apparent diffusion coefficient for each dye. The mean diffusion coefficient for the NR dye was approximately two-fold larger than that of NR-OH. Slower diffusion by the latter could result from its additional hydrogen bonding interactions with polar triglycine, amine, and glucose moieties near the nanotube surfaces. The knowledge gained in these studies will allow for the development of nanotubes that are better engineered for applications in the controlled transport and release of uncharged, dipolar drug molecules.

The feasibility and application of PPy in cathodic polarization antifouling.

Science.gov (United States)

Jia, Meng-Yang; Zhang, Zhi-Ming; Yu, Liang-Min; Wang, Jia; Zheng, Tong-Tong

2018-04-01

Cathodic polarization antifouling deserves attention because of its environmentally friendly nature and good sustainability. It has been proven that cathodic voltages applied on metal substrates exhibit outstanding antifouling effects. However, most metals immersed in marine environment are protected by insulated anticorrosive coatings, restricting the cathodic polarization applied on metals. This study developed a conducting polypyrrole (PPy)/acrylic resin coating (σ = 0.18 Scm -1 ), which can be applied in cathodic polarization antifouling. The good stability and electro-activity of PPy in the negative polarity zone in alkalescent NaCl solution were verified by linear sweep voltammetry (LSV), chronoamperometry (CA), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), demonstrating the feasibility of PPy as cathodic polarization material. Furthermore, the antifouling effects of PPy/acrylicresin coating on 24-h old Escherichia coli bacteria (E. coli) which formed on PPy/acrylic resin-coated plastic plate were measured under different cathodic potentials and treatment time, characterized by fluorescent microscope. The results suggest that at cathodic potential around -0.5 V (vs. saturated calomel electrode (SCE)), there was little trace of attached bacteria on the substrate after 20 min of treatment. PPy/acrylicresin-coated substrates were also subjected to repeated cycles of biofilm formation and electrochemical removal, where high removal efficiencies were maintained throughout the total polarization process. Under these conditions, the generation of hydrogen peroxide is believed to be responsible for the antifouling effects because of causing oxidative damage to cells, suggesting the potential of the proposed technology for application on insulated surfaces in various industrial settings. Copyright © 2018 Elsevier B.V. All rights reserved.

QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

Directory of Open Access Journals (Sweden)

Merete Krog Raarup

2011-05-01

Full Text Available This paper discusses recent advances in confocal laser scanning microscopy (CLSM for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer, FLIM (Fluorescence Lifetime Imaging Microscopy, FCS (Fluorescence Correlation Spectroscopy and FRAP (Fluorescence Recovery After Photobleaching are introduced and their applicability for quantitative imaging of biomolecular (co-localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

Visualization of the Activity of Rac1 Small GTPase in a Cell

International Nuclear Information System (INIS)

Higashi, Morihiro; Yu, Jianyong; Tsuchiya, Hiroshi; Saito, Teruyoshi; Oyama, Toshinao; Kawana, Hidetada; Kitagawa, Motoo; Tamaru, Jun-ichi; Harigaya, Kenichi

2010-01-01

Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used as intermolecular probes of FRET. The fluorophores were separated with linear unmixing method. The fluorescence resonance energy transfer efficiency was measured by acceptor photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a cell. The present findings indicate that this approach is sensitive enough to achieve results similar to those from ratiometric fluorescence resonance energy transfer analysis

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

Energy Technology Data Exchange (ETDEWEB)

Lemelle, A; Veksler, B; Piletsky, S A; Meglinski, I [Cranfield Health, Cranfield University, Cranfield, MK43 0AL (United Kingdom); Kozhevnikov, I S; Akchurin, G G, E-mail: a.lemelle.s06@cranfield.ac.uk [Physics Faculty, Saratov State University, Saratov 410012 (Russian Federation)

2009-01-15

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres.

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

International Nuclear Information System (INIS)

Lemelle, A; Veksler, B; Piletsky, S A; Meglinski, I; Kozhevnikov, I S; Akchurin, G G

2009-01-01

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

Science.gov (United States)

Lemelle, A.; Veksler, B.; Kozhevnikov, I. S.; Akchurin, G. G.; Piletsky, S. A.; Meglinski, I.

2009-01-01

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres.

Live imaging of dense-core vesicles in primary cultured hippocampal neurons.

Science.gov (United States)

Kwinter, David M; Silverman, Michael A; Kwinter, David; Michael, Silverman

2009-05-29

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is

Producing fluorescent digital printing ink: Investigating the effect of type and amount of coumarin derivative dyes on the quality of ink

Energy Technology Data Exchange (ETDEWEB)

Ataeefard, Maryam, E-mail: ataeefard-m@icrc.ac.ir [Department of Printing Science and Technology, Institute for Color Science and Technology, P.O. Box 16765-654, Tehran (Iran, Islamic Republic of); Nourmohammadian, Farahnaz, E-mail: nour@icrc.ac.ir [Centre of Excellence for Colour Science and Technology, Institute for Colour Science and Technology, P.O. Box 16765-654, Tehran (Iran, Islamic Republic of); Department of Organic Colorants, Institute for Colour Science and Technology, P.O. Box 16765-654, Tehran (Iran, Islamic Republic of)

2015-11-15

The aim of this work is to produce a composite powder as a fluorescent ink for digital electrophotographic printing. Three benzoxazolyl and benzimidazolyl coumarin derivative dyes are used as fluorescent dyes that are incorporated into poly (styrene-co-a crylic acid) using eco-friendly emulsion aggregation (EA) approaches in several amounts with final application of fluorescent laser printing ink called toner. Fluorescence and daylight spectrophotometry is used for investigating the emission and reflectance properties of fluorescent toner. It was found that the relations between emission of fluorescent toners and the amount of dyes are non-linear. Particle size analysis, scanning electron microscopy, differential scanning calorimeter and thermal gravimetric analysis were used to study the size, shape, morphology and thermal properties of fluorescent toner particles. Results verify that the polarity of the dyes and their compatibility with the environment could affect the shape of the fluorescent toner. In addition, the results show that the fluorescent toner produced by the EA method has appropriate characteristics comparing to an industrial toner. - Highlights: Fluorescent digital printing ink produced via emulsion aggregation technique. Fluorescent ink for produced for electrophotographic printing. The relations between fluorescent emission and the amount of dyes are non-linear. Different dyes, show different behavior.

Producing fluorescent digital printing ink: Investigating the effect of type and amount of coumarin derivative dyes on the quality of ink

International Nuclear Information System (INIS)

Ataeefard, Maryam; Nourmohammadian, Farahnaz

2015-01-01

The aim of this work is to produce a composite powder as a fluorescent ink for digital electrophotographic printing. Three benzoxazolyl and benzimidazolyl coumarin derivative dyes are used as fluorescent dyes that are incorporated into poly (styrene-co-a crylic acid) using eco-friendly emulsion aggregation (EA) approaches in several amounts with final application of fluorescent laser printing ink called toner. Fluorescence and daylight spectrophotometry is used for investigating the emission and reflectance properties of fluorescent toner. It was found that the relations between emission of fluorescent toners and the amount of dyes are non-linear. Particle size analysis, scanning electron microscopy, differential scanning calorimeter and thermal gravimetric analysis were used to study the size, shape, morphology and thermal properties of fluorescent toner particles. Results verify that the polarity of the dyes and their compatibility with the environment could affect the shape of the fluorescent toner. In addition, the results show that the fluorescent toner produced by the EA method has appropriate characteristics comparing to an industrial toner. - Highlights: Fluorescent digital printing ink produced via emulsion aggregation technique. Fluorescent ink for produced for electrophotographic printing. The relations between fluorescent emission and the amount of dyes are non-linear. Different dyes, show different behavior

The motional stark effect with laser-induced fluorescence diagnostic

Science.gov (United States)

Foley, E. L.; Levinton, F. M.

2010-05-01

The motional Stark effect (MSE) diagnostic is the worldwide standard technique for internal magnetic field pitch angle measurements in magnetized plasmas. Traditionally, it is based on using polarimetry to measure the polarization direction of light emitted from a hydrogenic species in a neutral beam. As the beam passes through the magnetized plasma at a high velocity, in its rest frame it perceives a Lorentz electric field. This field causes the H-alpha emission to be split and polarized. A new technique under development adds laser-induced fluorescence (LIF) to a diagnostic neutral beam (DNB) for an MSE measurement that will enable radially resolved magnetic field magnitude as well as pitch angle measurements in even low-field (experiments. An MSE-LIF system will be installed on the National Spherical Torus Experiment (NSTX) at the Princeton Plasma Physics Laboratory. It will enable reconstructions of the plasma pressure, q-profile and current as well as, in conjunction with the existing MSE system, measurements of radial electric fields.

Non-typical fluorescence studies of excited and ground state proton and hydrogen transfer

KAUST Repository

Gil, Michał; Kijak, Michał; Piwonski, Hubert Marek; Herbich, Jerzy; Waluk, Jacek

2017-01-01

Fluorescence studies of tautomerization have been carried out for various systems that exhibit single and double proton or hydrogen translocation in various environments, such as liquid and solid condensed phases, ultracold supersonic jets, and finally, polymer matrices with single emitters.We focus on less explored areas of application of fluorescence for tautomerization studies, using porphycene, a porphyrin isomer, as an example. Fluorescence anisotropy techniques allow investigations of self-exchange reactions, where the reactant and product are formally identical. Excitation with polarized light makes it possible to monitor tautomerization in single molecules and to detect their three-dimensional orientation. Analysis of fluorescence from single vibronic levels of jet-isolated porphycene not only demonstrates coherent tunneling of two internal protons, but also indicates that the process is vibrational mode-specific. Next, we present bifunctional proton donoracceptor systems, molecules that are able, depending on the environment, to undergo excited state single intramolecular or double intermolecular proton transfer. For molecules that have donor and acceptor groups located in separate moieties linked by a single bond, excited state tautomerization can be coupled to mutual twisting of the two subunits.

Non-typical fluorescence studies of excited and ground state proton and hydrogen transfer

KAUST Repository

Gil, Michał

2017-02-03

Fluorescence studies of tautomerization have been carried out for various systems that exhibit single and double proton or hydrogen translocation in various environments, such as liquid and solid condensed phases, ultracold supersonic jets, and finally, polymer matrices with single emitters.We focus on less explored areas of application of fluorescence for tautomerization studies, using porphycene, a porphyrin isomer, as an example. Fluorescence anisotropy techniques allow investigations of self-exchange reactions, where the reactant and product are formally identical. Excitation with polarized light makes it possible to monitor tautomerization in single molecules and to detect their three-dimensional orientation. Analysis of fluorescence from single vibronic levels of jet-isolated porphycene not only demonstrates coherent tunneling of two internal protons, but also indicates that the process is vibrational mode-specific. Next, we present bifunctional proton donoracceptor systems, molecules that are able, depending on the environment, to undergo excited state single intramolecular or double intermolecular proton transfer. For molecules that have donor and acceptor groups located in separate moieties linked by a single bond, excited state tautomerization can be coupled to mutual twisting of the two subunits.

Detection of ethanol in alcoholic beverages or vapor phase using fluorescent molecules embedded in a nanofibrous polymer.

Science.gov (United States)

Akamatsu, Masaaki; Mori, Taizo; Okamoto, Ken; Komatsu, Hirokazu; Kumagai, Ken; Shiratori, Seimei; Yamamura, Masaki; Nabeshima, Tatsuya; Sakai, Hideki; Abe, Masahiko; Hill, Jonathan P; Ariga, Katsuhiko

2015-03-25

An alcohol sensor was developed using the solid-state fluorescence emission of terphenyl-ol (TPhOH) derivatives. Admixtures of TPhOH and sodium carbonate exhibited bright sky-blue fluorescence in the solid state upon addition of small quantities of ethanol. A series of terphenol derivatives was synthesized, and the effects of solvent polarities and the structures of these π-conjugated systems on their fluorescence were systematically investigated by using fluorescence spectroscopy. In particular, π-extended TPhOHs and TPhOHs containing electron-withdrawing groups exhibited significant solvatochromism, and fluorescence colors varied from blue to red. Detection of ethanol contents in alcohol beverages (detection limit ∼ 5 v/v %) was demonstrated using different TPhOHs revealing the effect of molecular structure on sensing properties. Ethanol contents in alcoholic beverages could be estimated from the intensity of the fluorescence elicited from the TPhOHs. Moreover, when terphenol and Na2CO3 were combined with a water-absorbent polymer, ethanol could be detected at lower concentrations. Detection of ethanol vapor (8 v/v % in air) was also accomplished using a nanofibrous polymer scaffold as the immobilized sensing film.

Quantization of bovine serum albumin by fluorescence enhancement effects and corresponding binding of macrocyclic host-protein assembly.

Science.gov (United States)

Bardhan, Munmun; Misra, Tapas; Ganguly, Tapan

2012-01-05

The present paper reports the investigations on the spectroscopic behavior of the binary complexes of the dye aurintricarboxylic acid (ATA) with protein bovine serum albumin (BSA) and 18-crown 6 (CW) (ATA·BSA, ATA·CW) and the ternary complex ATA·CW·BSA by using UV-vis steady state and time resolved fluorescence spectroscopy. The primary aim of the work is to determine the protein (BSA) quantization by fluorescence enhancement method and investigate the 'enhancer' activity of crown ether (CW) on it to increase the resolution. Steady state and time resolved fluorescence measurements demonstrated how fluorescence intensity of ATA could be used for the determination of the protein BSA in aqueous solution. The binding of dye (probe/fluorescent medicinal molecule) with protein and the denaturing effect in the polar environment of acetonitrile of the dye protein complex act as drug binding as well as drug release activity. Apart from its basic research point of view, the present study also possesses significant importance and applications in the field of medicinal chemistry. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of a fluorescence polarographic immunoassay with increased sensitivity for measurement of low concentrations of tobramycin in serum

NARCIS (Netherlands)

Touw, D J; de Graaf, A I; de Goede, P

The limits of quantitation of the assay of tobramycin in serum by the fluorescence polarization immunoassay system marketed by Abbott Laboratories (TDxFLx system) are 0.1 and 10.0 mg/L. For some pharmacokinetic studies, however, a more sensitive analysis is needed. The sensitivity of the TDxFLx

Monitoring the corrosion process of Al alloys through pH induced fluorescence

International Nuclear Information System (INIS)

Pidaparti, R M; Neblett, E B; Miller, S A; Alvarez, J C

2008-01-01

A sensing and monitoring set-up based on electrochemical pH induced fluorescence to systematically control the electrochemical corrosion process has been developed for possible applications in the field of localized corrosion. The sensing and monitoring concept is based on exposing the corroding metal surface to solutions that contain selected redox chemicals which will react in local regions where anodic or cathodic polarizations occur. Redox couples that produce or consume protons in their electrochemical reactions were used so that local pH gradients can indicate electrochemical activity by inducing fluorescence in dyes. This approach has been applied to study the corrosion initiation in aircraft aluminum metal 2024-T3 in a controlled electrochemical cell. Preliminary results obtained suggest that monitoring of localized corrosion based on pH can be achieved for field applications

Rapid creation of distant entanglement by multi-photon resonant fluorescence

Science.gov (United States)

Cohen, Guy Z.; Sham, L. J.

2014-03-01

We study a simple, effective and robust method for entangling two separate stationary quantum dot spin qubits with high fidelity using multi-photon Gaussian state. The fluorescence signals from the two dots interfere at a beam splitter. The bosonic nature of photons leads, in analogy with the Hong-Ou-Mandel (HOM) effect, to selective pairing of photon holes (photon absences in the fluorescent signals). By the HOM effect, two photon holes with the same polarization end up at the same beam splitter output. As a result, two odd photon number detections at the outgoing beams, which must correspond to two photon holes with different polarizations, herald entanglement creation. The robustness of the Gaussian states is evidenced by the ability to compensate for photon absorption and noise by a moderate increase in the number of photons at the input. We calculate the entanglement generation rate in the ideal, non-ideal and near-ideal detector regimes and find substantial improvement over single-photon schemes in all three regimes. Fast and efficient spin-spin entanglement creation can form the basis for a scalable quantum dot quantum computing network. Our predictions can be tested using current experimental capabilities. This research was supported by the U.S. Army Research Office MURI award W911NF0910406, by NSF grant PHY-1104446 and by ARO (IARPA, W911NF-08-1-0487). The authors thank D. G. Steel for useful discussions.

Polarization of lanthanum nucleus by dynamic polarization method

International Nuclear Information System (INIS)

Adachi, Toshikazu; Ishimoto, Shigeru; Masuda, Yasuhiro; Morimoto, Kimio

1989-01-01

Preliminary studies have been carried out concerning the application of a dynamic polarization method to polarizing lanthanum fluoride single crystal to be employed as target in experiments with time reversal invariance. The present report briefly outlines the dynamic polarization method and describes some preliminary studies carried out so far. Dynamic polarization is of particular importance because no techniques are currently available that can produce highly polarized static nucleus. Spin interaction between electrons and protons (nuclei) plays a major role in the dynamic polarization method. In a thermal equilibrium state, electrons are polarized almost completely while most protons are not polarized. Positively polarized proton spin is produced by applying microwave to this system. The most hopeful candidate target material is single crystal of LaF 3 containing neodymium because the crystal is chemically stable and easy to handle. The spin direction is of great importance in experiments with time reversal invariance. The spin of neutrons in the target can be cancelled by adjusting the external magnetic field applied to a frozen polarized target. In a frozen spin state, the polarity decreases slowly with a relaxation time that depends on the external magnetic field and temperature. (N.K.)

Polarized neutrons

International Nuclear Information System (INIS)

Williams, W.G.

1988-01-01

The book on 'polarized neutrons' is intended to inform researchers in condensed matter physics and chemistry of the diversity of scientific problems that can be investigated using polarized neutron beams. The contents include chapters on:- neutron polarizers and instrumentation, polarized neutron scattering, neutron polarization analysis experiments and precessing neutron polarization. (U.K.)

Inner filter effect of gold nanoparticles on the fluorescence of rare-earth phosphate nanocrystals and its application for determination of biological aminothiols

Energy Technology Data Exchange (ETDEWEB)

Chen, Hong-Qi; Wu, Yong; Yuan, Fei; Xu, Juan; Zhang, Yi-Yan; Wang, Lun, E-mail: wanglun@mail.ahnu.edu.cn

2013-09-15

A simple, sensitive fluorescent method for detecting biological aminothiols has been developed based on the inner filter effect principle that utilizes CePO{sub 4}:Tb{sup 3+} luminescent nanoparticles as the donor and gold nanoparticles (AuNPs) as the energy receptor. Stable, water-soluble and well-dispersible CePO{sub 4}:Tb{sup 3+} nanoparticles with low photobleaching features were synthesized conveniently by a facile solvothermal method. At the same time, AuNPs with a high extinction coefficient are expected to be capable of functioning as powerful receptor. Based on the complementary overlap between the emission spectrum of CePO{sub 4}:Tb{sup 3+} nanoparticles and the absorption spectrum of Au NPs, an inner filter effect system was constructed. In the presence of aminothiols (such as cysteine), AuNPs interacted with the aminothiols, thereby inducing the aggregation of AuNPs, which induced the fluorescence recovery. In the present work, we developed a turn-on fluorescent assay for the determination of biological aminothiols. Under the optimum conditions, the linear concentration ranges were 1.0×10{sup −7}–2.0×10{sup −6} M for cysteine, 5.0×10{sup −8}–5.0×10{sup −7} M for glutathione and 8.0×10{sup −8}–1.0×10{sup −6} M for homocysteine, respectively. The method is successfully applied to the quantification of biological aminothiols in synthetic samples. -- Highlights: • An inner filter effect method for detecting biological aminothiols has been developed. • CePO{sub 4}:Tb{sup 3+} nanoparticles were synthesized and used as the donor. • Gold nanoparticles (AuNPs) were synthesized and used as the energy receptor.

Determination of heavy metals in polar snow and ice by laser-excited atomic fluorescence spectrometry

International Nuclear Information System (INIS)

Bolshov, M.A.; Boutron, C.F.

1994-01-01

The new laser-excited atomic fluorescence spectrometry technique offers unrivalled sensitivity for the determination of trace metals in a wide variety of samples. This has allowed the direct determination of Pb, Cd and Bi in Antarctic and Greenland snow and ice down to the sub pg/g level. (authors). 11 refs., 2 figs

Anomalous doping of a molecular crystal monitored with confocal fluorescence microscopy: Terrylene in a p-terphenyl crystal

Science.gov (United States)

Białkowska, Magda; Deperasińska, Irena; Makarewicz, Artur; Kozankiewicz, Bolesław

2017-09-01

Highly terrylene doped single crystals of p-terphenyl, obtained by co-sublimation of both components, showed bright spots in the confocal fluorescence images. Polarization of the fluorescence excitation spectra, blinking and bleaching, and saturation behavior allowed us to attribute them to single molecules of terrylene anomalously embedded between two neighbor layers of the host crystal, in the (a,b) plane. Such an orientation of terrylene molecules results in much more efficient absorption and collection of the fluorescence photons than in the case of previously investigated molecules embedded in the substitution sites. The above conclusion was supported by quantum chemistry calculations. We postulate that the kind of doping considered in this work should be possible in other molecular crystals where the host molecules are organized in a herringbone pattern.

Theoretical investigation of confocal microscopy using an elliptically polarized cylindrical vector laser beam: Visualization of quantum emitters near interfaces

Science.gov (United States)

Boichenko, Stepan

2018-04-01

We theoretically study laser-scanning confocal fluorescence microscopy using elliptically polarized cylindrical vector excitation light as a tool for visualization of arbitrarily oriented single quantum dipole emitters located (1) near planar surfaces enhancing fluorescence, (2) in a thin supported polymer film, (3) in a freestanding polymer film, and (4) in a dielectric planar microcavity. It is shown analytically that by using a tightly focused azimuthally polarized beam, it is possible to exclude completely the orientational dependence of the image intensity maximum of a quantum emitter that absorbs light as a pair of incoherent independent linear dipoles. For linear dipole quantum emitters, the orientational independence degree higher than 0.9 can normally be achieved (this quantity equal to 1 corresponds to completely excluded orientational dependence) if the collection efficiency of the microscope objective and the emitter's total quantum yield are not strongly orientationally dependent. Thus, the visualization of arbitrarily oriented single quantum emitters by means of the studied technique can be performed quite efficiently.

Synthesis and Fluorescence Spectra of Triazolylcoumarin Fluorescent Dyes

Institute of Scientific and Technical Information of China (English)

PENG Xian-fu; LI Hong-qi

2009-01-01

Much attention is devoted to fluorescent dyes especially those with potential in versatile applications. Reactions under "click" conditions between nonfluorescent 3 - azidocoumarins and terminal alkynes produced 3 -(1, 2, 3- triazol- 1 - yl)cournarins, a novel type of fluorescent dyes with intense fluorescence. The structures of the new coumarins were characterized by 1H NMR, MS, and IR spectra. Fluorescence spectra measurement demonstrated excellent fluorescence performance of the triazolylcoumarins and this click reaction is a promising candidate for bioconjugation and bioimaging applications since both azide and alkynes are quite inert to biological systems.

Online observation of emulsion polymerization by fluorescence technique

CERN Document Server

Rudschuck, S; Fuhrmann, J

1999-01-01

An online observation of local polarity via fluorescence spectroscopy was used to study the formation and growth of polymer particles during an emulsifier-free heterogeneous polymerization. The reaction mixture consisted of styrene dispersed in water and the polymerization was initiated by a macro-initiator (hydrolyzed propene-maleic acid copolymer with t-butyl perester groups). Pyrenyl probes were attached to the backbone of the initiator to analyze the heterogeneous reaction. The experimental results allow a clear distinction of different time regions during the heterogeneous polymerization. Information about the heating period, the latex formation, the particle growth and the final stage of the polymerization process (gel point) were obtained. (11 refs).

Lateral mobility of plasma membrane proteins in dividing eggs of the loach (Misgurnus fossilis): Regional differences and changes during the cell cycle.

Science.gov (United States)

Bozhkova, V P; Budayova, M; Kvasnicka, P; Cigankova, N; Chorvat, D

1994-12-01

Regional differences in lateral diffusion rates of fluorescence-labeled proteins have been studied in the plasma membrane of dividing eggs of the loach (Misgurnus fossilis) by fluorescence recovery after photobleaching (FRAP). Apparent animal-vegetal differences in fluorescence intensity, lateral diffusion coefficients, and fractions of mobile proteins have been found, with all these quantities being higher in the animal pole region than in the yolk region. Cyclic changes in protein diffusion coefficients and mobile fractions during the first few cell cycles have also been recorded. Soon after the end of a cleavage, the diffusion coefficient reaches its minimal value and increases rapidly before the next cleavage.

Improvement of Orange II Photobleaching by Moderate Ga3+ Doping of Titania and Detrimental Effect of Structural Disorder on Ga Overloading

Directory of Open Access Journals (Sweden)

Václav Štengl

2014-01-01

Full Text Available Highly photoactive Ga3+-doped anatase modification of titania was prepared by homogeneous hydrolysis of aqueous solutions mixture of titanium oxo-sulphate TiOSO4 and gallium(III nitrate with urea. Incorporation of Ga3+ into the anatase lattice has a clear positive effect on the photocatalytic activity under UV and Vis light irradiation up to a certain extent of Ga. Ga3+ doping decreased the size of the crystallites, increased surface area, and affected texture of the samples. Higher amount of gallium leads to the formation of a nondiffractive phase, probably photocatalytically inactive. The titania sample with 2.18 wt.% Ge3+ had the highest activity during the photocatalysed degradation in the UV and visible light regions; the total bleaching of dye Orange II was achieved within 29 minutes. Ga concentration larger than 5% (up to 15% significantly inhibited the growth of the anatase crystal domains which formed the nondiffractive phase content and led to remarkable worsening of the photobleaching efficiency.

Workshop on polarized neutron filters and polarized pulsed neutron experiments

International Nuclear Information System (INIS)

Itoh, Shinichi

2004-07-01

The workshop was held in KEK by thirty-three participants on April 26, 2004. The polarized neutron filter method was only discussed. It consists of three parts; the first part was discussed on the polarized neutron methods, the second part on the polarized neutron experiments and the third on the pulse neutron spectrometer and polarized neutron experiments. The six papers were presented such as the polarized 3 He neutron spin filter, neutron polarization by proton polarized filter, soft master and neutron scattering, polarized neutron in solid physics, polarization experiments by chopper spectroscope and neutron polarization system in superHRPD. (S.Y.)

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

Science.gov (United States)

Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

2017-07-01

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Fluorophore appended saccharide cyclophane: self-association, fluorescent properties, heterodimers with cyclodextrins, and cross-linking behavior with peanut agglutinin of dansyl-modified saccharide cyclophane.

Science.gov (United States)

Hayashida, Osamu; Hamachi, Itaru

2004-05-14

A saccharide cyclophane bearing an environment-sensitive fluorophore (1) was prepared by introducing not only three branches with a terminal galactose residue but also one with a dansyl moiety into a tetraaza[6.1.6.1]paracyclophane skeleton. Self-association behavior of the dansyl-appended saccharide cyclophane was characterized in aqueous media by fluorescence spectroscopy and dynamic light scattering measurements. At least in the concentrations below 1.0 x 10(-5) M, saccharide cyclophane 1 existed in a monomeric state, whereas it tended to form self-aggregated complexes in the higher concentration. Solvent polarity dependency on the emission spectra of 1 was examined by fluorescence spectroscopy. With increasing dioxane contents in dioxane/water solvents, the fluorescence intensity originating from the dansyl moiety of 1 increased along with a concomitant blue shift of the fluorescence maximum (lambda(em)). In the monomeric state of 1 in water, the dansyl moiety of 1 was not fully included into its cyclophane cavity but partially exposed to the bulk aqueous phase. In the higher concentration ranges in an aggregate state, however, the dansyl group of 1 was located in the apolar cyclophane cavity whose microenvironment was equivalent to the polarity of 1-butanol evaluated on the basis of a correlation between lambda(em) and solvent polarity. This indicates an intermolecular inclusion of the dansyl moiety within the cyclophane. When cyclodextrin (CD) was mixed with 1, the dansyl group of 1 was bound to an internal cavity of CD such as gamma-CD, beta-CD, 6-O-alpha-glucosyl-beta-CD, and 6-O-alpha-maltosyl-beta-CD with binding constants of 7.5 x 10(2), 7.8 x 10(2), 7.7 x 10(2), and 6.0 x 10(2) M(-1), respectively. Such a supramolecular assembling of dansyl-modified cyclophane 1 and CDs caused changes of the fluorescence spectra as well as appearance of induced CD bands in aqueous media. Furthermore, saccharide cyclophane 1 was selectively bound to peanut agglutinin

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

International Nuclear Information System (INIS)

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

2000-01-01

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

Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.

Directory of Open Access Journals (Sweden)

Zhou Han

Full Text Available ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein. Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼ 1%. The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

Studies on the metabolism and toxicological detection of the amphetamine-like anorectic fenproporex in human urine by gas chromatography-mass spectrometry and fluorescence polarization immunoassay.

Science.gov (United States)

Kraemer, T; Theis, G A; Weber, A A; Maurer, H H

2000-01-28

Studies on the metabolism and the toxicological analysis of fenproporex (R,S-3-[(1-phenyl-2-propyl)-amino]-propionitrile, FP) using GC-MS and fluorescence polarization immunoassay are described. The metabolites were identified in urine samples of volunteers by GC-MS after cleavage of conjugates, extraction and acetylation. Besides unchanged FP, fourteen metabolites, including amphetamine, could be identified. Two partially overlapping metabolic pathways could be postulated: ring degradation by one- and two-fold aromatic hydroxylation followed by methylation and side chain degradation by N-dealkylation to amphetamine (AM). A minor pathway leads via beta-hydroxylation of AM to norephedrine. For GC-MS detection, the systematic toxicological analysis procedure including acid hydrolysis, extraction at pH 8-9 and acetylation was suitable (detection limits 50 ng/ml for FP and 100 ng/ml for AM). Excretion studies showed, that only AM but neither FP nor its specific metabolites were detectable 30-60 h after ingestion of 20 mg of FP. Therefore, misinterpretation can occur. The Abbott TDx FPIA amphetamine/methamphetamine II gave positive results up to 58 h. All the positive immunoassay results could be confirmed by the described GC-MS procedure.

Fluorescence Image Segmentation by using Digitally Reconstructed Fluorescence Images

OpenAIRE

Blumer, Clemens; Vivien, Cyprien; Oertner, Thomas G; Vetter, Thomas

2011-01-01

In biological experiments fluorescence imaging is used to image living and stimulated neurons. But the analysis of fluorescence images is a difficult task. It is not possible to conclude the shape of an object from fluorescence images alone. Therefore, it is not feasible to get good manual segmented nor ground truth data from fluorescence images. Supervised learning approaches are not possible without training data. To overcome this issues we propose to synthesize fluorescence images and call...

Neutron polarization in polarized 3He targets

International Nuclear Information System (INIS)

Friar, J.L.; Gibson, B.F.; Payne, G.L.; Bernstein, A.M.; Chupp, T.E.

1990-01-01

Simple formulas for the neutron and proton polarizations in polarized 3 He targets are derived assuming (1) quasielastic final states; (2) no final-state interactions; (3) no meson-exchange currents; (4) large momentum transfers; (5) factorizability of 3 He SU(4) response-function components. Numerical results from a wide variety of bound-state solutions of the Faddeev equations are presented. It is found that this simple model predicts the polarization of neutrons in a fully polarized 3 He target to be 87%, while protons should have a slight residual polarization of -2.7%. Numerical studies show that this model works very well for quasielastic electron scattering

A polarizable embedding DFT study of one-photon absorption in fluorescent proteins

DEFF Research Database (Denmark)

Beerepoot, Maarten; Steindal, Arnfinn H.; Kongsted, Jacob

2013-01-01

mutants (BFP, eGFP, YFP and eCFP). The observed trends in excitation energies among the FPs are reproduced by our approach when performing calculations directly on the crystal structures or when using structures extracted from a molecular dynamics simulations. However, in the former case, QM/MM geometry......A theoretical study of the one-photon absorption of five fluorescent proteins (FPs) is presented. The absorption properties are calculated using a polarizable embedding approach combined with density functional theory (PE-DFT) on the wild-type green fluorescent protein (wtGFP) and several of its...... optimization of the chromophores within a frozen protein environment is needed in order to reproduce the experimental trends. Explicit account of polarization in the force field is not needed to yield the correct trend between the different FPs, but is necessary for reproducing the experimentally observed red...

On the performance of bioanalytical fluorescence correlation spectroscopy measurements in a multiparameter photon-counting microscope

Energy Technology Data Exchange (ETDEWEB)

Mazouchi, Amir; Liu Baoxu; Bahram, Abdullah [Department of Physics, Institute for Optical Sciences, University of Toronto, Toronto (Canada); Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, ON, L5L 1C6 (Canada); Gradinaru, Claudiu C., E-mail: claudiu.gradinaru@utoronto.ca [Department of Physics, Institute for Optical Sciences, University of Toronto, Toronto (Canada); Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, ON, L5L 1C6 (Canada)

2011-02-28

Fluorescence correlation spectroscopy (FCS) data acquisition and analysis routines were developed and implemented in a home-built, multiparameter photon-counting microscope. Laser excitation conditions were investigated for two representative fluorescent probes, Rhodamine110 and enhanced green fluorescent protein (EGFP). Reliable local concentrations and diffusion constants were obtained by fitting measured FCS curves, provided that the excitation intensity did not exceed 20% of the saturation level for each fluorophore. Accurate results were obtained from FCS measurements for sample concentrations varying from pM to {mu}M range, as well as for conditions of high background signals. These experimental constraints were found to be determined by characteristics of the detection system and by the saturation behavior of the fluorescent probes. These factors actually limit the average number of photons that can be collected from a single fluorophore passing through the detection volume. The versatility of our setup and the data analysis capabilities were tested by measuring the mobility of EGFP in the nucleus of Drosophila cells under conditions of high concentration and molecular crowding. As a bioanalytical application, we studied by FCS the binding affinity of a novel peptide-based drug to the cancer-regulating STAT3 protein and corroborated the results with fluorescence polarization analysis derived from the same photon data.

When measured spin polarization is not spin polarization

International Nuclear Information System (INIS)

Dowben, P A; Wu Ning; Binek, Christian

2011-01-01

Spin polarization is an unusually ambiguous scientific idiom and, as such, is rarely well defined. A given experimental methodology may allow one to quantify a spin polarization but only in its particular context. As one might expect, these ambiguities sometimes give rise to inappropriate interpretations when comparing the spin polarizations determined through different methods. The spin polarization of CrO 2 and Cr 2 O 3 illustrate some of the complications which hinders comparisons of spin polarization values. (viewpoint)

Anchored LH2 complexes in 2D polarization imaging.

Science.gov (United States)

Tubasum, Sumera; Sakai, Shunsuke; Dewa, Takehisa; Sundström, Villy; Scheblykin, Ivan G; Nango, Mamoru; Pullerits, Tõnu

2013-09-26

Protein is a soft material with inherently large structural disorder. Consequently, the bulk spectroscopies of photosynthetic pigment protein complexes provide averaged information where many details are lost. Here we report spectroscopy of single light-harvesting complexes where fluorescence excitation and detection polarizations are both independently rotated. Two samples of peripheral antenna (LH2) complexes from Rhodopseudomonas acidophila were studied. In one, the complexes were embedded in polyvinyl alcohol (PVA) film; in the other, they were anchored on the glass surface and covered by the PVA film. LH2 contains two rings of pigment molecules-B800 and B850. The B800 excitation polarization properties of the two samples were found to be very similar, indicating that orientation statistics of LH2s are the same in these two very different preparations. At the same time, we found a significant difference in B850 emission polarization statistics. We conclude that the B850 band of the anchored sample is substantially more disordered. We argue that both B800 excitation and B850 emission polarization properties can be explained by the tilt of the anchored LH2s due to the spin-casting of the PVA film on top of the complexes and related shear forces. Due to the tilt, the orientation statistics of two samples become similar. Anchoring is expected to orient the LH2s so that B850 is closer to the substrate. Consequently, the tilt-related strain leads to larger deformation and disorder in B850 than in B800.

Direct noninvasive observation of near infrared photobleaching of autofluorescence in human volar side fingertips in vivo

Science.gov (United States)

Deng, Bin; Wright, Colin; Lewis-Clark, Eric; Shaheen, G.; Geier, Roman; Chaiken, J.

2010-02-01

Human transdermal in vivo spectroscopic applications for tissue analysis involving near infrared (NIR) light often must contend with broadband NIR fluorescence that, depending on what kind of spectroscopy is being employed, can degrade signal to noise ratios and dynamic range. Such NIR fluorescence, i.e. "autofluorescence" is well known to originate in blood tissues and various other endogenous materials associated with the static tissues. Results of recent experiments on human volar side fingertips in vivo are beginning to provide a relative ordering of the contributions from various sources. Preliminary results involving the variation in the bleaching effect across different individuals suggest that for 830 nm excitation well over half of the total fluorescence comes from the static tissues and remainder originates with the blood tissues, i.e. the plasma and the hematocrit. Of the NIR fluorescence associated with the static tissue, over half originates with products of well-known post-enzymatic glycation reactions, i.e. Maillard chemistry, in the skin involving glucose and other carbohydrates and skin proteins like collagen and cytosol proteins.

Lie Group Analysis of the Photo-Induced Fluorescence of Drosophila Oogenesis with the Asymmetrically Localized Gurken Protein.

Directory of Open Access Journals (Sweden)

Jen-Cheng Wang

Full Text Available Lie group analysis of the photo-induced fluorescence of Drosophila oogenesis with the asymmetrically localized Gurken protein has been performed systematically to assess the roles of ligand-receptor complexes in follicle cells. The (2×2 matrix representations resulting from the polarized tissue spectra were employed to characterize the asymmetrical Gurken distributions. It was found that the fluorescence of the wild-type egg shows the Lie point symmetry X 23 at early stages of oogenesis. However, due to the morphogen regulation by intracellular proteins and extracellular proteins, the fluorescence of the embryogenesis with asymmetrically localized Gurken expansions exhibits specific symmetry features: Lie point symmetry Z 1 and Lie point symmetry X 1. The novel approach developed herein was successfully used to validate that the invariant-theoretical characterizations are consonant with the observed asymmetric fluctuations during early embryological development.

Fluorescence of soil humic acids and their fractions obtained by tandem size exclusion chromatography-polyacrylamide gel electrophoresis

Energy Technology Data Exchange (ETDEWEB)

Trubetskaya, O. [Russian Academy of Sciences, Moscow Region (Russian Federation). Shemyakin and Ovchinnikov Inst. of Bioorganic Chemistry; Trubetskoj, O. [Russian Academy of Sciences, Moscow Region (Russian Federation). Inst. of Basic Biological Problems; Guyot, G.; Richard, C. [UMR CNRS 6505, Aubiere (France). Lab. de Photochimie Moleculaire et Macromoleculaire; Andreux, F. [Centre des Sciences de la Terre, Dijon (France)

2002-07-01

Humic acids (HAs) extracted from soils of different origin (chernozem, ferralsol and ranker) and their fractions (A, B and C+D) obtained by tandem size exclusion chromatography-polyacrylamide gel electrophoresis were investigated by steady-state fluorescence spectroscopy in the emission mode. Independently of HA source, high molecular size fractions A and B are shown to be weakly fluorescent. The main fluorophores, especially those emitting at long wavelength (around 500-510 nm), are contained in the polar and low molecular size fractions C+D. As indicated by the observed pH effect, aromatic structures bearing carboxylate and OH substituents may be involved in these longer wavelength emissions. [author].

Trapping, manipulation and rapid rotation of NBD-C8 fluorescent single microcrystals in optical tweezers

International Nuclear Information System (INIS)

GALAUP, Jean-Pierre; RODRIGUEZ-OTAZO, Mariela; AUGIER-CALDERIN, Angel; LAMERE; Jean-Francois; FERY-FORGUES, Suzanne

2009-01-01

We have built an optical tweezers experiment based on an inverted microscope to trap and manipulate single crystals of micro or sub-micrometer size made from fluorescent molecules of 4-octylamino-7-nitrobenzoxadiazole (NBD-C8). These single crystals have parallelepiped shapes and exhibit birefringence properties evidenced through optical experiments between crossed polarizers in a polarizing microscope. The crystals are uniaxial with their optical axis oriented along their largest dimension. Trapped in the optical trap, the organic micro-crystals are oriented in such a way that their long axis is along the direction of the beam propagation, and their short axis follows the direction of the linear polarization. Therefore, with linearly polarized light, simply rotating the light polarization can orient the crystal. When using circularly or only elliptically polarized light, the crystal can spontaneously rotate and reach rotation speed of several hundreds of turns per second. A surprising result has been observed: when the incident power is growing up, the rotation speed increases to reach a maximum value and then decreases even when the power is still growing up. Moreover, this evolution is irreversible. Different possible explanations can be considered. The development of a 3D control of the crystals by dynamical holography using liquid crystal spatial modulators will be presented and discussed on the basis of the most recent results obtained. (Author)

Closed-Form Formulas vs. PDE Based Numerical Solution for the FRAP Data Processing: Theoretical and Practical Comparison

Czech Academy of Sciences Publication Activity Database

Papáček, Š.; Macdonald, B.; Matonoha, Ctirad

2017-01-01

Roč. 73, č. 8 (2017), s. 1673-1683 ISSN 0898-1221 Grant - others:GA MŠk(CZ) ED2.1.00/01.0024; GA MŠk(CZ) LO1205 Institutional support: RVO:67985807 Keywords : fluorescence recovery after photobleaching (FRAP) * parameter estimation * sensitivity analysis * partial differential equation (PDE) Subject RIV: BA - General Mathematics OBOR OECD: Applied mathematics Impact factor: 1.531, year: 2016

Studies of the fluorescent excited state of impurities in ionic crystals

International Nuclear Information System (INIS)

Romestain, Robert

1972-01-01

The author of this research thesis first presents experimental methods used in this research: principles (recall on the optical spectrum of an impurity in a solid, use of fluorescence polarization) and techniques (sample preparation, liquid helium cryostat, application of a disturbance, optical detection). Then, he reports the study of the Mn ++ ion in a tetrahedron crystalline field, the study of the Jahn Teller effect on the excited state of the F + centre in CaO, and the study by double resonance of a specific excited state of this same centre in CaO

Effect of Solvation on Electron Detachment and Excitation Energies of a Green Fluorescent Protein Chromophore Variant.

Science.gov (United States)

Bose, Samik; Chakrabarty, Suman; Ghosh, Debashree

2016-05-19

Hybrid quantum mechanics/molecular mechanics (QM/MM) is applied to the fluorinated green fluorescent protein (GFP) chromophore (DFHBDI) in its deprotonated form to understand the solvatochromic shifts in its vertical detachment energy (VDE) and vertical excitation energy (VEE). This variant of the GFP chromophore becomes fluorescent in an RNA environment and has a wide range of applications in biomedical and biochemical fields. From microsolvation studies, we benchmark (with respect to full QM) the accuracy of our QM/MM calculations with effective fragment potential (EFP) as the MM method of choice. We show that while the solvatochromic shift in the VEE is minimal (0.1 eV blue shift) and its polarization component is only 0.03 eV, the effect of the solvent on the VDE is quite large (3.85 eV). We also show by accurate calculations on the solvatochromic shift of the VDE that polarization accounts for ∼0.23 eV and therefore cannot be neglected. The effect of the counterions on the VDE of the deprotonated chromophore in solvation is studied in detail, and a charge-smearing scheme is suggested for charged chromophores.

Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

Science.gov (United States)

Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

2017-02-01

Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

Integrated organic electronic based optochemical sensors using polarization filters

International Nuclear Information System (INIS)

Kraker, Elke; Haase, Anja; Lamprecht, Bernhard; Jakopic, Georg; Konrad, Christian; Koestler, Stefan

2008-01-01

A compact, integrated photoluminescence based oxygen and pH sensor, utilizing an organic light emitting device (OLED) as the light source and an organic photodiode (OPD) as the detection unit, is described. The main challenge in such an integrated sensor is the suppression of the excitation light at the detector, which is typically by many orders of magnitude higher in intensity than the emitted fluorescence. In our approach, we refrain from utilizing edge filters which require narrow band excitation sources and dyes with an adequate large Stokes shift. We rather developed an integrated sensor concept relying on two polarizers to separate the emission and excitation light. One polarizer is located right after the OLED, while the other one, oriented at 90 deg. to the first, is placed in front of the OPD. The main advantage of this solution is that any combination of excitation and emission light is acceptable, even if the two signals overlap spectrally. This is especially important for the use of OLEDs as the excitation sources, as these devices typically exhibit a broad spectral emission

Magnetic modulation of exciplex fluorescence of pyrene solutions with azacrown-ether excess in the presence of ions of alkali and alkaline earth metals

International Nuclear Information System (INIS)

Borisenko, V.N.; Petrov, N.Kh.; Gromov, S.P.; Alfimov, M.V.

1997-01-01

Photoexcitation of polar pyrene solutions with excess of phenylaza-15-crown-5 as a donor results to intermolecular electron transfer with formation of ion-radical pairs, recombination of which produces fluorescent exciplex. Charge exchange between molecules of crown ether and its cation-radicals is practically absent at that. Magnetic effect, observed for fluorescence, decreases, when adding diamagnetic lithium and calcium ions to exiplex pyrene/crown-ether system. This can be explained by formation of paramagnetic complexes. 15 refs., 5 figs

Research on generating various polarization-modes in polarized illumination system

Science.gov (United States)

Huang, Jinping; Lin, Wumei; Fan, Zhenjie

2013-08-01

With the increase of the numerical aperture (NA), the polarization of light affects the imaging quality of projection lens more significantly. On the contrary, according to the mask pattern, the resolution of projection lens can be improved by using the polarized illumination. That is to say, using the corresponding polarized beam (or polarization-mode) along with the off-axis illumination will improve the resolution and the imaging quality of the of projection lens. Therefore, the research on the generation of various polarization modes and its conversion methods become more and more important. In order to realize various polarization modes in polarized illumination system, after read a lot of references, we provide a way that fitting for the illumination system with the wavelength of 193nm.Six polarization-modes and a depolarized mode are probably considered. Wave-plate stack is used to generate linearly polarization-mode, which have a higher degree polarization. In order to generate X-Y and Y-X polarization mode, the equipment consisting of four sectors of λ/2 wave plate was used. We combined 16 sectors of λ/2 wave plate which have different orientations of the "slow" axis to generate radial and azimuthal polarization. Finally, a multi-polarization control device was designed. Using the kind of multi-polarization control device which applying this method could help to choose the polarization modes conveniently and flexibility for the illumination system.

Polarization properties of linearly polarized parabolic scaling Bessel beams

Energy Technology Data Exchange (ETDEWEB)

Guo, Mengwen; Zhao, Daomu, E-mail: zhaodaomu@yahoo.com

2016-10-07

The intensity profiles for the dominant polarization, cross polarization, and longitudinal components of modified parabolic scaling Bessel beams with linear polarization are investigated theoretically. The transverse intensity distributions of the three electric components are intimately connected to the topological charge. In particular, the intensity patterns of the cross polarization and longitudinal components near the apodization plane reflect the sign of the topological charge. - Highlights: • We investigated the polarization properties of modified parabolic scaling Bessel beams with linear polarization. • We studied the evolution of transverse intensity profiles for the three components of these beams. • The intensity patterns of the cross polarization and longitudinal components can reflect the sign of the topological charge.

Statistical inference in single molecule measurements of protein adsorption

Science.gov (United States)

Armstrong, Megan J.; Tsitkov, Stanislav; Hess, Henry

2018-02-01

Significant effort has been invested into understanding the dynamics of protein adsorption on surfaces, in particular to predict protein behavior at the specialized surfaces of biomedical technologies like hydrogels, nanoparticles, and biosensors. Recently, the application of fluorescent single molecule imaging to this field has permitted the tracking of individual proteins and their stochastic contribution to the aggregate dynamics of adsorption. However, the interpretation of these results is complicated by (1) the finite time available to observe effectively infinite adsorption timescales and (2) the contribution of photobleaching kinetics to adsorption kinetics. Here, we perform a protein adsorption simulation to introduce specific survival analysis methods that overcome the first complication. Additionally, we collect single molecule residence time data from the adsorption of fibrinogen to glass and use survival analysis to distinguish photobleaching kinetics from protein adsorption kinetics.

Near infrared spectral polarization imaging of prostate cancer tissues using Cybesin: a receptor-targeted contrast agent

Science.gov (United States)

Pu, Yang; Wang, W. B.; Tang, G. C.; Liang, Kexian; Achilefu, S.; Alfano, R. R.

2013-03-01

Cybesin, a smart contrast agent to target cancer cells, was investigated using a near infrared (NIR) spectral polarization imaging technique for prostate cancer detection. The approach relies on applying a contrast agent that can target cancer cells. Cybesin, as a small ICG-derivative dye-peptide, emit fluorescence between 750 nm and 900 nm, which is in the "tissue optical window". Cybesin was reported targeting the over-expressed bombesin receptors in cancer cells in animal model and the human prostate cancers over-expressing bombesin receptors. The NIR spectral polarization imaging study reported here demonstrated that Cybesin can be used as a smart optical biomarker and as a prostate cancer receptor targeted contrast agent.

Polarized nuclear target based on parahydrogen induced polarization

OpenAIRE

Budker, D.; Ledbetter, M. P.; Appelt, S.; Bouchard, L. S.; Wojtsekhowski, B.

2012-01-01

We discuss a novel concept of a polarized nuclear target for accelerator fixed-target scattering experiments, which is based on parahydrogen induced polarization (PHIP). One may be able to reach a 33% free-proton polarization in the ethane molecule. The potential advantages of such a target include operation at zero magnetic field, fast ($\\sim$100 Hz) polarization reversal, and operation with large intensity of an electron beam.

Medically relevant assays with a simple smartphone and tablet based fluorescence detection system.

Science.gov (United States)

Wargocki, Piotr; Deng, Wei; Anwer, Ayad G; Goldys, Ewa M

2015-05-20

Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera) and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

Directory of Open Access Journals (Sweden)

Piotr Wargocki

2015-05-01

Full Text Available Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

Science.gov (United States)

Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

2009-01-01

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Fluorescent cellulose nanocrystals via supramolecular assembly of terpyridine-modified cellulose nanocrystals and terpyridine-modified perylene

International Nuclear Information System (INIS)

Hassan, Mohammad L.; Moorefield, Charles M.; Elbatal, Hany S.; Newkome, George R.; Modarelli, David A.; Romano, Natalie C.

2012-01-01

Highlights: ► Surfaces of cellulose nanocrystals were modified with terpyridine ligands. ► Fluorescent nanocrystals could be obtained via self-assembly of terpyridine-modified perylene dye onto the terpyridine-modified cellulose nanocrystals. ► Further self-assembly of azide-functionalized terpyridine onto the fluorescent cellulose nanocrystals was possible to obtain nanocellulosic material with expected use in bioimaging. - Abstract: Due to their natural origin, biocompatibility, and non-toxicity, cellulose nanocrystals are promising candidates for applications in nanomedicine. Highly fluorescent nanocellulosic material was prepared via surface modification of cellulose nanocrystals with 2,2′:6′,2″-terpyridine side chains followed by supramolecular assembly of terpyridine-modified perylene dye onto the terpyridine-modified cellulose nanocrystals (CTP) via Ru III /Ru II reduction. The prepared terpyridine-modified cellulose-Ru II -terpyridine-modified perylene (CTP-Ru II -PeryTP) fluorescent nanocrystals were characterized using cross-polarized/magic angle spin 13 C nuclear magnetic resonance (CP/MAS 13 C NMR), Fourier transform infrared (FTIR), UV–visible, and fluorescence spectroscopy. In addition, further self-assembly of terpyridine units with azide functional groups onto CTP-Ru II -PeryTP was possible via repeating the Ru III /Ru II reduction protocol to prepare supramolecular fluorescent nanocrystals with azide functionality (CTP-Ru II -PeryTP-Ru II -AZTP). The prepared derivative may have potential application in bio-imaging since the terminal azide groups can be easily reacted with antigens via “Click” chemistry reaction.

Time resolved fluorescence anisotropy of basic dyes bound to poly(methacrylic acid in solution

Directory of Open Access Journals (Sweden)

Oliveira Hueder Paulo M. de

2003-01-01

Full Text Available Solutions of atactic poly(methacrylic acid, PMAA, with molecular weights in the range of (1.6 to 3.4 x 10(5 g mol-1, and labeled with the fluorescent dyes 9-aminoacridine or Nile blue were studied by photophysical measurements as a function of solvent viscosity and polarity. The conformational behavior of the PMAA chain segments around the fluorescent probe was reported by the change in the rotational diffusion of the dyes. Ethylene glycol swells the polymer chain compared with the more contracted conformation of PMAA in 50% water/ethylene glycol. The change in the rotational relaxation time of the dye bound to PMAA with the decrease of water content in the solvent mixture indicates a progressive expansion of polymer chain to a more open coil form in solution.

Actin filaments and microtubules are involved in different membrane traffic pathways that transport sphingolipids to the apical surface of polarized HepG2 cells

NARCIS (Netherlands)

Zegers, MMP; Zaal, KJM; van Ijzendoorn, SCD; Klappe, K; Hoekstra, D

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and

Time-resolved fluorescence study of exciplex formation in diastereomeric naproxen-pyrrolidine dyads.

Science.gov (United States)

Khramtsova, Ekaterina A; Plyusnin, Viktor F; Magin, Ilya M; Kruppa, Alexander I; Polyakov, Nikolay E; Leshina, Tatyana V; Nuin, Edurne; Marin, M Luisa; Miranda, Miguel A

2013-12-19

The influence of chirality on the elementary processes triggered by excitation of the (S,S)- and (R,S)- diastereoisomers of naproxen-pyrrolidine (NPX-Pyr) dyads has been studied by time-resolved fluorescence in acetonitrile-benzene mixtures. In these systems, the quenching of the (1)NPX*-Pyr singlet excited state occurs through electron transfer and exciplex formation. Fluorescence lifetimes and quantum yields revealed a significant difference (around 20%) between the (S,S)- and (R,S)- diastereomers. In addition, the quantum yields of exciplexes differed by a factor of 2 regardless of solvent polarity. This allows us to suggest a similar influence of the chiral centers on the local charge transfer resulting in exciplex and full charge separation that leads to ion-biradicals. A simplified scheme is proposed to estimate a set of rate constant values (k1-k5) for the elementary stages in each solvent system.

Biomedical applications of nanodiamonds in imaging and therapy.

Science.gov (United States)

Perevedentseva, Elena; Lin, Yu-Chung; Jani, Mona; Cheng, Chia-Liang

2013-12-01

Nanodiamonds have attracted remarkable scientific attention for bioimaging and therapeutic applications owing to their low toxicity with many cell lines, convenient surface properties and stable fluorescence without photobleaching. Newer techniques are being applied to enhance fluorescence. Interest is also growing in exploring the possibilities for modifying the nanodiamond surface and functionalities by attaching various biomolecules of interest for interaction with the targets. The potential of Raman spectroscopy and fluorescence properties of nanodiamonds has been explored for bioimaging and drug delivery tracing. The interest in nanodiamonds' biological/medical application appears to be continuing with enhanced focus. In this review an attempt is made to capture the scope, spirit and recent developments in the field of nanodiamonds for biomedical applications.

Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans.

Directory of Open Access Journals (Sweden)

Romain Vauchelles

Full Text Available The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP together with fluorescence loss in photobleaching (FLIP studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

Fluorescence spectroscopy

DEFF Research Database (Denmark)

Bagatolli, Luis

2016-01-01

Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses the foundati......Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses...

Fusion of a polarized projectile with a polarized target

International Nuclear Information System (INIS)

Christley, J.A.; Johnson, R.C.; Thompson, I.J.

1995-01-01

The fusion cross sections for a polarized target with both unpolarized and polarized projectiles are studied. Expressions for the observables are given for the case when both nuclei are polarized. Calculations for fusion of an aligned 165 Ho target with 16 O and polarized 7 Li beams are presented

Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

Science.gov (United States)

Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

An organic fluorophore-nanodiamond hybrid sensor for photostable imaging and orthogonal, on-demand biosensing.

Science.gov (United States)

Purdey, Malcolm S; Capon, Patrick K; Pullen, Benjamin J; Reineck, Philipp; Schwarz, Nisha; Psaltis, Peter J; Nicholls, Stephen J; Gibson, Brant C; Abell, Andrew D

2017-11-21

Organic fluorescent probes are widely used to detect key biomolecules; however, they often lack the photostability required for extended intracellular imaging. Here we report a new hybrid nanomaterial (peroxynanosensor, PNS), consisting of an organic fluorescent probe bound to a nanodiamond, that overcomes this limitation to allow concurrent and extended cell-based imaging of the nanodiamond and ratiometric detection of hydrogen peroxide. Far-red fluorescence of the nanodiamond offers continuous monitoring without photobleaching, while the green fluorescence of the organic fluorescent probe attached to the nanodiamond surface detects hydrogen peroxide on demand. PNS detects basal production of hydrogen peroxide within M1 polarised macrophages and does not affect macrophage growth during prolonged co-incubation. This nanosensor can be used for extended bio-imaging not previously possible with an organic fluorescent probe, and is spectrally compatible with both Hoechst 33342 and MitoTracker Orange stains for hyperspectral imaging.

Update of the Polar SWIFT model for polar stratospheric ozone loss (Polar SWIFT version 2)

Science.gov (United States)

Wohltmann, Ingo; Lehmann, Ralph; Rex, Markus

2017-07-01

The Polar SWIFT model is a fast scheme for calculating the chemistry of stratospheric ozone depletion in polar winter. It is intended for use in global climate models (GCMs) and Earth system models (ESMs) to enable the simulation of mutual interactions between the ozone layer and climate. To date, climate models often use prescribed ozone fields, since a full stratospheric chemistry scheme is computationally very expensive. Polar SWIFT is based on a set of coupled differential equations, which simulate the polar vortex-averaged mixing ratios of the key species involved in polar ozone depletion on a given vertical level. These species are O3, chemically active chlorine (ClOx), HCl, ClONO2 and HNO3. The only external input parameters that drive the model are the fraction of the polar vortex in sunlight and the fraction of the polar vortex below the temperatures necessary for the formation of polar stratospheric clouds. Here, we present an update of the Polar SWIFT model introducing several improvements over the original model formulation. In particular, the model is now trained on vortex-averaged reaction rates of the ATLAS Chemistry and Transport Model, which enables a detailed look at individual processes and an independent validation of the different parameterizations contained in the differential equations. The training of the original Polar SWIFT model was based on fitting complete model runs to satellite observations and did not allow for this. A revised formulation of the system of differential equations is developed, which closely fits vortex-averaged reaction rates from ATLAS that represent the main chemical processes influencing ozone. In addition, a parameterization for the HNO3 change by denitrification is included. The rates of change of the concentrations of the chemical species of the Polar SWIFT model are purely chemical rates of change in the new version, whereas in the original Polar SWIFT model, they included a transport effect caused by the

Cytoplasmic electric fields and electroosmosis: possible solution for the paradoxes of the intracellular transport of biomolecules.

Science.gov (United States)

Andreev, Victor P

2013-01-01

The objective of the paper is to show that electroosmotic flow might play an important role in the intracellular transport of biomolecules. The paper presents two mathematical models describing the role of electroosmosis in the transport of the negatively charged messenger proteins to the negatively charged nucleus and in the recovery of the fluorescence after photobleaching. The parameters of the models were derived from the extensive review of the literature data. Computer simulations were performed within the COMSOL 4.2a software environment. The first model demonstrated that the presence of electroosmosis might intensify the flux of messenger proteins to the nucleus and allow the efficient transport of the negatively charged phosphorylated messenger proteins against the electrostatic repulsion of the negatively charged nucleus. The second model revealed that the presence of the electroosmotic flow made the time of fluorescence recovery dependent on the position of the bleaching spot relative to cellular membrane. The magnitude of the electroosmotic flow effect was shown to be quite substantial, i.e. increasing the flux of the messengers onto the nucleus up to 4-fold relative to pure diffusion and resulting in the up to 3-fold change in the values of fluorescence recovery time, and therefore the apparent diffusion coefficient determined from the fluorescence recovery after photobleaching experiments. Based on the results of the modeling and on the universal nature of the electroosmotic flow, the potential wider implications of electroosmotic flow in the intracellular and extracellular biological processes are discussed. Both models are available for download at ModelDB.

Spatial and temporal single-cell volume estimation by a fluorescence imaging technique with application to astrocytes in primary culture

Science.gov (United States)

Khatibi, Siamak; Allansson, Louise; Gustavsson, Tomas; Blomstrand, Fredrik; Hansson, Elisabeth; Olsson, Torsten

1999-05-01

Cell volume changes are often associated with important physiological and pathological processes in the cell. These changes may be the means by which the cell interacts with its surrounding. Astroglial cells change their volume and shape under several circumstances that affect the central nervous system. Following an incidence of brain damage, such as a stroke or a traumatic brain injury, one of the first events seen is swelling of the astroglial cells. In order to study this and other similar phenomena, it is desirable to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic cell shape changes in a quantitative and robust way. We have developed a technique to monitor and to quantify the spatial and temporal volume changes in a single cell in primary culture. The technique is based on two- and three-dimensional fluorescence imaging. The temporal information is obtained from a sequence of microscope images, which are analyzed in real time. The spatial data is collected in a sequence of images from the microscope, which is automatically focused up and down through the specimen. The analysis of spatial data is performed off-line and consists of photobleaching compensation, focus restoration, filtering, segmentation and spatial volume estimation.

Quantifying migration and polarization of murine mesenchymal stem cells on different bone substitutes by confocal laser scanning microscopy.

Science.gov (United States)

Roldán, J C; Chang, E; Kelantan, M; Jazayeri, L; Deisinger, U; Detsch, R; Reichert, T E; Gurtner, G C

2010-12-01

Cell migration is preceded by cell polarization. The aim of the present study was to evaluate the impact of the geometry of different bone substitutes on cell morphology and chemical responses in vitro. Cell polarization and migration were monitored temporally by using confocal laser scanning microscopy (CLSM) to follow green fluorescent protein (GFP)±mesenchymal stem cells (MSCs) on anorganic cancellous bovine bone (Bio-Oss(®)), β-tricalcium phosphate (β-TCP) (chronOS(®)) and highly porous calcium phosphate ceramics (Friedrich-Baur-Research-Institute for Biomaterials, Germany). Differentiation GFP±MSCs was observed using pro-angiogenic and pro-osteogenic biomarkers. At the third day of culture polarized vs. non-polarized cellular sub-populations were clearly established. Biomaterials that showed more than 40% of polarized cells at the 3rd day of culture, subsequently showed an enhanced cell migration compared to biomaterials, where non-polarized cells predominated (ppolarization predominated at the 7th day of culture (p=0.001). This model opens an interesting approach to understand osteoconductivity at a cellular level. MSCs are promising in bone tissue engineering considering the strong angiogenic effect before differentiation occurs. Copyright © 2010 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Axon Initial Segment Cytoskeleton: Architecture, Development, and Role in Neuron Polarity

Science.gov (United States)

Svitkina, Tatyana M.

2016-01-01

The axon initial segment (AIS) is a specialized structure in neurons that resides in between axonal and somatodendritic domains. The localization of the AIS in neurons is ideal for its two major functions: it serves as the site of action potential firing and helps to maintain neuron polarity. It has become increasingly clear that the AIS cytoskeleton is fundamental to AIS functions. In this review, we discuss current understanding of the AIS cytoskeleton with particular interest in its unique architecture and role in maintenance of neuron polarity. The AIS cytoskeleton is divided into two parts, submembrane and cytoplasmic, based on localization, function, and molecular composition. Recent studies using electron and subdiffraction fluorescence microscopy indicate that submembrane cytoskeletal components (ankyrin G, βIV-spectrin, and actin filaments) form a sophisticated network in the AIS that is conceptually similar to the polygonal/triangular network of erythrocytes, with some important differences. Components of the AIS cytoplasmic cytoskeleton (microtubules, actin filaments, and neurofilaments) reside deeper within the AIS shaft and display structural features distinct from other neuronal domains. We discuss how the AIS submembrane and cytoplasmic cytoskeletons contribute to different aspects of AIS polarity function and highlight recent advances in understanding their AIS cytoskeletal assembly and stability. PMID:27493806

Detonation nanodiamonds biofunctionalization and immobilization to titanium alloy surfaces as first steps towards medical application

OpenAIRE

Gon?alves, Juliana P L; Shaikh, Afnan Q; Reitzig, Manuela; Kovalenko, Daria A; Michael, Jan; Beutner, Ren?; Cuniberti, Gianaurelio; Scharnweber, Dieter; Opitz, J?rg

2014-01-01

Summary Due to their outstanding properties nanodiamonds are a promising nanoscale material in various applications such as microelectronics, polishing, optical monitoring, medicine and biotechnology. Beyond the typical diamond characteristics like extreme hardness or high thermal conductivity, they have additional benefits as intrinsic fluorescence due to lattice defects without photobleaching, obtained during the high pressure high temperature process. Further the carbon surface and its var...

Neutron polarization

International Nuclear Information System (INIS)

Firk, F.W.K.

1976-01-01

Some recent experiments involving polarized neutrons are discussed; they demonstrate how polarization studies provide information on fundamental aspects of nuclear structure that cannot be obtained from more traditional neutron studies. Until recently, neutron polarization studies tended to be limited either to very low energies or to restricted regions at higher energies, determined by the kinematics of favorable (p, vector n) and (d, vector n) reactions. With the advent of high intensity pulsed electron and proton accelerators and of beams of vector polarized deuterons, this is no longer the case. One has entered an era in which neutron polarization experiments are now being carried out, in a routine way, throughout the entire range from thermal energies to tens-of-MeV. The significance of neutron polarization studies is illustrated in discussions of a wide variety of experiments that include the measurement of T-invariance in the β-decay of polarized neutrons, a search for the effects of meson exchange currents in the photo-disintegration of the deuteron, the determination of quantum numbers of states in the fission of aligned 235 U and 237 Np induced by polarized neutrons, and the double- and triple-scattering of fast neutrons by light nuclei

Thermal and polarized spectroscopic characteristics of Nd3+:LiLa(WO4)2 crystal

International Nuclear Information System (INIS)

Huang Xinyang; Fang Qin; Yu Quanmiao; Lue Xingdong; Zhang Lizhen; Lin Zhoubin; Wang Guofu

2009-01-01

The thermal and polarized spectroscopic characteristics of Nd 3+ :LiLa(WO 4 ) 2 crystals have been investigated. The hardness of Nd 3+ :LiLa(WO 4 ) 2 crystal is 389.7 VDH. The specific heat at 330 K is 0.47 J g -1 K -1 . The thermal expansion coefficients for c- and a-axes are 7.70 x 10 -6 and 14.63 x 10 -5 deg. C -1 , respectively. The spectral parameters have been determined by Judd-Ofelt theory and Fuechtbauer-Ladenburg formula. The intensity parameters Ω t obtained are Ω 2 = 12.914 x 10 -20 cm 2 , Ω 4 = 3.79 x 10 -20 cm 2 , Ω 6 = 3.72 x 10 -20 cm 2 . The radiative and fluorescence lifetimes are 145 and 165 μs, respectively. The quantum efficiency is 91.9%. The absorption band at 805 nm has an FWHM of around 20 nm for both π- and σ-polarizations. The absorption cross-sections at 805 nm are 2.91 and 2.83 x 10 -20 cm 2 for π- and σ-polarizations, respectively. The stimulated emission cross-sections are 8.899 and 7.787 x 10 -20 cm 2 for π- and σ-polarizations, respectively

Three-photon polarization ququarts: polarization, entanglement and Schmidt decompositions

International Nuclear Information System (INIS)

Fedorov, M V; Miklin, N I

2015-01-01

We consider polarization states of three photons, propagating collinearly and having equal given frequencies but with arbitrary distributed horizontal or vertical polarizations of photons. A general form of such states is a superposition of four basic three-photon polarization modes, to be referred to as the three-photon polarization ququarts (TPPQ). All such states can be considered as consisting of one- and two-photon parts, which can be entangled with each other. The degrees of entanglement and polarization, as well as the Schmidt decomposition and Stokes vectors of TPPQ are found and discussed. (paper)