Genetic barcoding with fluorescent proteins for multiplexed applications.

Science.gov (United States)

Smurthwaite, Cameron A; Williams, Wesley; Fetsko, Alexandra; Abbadessa, Darin; Stolp, Zachary D; Reed, Connor W; Dharmawan, Andre; Wolkowicz, Roland

2015-04-14

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded 'indefinitely'. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Theoretical lifetimes and fluorescence yields for multiply-ionized fluorine

International Nuclear Information System (INIS)

Tunnell, T.W.; Can, C.; Bhalla, C.P.

1978-01-01

Theoretical lifetimes and multiplet partial fluorescence yields for various fluorine ions with a single K-shell vacancy were calculated. For few-electron systems, the lifetimes and line fluorescence yields were computed in the intermediate coupling scheme with the inclusion of the effects arising from configuration interactions. 6 references

Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads

Directory of Open Access Journals (Sweden)

Dae Hong Jeong

2012-03-01

Full Text Available Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.

Fluorescence lifetime evaluation of whole soils from the Amazon rainforest.

Science.gov (United States)

Nicolodelli, Gustavo; Tadini, Amanda Maria; Nogueira, Marcelo Saito; Pratavieira, Sebastião; Mounier, Stephane; Huaman, Jose Luis Clabel; Dos Santos, Cléber Hilário; Montes, Célia Regina; Milori, Débora Marcondes Bastos Pereira

2017-08-20

Time-resolved fluorescence spectroscopy (TRFS) is a new tool that can be used to investigate processes of interaction between metal ions and organic matter (OM) in soils, providing a specific analysis of the structure and dynamics of macromolecules. To the best of our knowledge, there are no studies in the literature reporting the use of this technique applied to whole/non-fractionated soil samples, making it a potential method for use in future studies. This work describes the use of TRFS to evaluate the fluorescence lifetimes of OM of whole soils from the Amazon region. Analysis was made of pellets of soils from an oxisol-spodosol system, collected in São Gabriel da Cachoeira (Amazonas, Brazil). The fluorescence lifetimes in the oxisol-spodosol system were attributed to two different fluorophores. One was related to complexation of an OM fraction with metals, resulting in a shorter fluorophore lifetime. A short fluorescence lifetime (2-12 ns) could be associated with simpler structures of the OM, while a long lifetime (19-66 ns) was associated with more complex OM structures. This new TRFS technique for analysis of the fluorescence lifetime in whole soil samples complies with the principles of green chemistry.

Time variation of fluorescence lifetime in enhanced cyan fluorescence protein

International Nuclear Information System (INIS)

Lee, Soonhyouk; Kim, Soo Yong; Park, Kyoungsook; Jeong, Jinyoung; Chung, Bong Hyun; Kim, Sok Won

2010-01-01

The lifetime variations of enhanced cyan fluorescence protein (ECFP) in relatively short integration time bins were studied via time-correlated single photon counting (TCSPC) measurement. We observed that minimum photon counts are necessary for the lifetime estimation to achieve a certain range of variance. The conditions to decrease the variance of lifetime were investigated and the channel width of the measurement of TCSPC data was found to be another important factor for the variance of lifetime. Though the lifetime of ECFP is best fit by a double exponential, a mono exponential fit for the same integration time is more stable. The results may be useful in the analysis of photophysical dynamics for ensemble molecules in short measurement time windows.

Ns-scaled time-gated fluorescence lifetime imaging for forensic document examination

Science.gov (United States)

Zhong, Xin; Wang, Xinwei; Zhou, Yan

2018-01-01

A method of ns-scaled time-gated fluorescence lifetime imaging (TFLI) is proposed to distinguish different fluorescent substances in forensic document examination. Compared with Video Spectral Comparator (VSC) which can examine fluorescence intensity images only, TFLI can detect questioned documents like falsification or alteration. TFLI system can enhance weak signal by accumulation method. The two fluorescence intensity images of the interval delay time tg are acquired by ICCD and fitted into fluorescence lifetime image. The lifetimes of fluorescence substances are represented by different colors, which make it easy to detect the fluorescent substances and the sequence of handwritings. It proves that TFLI is a powerful tool for forensic document examination. Furthermore, the advantages of TFLI system are ns-scaled precision preservation and powerful capture capability.

Fluorophore:dendrimer ratio impacts cellular uptake and intracellular fluorescence lifetime.

Science.gov (United States)

Dougherty, Casey A; Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

2015-02-18

G5-NH2-TAMRAn (n = 1-4, 5+, and 1.5(avg)) were prepared with n = 1-4 as a precise dye:dendrimer ratio, 5+ as a mixture of dendrimers with 5 or more dye per dendrimer, and 1.5(avg) as a Poisson distribution of dye:dendrimer ratios with a mean of 1.5 dye per dendrimer. The absorption intensity increased sublinearly with n whereas the fluorescence emission and lifetime decreased with an increasing number of dyes per dendrimer. Flow cytometry was employed to quantify uptake into HEK293A cells. Dendrimers with 2-4 dyes were found to have greater uptake than dendrimer with a single dye. Fluorescence lifetime imaging microscopy (FLIM) showed that the different dye:dendrimer ratio alone was sufficient to change the fluorescence lifetime of the material observed inside cells. We also observed that the lifetime of G5-NH2-TAMRA5+ increased when present in the cell as compared to solution. However, cells treated with G5-NH2-TAMRA1.5(avg) did not exhibit the high lifetime components present in G5-NH2-TAMRA1 and G5-NH2-TAMRA5+. In general, the effects of the dye:dendrimer ratio on fluorescence lifetime were of similar magnitude to environmentally induced lifetime shifts.

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

Science.gov (United States)

Bogh, Sidsel A.; Bora, Ilkay; Rosenberg, Martin; Thyrhaug, Erling; Laursen, Bo W.; Just Sørensen, Thomas

2015-12-01

Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r 0  =  0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (ϕ fl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕ fl, a ~20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.

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

Science.gov (United States)

Ishii, Kunihiko; Tahara, Tahei

2013-10-03

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

Fluorescence lifetime assays: current advances and applications in drug discovery.

Science.gov (United States)

Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

2011-06-01

Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

Science.gov (United States)

Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

2014-01-01

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.

Fluorescence lifetime measurement of radical ions

International Nuclear Information System (INIS)

Ichinose, Nobuyuki; Kinugasa, Jun-ichiro; Hagiri, Masahide; Nakayama, Toshihiro; Murakami, Hiroshi; Kishimoto, Maki; Daido, Hiroyuki

2004-01-01

One-photonic excitation of a charge transfer complex of hexamethoxybenzene (HMB) and nitrosonium tetrafluoroborate (NO + BF 4 - ) in acetonitrile afforded fluorescences emission from excited radical cation of HMB (HMB + *). Lifetime of the excited radical ion species was measured to be 7 ps by the pump-probe transient absorption technique. The lifetime was much shorter than that of free radical ion (63 ps), indicating the presence of an interaction between HMB + * and NO in the excited complex. (author)

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging.

Science.gov (United States)

Poëa-Guyon, Sandrine; Pasquier, Hélène; Mérola, Fabienne; Morel, Nicolas; Erard, Marie

2013-05-01

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

Portable fluorescence lifetime spectroscopy system for in-situ interrogation of biological tissues

Science.gov (United States)

Saito Nogueira, Marcelo; Cosci, Alessandro; Teixeira Rosa, Ramon Gabriel; Salvio, Ana Gabriela; Pratavieira, Sebastião; Kurachi, Cristina

2017-12-01

Fluorescence spectroscopy and lifetime techniques are potential methods for optical diagnosis and characterization of biological tissues with an in-situ, fast, and noninvasive interrogation. Several diseases may be diagnosed due to differences in the fluorescence spectra of targeted fluorophores, when, these spectra are similar, considering steady-state fluorescence, others may be detected by monitoring their fluorescence lifetime. Despite this complementarity, most of the current fluorescence lifetime systems are not robust and portable, and not being feasible for clinical applications. We describe the assembly of a fluorescence lifetime spectroscopy system in a suitcase, its characterization, and validation with clinical measurements of skin lesions. The assembled system is all encased and robust, maintaining its mechanical, electrical, and optical stability during transportation, and is feasible for clinical measurements. The instrument response function measured was about 300 ps, and the system is properly calibrated. At the clinical study, the system showed to be reliable, and the achieved spectroscopy results support its potential use as an auxiliary tool for skin diagnostics.

Remote UV Fluorescence Lifetime Spectrometer, Phase II

Data.gov (United States)

National Aeronautics and Space Administration — The goal of this project is to develop, demonstrate, and deliver to NASA an innovative, portable, and power efficient Remote UV Fluorescence Lifetime Spectrometer...

Quantitative fluorescence lifetime spectroscopy in turbid media: comparison of theoretical, experimental and computational methods

International Nuclear Information System (INIS)

Vishwanath, Karthik; Mycek, Mary-Ann; Pogue, Brian

2002-01-01

A Monte Carlo model developed to simulate time-resolved fluorescence propagation in a semi-infinite turbid medium was validated against previously reported theoretical and computational results. Model simulations were compared to experimental measurements of fluorescence spectra and lifetimes on tissue-simulating phantoms for single and dual fibre-optic probe geometries. Experiments and simulations using a single probe revealed that scattering-induced artefacts appeared in fluorescence emission spectra, while fluorescence lifetimes were unchanged. Although fluorescence lifetime measurements are generally more robust to scattering artefacts than are measurements of fluorescence spectra, in the dual-probe geometry scattering-induced changes in apparent lifetime were predicted both from diffusion theory and via Monte Carlo simulation, as well as measured experimentally. In all cases, the recovered apparent lifetime increased with increasing scattering and increasing source-detector separation. Diffusion theory consistently underestimated the magnitude of these increases in apparent lifetime (predicting a maximum increase of ∼15%), while Monte Carlo simulations and experiment were closely matched (showing increases as large as 30%). These results indicate that quantitative simulations of time-resolved fluorescence propagation in turbid media will be important for accurate recovery of fluorophore lifetimes in biological spectroscopy and imaging applications. (author)

Portable fluorescence lifetime spectroscopy system for in-situ interrogation of biological tissues.

Science.gov (United States)

Saito Nogueira, Marcelo; Cosci, Alessandro; Teixeira Rosa, Ramon Gabriel; Salvio, Ana Gabriela; Pratavieira, Sebastião; Kurachi, Cristina

2017-10-01

Fluorescence spectroscopy and lifetime techniques are potential methods for optical diagnosis and characterization of biological tissues with an in-situ, fast, and noninvasive interrogation. Several diseases may be diagnosed due to differences in the fluorescence spectra of targeted fluorophores, when, these spectra are similar, considering steady-state fluorescence, others may be detected by monitoring their fluorescence lifetime. Despite this complementarity, most of the current fluorescence lifetime systems are not robust and portable, and not being feasible for clinical applications. We describe the assembly of a fluorescence lifetime spectroscopy system in a suitcase, its characterization, and validation with clinical measurements of skin lesions. The assembled system is all encased and robust, maintaining its mechanical, electrical, and optical stability during transportation, and is feasible for clinical measurements. The instrument response function measured was about 300 ps, and the system is properly calibrated. At the clinical study, the system showed to be reliable, and the achieved spectroscopy results support its potential use as an auxiliary tool for skin diagnostics. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Xanthophyll cycle-dependent quenching of photosystem II chlorophyll a fluorescence: Formation of a quenching complex with a short fluorescence lifetime

Energy Technology Data Exchange (ETDEWEB)

Gilmore, A.M.; Hazlett, T.L.; Govindjee [Univ. of Illinois, Urbana, IL (United States)

1995-03-14

Excess light triggers protective nonradiative dissipation of excitation energy in photosystem II through the formation of a trans-thylakoid pH gradient that in turn stimulates formation of zeaxanthin and antheraxanthin. These xanthophylls when combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus, quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity to understand the mechanism of this process. Increasing the xanthophyll concentration in the presence of a pH gradient (quenched conditions) decreases the fractional intensity of a fluorescence lifetime component centered at {approx}2 ns and increases a component at {approx}0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly affect the fluorescence lifetimes in either the quenched or unquenched sample conditions. However, there are differences in fluorescence lifetimes between the quenched and unquenched states that are due to pH-related, but nonxanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophyllact on antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect of the pH gradient and xanthophyll concentration, resulting in the formation of a quenching complex with a short ({approx}0.4 ns) fluorescence lifetime. 33 refs., 6 figs., 2 tabs.

Mesh adaptation technique for Fourier-domain fluorescence lifetime imaging

International Nuclear Information System (INIS)

Soloviev, Vadim Y.

2006-01-01

A novel adaptive mesh technique in the Fourier domain is introduced for problems in fluorescence lifetime imaging. A dynamical adaptation of the three-dimensional scheme based on the finite volume formulation reduces computational time and balances the ill-posed nature of the inverse problem. Light propagation in the medium is modeled by the telegraph equation, while the lifetime reconstruction algorithm is derived from the Fredholm integral equation of the first kind. Stability and computational efficiency of the method are demonstrated by image reconstruction of two spherical fluorescent objects embedded in a tissue phantom

Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

Science.gov (United States)

van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees

2008-01-01

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations.

Science.gov (United States)

Hosny, Neveen A; Lee, David A; Knight, Martin M

2012-01-01

Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)(3)](2+), was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)(3)](2+) characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

High resolution multiplexed functional imaging in live embryos (Conference Presentation)

Science.gov (United States)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Fourier multiplexed fluorescence lifetime imaging (FmFLIM) scanning laser optical tomography (FmFLIM-SLOT) combines FmFLIM and Scanning laser optical tomography (SLOT) to perform multiplexed 3D FLIM imaging of live embryos. The system had demonstrate multiplexed functional imaging of zebrafish embryos genetically express Foster Resonant Energy Transfer (FRET) sensors. However, previous system has a 20 micron resolution because the focused Gaussian beam diverges quickly from the focused plane, makes it difficult to achieve high resolution imaging over a long projection depth. Here, we present a high-resolution FmFLIM-SLOT system with achromatic Bessel beam, which achieves 3 micron resolution in 3D deep tissue imaging. In Bessel-FmFLIM-SLOT, multiple laser excitation lines are firstly intensity modulated by a Michelson interferometer with a spinning polygon mirror optical delay line, which enables Fourier multiplexed multi-channel lifetime measurements. Then, a spatial light modulator and a prism are used to transform the modulated Gaussian laser beam to an achromatic Bessel beam. The achromatic Bessel beam scans across the whole specimen with equal angular intervals as sample rotated. After tomography reconstruction and the frequency domain lifetime analysis method, both the 3D intensity and lifetime image of multiple excitation-emission can be obtained. Using Bessel-FmFLIM-SLOT system, we performed cellular-resolution FLIM tomography imaging of live zebrafish embryo. Genetically expressed FRET sensors in these embryo will allow non-invasive observation of multiple biochemical processes in vivo.

In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue.

Science.gov (United States)

Kantelhardt, Sven R; Kalasauskas, Darius; König, Karsten; Kim, Ella; Weinigel, Martin; Uchugonova, Aisada; Giese, Alf

2016-05-01

High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.

Miniaturized side-viewing imaging probe for fluorescence lifetime imaging (FLIM): validation with fluorescence dyes, tissue structural proteins and tissue specimens

OpenAIRE

Elson, DS; Jo, JA; Marcu, L

2007-01-01

We report a side viewing fibre-based endoscope that is compatible with intravascular imaging and fluorescence lifetime imaging microscopy (FLIM). The instrument has been validated through testing with fluorescent dyes and collagen and elastin powders using the Laguerre expansion deconvolution technique to calculate the fluorescence lifetimes. The instrument has also been tested on freshly excised unstained animal vascular tissues.

In vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment.

Science.gov (United States)

Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

2014-07-01

Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in vivo fluorescence lifetime imaging with HER2-targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pretreatment size. Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (∼0.13 ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment), the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03 ns. The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. ©2014 American Association for Cancer Research.

In-vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment

Science.gov (United States)

Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

2015-01-01

Purpose Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in-vivo fluorescence lifetime imaging with HER2 targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. Experimental Design HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice, bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pre-treatment size. Results Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (~0.13ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment) the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03ns. Conclusions The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in-vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. PMID:24671949

Multiphoton fluorescence lifetime imaging of chemotherapy distribution in solid tumors

Science.gov (United States)

Carlson, Marjorie; Watson, Adrienne L.; Anderson, Leah; Largaespada, David A.; Provenzano, Paolo P.

2017-11-01

Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples

Science.gov (United States)

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal and polyclonal anti...

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

International Nuclear Information System (INIS)

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

2013-01-01

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

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

Science.gov (United States)

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

2013-06-01

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

Plasmonic-based instrument response function for time-resolved fluorescence: toward proper lifetime analysis

Energy Technology Data Exchange (ETDEWEB)

Szlazak, Radoslaw; Tutaj, Krzysztof; Grudzinski, Wojciech; Gruszecki, Wieslaw I.; Luchowski, Rafal, E-mail: rafal.luchowski@umcs.pl [Maria Curie-Sklodowska University, Department of Biophysics, Institute of Physics (Poland)

2013-06-15

In this report, we investigated the so-called plasmonic platforms prepared to target ultra-short fluorescence and accurate instrumental response function in a time-domain spectroscopy and microscopy. The interaction of metallic nanoparticles with nearby fluorophores results in the increase of the dye fluorescence quantum yield, photostability and decrease of the lifetime parameter. The mentioned properties of platforms were applied to achieve a picosecond fluorescence lifetime (21 ps) of erythrosin B, used later as a better choice for deconvolution of fluorescence decays measured with 'color' sensitive photo-detectors. The ultra-short fluorescence standard based on combination of thin layers of silver film, silver colloidal nanoparticles (about 60 nm in diameter), and top layer of erythrosin B embedded in 0.2 % poly(vinyl) alcohol. The response functions were monitored on two photo-detectors; microchannel plate photomultiplier and single photon avalanche photodiode as a Rayleigh scattering and ultra-short fluorescence. We demonstrated that use of the plasmonic base fluorescence standard as an instrumental response function results in the absence of systematic error in lifetime measurements and analysis.

Chlorophyll fluorescence lifetime imaging provides new insight into the chlorosis induced by plant virus infection.

Science.gov (United States)

Lei, Rong; Jiang, Hongshan; Hu, Fan; Yan, Jin; Zhu, Shuifang

2017-02-01

Leaf chlorosis induced by plant virus infection has a short fluorescence lifetime, which reflects damaged photosynthetic complexes and degraded chloroplasts. Plant viruses often induce chlorosis and necrosis, which are intimately related to photosynthetic functions. Chlorophyll fluorescence lifetime measurement is a valuable noninvasive tool for analyzing photosynthetic processes and is a sensitive indicator of the environment surrounding the fluorescent molecules. In this study, our central goal was to explore the effect of viral infection on photosynthesis by employing chlorophyll fluorescence lifetime imaging (FLIM), steady-state fluorescence, non-photochemical quenching (NPQ), transmission electron microscopy (TEM), and pigment analysis. The data indicated that the chlorophyll fluorescence lifetime of chlorotic leaves was significantly shorter than that of healthy control leaves, and the fitted short lifetime component of chlorophyll fluorescence of chlorotic leaves was dominant. This dominant short lifetime component may result from damage to the structure of thylakoid, which was confirmed by TEM. The NPQ value of chlorotic leaves was slightly higher than that of healthy green leaves, which can be explained by increased neoxanthin, lutein and violaxanthin content relative to chlorophyll a. The difference in NPQ is slight, but FLIM can provide simple and direct characterization of PSII structure and photosynthetic function. Therefore, this technique shows great potential as a simple and rapid method for studying mechanisms of plant virus infection.

Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy

NARCIS (Netherlands)

van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

2008-01-01

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH

Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

Science.gov (United States)

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

2011-07-01

Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

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...

Multiplex and high-throughput DNA detection using surface plasmon mediated fluorescence

Science.gov (United States)

Mei, Zhong

The overall objective of this research project was to develop a user-friendly and sensitive biosensor for nucleic acid aptamers with multiplexing and high-throughput capability. The sensing was based on the fluorescence signals emitted by the fluorophores coupling with plamonic nanoparticle (gold nanorod) deposited on a patterned substrate. Gold nanorods (GNRs) were synthesized using a binary mixture of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NaOL) in seed mediated growth method. Polytetrafluoroethylene (PTFE) printed glass slides were selectively coated with a gold thin-film to define hydrophilic areas for GNR deposition. Due to the wettablity contrast, GNR solution dropped on the slide was induced to assemble exclusively in the hydrophilic spots. By controlling temperature and humidity of the evaporation process, vertically-standing GNR arrays were achieved on the pattered slide. Fluorescence was conjugated to GNR surface via DNA double strand with tunable length. Theoretical simulation predicted a flat layer ( 30 nm thick) of uniform "hot spots" presented on the GNR tips, which could modify the nearby fluorescence. Experimentally, the vertical GNR arrays yielded metallic enhanced fluorescence (MEF) effect, which was dependent on the spectrum overlap and GNR-fluorophore distance. Specifically, the maximum enhancement of Quasar 670 and Alexa 750 was observed when it was coupled with GNR664 (plasmonic wavelength 664 nm) and GNR778 respectively at a distance of 16 nm, while the carboxyfluorescein (FAM) was at maximal intensity when attached to gold nanosphere520. This offers an opportunity for multiplexed DNA sensing. Based on this, we developed a novel GNR mediated fluorescence biosensor for DNA detection. Fluorescence labeled haipin-DNA probes were introduced to designated spots of GNR array with the matching LSPR wavelengths on the substrate. The fluorescence was quenched originally because of Forster resonance energy transfer (FRET) effect

Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo

Science.gov (United States)

Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V.; Roberts, Michael S.

2013-02-01

There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

Standard reference for instrument response function in fluorescence lifetime measurements in visible and near infrared

International Nuclear Information System (INIS)

Chib, Rahul; Shah, Sunil; Gryczynski, Zygmunt; Fudala, Rafal; Borejdo, Julian; Gryczynski, Ignacy; Zelent, Bogumil; Corradini, Maria G; Ludescher, Richard D

2016-01-01

Allura red (AR) fluorophore, a common dye in the food industry, displays a broad emission spectrum in water (visible-to-near infrared region of the electromagnetic spectrum) and has a remarkably short fluorescence lifetime of about 10 ps. This short lifetime does not depend on the emission (observation) wavelength. We examined time responses of AR fluorescence across emission wavelengths from 550 nm to 750 nm and found that it is an ideal candidate for impulse response functions in fluorescence lifetime measurements. (technical note)

Fluorescence lifetime spectroscopy in multiple-scattering environments: an application to biotechnology

Science.gov (United States)

Cerussi, Albert E.; Gratton, Enrico; Fantini, Sergio

1999-07-01

Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in the milk, which is an application vital to the tremendous economic burden of bovine subclinical mastitis detection. Furthermore, we may in principle use the spirit of this technique as a quantitative monitor of the binding of fluorescent drugs inside tissues. This is a first step towards lifetime spectroscopy in tissues.

CVD grown 2D MoS{sub 2} layers: A photoluminescence and fluorescence lifetime imaging study

Energy Technology Data Exchange (ETDEWEB)

Oezden, Ayberk; Madenoglu, Buesra [Department of Materials Science and Engineering, Faculty of Engineering, Anadolu University, Eskisehir (Turkey); Sar, Hueseyin; Ay, Feridun; Perkgoez, Nihan Kosku [Department of Electrical and Electronics Engineering, Faculty of Engineering, Anadolu University, Eskisehir (Turkey); Yeltik, Aydan [Department of Physics, UNAM Institute of Materials Science and Nanotechnology, Bilkent University, Ankara (Turkey); Sevik, Cem [Department of Mechanical Engineering, Faculty of Engineering, Anadolu University, Eskisehir (Turkey)

2016-11-15

In this letter, we report on the fluorescence lifetime imaging and accompanying photoluminescence properties of a chemical vapour deposition (CVD) grown atomically thin material, MoS{sub 2}. μ-Raman, μ-photoluminescence (PL) and fluorescence lifetime imaging microscopy (FLIM) are utilized to probe the fluorescence lifetime and photoluminescence properties of individual flakes of MoS{sub 2} films. Usage of these three techniques allows identification of the grown layers, grain boundaries, structural defects and their relative effects on the PL and fluorescence lifetime spectra. Our investigation on individual monolayer flakes reveals a clear increase of the fluorescence lifetime from 0.3 ns to 0.45 ns at the edges with respect to interior region. On the other hand, investigation of the film layer reveals quenching of PL intensity and lifetime at the grain boundaries. These results could be important for applications where the activity of edges is important such as in photocatalytic water splitting. Finally, it has been demonstrated that PL mapping and FLIM are viable techniques for the investigation of the grain-boundaries. (copyright 2016 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

Azadioxatriangulenium (ADOTA+): A long fluorescence lifetime fluorophore for large biomolecule binding assay

Science.gov (United States)

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

2013-01-01

Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes. PMID:24058730

Generation of a new spectral format, the lifetime synchronous spectrum (LiSS), using phase-resolved fluorescence spectroscopy

International Nuclear Information System (INIS)

Shaver, J.M.; McGown, L.B.

1994-01-01

A new fluorescence spectral format is introduced in which fluorescence lifetime is shown as a function of synchronously scanned wavelength to generate a Lifetime Synchronous Spectrum (LiSS). Lifetimes are determined in the frequency domain with the use of Phase-Resolved Fluorescence Spectroscopy (PRFS) to obtain the phase of the fluorescence signal. Theory and construction of the LiSS are presented and experimental results are shown for solutions of single components and simple binary and ternary mixtures. These results show how the lifetime information in the LiSS augments the steady-state intensity information of a standard synchronous spectrum, providing unique information for identification of components and resolution of overlapping spectral peaks. The LiSS technique takes advantage of noise reduction inherent in the extraction of lifetime from PRFS in addition to standard spectral smoothing techniques. The precision of phase determination through PRFS is found to be comparable to that of direct phase measurements at normal fluorescence intensities and superior for low-intensity signals

Measurements of fluorescence lifetime of group III metalo-8-quinolinolates and their use in analytical chemistry

Energy Technology Data Exchange (ETDEWEB)

Nishikawa, Y; Hiraki, K; Morishige, K; Takahashi, K [Kinki Univ., Higashi-Osaka, Osaka (Japan). Faculty of Science and Technology; Shigematsu, T

1976-07-01

8-Quinolinolates of aluminum, gallium, and indium in chloroform exhibit strong yellowish green fluorescence with an emission maximum at 510, 526, and 528 nm, respectively. The time resolved fluorescence spectra and the fluorescence lifetime properties of these chelates were measured with a time-resolved spectrofluorometer. The fluorescence intensity of these chelates decays exponentially with time t, and obeys the following equation: F=F/sub 0/e-t/tau=F/sub 0/e-k sub(f).t where F/sub 0/ and F are the fluorescence intensity when the exciting light is irradiating and shut off, respectively; tau and k sub(f) being the lifetime and the rate constant for the process of fluorescence emission. The lifetimes of these chelates in chloroform solution at the ordinary temperature were 17.8, 10.1, and 8.4 ns for Al(C/sub 9/H/sub 6/ON)/sub 3/, Ga(C/sub 9/H/sub 6/ON)/sub 3/, and In(C/sub 9/H/sub 6/ON)/sub 3/, respectively. Thus, 8-quinolinolates of group III metals emit the same type radiation with different lifetimes. Between Al-chelate and In-chelate, there were significant difference in the lifetime by 9.4 ns. Then, the logarithmic plot of the composite fluorescence intensity against time is the overlap of some straight lines with different slopes which indicate k sub(f) of various decay processes. The linear portion of the logarithmic plot of the composite fluorescence intensity corresponded to the longer lifetime component (Al-chelate), and by substracting this component from the whole one, the straight line due to the shorter lifetime component (In-chelate) is obtained. Aluminum and indium contents were then determined by comparing the fluorescence intensity of the sample with that of the standard at a definite time (extrapolated to t=0). By using this composite decay curve, the composition of mixtures of nx10/sup -4/ mol/l of Al and In-chelates in chloroform could be determined.

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

Energy Technology Data Exchange (ETDEWEB)

Mueller-Harvey, Irene, E-mail: i.mueller-harvey@reading.ac.uk [Chemistry and Biochemistry Laboratory, Food Production and Quality Research Division, School of Agriculture, Policy and Development, University of Reading, P O Box 236, Reading RG6 6AT (United Kingdom); Feucht, Walter, E-mail: walter.feucht@gmail.com [Department of Plant Sciences, Technical University of Munich (TUM), Wissenschaftszentrum Weihenstephan (WZW), D-85354 Freising (Germany); Polster, Juergen, E-mail: j.polster@wzw.tum.de [Department of Physical Biochemistry, Technical University of Munich (TUM), Wissenschaftszentrum Weihenstephan (WZW), D-85354 Freising (Germany); Trnkova, Lucie, E-mail: lucie.trnkova@uhk.cz [University of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62, 50003 Hradec Kralove (Czech Republic); Burgos, Pierre, E-mail: pierre.burgos@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom); Parker, Anthony W., E-mail: tony.parker@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom); Botchway, Stanley W., E-mail: stan.botchway@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom)

2012-03-16

Highlights: Black-Right-Pointing-Pointer This fluorescence lifetime imaging microscopy (FLIM) technique for flavanols overcomes autofluorescence interference in cells. Black-Right-Pointing-Pointer Plant flavanols differed in their lifetimes. Black-Right-Pointing-Pointer Dissolved and bound flavanols revealed contrasting lifetime changes. Black-Right-Pointing-Pointer This technique will allow studying of flavanol trafficking in live cells. - Abstract: Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were {approx}1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime ({tau}{sub 2} = 1.9-3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there

The LB Films of Dansyl Chloride Labeled Octadecylamine and Its Fluorescence Lifetime

Institute of Scientific and Technical Information of China (English)

无

2000-01-01

Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) in order to simplify and understand the LB films of fluorescent probe labeling proteins.Its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique.Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.

Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

Science.gov (United States)

Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

2014-03-01

Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

Science.gov (United States)

Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V.; Knutson, Jay R.; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

2012-01-01

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers.

Directory of Open Access Journals (Sweden)

Yasaman Ardeshirpour

Full Text Available One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.

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.

RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

Science.gov (United States)

Moffitt, Jeffrey R.; Zhuang, Xiaowei

2016-01-01

Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

A Fast Global Fitting Algorithm for Fluorescence Lifetime Imaging Microscopy Based on Image Segmentation

OpenAIRE

Pelet, S.; Previte, M.J.R.; Laiho, L.H.; So, P.T. C.

2004-01-01

Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained ana...

Fluorescence lifetime, dipole orientation and bilayer polymer films

Science.gov (United States)

Ho, Xuan Long; Chen, Po-Jui; Woon, Wei-Yen; White, Jonathon David

2017-10-01

Bilayer films consisting of the optically transparent polymers, polystyrene (PS) and poly(methyl methacrylate) (PMMA) were spin-cast on glass substrates. The upper 13.5 nm layer (PS) was lightly doped with Rhodamine-6 G (RH6G) or MEH-PPV. While the fluorescence of MEH-PPV was independent of PMMA thickness, the lifetime of RH6G increased 3-fold as the underlying PMMA thickness increased from 0 to 500 nm while the collected flux decreased suggesting a reorientation of the smaller molecule's dipole with respect to the air-polymer interface with PMMA thickness. This suggests lifetime may find application for nondestructive thickness measurements of transparent films with sub-micron lateral resolution and large range.

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

Science.gov (United States)

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

2017-09-01

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

Multiplexed phase-space imaging for 3D fluorescence microscopy.

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Liu, Hsiou-Yuan; Zhong, Jingshan; Waller, Laura

2017-06-26

Optical phase-space functions describe spatial and angular information simultaneously; examples of optical phase-space functions include light fields in ray optics and Wigner functions in wave optics. Measurement of phase-space enables digital refocusing, aberration removal and 3D reconstruction. High-resolution capture of 4D phase-space datasets is, however, challenging. Previous scanning approaches are slow, light inefficient and do not achieve diffraction-limited resolution. Here, we propose a multiplexed method that solves these problems. We use a spatial light modulator (SLM) in the pupil plane of a microscope in order to sequentially pattern multiplexed coded apertures while capturing images in real space. Then, we reconstruct the 3D fluorescence distribution of our sample by solving an inverse problem via regularized least squares with a proximal accelerated gradient descent solver. We experimentally reconstruct a 101 Megavoxel 3D volume (1010×510×500µm with NA 0.4), demonstrating improved acquisition time, light throughput and resolution compared to scanning aperture methods. Our flexible patterning scheme further allows sparsity in the sample to be exploited for reduced data capture.

Frequency division multiplexed multi-color fluorescence microscope system

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Le, Vu Nam; Yang, Huai Dong; Zhang, Si Chun; Zhang, Xin Rong; Jin, Guo Fan

2017-10-01

Grayscale camera can only obtain gray scale image of object, while the multicolor imaging technology can obtain the color information to distinguish the sample structures which have the same shapes but in different colors. In fluorescence microscopy, the current method of multicolor imaging are flawed. Problem of these method is affecting the efficiency of fluorescence imaging, reducing the sampling rate of CCD etc. In this paper, we propose a novel multiple color fluorescence microscopy imaging method which based on the Frequency division multiplexing (FDM) technology, by modulating the excitation lights and demodulating the fluorescence signal in frequency domain. This method uses periodic functions with different frequency to modulate amplitude of each excitation lights, and then combine these beams for illumination in a fluorescence microscopy imaging system. The imaging system will detect a multicolor fluorescence image by a grayscale camera. During the data processing, the signal obtained by each pixel of the camera will be processed with discrete Fourier transform, decomposed by color in the frequency domain and then used inverse discrete Fourier transform. After using this process for signals from all of the pixels, monochrome images of each color on the image plane can be obtained and multicolor image is also acquired. Based on this method, this paper has constructed and set up a two-color fluorescence microscope system with two excitation wavelengths of 488 nm and 639 nm. By using this system to observe the linearly movement of two kinds of fluorescent microspheres, after the data processing, we obtain a two-color fluorescence dynamic video which is consistent with the original image. This experiment shows that the dynamic phenomenon of multicolor fluorescent biological samples can be generally observed by this method. Compared with the current methods, this method can obtain the image signals of each color at the same time, and the color video's frame

Assessment of post-implantation integration of engineered tissues using fluorescence lifetime spectroscopy

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Elahi, Sakib F.; Lee, Seung Y.; Lloyd, William R.; Chen, Leng-Chun; Kuo, Shiuhyang; Zhou, Ying; Kim, Hyungjin M.; Kennedy, Robert; Marcelo, Cynthia; Feinberg, Stephen E.; Mycek, Mary-Ann

2018-02-01

Clinical translation of engineered tissue constructs requires noninvasive methods to assess construct health and viability after implantation in patients. However, current practices to monitor post-implantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). As label-free fluorescence lifetime sensing can noninvasively characterize pre-implantation construct viability, we employed a handheld fluorescence lifetime spectroscopy probe to quantitatively and noninvasively assess tissue constructs that were implanted in a murine model. We designed the system to be suitable for intravital measurements: portability, localization with precise maneuverability, and rapid data acquisition. Our model tissue constructs were manufactured from primary human cells to simulate patient variability and were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess pre-implantation construct health. In vivo optical sensing assessed tissue integration of constructs at one-week and three-weeks post-implantation. At one-week post-implantation, optical parameters correlated with in vitro pre-implantation secretion levels of all three cytokines (p clinical optical diagnostic tools based on label-free fluorescence lifetime sensing of endogenous tissue fluorophores could noninvasively monitor post-implantation integration of engineered tissues.

Time gated fluorescence lifetime imaging and micro-volume spectroscopy using two-photon excitation

NARCIS (Netherlands)

Sytsma, J.; Vroom, J.M.; de Grauw, C.J.; Gerritsen, H.C.

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes.

Fluorescence intensity and lifetime-based cyanide sensitive probes for physiological safeguard

International Nuclear Information System (INIS)

Badugu, Ramachandram; Lakowicz, Joseph R.; Geddes, Chris D.

2004-01-01

We characterize six new fluorescent probes that show both intensity and lifetime changes in the presence of free uncomplexed aqueous cyanide, allowing for fluorescence based cyanide sensing up to physiological safeguard levels, i.e. 2 to the anionic R-B - (CN) 3 form, a new cyanide binding mechanism which we have recently reported. The presence of an electron deficient quaternary heterocyclic nitrogen nucleus, and the electron rich cyanide bound form, provides for the intensity changes observed. We have determined the disassociation constants of the probes to be in the range ∼15-84 μM 3 . In addition we have synthesized control compounds which do not contain the boronic acid moiety, allowing for a rationale of the cyanide responses between the probe isomers to be made. The lifetime of the cyanide bound probes are significantly shorter than the free R-B(OH) 2 probe forms, providing for the opportunity of lifetime based cyanide sensing up to physiologically lethal levels. Finally, while fluorescent probes containing the boronic acid moiety have earned a well-deserved reputation for monosaccharide sensing, we show that strong bases such as CN - and OH - preferentially bind as compared to glucose, enabling the potential use of these probes for cyanide safeguard and determination in physiological fluids, especially given that physiologies do not experience any notable changes in pH

Olive oil DNA fingerprinting by multiplex SNP genotyping on fluorescent microspheres.

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Kalogianni, Despina P; Bazakos, Christos; Boutsika, Lemonia M; Targem, Mehdi Ben; Christopoulos, Theodore K; Kalaitzis, Panagiotis; Ioannou, Penelope C

2015-04-01

Olive oil cultivar verification is of primary importance for the competitiveness of the product and the protection of consumers and producers from fraudulence. Single-nucleotide polymorphisms (SNPs) have emerged as excellent DNA markers for authenticity testing. This paper reports the first multiplex SNP genotyping assay for olive oil cultivar identification that is performed on a suspension of fluorescence-encoded microspheres. Up to 100 sets of microspheres, with unique "fluorescence signatures", are available. Allele discrimination was accomplished by primer extension reaction. The reaction products were captured via hybridization on the microspheres and analyzed, within seconds, by a flow cytometer. The "fluorescence signature" of each microsphere is assigned to a specific allele, whereas the signal from a reporter fluorophore denotes the presence of the allele. As a model, a panel of three SNPs was chosen that enabled identification of five common Greek olive cultivars (Adramytini, Chondrolia Chalkidikis, Kalamon, Koroneiki, and Valanolia).

Multiphoton Laser Microscopy and Fluorescence Lifetime Imaging for the Evaluation of the Skin

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Stefania Seidenari

2012-01-01

Full Text Available Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging. Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM, is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development of multiphoton laser microscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena.

Enhancement of early cervical cancer diagnosis with epithelial layer analysis of fluorescence lifetime images.

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Jun Gu

Full Text Available This work reports the use of layer analysis to aid the fluorescence lifetime diagnosis of cervical intraepithelial neoplasia (CIN from H&E stained cervical tissue sections. The mean and standard deviation of lifetimes in single region of interest (ROI of cervical epithelium were previously shown to correlate to the gold standard histopathological classification of early cervical cancer. These previously defined single ROIs were evenly divided into layers for analysis. A 10-layer model revealed a steady increase in fluorescence lifetime from the inner to the outer epithelial layers of healthy tissue sections, suggesting a close association with cellular maturity. The shorter lifetime and minimal lifetime increase towards the epithelial surface of CIN-affected regions are in good agreement with the absence of cellular maturation in CIN. Mean layer lifetimes in the top-half cervical epithelium were used as feature vectors for extreme learning machine (ELM classifier discriminations. It was found that the proposed layer analysis technique greatly improves the sensitivity and specificity to 94.6% and 84.3%, respectively, which can better supplement the traditional gold standard cervical histopathological examinations.

FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye.

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Matthias Klemm

Full Text Available Fluorescence lifetime imaging ophthalmoscopy (FLIO is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX's applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation.

FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye.

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Klemm, Matthias; Schweitzer, Dietrich; Peters, Sven; Sauer, Lydia; Hammer, Martin; Haueisen, Jens

2015-01-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX's applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation.

A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation.

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Pelet, S; Previte, M J R; Laiho, L H; So, P T C

2004-10-01

Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained analysis. Convergence speed can be greatly accelerated by providing appropriate initial guesses. Realizing that the image morphology often correlates with fluorophore distribution, a global fitting algorithm has been developed to assign initial guesses throughout an image based on a segmentation analysis. This algorithm was tested on both simulated data sets and time-domain lifetime measurements. We have successfully measured fluorophore distribution in fibroblasts stained with Hoechst and calcein. This method further allows second harmonic generation from collagen and elastin autofluorescence to be differentiated in fluorescence lifetime imaging microscopy images of ex vivo human skin. On our experimental measurement, this algorithm increased convergence speed by over two orders of magnitude and achieved significantly better fits. Copyright 2004 Biophysical Society

Diagnosis of basal cell carcinoma by two photon excited fluorescence combined with lifetime imaging

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Fan, Shunping; Peng, Xiao; Liu, Lixin; Liu, Shaoxiong; Lu, Yuan; Qu, Junle

2014-02-01

Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

Time-Domain Fluorescence Lifetime Imaging Techniques Suitable for Solid-State Imaging Sensor Arrays

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Robert K. Henderson

2012-05-01

Full Text Available We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD-based cameras for fluorescence lifetime imaging microscopy (FLIM by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 μm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast.

Rotational multispectral fluorescence lifetime imaging and intravascular ultrasound: bimodal system for intravascular applications

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Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Gorpas, Dimitris; Fatakdawala, Hussain; Marcu, Laura

2014-01-01

Abstract. We report the development and validation of a hybrid intravascular diagnostic system combining multispectral fluorescence lifetime imaging (FLIm) and intravascular ultrasound (IVUS) for cardiovascular imaging applications. A prototype FLIm system based on fluorescence pulse sampling technique providing information on artery biochemical composition was integrated with a commercial IVUS system providing information on artery morphology. A customized 3-Fr bimodal catheter combining a rotational side-view fiberoptic and a 40-MHz IVUS transducer was constructed for sequential helical scanning (rotation and pullback) of tubular structures. Validation of this bimodal approach was conducted in pig heart coronary arteries. Spatial resolution, fluorescence detection efficiency, pulse broadening effect, and lifetime measurement variability of the FLIm system were systematically evaluated. Current results show that this system is capable of temporarily resolving the fluorescence emission simultaneously in multiple spectral channels in a single pullback sequence. Accurate measurements of fluorescence decay characteristics from arterial segments can be obtained rapidly (e.g., 20 mm in 5 s), and accurate co-registration of fluorescence and ultrasound features can be achieved. The current finding demonstrates the compatibility of FLIm instrumentation with in vivo clinical investigations and its potential to complement conventional IVUS during catheterization procedures. PMID:24898604

Multicolor fluorescent biosensor for multiplexed detection of DNA.

Science.gov (United States)

Hu, Rong; Liu, Tao; Zhang, Xiao-Bing; Huan, Shuang-Yan; Wu, Cuichen; Fu, Ting; Tan, Weihong

2014-05-20

Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

Study on the effect of deposition rate and concentration of Eu on the fluorescent lifetime of CsI: Tl thin film

Energy Technology Data Exchange (ETDEWEB)

Xie, Yijun; Guo, Lina [University of Electronic Science and Technology of China, State Key Laboratory of Electronic Thin Films and Integrated Devices, School of Optoelectronic Information, Chengdu 610054 (China); Liu, Shuang, E-mail: shuangliu@uestc.edu.cn [University of Electronic Science and Technology of China, State Key Laboratory of Electronic Thin Films and Integrated Devices, School of Optoelectronic Information, Chengdu 610054 (China); Wang, Qianfeng; Zhang, Shangjian; Liu, Yong [University of Electronic Science and Technology of China, State Key Laboratory of Electronic Thin Films and Integrated Devices, School of Optoelectronic Information, Chengdu 610054 (China); Zhong, Zhiyong [University of Electronic Science and Technology of China, State Key Laboratory of Electronic Thin Films and Integrated Devices, Chengdu 610054 (China)

2017-06-21

Although there are many new scintillators being developed recently, CsI: Tl is still very efficient among them. The fluorescent lifetime is a very important parameter of CsI: Tl thin film and two series of experiments have been conducted to learn about it. Our experiments, however, have demonstrated that the deposition rate and the codoping of Eu{sup 2+} will significantly influence its fluorescent lifetime. In order to increase the efficiency of the imaging system, we intend to obtain a higher fluorescent lifetime for CsI: Tl thin film by controlling these two conditions. - Highlights: • We used vacuum vapor deposition method to grow the high-quality thin films. • The relationship between the deposition rate and the fluorescent lifetime of CsI: Tl thin film was tested. • Concentration of Eu on fluorescent lifetime of the CsI: Tl thin film was studied.

Community detection for fluorescent lifetime microscopy image segmentation

Science.gov (United States)

Hu, Dandan; Sarder, Pinaki; Ronhovde, Peter; Achilefu, Samuel; Nussinov, Zohar

2014-03-01

Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes.1 In the current paper, we further explore this method in FLT images of ex vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or "solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1:25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.

Fluorescence lifetime microscopy for monitoring cell adhesion using metal induced energy transfer

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Hwang, Wonsang; Seo, JinWon; Song, Jun ho; Kim, DongEun; Won, YoungJae; Choi, In-Hong; Yoo, Kyung-Hwa; Kim, Dug Young

2018-02-01

A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.

Monitoring macular pigment changes in macular holes using fluorescence lifetime imaging ophthalmoscopy.

Science.gov (United States)

Sauer, Lydia; Peters, Sven; Schmidt, Johanna; Schweitzer, Dietrich; Klemm, Matthias; Ramm, Lisa; Augsten, Regine; Hammer, Martin

2017-08-01

To investigate the impact of macular pigment (MP) on fundus autofluorescence (FAF) lifetimes in vivo by characterizing full-thickness idiopathic macular holes (MH) and macular pseudo-holes (MPH). A total of 37 patients with MH and 52 with MPH were included. Using the fluorescence lifetime imaging ophthalmoscope (FLIO), based on a Heidelberg Engineering Spectralis system, a 30° retinal field was investigated. FAF decays were detected in a short (498-560 nm; ch1) and long (560-720 nm; ch2) wavelength channel. τ m , the mean fluorescence lifetime, was calculated from a three-exponential approximation of the FAF decays. Macular coherence tomography scans were recorded, and macular pigment's optical density (MPOD) was measured (one-wavelength reflectometry). Two MH subgroups were analysed according to the presence or absence of an operculum above the MH. A total of 17 healthy fellow eyes were included. A longitudinal FAF decay examination was conducted in nine patients, which were followed up after surgery and showed a closed MH. In MH without opercula, significant τ m differences (p hole area (MHa) and surrounding areas (MHb) (ch1: MHa 238 ± 64 ps, MHb 181 ± 78 ps; ch2: MHa 275 ± 49 ps, MHb 223 ± 48 ps), as well as between MHa and healthy eyes or closed MH. Shorter τ m , adjacent to the hole, can be assigned to areas with equivalently higher MPOD. Opercula containing MP also show short τ m . In MPH, the intactness of the Hele fibre layer is associated with shortest τ m . Shortest τ m originates from MP-containing retinal layers, especially from the Henle fibre layer. Fluorescence lifetime imaging ophthalmoscope (FLIO) provides information on the MP distribution, the pathogenesis and topology of MH. Macular pigment (MP) fluorescence may provide a biomarker for monitoring pathological changes in retinal diseases. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine

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Alexander Boreham

2016-12-01

Full Text Available The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine.

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Boreham, Alexander; Brodwolf, Robert; Walker, Karolina; Haag, Rainer; Alexiev, Ulrike

2016-12-24

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

Fluorescence lifetime imaging of endogenous molecules in live mouse cancer models (Conference Presentation)

Science.gov (United States)

Svindrych, Zdenek; Wang, Tianxiong; Hu, Song; Periasamy, Ammasi

2017-02-01

NADH and FAD are important endogenous fluorescent coenzymes participating in key enzymatic reactions of cellular metabolism. While fluorescence intensities of NADH and FAD have been used to determine the redox state of cells and tissues, this simple approach breaks down in the case of deep-tissue intravital imaging due to depth- and wavelength-dependent light absorption and scattering. To circumvent this limitation, our research focuses on fluorescence lifetimes of two-photon excited NADH and FAD emission to study the metabolic state of live tissues. In our custom-built scanning microscope we combine tunable femtosecond Ti:sapphire laser (operating at 740 nm for NADH excitation and 890 nm for FAD excitation), two GaAsP hybrid detectors for registering individual fluorescence photons and two Becker and Hickl time correlator boards for high precision lifetime measurements. Together with our rigorous FLIM analysis approach (including image segmentation, multi-exponential decay fitting and detailed statistical analysis) we are able to detect metabolic changes in cancer xenografts (human pancreatic cancer MPanc96 cells injected subcutaneously into the ear of an immunodeficient nude mouse), relative to surrounding healthy tissue. Advantageously, with the same instrumentation we can also take high-resolution and high-contrast images of second harmonic signal (SHG) originating from collagen fibers of both the healthy skin and the growing tumor. The combination of metabolic measurements (NADH and FAD lifetime) and morphological information (collagen SHG) allows us to follow the tumor growth in live mouse model and the changes in tumor microenvironment.

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

Science.gov (United States)

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

1999-05-01

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

An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy

International Nuclear Information System (INIS)

Dunsby, C; Lanigan, P M P; McGinty, J; Elson, D S; Requejo-Isidro, J; Munro, I; Galletly, N; McCann, F; Treanor, B; Oenfelt, B; Davis, D M; Neil, M A A; French, P M W

2004-01-01

Fluorescence imaging is used widely in microscopy and macroscopic imaging applications for fields ranging from biomedicine to materials science. A critical component for any fluorescence imaging system is the excitation source. Traditionally, wide-field systems use filtered thermal or arc-generated white light sources, while point scanning confocal microscope systems require spatially coherent (point-like) laser sources. Unfortunately, the limited range of visible wavelengths available from conventional laser sources constrains the design and usefulness of fluorescent probes in confocal microscopy. A 'hands-off' laser-like source, electronically tunable across the visible spectrum, would be invaluable for fluorescence imaging and provide new opportunities, e.g. automated excitation fingerprinting and in situ measurement of excitation cross-sections. Yet more information can be obtained using fluorescence lifetime imaging (FLIM), which requires that the light source be pulsed or rapidly modulated. We show how a white light continuum, generated by injecting femtosecond optical radiation into a micro-structured optical fibre, coupled with a simple prism-based tunable filter arrangement, can fulfil all these roles as a continuously electronically tunable (435-1150 nm) visible ultrafast light source in confocal, wide-field and FLIM systems

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo.

Science.gov (United States)

Yaseen, Mohammad A; Sutin, Jason; Wu, Weicheng; Fu, Buyin; Uhlirova, Hana; Devor, Anna; Boas, David A; Sakadžić, Sava

2017-05-01

Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics.

In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles.

Science.gov (United States)

Gu, Luo; Hall, David J; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J; Howell, Stephen B; Sailor, Michael J

2013-01-01

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

A Compact Fluorescence Lifetime Excitation-Emission Spectrometer (FLEXEMS) for Detecting Trace Organics, Phase II

Data.gov (United States)

National Aeronautics and Space Administration — In this Small Business Innovative Research (SBIR) effort, Leiden Measurement Technology (LMT) proposes to design and build the Fluorescence Lifetime Excitation...

A Compact Fluorescence Lifetime Excitation-Emission Spectrometer (FLEXEMS) for Detecting Trace Organics, Phase I

Data.gov (United States)

National Aeronautics and Space Administration — In this Small Business Innovative Research (SBIR) effort, Leiden Measurement Technology (LMT) proposes to design and build the Fluorescence Lifetime Excitation...

Using non-empirically tuned range-separated functionals with simulated emission bands to model fluorescence lifetimes.

Science.gov (United States)

Wong, Z C; Fan, W Y; Chwee, T S; Sullivan, Michael B

2017-08-09

Fluorescence lifetimes were evaluated using TD-DFT under different approximations for the emitting molecule and various exchange-correlation functionals, such as B3LYP, BMK, CAM-B3LYP, LC-BLYP, M06, M06-2X, M11, PBE0, ωB97, ωB97X, LC-BLYP*, and ωB97X* where the range-separation parameters in the last two functionals were tuned in a non-empirical fashion. Changes in the optimised molecular geometries between the ground and electronically excited states were found to affect the quality of the calculated lifetimes significantly, while the inclusion of vibronic features led to further improvements over the assumption of a vertical electronic transition. The LC-BLYP* functional was found to return the most accurate fluorescence lifetimes with unsigned errors that are mostly within 1.5 ns of experimental values.

Alterations in cerebral metabolism observed in living rodents using fluorescence lifetime microscopy of intrinsic NADH (Conference Presentation)

Science.gov (United States)

Yaseen, Mohammad A.; Sakadžić, Sava; Sutin, Jason; Wu, Weicheng; Fu, Buyin; Boas, David A.

2017-02-01

Monitoring cerebral energy metabolism at a cellular level is essential to improve our understanding of healthy brain function and its pathological alterations. In this study, we resolve specific alterations in cerebral metabolism utilizing minimally-invasive 2-Photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence, collected in vivo from anesthetized rats and mice. Time-resolved lifetime measurements enables distinction of different components contributing to NADH autofluorescence. These components reportedly represent different enzyme-bound formulations of NADH. Our observations from this study confirm the hypothesis that NADH FLIM can identify specific alterations in cerebral metabolism. Using time-correlated single photon counting (TCSPC) equipment and a custom-built multimodal imaging system, 2-photon fluorescence lifetime imaging (FLIM) was performed in cerebral tissue with high spatial and temporal resolution. Multi-exponential fits for NADH fluorescence lifetimes indicate 4 distinct components, or 'species.' We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in anaerobic glycolysis and aerobic oxidative metabolism. Classification models developed with experimental data correctly predict the metabolic impairments associated with bicuculline-induced focal seizures in separate experiments. Compared to traditional intensity-based NADH measurements, lifetime imaging of NADH is less susceptible to the adverse effects of overlying blood vessels. Evaluating NADH measurements will ultimately lead to a deeper understanding of cerebral energetics and its pathology-related alterations. Such knowledge will likely aid development of therapeutic strategies for neurodegenerative diseases such as Alzheimer's Disease, Parkinson's disease, and stroke.

Time-resolved laser fluorescence spectroscopy of organic ligands by europium: Fluorescence quenching and lifetime properties

Science.gov (United States)

Nouhi, A.; Hajjoul, H.; Redon, R.; Gagné, J. P.; Mounier, S.

2018-03-01

Time-resolved Laser Fluorescence Spectroscopy (TRLFS) has proved its usefulness in the fields of biophysics, life science and geochemistry to characterize the fluorescence probe molecule with its chemical environment. The purpose of this study is to demonstrate the applicability of this powerful technique combined with Steady-State (S-S) measurements. A multi-mode factor analysis, in particular CP/PARAFAC, was used to analyze the interaction between Europium (Eu) and Humic substances (HSs) extracted from Saint Lawrence Estuary in Canada. The Saint Lawrence system is a semi-enclosed water stream with connections to the Atlantic Ocean and is an excellent natural laboratory. CP/PARAFAC applied to fluorescence S-S data allows introspecting ligands-metal interactions and the one-site 1:1 modeling gives information about the stability constants. From the spectral signatures and decay lifetimes data given by TRLFS, one can deduce the fluorescence quenching which modifies the fluorescence and discuss its mechanisms. Results indicated a relatively strong binding ability between europium and humic substances samples (Log K value varies from 3.38 to 5.08 at pH 7.00). Using the Stern-Volmer plot, it has been concluded that static and dynamic quenching takes places in the case of salicylic acid and europium interaction while for HSs interaction only a static quenching is observed.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

Science.gov (United States)

Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

2015-12-15

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Online multispectral fluorescence lifetime values estimation and overlay onto tissue white-light video frames

Science.gov (United States)

Gorpas, Dimitris; Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Marcu, Laura

2016-03-01

Fluorescence lifetime imaging has been shown to be a robust technique for biochemical and functional characterization of tissues and to present great potential for intraoperative tissue diagnosis and guidance of surgical procedures. We report a technique for real-time mapping of fluorescence parameters (i.e. lifetime values) onto the location from where the fluorescence measurements were taken. This is achieved by merging a 450 nm aiming beam generated by a diode laser with the excitation light in a single delivery/collection fiber and by continuously imaging the region of interest with a color CMOS camera. The interrogated locations are then extracted from the acquired frames via color-based segmentation of the aiming beam. Assuming a Gaussian profile of the imaged aiming beam, the segmentation results are fitted to ellipses that are dynamically scaled at the full width of three automatically estimated thresholds (50%, 75%, 90%) of the Gaussian distribution's maximum value. This enables the dynamic augmentation of the white-light video frames with the corresponding fluorescence decay parameters. A fluorescence phantom and fresh tissue samples were used to evaluate this method with motorized and hand-held scanning measurements. At 640x512 pixels resolution the area of interest augmented with fluorescence decay parameters can be imaged at an average 34 frames per second. The developed method has the potential to become a valuable tool for real-time display of optical spectroscopy data during continuous scanning applications that subsequently can be used for tissue characterization and diagnosis.

Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate.

Science.gov (United States)

Eibl, Matthias; Karpf, Sebastian; Weng, Daniel; Hakert, Hubertus; Pfeiffer, Tom; Kolb, Jan Philip; Huber, Robert

2017-07-01

Two-photon-excited fluorescence lifetime imaging microscopy (FLIM) is a chemically specific 3-D sensing modality providing valuable information about the microstructure, composition and function of a sample. However, a more widespread application of this technique is hindered by the need for a sophisticated ultra-short pulse laser source and by speed limitations of current FLIM detection systems. To overcome these limitations, we combined a robust sub-nanosecond fiber laser as the excitation source with high analog bandwidth detection. Due to the long pulse length in our configuration, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. In this paper, we show high quality FLIM images acquired at a pixel rate of 1 MHz. This approach is a promising candidate for an easy-to-use and benchtop FLIM system to make this technique available to a wider research community.

Long-term fluorescence lifetime imaging of a genetically encoded sensor for caspase-3 activity in mouse tumor xenografts

Science.gov (United States)

Zherdeva, Victoria; Kazachkina, Natalia I.; Shcheslavskiy, Vladislav; Savitsky, Alexander P.

2018-03-01

Caspase-3 is known for its role in apoptosis and programmed cell death regulation. We detected caspase-3 activation in vivo in tumor xenografts via shift of mean fluorescence lifetimes of a caspase-3 sensor. We used the genetically encoded sensor TR23K based on the red fluorescent protein TagRFP and chromoprotein KFP linked by 23 amino acid residues (TagRFP-23-KFP) containing a specific caspase cleavage DEVD motif to monitor the activity of caspase-3 in tumor xenografts by means of fluorescence lifetime imaging-Forster resonance energy transfer. Apoptosis was induced by injection of paclitaxel for A549 lung adenocarcinoma and etoposide and cisplatin for HEp-2 pharynx adenocarcinoma. We observed a shift in lifetime distribution from 1.6 to 1.9 ns to 2.1 to 2.4 ns, which indicated the activation of caspase-3. Even within the same tumor, the lifetime varied presumably due to the tumor heterogeneity and the different depth of tumor invasion. Thus, processing time-resolved fluorescence images allows detection of both the cleaved and noncleaved states of the TR23K sensor in real-time mode during the course of several weeks noninvasively. This approach can be used in drug screening, facilitating the development of new anticancer agents as well as improvement of chemotherapy efficiency and its adaptation for personal treatment.

Efficacy of photodynamic therapy against larvae of Aedes aegypti: confocal microscopy and fluorescence-lifetime imaging

Science.gov (United States)

de Souza, L. M.; Pratavieira, S.; Inada, N. M.; Kurachi, C.; Corbi, J.; Guimarães, F. E. G.; Bagnato, V. S.

2014-03-01

Recently a few demonstration on the use of Photodynamic Reaction as possibility to eliminate larvae that transmit diseases for men has been successfully demonstrated. This promising tool cannot be vastly used due to many problems, including the lake of investigation concerning the mechanisms of larvae killing as well as security concerning the use of photosensitizers in open environment. In this study, we investigate some of the mechanisms in which porphyrin (Photogem) is incorporated on the Aedes aegypti larvae previously to illumination and killing. Larvae at second instar were exposed to the photosensitizer and after 30 minutes imaged by a confocal fluorescence microscope. It was observed the presence of photosensitizer in the gut and at the digestive tract of the larva. Fluorescence-Lifetime Imaging showed greater photosensitizer concentration in the intestinal wall of the samples, which produces a strong decrease of the Photogem fluorescence lifetime. For Photodynamic Therapy exposition to different light doses and concentrations of porphyrin were employed. Three different light sources (LED, Fluorescent lamp, Sun light) also were tested. Sun light and fluorescent lamp shows close to 100% of mortality after 24 hrs. of illumination. These results indicate the potential use of photodynamic effect against the LARVAE of Aedes aegypti.

Fluorescence lifetime of emitters with broad homogeneous linewidths modified in opal photonic crystals

DEFF Research Database (Denmark)

Nikolaev, Ivan S.; Lodahl, Peter; Vos, Willem L.

2008-01-01

We have investigated the dynamics of spontaneous emission from dye molecules embedded in opal photonic crystals. Fluorescence lifetimes of Rhodamine 6G (R6G) dye were measured as a function of both optical frequency and crystal lattice parameter of the polystyrene opals. Due to the broad...

Fluorescence lifetime measurement with confocal endomicroscopy for direct analysis of tissue biochemistry in vivo

Directory of Open Access Journals (Sweden)

Youngjae Won

2016-08-01

Full Text Available Confocal endomicroscopy is a powerful tool for in vivo real-time imaging at cellular resolution inside a living body without tissue resection. Microscopic fluorescence lifetime measurement can provide information about localized biochemical conditions such as pH and the concentrations of oxygen and calcium. We hypothesized that combining these techniques could assist accurate cancer discrimination by providing both biochemical and morphological information. We designed a dual-mode experimental setup for confocal endomicroscopic imaging and fluorescence lifetime measurement and applied it to a mouse xenograft model of activated human pancreatic cancer generated by subcutaneous injection of AsPC-1 tumor cells. Using this method with pH-sensitive sodium fluorescein injection, we demonstrated discrimination between normal and cancerous tissues in a living mouse. With further development, this method may be useful for clinical cancer detection.

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.

Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

Directory of Open Access Journals (Sweden)

Qiuying Huang

2011-04-01

Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

Science.gov (United States)

Laine, Romain; Stuckey, Daniel W.; Manning, Hugh; Warren, Sean C.; Kennedy, Gordon; Carling, David

2012-01-01

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that m

Fluorescently labelled multiplex lateral flow immunoassay based on cadmium-free quantum dots.

Science.gov (United States)

Beloglazova, Natalia V; Sobolev, Aleksander M; Tessier, Mickael D; Hens, Zeger; Goryacheva, Irina Yu; De Saeger, Sarah

2017-03-01

A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures. They consisted of core/shell InP/ZnS QDs enrobed in a silica shell that allowed the water solubility (QD@SiO 2 ). Then we applied the QD@SiO 2 as novel, silica shell-encapsulated fluorescent labels in immunoassays for rapid multiplexed screening. Two mycotoxins, zearalenone and deoxynivalenol, were simultaneously detected in maize and wheat, since the two QD@SiO 2 labelled conjugates emit at two different, individually detectable wavelengths. The cutoff values for the simultaneous determination were 50 and 500μgkg -1 for zearalenone and deoxynivalenol, respectively, in both maize and wheat. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the result. Copyright © 2017 Elsevier Inc. All rights reserved.

Using multiphoton fluorescence lifetime imaging to characterize liver damage and fluorescein disposition in liver in vivo

Science.gov (United States)

Thorling, Camilla A.; Studier, Hauke; Crawford, Darrell; Roberts, Michael S.

2016-03-01

Liver disease is the fifth most common cause of death and unlike many other major causes of mortality, liver disease rates are increasing rather than decreasing. There is no ideal measurement of liver disease and although biopsies are the gold standard, this only allows for a spot examination and cannot follow dynamic processes of the liver. Intravital imaging has the potential to extract detailed information over a larger sampling area continuously. The aim of this project was to investigate whether multiphoton and fluorescence lifetime imaging microscopy could detect early liver damage and to assess whether it could detect changes in metabolism of fluorescein in normal and diseased livers. Four experimental groups were used in this study: 1) control; 2) ischemia reperfusion injury; 3) steatosis and 4) steatosis with ischemia reperfusion injury. Results showed that multiphoton microscopy could visualize morphological changes such as decreased fluorescence of endogenous fluorophores and the presence of lipid droplets, characteristic of steatosis. Fluorescence lifetime imaging microscopy showed increase in NADPH in steatosis with and without ischemia reperfusion injury and could detect changes in metabolism of fluorescein to fluorescein monoglurcuronide, which was impaired in steatosis with ischemia reperfusion injury. These results concluded that the combination of multiphoton microscopy and fluorescence lifetime imaging is a promising method of assessing early stage liver damage and that it can be used to study changes in drug metabolism in the liver as an indication of liver disease and has the potential to replace the traditional static liver biopsy currently used.

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method

Science.gov (United States)

Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae

2016-01-01

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection. PMID:28018724

A fusion-spliced near-field optical fiber probe using photonic crystal fiber for nanoscale thermometry based on fluorescence-lifetime measurement of quantum dots.

Science.gov (United States)

Fujii, Takuro; Taguchi, Yoshihiro; Saiki, Toshiharu; Nagasaka, Yuji

2011-01-01

We have developed a novel nanoscale temperature-measurement method using fluorescence in the near-field called fluorescence near-field optics thermal nanoscopy (Fluor-NOTN). Fluor-NOTN enables the temperature distributions of nanoscale materials to be measured in vivo/in situ. The proposed method measures temperature by detecting the temperature dependent fluorescence lifetimes of Cd/Se quantum dots (QDs). For a high-sensitivity temperature measurement, the auto-fluorescence generated from a fiber probe should be reduced. In order to decrease the noise, we have fabricated a novel near-field optical-fiber probe by fusion-splicing a photonic crystal fiber (PCF) and a conventional single-mode fiber (SMF). The validity of the novel fiber probe was assessed experimentally by evaluating the auto-fluorescence spectra of the PCF. Due to the decrease of auto-fluorescence, a six- to ten-fold increase of S/N in the near-field fluorescence lifetime detection was achieved with the newly fabricated fusion-spliced near-field optical fiber probe. Additionally, the near-field fluorescence lifetime of the quantum dots was successfully measured by the fabricated fusion-spliced near-field optical fiber probe at room temperature, and was estimated to be 10.0 ns.

Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

Science.gov (United States)

Rudkouskaya, Alena; Sinsuebphon, Nattawut; Intes, Xavier; Mazurkiewicz, Joseph E.; Barroso, Margarida

2017-02-01

To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

Carrier Lifetimes in Fluorescent 6H-SiC for LEDs Application

DEFF Research Database (Denmark)

Grivickas, Vytautas; Gulbinas, Karolis; Jokubavičius, Valdas

Recently it was shown a new approach based on all-semiconductor material technology which is composed with a near ultra-violet GaN LED excitation source and fluorescent silicon carbide (f-6H-SiC) substrate which generates a visible broad spectral light by N and B dopants and an efficient donor...... to acceptor pair recombination [1,2]. This combination can achieve higher electric-light conversion efficiency and high color rendering in comparison with today’s used blue GaN LED based and phosphors. The devices are promising candidates for general lightning applications and may obtain stability...... under co-linear and orthogonal probe geometry was used to measure carrier lifetimes in the layers under variable injection conditions. Same results are shown in Fig. 1 exaggerating the fact that longer electron lifetime responsible for higher emission and n-type doping should prevail the p-type doping...

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

Science.gov (United States)

Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

2018-05-01

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

CONSTRAINING THE LIFETIME AND OPENING ANGLE OF QUASARS USING FLUORESCENT Ly α EMISSION: THE CASE OF Q0420–388

International Nuclear Information System (INIS)

Borisova, Elena; Lilly, Simon J.; Cantalupo, Sebastiano; Prochaska, J. Xavier; Rakic, Olivera; Worseck, Gabor

2016-01-01

A toy model is developed to understand how the spatial distribution of fluorescent emitters in the vicinity of bright quasars could be affected by the geometry of the quasar bi-conical radiation field and by its lifetime. The model is then applied to the distribution of high-equivalent-width Ly α emitters (with rest-frame equivalent widths above 100 Å, threshold used in, e.g., Trainor and Steidel) identified in a deep narrow-band 36 × 36 arcmin 2 image centered on the luminous quasar Q0420–388. These emitters are found near the edge of the field and show some evidence of an azimuthal asymmetry on the sky of the type expected if the quasar is radiating in a bipolar cone. If these sources are being fluorescently illuminated by the quasar, the two most distant objects require a lifetime of at least 15 Myr for an opening angle of 60° or more, increasing to more than 40 Myr if the opening angle is reduced to a minimum of 30°. However, some other expected signatures of boosted fluorescence are not seen at the current survey limits, e.g., a fall off in Ly α brightness, or equivalent width, with distance. Furthermore, to have most of the Ly α emission of the two distant sources to be fluorescently boosted would require the quasar to have been significantly brighter in the past. This suggests that these particular sources may not be fluorescent, invalidating the above lifetime constraints. This would cast doubt on the use of this relatively low equivalent width threshold and thus also on the lifetime analysis in Trainor and Steidel.

CONSTRAINING THE LIFETIME AND OPENING ANGLE OF QUASARS USING FLUORESCENT Ly α EMISSION: THE CASE OF Q0420–388

Energy Technology Data Exchange (ETDEWEB)

Borisova, Elena; Lilly, Simon J.; Cantalupo, Sebastiano [Institute for Astronomy, ETH Zurich, Zurich, CH-8093 (Switzerland); Prochaska, J. Xavier [UCO/Lick Observatory, UC Santa Cruz, Santa Cruz, CA 95064 (United States); Rakic, Olivera; Worseck, Gabor, E-mail: borisova@phys.ethz.ch [Max-Planck-Institut für Astronomie, Heidelberg, D-69117 (Germany)

2016-10-20

A toy model is developed to understand how the spatial distribution of fluorescent emitters in the vicinity of bright quasars could be affected by the geometry of the quasar bi-conical radiation field and by its lifetime. The model is then applied to the distribution of high-equivalent-width Ly α emitters (with rest-frame equivalent widths above 100 Å, threshold used in, e.g., Trainor and Steidel) identified in a deep narrow-band 36 × 36 arcmin{sup 2} image centered on the luminous quasar Q0420–388. These emitters are found near the edge of the field and show some evidence of an azimuthal asymmetry on the sky of the type expected if the quasar is radiating in a bipolar cone. If these sources are being fluorescently illuminated by the quasar, the two most distant objects require a lifetime of at least 15 Myr for an opening angle of 60° or more, increasing to more than 40 Myr if the opening angle is reduced to a minimum of 30°. However, some other expected signatures of boosted fluorescence are not seen at the current survey limits, e.g., a fall off in Ly α brightness, or equivalent width, with distance. Furthermore, to have most of the Ly α emission of the two distant sources to be fluorescently boosted would require the quasar to have been significantly brighter in the past. This suggests that these particular sources may not be fluorescent, invalidating the above lifetime constraints. This would cast doubt on the use of this relatively low equivalent width threshold and thus also on the lifetime analysis in Trainor and Steidel.

The increase of NADH fluorescence lifetime is associated with the metabolic change during osteogenic differentiation of human mesenchymal stem cells (hMSCs)

Science.gov (United States)

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

2011-03-01

Fluorescence lifetime of NADH had been used as an optical marker for monitoring cellular metabolism. In our pervious studies, we have demonstrated that NADH lifetime of hMSCs increase gradually with time of osteogenic differentiation. In this study, we measured NADH lifetime of hMSCs from a different donor as well as the corresponding metabolic indices such as ATP level, oxygen consumption and lactate release. We also measure the quantity of Complex I, III, IV and V. The results show that during differentiation more oxygen consumed, higher ATP level expressed and less lactate released, and the increase of NADH lifetime was associated with ATP level. Higher expression of the total Complex protein was observed at 3 and 4 weeks after differentiation than controls. However, Complex I expression did not show significant correlation with the increase of NADH fluorescence lifetime. In summary, we demonstrated that the change of NADH lifetime was associated with the metabolic change during osteogenic differentiation of hMSCs. The increase of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria after differentiation.

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex

Science.gov (United States)

Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

2016-02-01

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

Multiplexed measurements by time resolved spectroscopy using colloidal CdSe/ZnS quantum dots

Energy Technology Data Exchange (ETDEWEB)

Kaiser, U.; Jimenez de Aberasturi, D.; Malinowski, R.; Amin, F.; Parak, W. J.; Heimbrodt, W., E-mail: Wolfram.Heimbrodt@physik.uni-marburg.de [Department of Physics and Materials Sciences Center, Philipps-University of Marburg, Renthof 5, D-35032 Marburg (Germany)

2014-01-27

Multiplexed measurements of analytes in parallel is a topical demand in bioanalysis and bioimaging. An interesting alternative to commonly performed spectral multiplexing is lifetime multiplexing. In this Letter, we present a proof of principle of single-color lifetime multiplexing by coupling the same fluorophore to different nanoparticles. The effective lifetime of the fluorophores can be tuned by more than one order of magnitude due to resonance energy transfer from donor states. Measurements have been done on a model systems consisting of ATTO-590 dye molecules linked to either gold particles or to CdSe/ZnS core shell quantum dots. Both systems show the same luminescence spectrum of ATTO-590 dye emission in continuous wave excitation, but can be distinguished by means of time resolved measurements. The dye molecules bound to gold particles exhibit a mono-exponential decay with a lifetime of 4.5 ns, whereas the dye molecules bound to CdSe/ZnS dots show a nonexponential decay with a slow component of about 135 ns due to the energy transfer from the quantum dots. We demonstrate the fundamental possibility to determine the mixing ratio for dyes with equal luminescence spectra but very different transients. This opens up a pathway independent of the standard optical multiplexing with many different fluorophores emitting from the near ultraviolet to the near infrared spectral region.

FUNDUS AUTOFLUORESCENCE LIFETIMES AND CENTRAL SEROUS CHORIORETINOPATHY.

Science.gov (United States)

Dysli, Chantal; Berger, Lieselotte; Wolf, Sebastian; Zinkernagel, Martin S

2017-11-01

To quantify retinal fluorescence lifetimes in patients with central serous chorioretinopathy (CSC) and to identify disease specific lifetime characteristics over the course of disease. Forty-seven participants were included in this study. Patients with central serous chorioretinopathy were imaged with fundus photography, fundus autofluorescence, optical coherence tomography, and fluorescence lifetime imaging ophthalmoscopy (FLIO) and compared with age-matched controls. Retinal autofluorescence was excited using a 473-nm blue laser light and emitted fluorescence light was detected in 2 distinct wavelengths channels (498-560 nm and 560-720 nm). Clinical features, mean retinal autofluorescence lifetimes, autofluorescence intensity, and corresponding optical coherence tomography (OCT) images were further analyzed. Thirty-five central serous chorioretinopathy patients with a mean visual acuity of 78 ETDRS letters (range, 50-90; mean Snellen equivalent: 20/32) and 12 age-matched controls were included. In the acute stage of central serous chorioretinopathy, retinal fluorescence lifetimes were shortened by 15% and 17% in the respective wavelength channels. Multiple linear regression analysis showed that fluorescence lifetimes were significantly influenced by the disease duration (P autofluorescence lifetimes, particularly in eyes with retinal pigment epithelial atrophy, were associated with poor visual acuity. This study establishes that autofluorescence lifetime changes occurring in central serous chorioretinopathy exhibit explicit patterns which can be used to estimate perturbations of the outer retinal layers with a high degree of statistical significance.

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex.

Science.gov (United States)

Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

2016-02-05

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases. Copyright © 2015 Elsevier B.V. All rights reserved.

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

Science.gov (United States)

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Upconversion Nanoparticles-Encoded Hydrogel Microbeads-Based Multiplexed Protein Detection

Science.gov (United States)

Shikha, Swati; Zheng, Xiang; Zhang, Yong

2018-06-01

Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex's xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

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

Fluorescence lifetime imaging of microviscosity changes during ER autophagy in live cells

Science.gov (United States)

He, Ying; Samanta, Soham; Gong, Wanjun; Liu, Wufan; Pan, Wenhui; Yang, Zhigang; Qu, Junle

2018-02-01

Unfolded or misfolded protein accumulation inside Endoplasmic Reticulum (ER) will cause ER stress and subsequently will activate cellular autophagy to release ER stress, which would ultimately result in microviscosity changes. However, even though, it is highly significant to gain a quantitative assessment of microviscosity changes during ER autophagy to study ER stress and autophagy behaviors related diseases, it has rarely been reported yet. In this work, we have reported a BODIPY based fluorescent molecular rotor that can covalently bind with vicinal dithiols containing nascent proteins in ER and hence can result in ER stress through the inhibition of the folding of nascent proteins. The change in local viscosity, caused by the release of the stress in cells through autophagy, was quantified by the probe using fluorescence lifetime imaging. This work basically demonstrates the possibility of introducing synthetic chemical probe as a promising tool to diagnose ER-viscosity-related diseases.

In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH.

Science.gov (United States)

Yaseen, Mohammad A; Sakadžić, Sava; Wu, Weicheng; Becker, Wolfgang; Kasischke, Karl A; Boas, David A

2013-02-01

Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism.

Multimodal optical coherence tomography and fluorescence lifetime imaging with interleaved excitation sources for simultaneous endogenous and exogenous fluorescence.

Science.gov (United States)

Shrestha, Sebina; Serafino, Michael J; Rico-Jimenez, Jesus; Park, Jesung; Chen, Xi; Zhaorigetu, Siqin; Walton, Brian L; Jo, Javier A; Applegate, Brian E

2016-09-01

Multimodal imaging probes a variety of tissue properties in a single image acquisition by merging complimentary imaging technologies. Exploiting synergies amongst the data, algorithms can be developed that lead to better tissue characterization than could be accomplished by the constituent imaging modalities taken alone. The combination of optical coherence tomography (OCT) with fluorescence lifetime imaging microscopy (FLIM) provides access to detailed tissue morphology and local biochemistry. The optical system described here merges 1310 nm swept-source OCT with time-domain FLIM having excitation at 355 and 532 nm. The pulses from 355 and 532 nm lasers have been interleaved to enable simultaneous acquisition of endogenous and exogenous fluorescence signals, respectively. The multimodal imaging system was validated using tissue phantoms. Nonspecific tagging with Alexa Flour 532 in a Watanbe rabbit aorta and active tagging of the LOX-1 receptor in human coronary artery, demonstrate the capacity of the system for simultaneous acquisition of OCT, endogenous FLIM, and exogenous FLIM in tissues.

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

Science.gov (United States)

Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

2015-05-01

Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

Preparation and properties of Nd{sup 3+}:SrAlF{sub 5} nanocrystals embedded fluorophosphate transparent glass-ceramic with long fluorescence lifetime

Energy Technology Data Exchange (ETDEWEB)

Zheng, Ruilin; Wang, Jinlong; Zhang, Liaolin; Liu, Chunxiao; Wei, Wei [Nanjing University of Posts and Telecommunications, School of Optoelectronic Engineering, Nanjing (China)

2016-07-15

Nd{sup 3+}:SrAlF{sub 5} nanocrystals embedded fluorophosphate glass-ceramics were prepared by the melt quenching and subsequent thermal treatment method. The formation of SrAlF{sub 5} nanocrystals in the glass was confirmed by X-ray diffraction and scanning electron microscope. The fluorescence intensity and lifetime of the glass-ceramics increased with the increase of size of nanocrystals. Importantly, by controlling growth of nanocrystals, an obvious enhancement of lifetime (725 μs) emerged in the glass-ceramics heat-treated at 510 C and the transmittance can reach to 72.2 % at 1049 nm. The enhanced fluorescence intensity and lifetime were ascribed to the comfortable local environment to the Nd{sup 3+} ion and scattering of the nanoparticle embedded into the glass matrix. (orig.)

Lifetime-based optical sensor for high-level pCO2 detection employing fluorescence resonance energy transfer

International Nuclear Information System (INIS)

Bueltzingsloewen, Christoph von; McEvoy, Aisling K.; McDonagh, Colette; MacCraith, Brian D.

2003-01-01

An optical sensor for the measurement of high levels of carbon dioxide in gas phase has been developed. It is based on fluorescence resonance energy transfer (FRET) between a long-lifetime ruthenium polypyridyl complex and the pH-active disazo dye Sudan III. The donor luminophore and the acceptor dye are both immobilised in a hydrophobic silica sol-gel/ethyl cellulose hybrid matrix material. Tetraoctylammonium hydroxide (TOA-OH) is used as an internal buffering system. Fluorescence lifetime is measured in the frequency domain, using low-cost phase modulation measurement technology. The use of Sudan III as an acceptor dye has enabled the sensor to have a dynamic range up to 100% carbon dioxide. The sensor displays 11.2 deg. phase shift between the limit of detection (LOD) of 0.06 and 100% CO 2 with a resolution of better than 2%. The encapsulation in the silica/polymer hybrid material has provided the sensor with good mechanical and chemical stability. The effect of molecular oxygen, humidity and temperature on the sensor performance was studied in detail

Nanoparticle discrimination based on wavelength and lifetime-multiplexed cathodoluminescence microscopy

NARCIS (Netherlands)

Garming, Mathijs W H; Weppelman, I Gerward C; de Boer, Pascal; Martínez, Felipe Perona; Schirhagl, Romana; Hoogenboom, Jacob P; Moerland, Robert J

2017-01-01

Nanomaterials can be identified in high-resolution electron microscopy images using spectrally-selective cathodoluminescence. Capabilities for multiplex detection can however be limited, e.g., due to spectral overlap or availability of filters. Also, the available photon flux may be limited due to

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

Science.gov (United States)

Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

2007-01-01

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging

Directory of Open Access Journals (Sweden)

Zuzana eBurdikova

2015-03-01

Full Text Available Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g. pH, redox potential due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM. In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening.

Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging.

Science.gov (United States)

Burdikova, Zuzana; Svindrych, Zdenek; Pala, Jan; Hickey, Cian D; Wilkinson, Martin G; Panek, Jiri; Auty, Mark A E; Periasamy, Ammasi; Sheehan, Jeremiah J

2015-01-01

Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM). In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening.

Segmented frequency-domain fluorescence lifetime measurements: minimizing the effects of photobleaching within a multi-component system.

Science.gov (United States)

Marwani, Hadi M; Lowry, Mark; Keating, Patrick; Warner, Isiah M; Cook, Robert L

2007-11-01

This study introduces a newly developed frequency segmentation and recombination method for frequency-domain fluorescence lifetime measurements to address the effects of changing fractional contributions over time and minimize the effects of photobleaching within multi-component systems. Frequency segmentation and recombination experiments were evaluated using a two component system consisting of fluorescein and rhodamine B. Comparison of experimental data collected in traditional and segmented fashion with simulated data, generated using different changing fractional contributions, demonstrated the validity of the technique. Frequency segmentation and recombination was also applied to a more complex system consisting of pyrene with Suwannee River fulvic acid reference and was shown to improve recovered lifetimes and fractional intensity contributions. It was observed that photobleaching in both systems led to errors in recovered lifetimes which can complicate the interpretation of lifetime results. Results showed clear evidence that the frequency segmentation and recombination method reduced errors resulting from a changing fractional contribution in a multi-component system, and allowed photobleaching issues to be addressed by commercially available instrumentation.

A novel lab-on-chip platform with integrated solid phase PCR and Supercritical Angle Fluorescence (SAF) microlens array for highly sensitive and multiplexed pathogen detection

DEFF Research Database (Denmark)

Hung, Tran Quang; Chin, Wai Hoe; Sun, Yi

2016-01-01

technology. In this paper, we addressed this challenge by combining the SP-PCR with super critical angle fluorescence (SAF) microlens array embedded in a microchip. We fabricated miniaturized SAF microlens array as part of a microfluidic chamber in thermoplastic material and performed multiplexed SP...

Comparitive study of fluorescence lifetime quenching of rhodamine 6G by MoS2 and Au-MoS2

Science.gov (United States)

Shakya, Jyoti; Kasana, Parath; Mohanty, T.

2018-04-01

Time resolved fluorescence study of Rhodamine 6G (R6G) in the presence of Molybdenum disulfide (MoS2) nanosheets and gold doped MoS2 (Au-MoS2) have been carried out and discussed. We have analyzed the fluorescence decay curves of R6G and it is observed that Au-MoS2 is a better fluorescence lifetime quencher as compare to MoS2 nanosheets. Also, the energy transfer efficiency and energy transfer rate from R6G to MoS2 and Au-MoS2 has been calculated and found higher for Au-MoS2.

Exciton-polaron quenching in organic thin-film transistors studied by fluorescence lifetime imaging microscopy

DEFF Research Database (Denmark)

Jensen, Per Baunegaard With; Leißner, Till; Osadnik, Andreas

Organic semiconductors show great potential in electronic and optical applications. However, a major challenge is the degradation of the semiconductor materials that cause a reduction in device performance. Here, we present our investigations of Organic Thin Film Transistors (OTFT) based...... that correlates with the local charge density indicates a pronounced exciton quenching by the injected charges. Subsequent FLIM measurements on previously biased OTFT devices show a general decrease in fluorescence lifetime suggesting degradation of the organic semiconductor. This is correlated with the results...

Mercury effects on Thalassiosira weissflogii: Applications of two-photon excitation chlorophyll fluorescence lifetime imaging and flow cytometry

International Nuclear Information System (INIS)

Wu Yun; Zeng Yan; Qu, Jianan Y.; Wang Wenxiong

2012-01-01

The toxic effects of inorganic mercury [Hg(II)] and methylmercury (MeHg) on the photosynthesis and population growth in a marine diatom Thalassiosira weissflogii were investigated using two methods: two-photon excitation fluorescence lifetime imaging (FLIM) and flow cytometry (FCM). For photosynthesis, Hg(II) exposure increased the average chlorophyll fluorescence lifetime, whereas such increment was not found under MeHg stress. This may be caused by the inhibitory effect of Hg(II) instead of MeHg on the electron transport chain. For population growth, modeled specific growth rate data showed that the reduction in population growth by Hg(II) mainly resulted from an increased number of injured cells, while the live cells divided at the normal rates. However, MeHg inhibitory effects on population growth were contributed by the reduced division rates of all cells. Furthermore, the cell images and the FCM data reflected the morphological changes of diatom cells under Hg(II)/MeHg exposure vividly and quantitatively. Our results demonstrated that the toxigenicity mechanisms between Hg(II) and MeHg were different in the algal cells.

Fluorescent multiplex cell flow systems and methods

KAUST Repository

Merzaban, Jasmeen; Abuelela, Ayman F.; Mohammad, Amal Jehad

2017-01-01

scanning system emits multiple electromagnetic wavelengths simultaneously it cause multiple fluorescent labels having different excitation wavelength maximums to fluoresce. The system can simultaneously capture real-time fluorescence images from at least

Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II.

Science.gov (United States)

Nemtseva, Elena V; Lashchuk, Olesya O; Gerasimova, Marina A; Melnik, Tatiana N; Nagibina, Galina S; Melnik, Bogdan S

2017-12-21

In most cases, intermediate states of multistage folding proteins are not 'visible' under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

Design, construction, and validation of a rotary multifunctional intravascular diagnostic catheter combining multispectral fluorescence lifetime imaging and intravascular ultrasound.

Science.gov (United States)

Bec, Julien; Xie, Hongtao; Yankelevich, Diego R; Zhou, Feifei; Sun, Yang; Ghata, Narugopal; Aldredge, Ralph; Marcu, Laura

2012-10-01

We report the development and validation of an intravascular rotary catheter for bimodal interrogation of arterial pathologies. This is based on a point-spectroscopy scanning time-resolved fluorescence spectroscopy technique enabling reconstruction of fluorescence lifetime images (FLIm) and providing information on arterial intima composition and intravascular ultrasound (IVUS) providing information on arterial wall morphology. The catheter design allows for independent rotation of the ultrasonic and optical channels within an 8 Fr outer diameter catheter sheath and integrates a low volume flushing channel for blood removal in the optical pathways. In the current configuration, the two channels consist of (a) a standard 3 Fr IVUS catheter with single element transducer (40 MHz) and (b) a side-viewing fiber optic (400 μm core). Experiments conducted in tissue phantoms showed the ability of the catheter to operate in an intraluminal setting and to generate coregistered FLIm and IVUS in one pull-back scan. Current results demonstrate the feasibility of the catheter for simultaneous bimodal interrogation of arterial lumen and for generation of robust fluorescence lifetime data under IVUS guidance. These results facilitate further development of a FLIm-IVUS technique for intravascular diagnosis of atherosclerotic cardiovascular diseases including vulnerable plaques.

Effect of pharmacologically induced retinal degeneration on retinal autofluorescence lifetimes in mice.

Science.gov (United States)

Dysli, Chantal; Dysli, Muriel; Zinkernagel, Martin S; Enzmann, Volker

2016-12-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) was used to investigate retinal autofluorescence lifetimes in mouse models of pharmacologically induced retinal degeneration over time. Sodium iodate (NaIO 3 , 35 mg/kg intravenously) was used to induce retinal pigment epithelium (RPE) degeneration with subsequent loss of photoreceptors (PR) whereas N-methyl-N-nitrosourea (MNU, 45 mg/kg intraperitoneally) was employed for degeneration of the photoreceptor cell layer alone. All mice were measured at day 3, 7, 14, and 28 after the respective injection of NaIO 3 , MNU or NaCl (control). Fluorescence lifetime imaging was performed using a fluorescence lifetime imaging ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). Fluorescence was excited at 473 nm and fluorescence lifetimes were measured in a short and a long spectral channel (498-560 nm and 560-720 nm). Corresponding optical coherence tomography (OCT) images were consecutively acquired and histology was performed at the end of the experiments. Segmentation of OCT images and histology verified the cell type-specific degeneration process over time. Retinal autofluorescence lifetimes increased from day 3 to day 28 in mice after NaIO 3 treatment. Finally, at day 28, fluorescence lifetimes were prolonged by 8% in the short and 61% in the long spectral channel compared to control animals (p = 0.21 and p = 0.004, respectively). In mice after MNU treatment, the mean retinal autofluorescence lifetimes were already decreased at day 3 and retinal lifetimes were finally shortened by 27% in the short and 51% in the long spectral channel at day 28 (p = 0.0028). In conclusion, degeneration of the RPE with subsequent photoreceptor degeneration by NaIO 3 lead to longer mean fluorescence lifetimes of the retina compared to control mice, whereas during specific degeneration of the photoreceptor layer induced by MNU shorter lifetimes were measured. Therefore, short retinal fluorescence lifetimes may originate

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

Science.gov (United States)

Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

2013-07-25

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of calcinations temperature on the luminescence intensity and fluorescent lifetime of Tb3+-doped hydroxyapatite (Tb-HA nanocrystallines

Directory of Open Access Journals (Sweden)

Hairong Yin

2017-06-01

Full Text Available Hydroxyapatite luminescent nanocrystallines doped with 6 mol.% Tb3+ (Tb-HA were prepared via chemical deposition method and calcined at different temperature, and the effects of calcinations temperature on the luminescence intensity and fluorescent lifetime were studied. TEM image of Tb-HA revealed that the shape of nanocrystallines changed from needle-like to short rod-like and sphere-like with the increase of calcinations temperature; while the particles sizes decreased from 190 nm to 110 nm. The crystallinity degree increased. The typical emission peaks attributed to Tb3+ ions were observed in emission spectra of 6 mol.% Tb-HA under 378 nm excitation. The luminescent intensity of Tb-HA, which showed the fluorescence quenching, firstly enhanced and then decreased at 700 °C; while the fluorescent lifetime increased firstly and then decreased after 600 °C. Furthermore, the ratio of intensity between 545 nm and 490 nm corresponding to electric-dipole and magnetic-dipole transition (IR: IO increases firstly and then decreases, which revealed that the proportion of substitute type and site of Ca2+ ions by Tb3+ ions were helpful to realize the substitute process and functional structure design.

Multiplex competitive microbead-based flow cytometric immunoassay using quantum dot fluorescent labels

International Nuclear Information System (INIS)

Yu, Hye-Weon; Kim, In S.; Niessner, Reinhard; Knopp, Dietmar

2012-01-01

Highlights: ► First time, duplex competitive bead-based flow cytometric immunoassay was developed using ODs. ► Antibody-coated QD detection probes and antigen-immobilized microspheres were synthesized. ► The two model target analytes were low molecular weight compounds of microbial and chemical origin. ► The determination of different water types was possible after simple filtration of samples. - Abstract: In answer to the ever-increasing need to perform the simultaneous analysis of environmental hazards, microcarrier-based multiplex technologies show great promise. Further integration with biofunctionalized quantum dots (QDs) creates new opportunities to extend the capabilities of multicolor flow cytometry with their unique fluorescence properties. Here, we have developed a competitive microbead-based flow cytometric immunoassay using QDs fluorescent labels for simultaneous detection of two analytes, bringing the benefits of sensitive, rapid and easy-of-manipulation analytical tool for environmental contaminants. As model target compounds, the cyanobacterial toxin microcystin-LR and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene were selected. The assay was carried out in two steps: the competitive immunological reaction of multiple targets using their exclusive sensing elements of QD/antibody detection probes and antigen-coated microsphere, and the subsequent flow cytometric analysis. The fluorescence of the QD-encoded microsphere was thus found to be inversely proportional to target analyte concentration. Under optimized conditions, the proposed assay performed well within 30 min for the identification and quantitative analysis of the two environmental contaminants. For microcystin-LR and benzo[a]pyrene, dose–response curves with IC 50 values of 5 μg L −1 and 1.1 μg L −1 and dynamic ranges of 0.52–30 μg L −1 and 0.13–10 μg L −1 were obtained, respectively. Recovery was 92.6–106.5% for 5 types of water samples like bottled

Magnetic field and temperature dependence of the fluorescence lifetime of Cr sup(3+) in GdA103

International Nuclear Information System (INIS)

Helman, J.S.; Caride, A.O.; Basso, H.C.; Terrile, M.C.; Carvalho, R.A.

1991-01-01

The fluorescence lifetime of Cr sup(3+) in GdA10 sub(3) was measured in the range 1.8 - 4.2 K in magnetic fields up to 6 T. The results show a remarkable dependence of the transition probabilities on magnetic order. A model based on the exchange interaction between Cr sup(3+) in highly excited states and the Gd sup(3+) ions is proposed. (author)

Differentiation of ocular fundus fluorophores by fluorescence lifetime imaging using multiple excitation and emission wavelengths

Science.gov (United States)

Hammer, M.; Schweitzer, D.; Schenke, S.; Becker, W.; Bergmann, A.

2006-10-01

Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH II provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.

The fluorescence lifetime of BRI1-GFP as probe for the noninvasive determination of the membrane potential in living cells

Science.gov (United States)

Elgass, K.; Caesar, K.; Schleifenbaum, F.; Meixner, A. J.; Harter, K.

2010-02-01

As the excited state lifetime of a fluorescent molecule depends on its environment, it is possible to use it as a probe for physico-chemical parameters of the surrounding medium. Whereas this is well known for many solid guest/host systems, only few reports of quantitative, temporal resolved in vivo studies to monitor the nano-environment for a protein-coupled chromophore such as GFP are known from literature. Here we present a novel approach to determine the membrane potential of living (plant) cells based on the fluorescence lifetime (FLT) analysis of membrane-located GFP. By using confocal sample scanning microscopy (CSSM) combined with fluorescence lifetime imaging microscopy, we recently showed that the phytohormone brassinolide (BL) induces cell wall expansion and a decrease in the FLT of the BRI1-GFP in living cells of Arabidopsis thaliana seedlings. BRI1 is the dominant functional receptor for BL in Arabidopsis and locates to the plasma membrane. Although the dependence of the FLT of GFP on its physico-chemical environment such as pH-value, refractive index and pressure has been reported, the observed FLT decrease of BRI1-GFP in response to BL application could not be explained by these parameters. However, our in vivo FLT and CSSM analyses indicate that the BLinduced change in the FLT of BRI1-GFP is caused by hyperpolarisation of the plasma membrane (Em). Thus, our results indicate that BRI1-GFP serves as sensitive and non-invasive probe for recording the Em of the plasma membrane in living plant cells with high spatio-temporal resolution.

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

Energy Technology Data Exchange (ETDEWEB)

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.

Demonstration of the lack of cytotoxicity of unmodified and folic acid modified graphene oxide quantum dots, and their application to fluorescence lifetime imaging of HaCaT cells.

Science.gov (United States)

Goreham, Renee V; Schroeder, Kathryn L; Holmes, Amy; Bradley, Siobhan J; Nann, Thomas

2018-01-24

The authors describe the synthesis of water-soluble and fluorescent graphene oxide quantum dots via acid exfoliation of graphite nanoparticles. The resultant graphene oxide quantum dots (GoQDs) were then modified with folic acid. Folic acid receptors are overexpressed in cancer cells and hence can bind to functionalized graphene oxide quantum dots. On excitation at 305 nm, the GoQDs display green fluorescence with a peak wavelength at ~520 nm. The modified GoQDs are non-toxic to macrophage cells even after prolonged exposure and high concentrations. Fluorescence lifetime imaging and multiphoton microscopy was used (in combination) to image HeCaT cells exposed to GoQDs, resulting in a superior method for bioimaging. Graphical abstract Schematic representation of graphene oxide quantum dots, folic acid modified graphene oxide quantum dots (red), and the use of fluorescence lifetime to discriminate against green auto-fluorescence of HeCaT cells.

Autofluorescence Lifetimes in Geographic Atrophy in Patients With Age-Related Macular Degeneration.

Science.gov (United States)

Dysli, Chantal; Wolf, Sebastian; Zinkernagel, Martin S

2016-05-01

To investigate fluorescence lifetime characteristics in patients with geographic atrophy (GA) in eyes with age-related macular degeneration and to correlate the measurements with clinical data and optical coherence tomography (OCT) findings. Patients with GA were imaged with a fluorescence lifetime imaging ophthalmoscope. Retinal autofluorescence lifetimes were measured in a short and a long spectral channel (498-560 nm and 560-720 nm). Mean retinal fluorescence lifetimes were analyzed within GA and the surrounding retina, and data were correlated with best corrected visual acuity and OCT measurements. Fluorescence lifetime maps of 41 eyes of 41 patients (80 ± 7 years) with GA were analyzed. Mean lifetimes within areas of atrophy were prolonged by 624 ± 276 ps (+152%) in the short spectral channel and 418 ± 186 ps (+83%) in the long spectral channel compared to the surrounding tissue. Autofluorescence lifetime abnormalities in GA occurred with particular patterns, similar to those seen in fundus autofluorescence intensity images. Within the fovea short mean autofluorescence lifetimes were observed, presumably representing macular pigment. Short lifetimes were preserved even in the absence of foveal sparing but were decreased in patients with advanced retinal atrophy in OCT. Short lifetimes in the fovea correlated with better best corrected visual acuity in both spectral channels. This study established that autofluorescence lifetime changes in GA present with explicit patterns. We hypothesize that the short lifetimes seen within the atrophy may be used to estimate damage induced by atrophy and to monitor disease progression in the context of natural history or interventional therapeutic studies.

Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

Science.gov (United States)

Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

2010-02-01

An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning.

Science.gov (United States)

Silva, Susana F; Domingues, José Paulo; Morgado, António Miguel

2018-01-01

Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed.

Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

Czech Academy of Sciences Publication Activity Database

Macháň, Radek; Kapusta, Peter; Hof, Martin

Roč. 406 , č. 20 (2014), s. 4797-4813 ISSN 1618-2642 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : Filtered fluorescence correlation spectroscopy * Fluorescence lifetime correlation spectroscopy * Fluorescence spectral correlation spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.436, year: 2014

Laser induced fluorescence lifetime characterization of Bacillus endospore species using time correlated single photon counting analysis with the multi-exponential fit method

Science.gov (United States)

Smith, Clint; Edwards, Jarrod; Fisher, Andmorgan

2010-04-01

Rapid detection of biological material is critical for determining presence/absence of bacterial endospores within various investigative programs. Even more critical is that if select material tests positive for bacillus endospores then tests should provide data at the species level. Optical detection of microbial endospore formers such as Bacillus sp. can be heavy, cumbersome, and may only identify at the genus level. Data provided from this study will aid in characterization needed by future detection systems for further rapid breakdown analysis to gain insight into a more positive signature collection of Bacillus sp. Literature has shown that fluorescence spectroscopy of endospores could be statistically separated from other vegetative genera, but could not be separated among one another. Results of this study showed endospore species separation is possible using laser-induce fluorescence with lifetime decay analysis for Bacillus endospores. Lifetime decays of B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain were investigated. Using the Multi-Exponential fit method data showed three distinct lifetimes for each species within the following ranges, 0.2-1.3 ns; 2.5-7.0 ns; 7.5-15.0 ns, when laser induced at 307 nm. The four endospore species were individually separated using principle component analysis (95% CI).

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise

DEFF Research Database (Denmark)

Sanchez, Juan Jose; Børsting, C; Balogh, K

2008-01-01

base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA...

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.

High speed fluorescence imaging with compressed ultrafast photography

Science.gov (United States)

Thompson, J. V.; Mason, J. D.; Beier, H. T.; Bixler, J. N.

2017-02-01

Fluorescent lifetime imaging is an optical technique that facilitates imaging molecular interactions and cellular functions. Because the excited lifetime of a fluorophore is sensitive to its local microenvironment,1, 2 measurement of fluorescent lifetimes can be used to accurately detect regional changes in temperature, pH, and ion concentration. However, typical state of the art fluorescent lifetime methods are severely limited when it comes to acquisition time (on the order of seconds to minutes) and video rate imaging. Here we show that compressed ultrafast photography (CUP) can be used in conjunction with fluorescent lifetime imaging to overcome these acquisition rate limitations. Frame rates up to one hundred billion frames per second have been demonstrated with compressed ultrafast photography using a streak camera.3 These rates are achieved by encoding time in the spatial direction with a pseudo-random binary pattern. The time domain information is then reconstructed using a compressed sensing algorithm, resulting in a cube of data (x,y,t) for each readout image. Thus, application of compressed ultrafast photography will allow us to acquire an entire fluorescent lifetime image with a single laser pulse. Using a streak camera with a high-speed CMOS camera, acquisition rates of 100 frames per second can be achieved, which will significantly enhance our ability to quantitatively measure complex biological events with high spatial and temporal resolution. In particular, we will demonstrate the ability of this technique to do single-shot fluorescent lifetime imaging of cells and microspheres.

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

Fluorescent multiplex cell flow systems and methods

KAUST Repository

Merzaban, Jasmeen

2017-06-01

Systems and methods are provided for simultaneously assaying cell adhesion or cell rolling for multiple cell specimens. One embodiment provides a system for assaying adhesion or cell rolling of multiple cell specimens that includes a confocal imaging system containing a parallel plate flow chamber, a pump in fluid communication with the parallel plate flow chamber via a flow chamber inlet line and a cell suspension in fluid communication with the parallel plate flow chamber via a flow chamber outlet line. The system also includes a laser scanning system in electronic communication with the confocal imaging system, and a computer in communication with the confocal imaging system and laser scanning system. In certain embodiments, the laser scanning system emits multiple electromagnetic wavelengths simultaneously it cause multiple fluorescent labels having different excitation wavelength maximums to fluoresce. The system can simultaneously capture real-time fluorescence images from at least seven cell specimens in the parallel plate flow chamber.

Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging

Directory of Open Access Journals (Sweden)

Mély Yves

2008-09-01

Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. Conclusion We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

Science.gov (United States)

Bray, Mark-Anthony; Singh, Shantanu; Han, Han; Davis, Chadwick T.; Borgeson, Blake; Hartland, Cathy; Kost-Alimova, Maria; Gustafsdottir, Sigrun M.; Gibson, Christopher C.; Carpenter, Anne E.

2016-01-01

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks. PMID:27560178

Fully time-resolved near-field scanning optical microscopy fluorescence imaging

International Nuclear Information System (INIS)

Kwak, Eun-Soo; Vanden Bout, David A.

2003-01-01

Time-correlated single photon counting has been coupled with near-field scanning optical microscopy (NSOM) to record complete fluorescence lifetime decays at each pixel in an NSOM image. The resulting three-dimensional data sets can be binned in the time dimension to create images of photons at particular time delays or images of the fluorescence lifetime. Alternatively, regions of interest identified in the topography and fluorescence images can be used to bin the data in the spatial dimensions resulting in high signal to noise fluorescence decays of particular regions of the sample. The technique has been demonstrated on films of poly(vinylalcohol), doped with the fluorescent dye, cascade blue (CB). The CB segregates into small circular regions of high concentration within the films during the drying process. The lifetime imaging shows that the spots have slightly faster excited state decays due to quenching of the luminescence as a result of the higher concentration. The technique is also used to image the fluorescence lifetime of an annealed film of poly(dihexylfluorene). The samples show high contrast in the total intensity fluorescence image, but the lifetime image reveals the sample to be extremely uniform

Precision lifetime measurements

International Nuclear Information System (INIS)

Tanner, C.E.

1994-01-01

Precision measurements of atomic lifetimes provide important information necessary for testing atomic theory. The authors employ resonant laser excitation of a fast atomic beam to measure excited state lifetimes by observing the decay-in-flight of the emitted fluorescence. A similar technique was used by Gaupp, et al., who reported measurements with precisions of less than 0.2%. Their program includes lifetime measurements of the low lying p states in alkali and alkali like systems. Motivation for this work comes from a need to test the atomic many-body-perturbation theory (MBPT) that is necessary for interpretation of parity nonconservation experiments in atomic cesium. The authors have measured the cesium 6p 2 P 1/2 and 6p 2 P 3/2 state lifetimes to be 34.934±0.094 ns and 30.499±0.070 ns respectively. With minor changes to the apparatus, they have extended their measurements to include the lithium 2p 2 P 1/2 and 2p 2 P 3/2 states

Time-resolved fluorescence spectroscopy

International Nuclear Information System (INIS)

Gustavsson, Thomas; Mialocq, Jean-Claude

2007-01-01

This article addresses the evolution in time of light emitted by a molecular system after a brief photo-excitation. The authors first describe fluorescence from a photo-physical point of view and discuss the characterization of the excited state. Then, they explain some basic notions related to fluorescence characterization (lifetime and decays, quantum efficiency, so on). They present the different experimental methods and techniques currently used to study time-resolved fluorescence. They discuss basic notions of time resolution and spectral reconstruction. They briefly present some conventional methods: intensified Ccd cameras, photo-multipliers and photodiodes associated with a fast oscilloscope, and phase modulation. Other methods and techniques are more precisely presented: time-correlated single photon counting (principle, examples, and fluorescence lifetime imagery), streak camera (principle, examples), and optical methods like the Kerr optical effect (principle and examples) and fluorescence up-conversion (principle and theoretical considerations, examples of application)

Multiplexing a high-throughput liability assay to leverage efficiencies.

Science.gov (United States)

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Fluorescent Quantification of DNA Based on Core-Shell Fe3O4@SiO2@Au Nanocomposites and Multiplex Ligation-Dependent Probe Amplification.

Science.gov (United States)

Fan, Jing; Yang, Haowen; Liu, Ming; Wu, Dan; Jiang, Hongrong; Zeng, Xin; Elingarami, Sauli; Ll, Zhiyang; Li, Song; Liu, Hongna; He, Nongyue

2015-02-01

In this research, a novel method for relative fluorescent quantification of DNA based on Fe3O4@SiO2@Au gold-coated magnetic nanocomposites (GMNPs) and multiplex ligation- dependent probe amplification (MLPA) has been developed. With the help of self-assembly, seed-mediated growth and chemical reduction method, core-shell Fe3O4@SiO2@Au GMNPs were synthesized. Through modified streptavidin on the GMNPs surface, we obtained a bead chip which can capture the biotinylated probes. Then we designed MLPA probes which were tagged with biotin or Cy3 and target DNA on the basis of human APP gene sequence. The products from the thermostable DNA ligase induced ligation reactions and PCR amplifications were incubated with SA-GMNPs. After washing, magnetic separation, spotting, the fluorescent scanning results showed our method can be used for the relative quantitative analysis of the target DNA in the concentration range of 03004~0.5 µM.

Anthracene Fluorescence Quenching by a Tetrakis (Ketocarboxamide Cavitand

Directory of Open Access Journals (Sweden)

Tibor Zoltan Janosi

2014-01-01

Full Text Available Quenching of both fluorescence lifetime and fluorescence intensity of anthracene was investigated in the presence of a newly derived tetrakis (ketocarboxamide cavitand at various concentrations. Time-correlated single photon counting method was applied for the lifetime measurements. A clear correlation between the fluorescence lifetime of anthracene as a function of cavitand concentration in dimethylformamide solution was observed. The bimolecular collisional quenching constant was derived from the decrease of lifetime. Fluorescence intensity was measured in the emission wavelength region around 400 nm as a result of excitation at 280 nm. Effective quenching was observed in the presence of the cavitand. The obtained Stern-Volmer plot displayed upward curvature. The results did not follow even extended Stern-Volmer behavior, often used to describe deviations from static bimolecular quenching. To explain our results we adopted the Smoluchowski model and obtained a reasonable estimate for the molecular radius of the cavitand in solution.

madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy

Science.gov (United States)

Yi, Jason; Manna, Asit; Barr, Valarie A.; Hong, Jennifer; Neuman, Keir C.; Samelson, Lawrence E.

2016-01-01

Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM. PMID:27708141

Multimodal fluorescence imaging spectroscopy

NARCIS (Netherlands)

Stopel, Martijn H W; Blum, Christian; Subramaniam, Vinod; Engelborghs, Yves; Visser, Anthonie J.W.G.

2014-01-01

Multimodal fluorescence imaging is a versatile method that has a wide application range from biological studies to materials science. Typical observables in multimodal fluorescence imaging are intensity, lifetime, excitation, and emission spectra which are recorded at chosen locations at the sample.

THESEUS: A wavelength division multiplexed/microwave subcarrier multiplexed optical network, its ATM switch applications and device requirements

Science.gov (United States)

Xin, Wei

1997-10-01

A Terabit Hybrid Electro-optical /underline[Se]lf- routing Ultrafast Switch (THESEUS) has been proposed. It is a self-routing wavelength division multiplexed (WDM) / microwave subcarrier multiplexed (SCM) asynchronous transfer mode (ATM) switch for the multirate ATM networks. It has potential to be extended to a large ATM switch as 1000 x 1000 without internal blocking. Among the advantages of the hybrid implementation are flexibility in service upgrade, relaxed tolerances on optical filtering, protocol simplification and less processing overhead. For a small ATM switch, the subcarrier can be used as output buffers to solve output contention. A mathematical analysis was conducted to evaluate different buffer configurations. A testbed has been successfully constructed. Multirate binary data streams have been switched through the testbed and error free reception ([<]10-9 bit error rate) has been achieved. A simple, intuitive theoretical model has been developed to describe the heterodyne optical beat interference. A new concept of interference time and interference length has been introduced. An experimental confirmation has been conducted. The experimental results match the model very well. It shows that a large portion of optical bandwidth is wasted due to the beat interference. Based on the model, several improvement approaches have been proposed. The photo-generated carrier lifetime of silicon germanium has been measured using time-resolved reflectivity measurement. Via oxygen ion implantation, the carrier lifetime has been reduced to as short as 1 ps, corresponding to 1 THz of photodetector bandwidth. It has also been shown that copper dopants act as recombination centers in the silicon germanium.

Multiplex detection of tumor markers with photonic suspension array

Energy Technology Data Exchange (ETDEWEB)

Zhao Yuanjin; Zhao Xiangwei [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Pei Xiaoping [Department of Hematology, Affiliated Zhongda Hospital, Southeast University, Nanjing 210009 (China); Hu Jing; Zhao Wenju [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Chen Baoan [Department of Hematology, Affiliated Zhongda Hospital, Southeast University, Nanjing 210009 (China); Gu Zhongze [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Laboratory of Environment and Biosafety, Research Institute of Southeast University in Suzhou, Dushu Lake Higher Education Town, Suzhou 215123 (China)], E-mail: gu@seu.edu.cn

2009-02-02

A novel photonic suspension array was developed for multiplex immunoassay. The carries of this array were silica colloidal crystal beads (SCCBs). The codes of these carriers are the characteristic reflection peak originated from their structural periodicity, and therefore they do not suffer from fading, bleaching, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are included, the fluorescence background of SCCBs is very low. With a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the four tumor markers, {alpha}-fetoprotein (AFP), carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125) and carcinoma antigen 19-9 (CA 19-9) could be assayed in the ranges of 1.0-500 ng mL{sup -1}, 1.0-500 ng mL{sup -1}, 1.0-500 U mL{sup -1} and 3.0-500 U mL{sup -1} with limits of detection of 0.68 ng mL{sup -1}, 0.95 ng mL{sup -1}, 0.99 U mL{sup -1} and 2.30 U mL{sup -1} at 3{sigma}, respectively. The proposed array showed acceptable accuracy, detection reproducibility, storage stability and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassay.

Quantitative time domain analysis of lifetime-based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions

DEFF Research Database (Denmark)

Alexandrov, Yuriy; Nikolic, Dino Solar; Dunsby, Christopher

2018-01-01

Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative...... into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic...... analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations...

Emerging biomedical applications of time-resolved fluorescence spectroscopy

Science.gov (United States)

Lakowicz, Joseph R.; Szmacinski, Henryk; Koen, Peter A.

1994-07-01

Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics, and chemical physics. Advances in laser technology, the development of long-wavelength probes, and the use of lifetime-based methods are resulting in the rapid migration of time-resolved fluorescence to the clinical chemistry lab, to the patient's bedside, to flow cytometers, to the doctor's office, and even to home health care. Additionally, time-resolved imaging is now a reality in fluorescence microscopy, and will provide chemical imaging of a variety of intracellular analytes and/or cellular phenomena. In this overview paper we attempt to describe some of the opportunities available using chemical sensing based on fluorescence lifetimes, and to predict those applications of lifetime-based sensing which are most likely in the near future.

Plasmonic enhancement of ultraviolet fluorescence

Science.gov (United States)

Jiao, Xiaojin

Plasmonics relates to the interaction between electromagnetic radiation and conduction electrons at metallic interfaces or in metallic nanostructures. Surface plasmons are collective electron oscillations at a metal surface, which can be manipulated by shape, texture and material composition. Plasmonic applications cover a broad spectrum from visible to near infrared, including biosensing, nanolithography, spectroscopy, optoelectronics, photovoltaics and so on. However, there remains a gap in this activity in the ultraviolet (UV, research. Motivating factors in the study of UV Plasmonics are the direct access to biomolecular resonances and native fluorescence, resonant Raman scattering interactions, and the potential for exerting control over photochemical reactions. This dissertation aims to fill in the gap of Plasmonics in the UV with efforts of design, fabrication and characterization of aluminium (Al) and magnesium (Mg) nanostructures for the application of label-free bimolecular detection via native UV fluorescence. The first contribution of this dissertation addresses the design of Al nanostructures in the context of UV fluorescence enhancement. A design method that combines analytical analysis with numerical simulation has been developed. Performance of three canonical plasmonic structures---the dipole antenna, bullseye nanoaperture and nanoaperture array---has been compared. The optimal geometrical parameters have been determined. A novel design of a compound bullseye structure has been proposed and numerically analyzed for the purpose of compensating for the large Stokes shift typical of UV fluorescence. Second, UV lifetime modification of diffusing molecules by Al nanoapertures has been experimentally demonstrated for the first time. Lifetime reductions of ~3.5x have been observed for the high quantum yield (QY) laser dye p-terphenyl in a 60 nm diameter aperture with 50 nm undercut. Furthermore, quantum-yield-dependence of lifetime reduction has been

Fluorescence kinetics of Trp-Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy.

Science.gov (United States)

Jia, Menghui; Yi, Hua; Chang, Mengfang; Cao, Xiaodan; Li, Lei; Zhou, Zhongneng; Pan, Haifeng; Chen, Yan; Zhang, Sanjun; Xu, Jianhua

2015-08-01

Ultrafast fluorescence dynamics of Tryptophan-Tryptophan (Trp-Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N'-aldehyde group-l-tryptophan-l-tryptophan (NBTrp2), l-tryptophan-l-tryptophan methyl ester (Trp2Me), and N-acetyl-l-tryptophan-l-tryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22ns, respectively. Due to the intramolecular interaction between two Trp residues, the "water relaxation" lifetime was observed around 4ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of ∼100ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20-30ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed. Copyright © 2015. Published by Elsevier B.V.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

Science.gov (United States)

Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2018-05-01

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

In vivo multiphoton and fluorescence lifetime imaging microscopy of the healthy and cholestatic liver

Science.gov (United States)

Kuznetsova, Daria S.; Dudenkova, Varvara V.; Rodimova, Svetlana A.; Bobrov, Nikolai V.; Zagainov, Vladimir E.; Zagaynova, Elena V.

2018-02-01

A cholestatic liver disease presents one of the most common liver diseases and can potentially progress to cirrhosis or even cholangiocarcinoma. Conventional techniques are insufficient to precisely describe the complex internal structure, heterogeneous cell populations and the dynamics of biological processes of the liver. Currently, the methods of multiphoton and fluorescence lifetime imaging microscopy are actively introducing to biomedical research. Those methods are extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. Multiphoton and FLIM microscopy do not need additional staining of samples or the incorporation of any markers to study metabolism, lipid composition, microstructure analysis, evaluation of fibrous structures. These parameters have pronounced changes in hepatocytes of liver with common pathological diseases. Thereby in this study we investigated metabolic changes in the healthy and cholestatic liver based on the fluorescence of the metabolic co-factors NAD(P)H and FAD by multiphoton microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH was presented. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.

A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.

Science.gov (United States)

Xu, Youchun; Yan, He; Zhang, Yan; Jiang, Kewei; Lu, Ying; Ren, Yonghong; Wang, Hui; Wang, Shan; Xing, Wanli

2015-07-07

Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing.

Exploiting fluorescence for multiplex immunoassays on protein microarrays

International Nuclear Information System (INIS)

Herbáth, Melinda; Balogh, Andrea; Matkó, János; Papp, Krisztián; Prechl, József

2014-01-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

QUANTIFYING THE SHORT LIFETIME WITH TCSPC-FLIM: FIRST MOMENT VERSUS FITTING METHODS

Directory of Open Access Journals (Sweden)

LINGLING XU

2013-10-01

Full Text Available Combing the time-correlated single photon counting (TCSPC with fluorescence lifetime imaging microscopy (FLIM provides promising opportunities in revealing important information on the microenvironment of cells and tissues, but the applications are thus far mainly limited by the accuracy and precision of the TCSPC-FLIM technique. Here we present a comprehensive investigation on the performance of two data analysis methods, the first moment (M1 method and the conventional least squares (Fitting method, in quantifying fluorescence lifetime. We found that the M1 method is more superior than the Fitting method when the lifetime is short (70 ~ 400 ps or the signal intensity is weak (<103 photons.

Droplet-based microscale colorimetric biosensor for multiplexed DNA analysis via a graphene nanoprobe

International Nuclear Information System (INIS)

Xiang Xia; Luo Ming; Shi Liyang; Ji Xinghu; He Zhike

2012-01-01

Graphical abstract: With a microvalve manipulate technique combined with droplet platform, a microscale fluorescence-based colorimetric sensor for multiplexed DNA analysis is developed via a graphene nanoprobe. Highlights: ► A quantitative detection for multiplexed DNA is first realized on droplet platform. ► The DNA detection is relied on a simple fluorescence-based colorimetric method. ► GO is served as a quencher for two different DNA fluorescent probes. ► This present work provides a rapid, sensitive, visual and convenient detection tool for droplet biosensor. - Abstract: The development of simple and inexpensive DNA detection strategy is very significant for droplet-based microfluidic system. Here, a droplet-based biosensor for multiplexed DNA analysis is developed with a common imaging device by using fluorescence-based colorimetric method and a graphene nanoprobe. With the aid of droplet manipulation technique, droplet size adjustment, droplet fusion and droplet trap are realized accurately and precisely. Due to the high quenching efficiency of graphene oxide (GO), in the absence of target DNAs, the droplet containing two single-stranded DNA probes and GO shows dark color, in which the DNA probes are labeled carboxy fluorescein (FAM) and 6-carboxy-X-rhodamine (ROX), respectively. The droplet changes from dark to bright color when the DNA probes form double helix with the specific target DNAs leading to the dyes far away from GO. This colorimetric droplet biosensor exhibits a quantitative capability for simultaneous detection of two different target DNAs with the detection limits of 9.46 and 9.67 × 10 −8 M, respectively. It is also demonstrated that this biosensor platform can become a promising detection tool in high throughput applications with low consumption of reagents. Moreover, the incorporation of graphene nanoprobe and droplet technique can drive the biosensor field one more step to some extent.

Characterization of a pulsed x-ray source for fluorescent lifetime measurements

International Nuclear Information System (INIS)

Blankespoor, S.C.; Derenzo, S.E.; Moses, W.W.; Rossington, C.S.; Ito, M.; Oba, K.

1994-01-01

To search for new, fast, inorganic scintillators, the authors have developed a bench-top pulsed x-ray source for determining fluorescent lifetimes and wavelengths of compounds in crystal or powdered form. This source uses a light-excited x-ray tube which produces x-rays when light from a laser diode strikes its photocathode. The x-ray tube has a tungsten anode, a beryllium exit window, a 30 kV maximum tube bias, and a 50 μA maximum average cathode current. The laser produces 3 x 10 7 photons at 650 nm per ∼100 ps pulse, with up to 10 7 pulses/sec. The time spread for the laser diode, x-ray tube, and a microchannel plate photomultiplier tube is less than 120 ps fwhm. The mean x-ray energy at tube biases of 20, 25, and 30 kV is 9.4, 10.3, and 11.1 keV, respectively. The authors measured 140, 230, and 330 x-ray photons per laser diode pulse per steradian, at tube biases of 20, 25, and 30 kV, respectively. Background x-rays due to dark current occur at a rate of 1 x 10 6 and 3 x 10 6 photons/sec/steradian at biases of 25 and 30 kV, respectively. Data characterizing the x-ray output with an aluminum filter in the x-ray beam are also presented

Principles of fluorescence techniques

CERN Document Server

2016-01-01

Fluorescence techniques are being used and applied increasingly in academics and industry. The Principles of Fluorescence Techniques course will outline the basic concepts of fluorescence techniques and the successful utilization of the currently available commercial instrumentation. The course is designed for students who utilize fluorescence techniques and instrumentation and for researchers and industrial scientists who wish to deepen their knowledge of fluorescence applications. Key scientists in the field will deliver theoretical lectures. The lectures will be complemented by the direct utilization of steady-state and lifetime fluorescence instrumentation and confocal microscopy for FLIM and FRET applications provided by leading companies.

Metabolic Mapping of Breast Cancer with Multiphoton Spectral and Lifetime Imaging

Science.gov (United States)

2007-03-01

2002. Spectrally resolved fluorescence lifetime imaging microscopy. Appl. Spec- trosc. 56 :155-166. 38. Becker, W., A. Bergmann, E. Haustein , Z...photon fluores- cence lifetime imaging microscopy of macrophage-mediated antigen processing. J. Microsc. 185 :339-353. 45. Lin, H.J., P. Herman , and

Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook

Czech Academy of Sciences Publication Activity Database

Kapusta, Peter; Macháň, Radek; Benda, A.; Hof, Martin

2012-01-01

Roč. 13, č. 10 (2012), s. 12890-12910 E-ISSN 1422-0067 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : fluorescence correlation spectroscopy (FCS) * time correlated single photon counting (TCSPC) * fluorescence cross-correlation spectroscopy (FCCS) Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.464, year: 2012

Second-harmonic generation and fluorescence lifetime imaging microscopy through a rodent mammary imaging window

Science.gov (United States)

Young, Pamela A.; Nazir, Muhammad; Szulczewski, Michael J.; Keely, Patricia J.; Eliceiri, Kevin W.

2012-03-01

Tumor-Associated Collagen Signatures (TACS) have been identified that manifest in specific ways during breast tumor progression and that correspond to patient outcome. There are also compelling metabolic changes associated with carcinoma invasion and progression. We have characterized the difference in the autofluorescent properties of metabolic co-factors, NADH and FAD, between normal and carcinoma breast cell lines. Also, we have shown in vitro that increased collagen density alters metabolic genes which are associated with glycolysis and leads to a more invasive phenotype. Establishing the relationship between collagen density, cellular metabolism, and metastasis in physiologically relevant cancer models is crucial for developing cancer therapies. To study cellular metabolism with respect to collagen density in vivo, we use multiphoton fluorescence excitation microscopy (MPM) in conjunction with a rodent mammary imaging window implanted in defined mouse cancer models. These models are ideal for the study of collagen changes in vivo, allowing determination of corresponding metabolic changes in breast cancer invasion and progression. To measure cellular metabolism, we collect fluorescence lifetime (FLIM) signatures of NADH and FAD, which are known to change based on the microenvironment of the cells. Additionally, MPM systems are capable of collecting second harmonic generation (SHG) signals which are a nonlinear optical property of collagen. Therefore, MPM, SHG, and FLIM are powerful tools with great potential for characterizing key features of breast carcinoma in vivo. Below we present the current efforts of our collaborative group to develop intravital approaches based on these imaging techniques to look at defined mouse mammary models.

Using the fluorescence of DBO to study the aggregation of asphaltenes

Energy Technology Data Exchange (ETDEWEB)

Yang, Zixin; Bohne, Cornelia [Department of Chemistry, University of Victoria (Canada)], email: xyang@uvic.ca

2010-07-01

Asphaltene, operationally defined as the fraction of bitumen that is insoluble in heptane but soluble in toluene, is the least characterized component of crude oil. It can aggregate at low concentrations, causing problems in the reservoir and during transport and processing. Fluorescence techniques have been employed to characterize asphaltenes, e.g. by adding external probes. DBO (2,3-diazabicyclo [2.2.2]oct-2-ene), a fluorescence probe molecule with a long fluorescence lifetime, was made sensitive to the presence of aliphatic C-H bonds. DBO was used as an external fluorescent probe to characterize the aggregation of Athabasca asphaltene. The lifetime of DBO was measured using single photon counting. Preliminary lifetime measurements show that AA-5 quenches the emission of DBO, leading to a shortening of the DBO lifetime. The abrupt decrease in lifetime may be related to the interaction of DBO with the AA-5 aggregate; further studies are being performed to test this hypothesis. In conclusion, DBO interacts with asphaltene components and has the potential for being used as a probe to study the asphaltene aggregation.

Automated detection of breast cancer in resected specimens with fluorescence lifetime imaging

Science.gov (United States)

Phipps, Jennifer E.; Gorpas, Dimitris; Unger, Jakob; Darrow, Morgan; Bold, Richard J.; Marcu, Laura

2018-01-01

Re-excision rates for breast cancer lumpectomy procedures are currently nearly 25% due to surgeons relying on inaccurate or incomplete methods of evaluating specimen margins. The objective of this study was to determine if cancer could be automatically detected in breast specimens from mastectomy and lumpectomy procedures by a classification algorithm that incorporated parameters derived from fluorescence lifetime imaging (FLIm). This study generated a database of co-registered histologic sections and FLIm data from breast cancer specimens (N  =  20) and a support vector machine (SVM) classification algorithm able to automatically detect cancerous, fibrous, and adipose breast tissue. Classification accuracies were greater than 97% for automated detection of cancerous, fibrous, and adipose tissue from breast cancer specimens. The classification worked equally well for specimens scanned by hand or with a mechanical stage, demonstrating that the system could be used during surgery or on excised specimens. The ability of this technique to simply discriminate between cancerous and normal breast tissue, in particular to distinguish fibrous breast tissue from tumor, which is notoriously challenging for optical techniques, leads to the conclusion that FLIm has great potential to assess breast cancer margins. Identification of positive margins before waiting for complete histologic analysis could significantly reduce breast cancer re-excision rates.

Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy

Science.gov (United States)

Osseiran, Sam; Roider, Elisabeth M.; Wang, Hequn; Suita, Yusuke; Murphy, Michael; Fisher, David E.; Evans, Conor L.

2017-12-01

Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.

Functionalization of optical nanotip arrays with an electrochemical microcantilever for multiplexed DNA detection.

Science.gov (United States)

Descamps, Emeline; Duroure, Nathalie; Deiss, Frédérique; Leichlé, Thierry; Adam, Catherine; Mailley, Pascal; Aït-Ikhlef, Ali; Livache, Thierry; Nicu, Liviu; Sojic, Neso

2013-08-07

Optical nanotip arrays fabricated on etched fiber bundles were functionalized with DNA spots. Such unconventional substrates (3D and non-planar) are difficult to pattern with standard microfabrication techniques but, using an electrochemical cantilever, up to 400 spots were electrodeposited on the nanostructured optical surface in 5 min. This approach allows each spot to be addressed individually and multiplexed fluorescence detection is demonstrated. Finally, remote fluorescence detection was performed by imaging through the optical fiber bundle itself after hybridisation with the complementary sequence.

Fluorescence resonance energy transfer imaging of CFP/YFP labeled NDH in cyanobacterium cell

International Nuclear Information System (INIS)

Ji Dongmei; Lv Wei; Huang Zhengxi; Xia Andong; Xu Min; Ma Weimin; Mi Hualing; Ogawa Teruo

2007-01-01

The laser confocal scanning microscopy combined with time-correlated single photon counting imaging technique to obtain fluorescence intensity and fluorescence lifetime images for fluorescence resonance energy transfer measurement is reported. Both the fluorescence lifetime imaging microscopy (FLIM) and intensity images show inhomogeneous cyan fluorescent protein and yellow fluorescent protein (CFP /YFP) expression or inhomogeneous energy transfer between CFP and YFP over whole cell. The results presented in this work show that FLIM could be a potential method to reveal the structure-function behavior of NAD(P)H dehydrogenase complexes in living cell

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

Science.gov (United States)

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

2014-07-09

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

An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.

Science.gov (United States)

Liu, Tao; Hu, Rong; Lv, Yi-Fan; Wu, Yuan; Liang, Hao; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong; Yu, Ru-Qin

2014-08-15

Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. Copyright © 2014 Elsevier B.V. All rights reserved.

Dual-excitation upconverting nanoparticle and quantum dot aptasensor for multiplexed food pathogen detection.

Science.gov (United States)

Kurt, Hasan; Yüce, Meral; Hussain, Babar; Budak, Hikmet

2016-07-15

In this report, a dual-excitation sensing method was developed using aptamer-functionalized quantum dots and upconverting nanoparticles, exhibiting Stokes and anti-Stokes type excitation profiles, respectively. Conjugation of the aptamer-functionalized luminescent nanoparticles with the magnetic beads, comprising short DNA sequences that were partially complementary to the aptamer sequences, enabled facile separation of the analyte-free conjugates for fluorescent measurement. UV-Visible spectroscopy, Circular Dichroism spectroscopy, Dynamic Light Scattering and Polyacrylamide Gel Electrophoresis techniques were used to characterize the aptamer probes developed. The target-specific luminescent conjugates were applied for multiplex detection of model food pathogens, Salmonella typhimurium, and Staphylococcus aureus, in which the fluorescent emission spectra were obtained under UV excitation at 325nm for quantum dots and NIR excitation at 980nm for upconverting nanoparticles, respectively. The dual-excitation strategy was aimed to minimize cross-talk between the luminescent signals for multiplexed detection, and yielded limit of detection values of 16 and 28cfumL(-1) for Staphylococcus aureus, and Salmonella typhimurium, respectively. By employing a greater number of quantum dots and upconverting nanoparticles with non-overlapping fluorescent emissions, the proposed methodology might be exploited further to detect several analytes, simultaneously. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Energy Technology Data Exchange (ETDEWEB)

Hirvonen, Liisa M.; Le Marois, Alix; Suhling, Klaus, E-mail: klaus.suhling@kcl.ac.uk [Department of Physics, King' s College London, Strand, London WC2R 2LS (United Kingdom); Becker, Wolfgang; Smietana, Stefan [Becker & Hickl GmbH, Nahmitzer Damm 30, 12277 Berlin (Germany); Milnes, James; Conneely, Thomas [Photek Ltd., 26 Castleham Rd, Saint Leonards-on-Sea TN38 9NS (United Kingdom); Jagutzki, Ottmar [Institut für Kernphysik, Max-von-Laue-Str. 1, 60438 Frankfurt (Germany)

2016-08-15

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

Spatial Multiplexing of Atom-Photon Entanglement Sources using Feedforward Control and Switching Networks.

Science.gov (United States)

Tian, Long; Xu, Zhongxiao; Chen, Lirong; Ge, Wei; Yuan, Haoxiang; Wen, Yafei; Wang, Shengzhi; Li, Shujing; Wang, Hai

2017-09-29

The light-matter quantum interface that can create quantum correlations or entanglement between a photon and one atomic collective excitation is a fundamental building block for a quantum repeater. The intrinsic limit is that the probability of preparing such nonclassical atom-photon correlations has to be kept low in order to suppress multiexcitation. To enhance this probability without introducing multiexcitation errors, a promising scheme is to apply multimode memories to the interface. Significant progress has been made in temporal, spectral, and spatial multiplexing memories, but the enhanced probability for generating the entangled atom-photon pair has not been experimentally realized. Here, by using six spin-wave-photon entanglement sources, a switching network, and feedforward control, we build a multiplexed light-matter interface and then demonstrate a ∼sixfold (∼fourfold) probability increase in generating entangled atom-photon (photon-photon) pairs. The measured compositive Bell parameter for the multiplexed interface is 2.49±0.03 combined with a memory lifetime of up to ∼51  μs.

Lifetime measurement of the 8s level in francium

International Nuclear Information System (INIS)

Gomez, E.; Sprouse, G.D.; Orozco, L.A.; Galvan, A. Perez

2005-01-01

We measure the lifetime of the 8s level of 210 Fr atoms on a magneto-optically trapped sample with time-correlated single-photon counting. The 7P 1/2 state serves as the resonant intermediate level for two-step excitation of the 8s level completed with a 1.3-μm laser. Analysis of the fluorescence decay through the 7P 3/2 level gives 53.30±0.44 ns for the 8s level lifetime

From morphology to biochemical state - intravital multiphoton fluorescence lifetime imaging of inflamed human skin

Science.gov (United States)

Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

2016-03-01

The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

Fluorescent nanoparticles for intracellular sensing: A review

International Nuclear Information System (INIS)

Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

2012-01-01

Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

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

Nanodiamond arrays on glass for quantification and fluorescence characterisation.

Science.gov (United States)

Heffernan, Ashleigh H; Greentree, Andrew D; Gibson, Brant C

2017-08-23

Quantifying the variation in emission properties of fluorescent nanodiamonds is important for developing their wide-ranging applicability. Directed self-assembly techniques show promise for positioning nanodiamonds precisely enabling such quantification. Here we show an approach for depositing nanodiamonds in pre-determined arrays which are used to gather statistical information about fluorescent lifetimes. The arrays were created via a layer of photoresist patterned with grids of apertures using electron beam lithography and then drop-cast with nanodiamonds. Electron microscopy revealed a 90% average deposition yield across 3,376 populated array sites, with an average of 20 nanodiamonds per site. Confocal microscopy, optimised for nitrogen vacancy fluorescence collection, revealed a broad distribution of fluorescent lifetimes in agreement with literature. This method for statistically quantifying fluorescent nanoparticles provides a step towards fabrication of hybrid photonic devices for applications from quantum cryptography to sensing.

Capillaries for use in a multiplexed capillary electrophoresis system

Science.gov (United States)

Yeung, E.S.; Chang, H.T.; Fung, E.N.

1997-12-09

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

Characterization of porcine eyes based on autofluorescence lifetime imaging

Science.gov (United States)

Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

2015-03-01

Multiphoton microscopy is a non-invasive imaging technique with ideal characteristics for biological applications. In this study, we propose to characterize three major structures of the porcine eye, the cornea, crystalline lens, and retina using two-photon excitation fluorescence lifetime imaging microscopy (2PE-FLIM). Samples were imaged using a laser-scanning microscope, consisting of a broadband sub-15 femtosecond (fs) near-infrared laser. Signal detection was performed using a 16-channel photomultiplier tube (PMT) detector (PML-16PMT). Therefore, spectral analysis of the fluorescence lifetime data was possible. To ensure a correct spectral analysis of the autofluorescence lifetime data, the spectra of the individual endogenous fluorophores were acquired with the 16-channel PMT and with a spectrometer. All experiments were performed within 12h of the porcine eye enucleation. We were able to image the cornea, crystalline lens, and retina at multiple depths. Discrimination of each structure based on their autofluorescence intensity and lifetimes was possible. Furthermore, discrimination between different layers of the same structure was also possible. To the best of our knowledge, this was the first time that 2PE-FLIM was used for porcine lens imaging and layer discrimination. With this study we further demonstrated the feasibility of 2PE-FLIM to image and differentiate three of the main components of the eye and its potential as an ophthalmologic technique.

A Novel Multiplex HRM Assay to Detect Clopidogrel Resistance.

Science.gov (United States)

Zhang, Lichen; Ma, Xiaowei; You, Guoling; Zhang, Xiaoqing; Fu, Qihua

2017-11-22

Clopidogrel is an antiplatelet medicine used to prevent blood clots in patients who have had a heart attack, stroke, or other symptoms. Variability in the clinical response to clopidogrel treatment has been attributed to genetic factors. In particular, five SNPs of rs4244285, rs4986893, rs12248560, rs662 and rs1045642 have been associated with resistance to clopidogrel therapy in Chinese population. This work involves the development of a multiplex high-resolution melting (HRM) assay to genotype all five of these loci in 2 tubes. Amplicons corresponding to distinct SNPs in a common tube were designed with the aid of uMelt prediction software to have different melting temperatures Tm by addition of a GC-rich tail to the 5' end of the certain primers. Two kinds of commercial methods, Digital Fluorescence Molecular Hybridization (DFMH) and Sanger sequencing, were used as a control. Three hundred sixteen DFMH pretested samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM. The sensitivity of HRM was 100% and the specificity was 99.93% reflecting detection of variants other than the known resistance SNPs. Multiplex HRM is an effective closed-tube, highly accurate, fast, and inexpensive method for genotyping the 5 clopidogrel resistance associated SNPs.

Solvent isotope effect on the fluorescence of azoalkanes

International Nuclear Information System (INIS)

Mirbach, M.J.; Mirbach, M.F.; Cherry, W.R.; Turro, N.J.; Engel, P.

1977-01-01

A study of fluorescence quantum yields and fluorescence lifetimes of two cyclic azoalkanes reveal a striking dependence of phisub(F) and tausub(F) on solvent and on isotopic substitution (OH → OD). A mechanism involving specific deactivation of the fluorescent state from a hydrogen bonded complex is proposed to rationalize the data. (orig./HK) [de

Toward photostable multiplex analyte detection on a single mode planar optical waveguide

Energy Technology Data Exchange (ETDEWEB)

Mukundan, Harshini [Los Alamos National Laboratory; Xei, Hongshi [Los Alamos National Laboratory; Anderson, Aaron S [Los Alamos National Laboratory; Grace, Wynne K [Los Alamos National Laboratory; Martinez, Jennifer S [NON LANL; Swanson, Basil [Los Alamos National Laboratory

2009-01-01

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.

Enhancement of uranyl fluorescence using trimesic acid: Ligand sensitization and co-fluorescence

Energy Technology Data Exchange (ETDEWEB)

Maji, S. [Chemistry Group, Materials Chemistry Division, Indira Gandhi Centre for Atomic Research, Kalpakkam 603102 (India); Viswanathan, K.S., E-mail: vish@igcar.gov.in [Chemistry Group, Materials Chemistry Division, Indira Gandhi Centre for Atomic Research, Kalpakkam 603102 (India)

2011-09-15

Trimesic acid (TMA) was shown to sensitize and enhance uranyl fluorescence in aqueous medium, with the enhancement being a maximum at pH 5.0. Fluorescence spectra and lifetime data together suggest that TMA complexes with uranyl (UO{sub 2}{sup 2+}). The fluorescence of UO{sub 2}{sup 2+} in its acid complex is further enhanced by more than two orders of magnitude following the addition of Y{sup 3+}; a process referred to as co-fluorescence, leading to the possibility of detecting uranium at sub ng/mL level. The present study demonstrates, for the first time, fluorescence enhancement of the uranyl species due to co-fluorescence. - Highlights: > Trimesic acid was shown to sensitize and enhance the fluorescence of uranium in aqueous medium. > This ligand also exhibited co-fluorescence of uranium with Y{sup 3+}. > To the best of our knowledge this is the first report of co-fluorescence in uranium. > The enhancement of uranium fluorescence, resulted in detection limits in the ng/mL regime.

Functional Multiplex PageRank

Science.gov (United States)

Iacovacci, Jacopo; Rahmede, Christoph; Arenas, Alex; Bianconi, Ginestra

2016-10-01

Recently it has been recognized that many complex social, technological and biological networks have a multilayer nature and can be described by multiplex networks. Multiplex networks are formed by a set of nodes connected by links having different connotations forming the different layers of the multiplex. Characterizing the centrality of the nodes in a multiplex network is a challenging task since the centrality of the node naturally depends on the importance associated to links of a certain type. Here we propose to assign to each node of a multiplex network a centrality called Functional Multiplex PageRank that is a function of the weights given to every different pattern of connections (multilinks) existent in the multiplex network between any two nodes. Since multilinks distinguish all the possible ways in which the links in different layers can overlap, the Functional Multiplex PageRank can describe important non-linear effects when large relevance or small relevance is assigned to multilinks with overlap. Here we apply the Functional Page Rank to the multiplex airport networks, to the neuronal network of the nematode C. elegans, and to social collaboration and citation networks between scientists. This analysis reveals important differences existing between the most central nodes of these networks, and the correlations between their so-called pattern to success.

Development of a multiplex PCR for the genetic analysis of paddlefish (Polyodon spathula Walbaum,1792 populations

Directory of Open Access Journals (Sweden)

K. Kurta

2017-12-01

Full Text Available Purpose. Paddlefish is commercially important species owing to its biological features and consumer characteristics, namely it produces valuable and delicious fish products, such as high quality meat and black caviar. Consequently, its cultivation under Ukrainian fish farm conditions and further realization in domestic and foreign markets are economically efficient. However, the paddlefish broodstock in Ukraine requires the efficient solution of increasing its productivity, identification and assessment of its genetic variation. Thus, the aim of our study was to develop and implement a multiplex PCR-analysis of paddlefish (Polyodon spathula for population-genetic monitoring of its artificial broodstocks in Ukraine. Methodology. A multiplex PCR was used for the study. The multiplex PCR development was performed for four microsatellite DNA markers: Psp12, Psp21, Psp26 and Psp28. Each investigated DNA loci, for which the multiplex PCR was optimized, was selected in such a way that the colored PCR products labeled with fluorescent dye did not overlap the length of the amplified fragments. Evaluation of the multiplex PCR effectiveness and processing of the data were performed by fragment analysis of DNA on the genetic analyzer ABI Prism 3130 (Applied Biosystem, USA. The size of the identified alleles was determined using the "Gene Mapper 3.7" program (Applied Biosystems, USA and LIZ-500 size standard (Applied Biosystems, USA. Results. Based on the results of capillary electrophoresis of multiplex PCR products, it was found that the amplified fragments for each of the four studied loci: Psp12, Psp21, Psp26 and Psp28 in one PCR reaction were within the expected size range. Data analysis on the electrophoregram demonstrated that Psp21 had the highest peak intensity at 611 fluorescent units (FU and the lowest peak intensity at 105 FU was observed for Psp26 locus. In the multiplex PCR after proper interpretation of the data we identified heterozygous

Multiplex PageRank.

Directory of Open Access Journals (Sweden)

Arda Halu

Full Text Available Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation.

Multiplex PageRank.

Science.gov (United States)

Halu, Arda; Mondragón, Raúl J; Panzarasa, Pietro; Bianconi, Ginestra

2013-01-01

Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation.

Absorbance and fluorescence studies on porphyrin Nanostructures ...

African Journals Online (AJOL)

The aim of this work was to study some photophysical properties of PNR for application as light harvester in dye sensitized solar cells. These properties included absorbance, fluorescence, and fluorescence quantum yield and lifetime. The results of Transmission Electron Microscope (TEM) images showed the formation of ...

Analogue multiplexer

International Nuclear Information System (INIS)

Gorshkov, V.A.; Kuznetsov, A.N.

1980-01-01

In systems of signal recording from several parallel spectrometric channels one can considerably reduce the total apparatus volume using a special unit - an analog multiplexer. A description of the multiplexer in the CAMAC system on the base of fast linear gating circuits which allows one analog-to-code converter to attend four spectrometric channels is given. On the example of the 4-channel spectrometer the logics of interaction of the multiple with analog-to-digital coxernver and signal recorder is shown. Electrical and functional multiplexer flow-sheets are given and its main characteristics are presented

Fluorescent nanoparticles for intracellular sensing: A review

Energy Technology Data Exchange (ETDEWEB)

Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

2012-11-02

Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.

Directory of Open Access Journals (Sweden)

Sandrine Prost

Full Text Available The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family. Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705, Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches.

Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

Science.gov (United States)

Uchugonova, Aisada

2017-06-01

The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

Steady state and time-resolved fluorescence spectroscopy of quinine sulfate dication bound to sodium dodecylsulfate micelles: Fluorescent complex formation

Energy Technology Data Exchange (ETDEWEB)

Joshi, Sunita; Pant, Debi D., E-mail: ddpant@pilani.bits-pilani.ac.in

2014-01-15

Interaction of quinine sulfate dication (QSD) with anionic, sodium dodecylsulphate (SDS) surfactant has been studied at different premicellar, micellar and postmicellar concentrations in aqueous phase using steady state, time-resolved fluorescence and fluorescence anisotropy techniques. At premicellar concentrations of SDS, the decrease in absorbance, appearance of an extra fluorescence band at lower wavelengths and tri-exponential decay behavior of fluorescence, are attributed to complex formation between QSD molecules and surfactant monomers. At postmicellar concentrations the red shift in fluorescence spectrum, increase in quantum yield and increase in fluorescence lifetimes are attributed to incorporation of solute molecules to micelles. At lower concentrations of SDS, a large shift in fluorescence is observed on excitation at the red edge of absorption spectrum and this is explained in terms of distribution of ion pairs of different energies in the ground state and the observed fluorescence lifetime behavior corroborates with this model. The temporal fluorescence anisotropy decay of QSD in SDS micelles allowed determination of restriction on the motion of the fluorophore. All the different techniques used in this study reveal that the photophysics of QSD is very sensitive to the microenvironments of SDS micelles and QSD molecules reside at the water-micelle interface. -- Highlights: • Probe molecule is very sensitive to microenvironment of micelles. • Highly fluorescent ion-pair formation has been observed. • Modulated photophysics of probe molecule in micellar solutions has been observed. • Probe molecules strongly bind with micelles and reside at probe–micelle interface.

A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

Science.gov (United States)

Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

2016-05-01

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

Science.gov (United States)

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors

Directory of Open Access Journals (Sweden)

Fadel Sayes

2018-04-01

Full Text Available Summary: The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II. : Sayes et al. develop an approach to express distinct fluorescent reporters that is based on the recognition of specific Mycobacterium tuberculosis MHC class II epitopes by highly discriminative T cell hybridomas. This multiplexed technology allows the study of secretion, subcellular location, and regulation patterns of these instrumental protein members. Keywords: mycobacterium tuberculosis, type VII secretion systems, intracellular bacteria, T-cell hybridomas, mycobacterial virulence factors, bacterial antigen presentation, lentiviral vectors, reporter T cells, in vivo antigen presentation, protein localization

From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients' bedside

Science.gov (United States)

Mess, Christian; Zens, Katharina; Gorzelanny, Christian; Metze, Dieter; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.; Huck, Volker

2017-02-01

Application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of skin diseases. By means of multiphoton excitation, endogenous biomolecules like NADH, collagen or elastin show autofluorescence or second harmonic generation. Thus, these molecules provide information about the subcellular morphology, epidermal architecture and physiological condition of the skin. To gain a deeper understanding of the linkage between cellular structure and physiological processes, non-invasive multiphotonbased intravital tomography (MPT) and fluorescence lifetime imaging (FLIM) were combined within the scopes of inflammatory skin, chronic wounds and drug delivery in clinical application. The optical biopsies generated via MPT were morphologically analyzed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Independent morphometric algorithms reliably showed a perinuclear accumulation in lesional skin in contrast to an even distribution in healthy skin. Confirmatively, MPT-FLIM showed an obvious metabolic shift in lesions. Moreover, detection of the onset and progression of inflammatory processes could be achieved. The feasibility of primary in vivo tracking of applied therapeutic agents further broadened our scope: We examined the permeation and subsequent distribution of agents directly visualized in patientś skin in short-term repetitive measurements. Furthermore, we performed MPT-FLIM follow-up investigations in the long-term course of therapy. Therefore, clinical MPT-FLIM application offers new insights into the pathophysiology and the individual therapeutic course of skin diseases, facilitating a better understanding of the processes of inflammation and wound healing.

Efficiency of semi-automated fluorescent multiplex PCRs with 11 microsatellite markers for genetic studies of deer populations.

Science.gov (United States)

Bonnet, A; Thévenon, S; Maudet, F; Maillard, J C

2002-10-01

Thirty bovine and eight ovine microsatellite primer pairs were tested on four tropical deer species: Eld's and Swamp deer (highly threatened) and Rusa and Vietnamese Sika deer (economically important). Thirty markers gave an amplified product in all four species (78.9%). The number of polymorphic microsatellite markers varied among the species from 14 in Eld's deer (47%) to 20 in Swamp deer (67%). Among them, 11 microsatellite loci were multiplexed in three polymerase chain reactions (PCRs) and labelled with three different fluorochromes that can be loaded in one gel-lane. To test the efficiency of the multiplex, primary genetic studies (mean number of alleles, expected heterozygosities and Fis values) were carried out on four deer populations. Parentage exclusion probability and probability of identity were computed and discussed on a Swamp deer population. These multiplexes PCRs were also tested on several other deer species and subspecies. The aim of this study is to establish a tool useful for genetic studies of population structure and diversity in four tropical deer species which with few modifications can be applied to other species of the genus Cervus.

Generating multiplex gradients of biomolecules for controlling cellular adhesion in parallel microfluidic channels.

Science.gov (United States)

Didar, Tohid Fatanat; Tabrizian, Maryam

2012-11-07

Here we present a microfluidic platform to generate multiplex gradients of biomolecules within parallel microfluidic channels, in which a range of multiplex concentration gradients with different profile shapes are simultaneously produced. Nonlinear polynomial gradients were also generated using this device. The gradient generation principle is based on implementing parrallel channels with each providing a different hydrodynamic resistance. The generated biomolecule gradients were then covalently functionalized onto the microchannel surfaces. Surface gradients along the channel width were a result of covalent attachments of biomolecules to the surface, which remained functional under high shear stresses (50 dyn/cm(2)). An IgG antibody conjugated to three different fluorescence dyes (FITC, Cy5 and Cy3) was used to demonstrate the resulting multiplex concentration gradients of biomolecules. The device enabled generation of gradients with up to three different biomolecules in each channel with varying concentration profiles. We were also able to produce 2-dimensional gradients in which biomolecules were distributed along the length and width of the channel. To demonstrate the applicability of the developed design, three different multiplex concentration gradients of REDV and KRSR peptides were patterned along the width of three parallel channels and adhesion of primary human umbilical vein endothelial cell (HUVEC) in each channel was subsequently investigated using a single chip.

Smart optical probes for near-infrared fluorescence imaging of Alzheimer's disease pathology

International Nuclear Information System (INIS)

Raymond, Scott B.; Bacskai, Brian J.; Skoch, Jesse; Hills, Ivory D.; Swager, Timothy M.; Nesterov, Evgueni E.

2008-01-01

Near-infrared fluorescent probes for amyloid-beta (Aβ) are an exciting option for molecular imaging in Alzheimer's disease research and may translate to clinical diagnostics. However, Aβ-targeted optical probes often suffer from poor specificity and slow clearance from the brain. We are designing smart optical probes that emit characteristic fluorescence signal only when bound to Aβ. We synthesized a family of dyes and tested Aβ-binding sensitivity with fluorescence spectroscopy and tissue-staining. Select compounds exhibited Aβ-dependent changes in fluorescence quantum yield, lifetime, and emission spectra that may be imaged microscopically or in vivo using new lifetime and spectral fluorescence imaging techniques. Smart optical probes that turn on when bound to Aβ will improve amyloid detection and may enable quantitative molecular imaging in vivo. (orig.)

Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

Science.gov (United States)

Aydin, Muhsin; Carter-Conger, Jacqueline; Gao, Ning; Gilmore, David F; Ricke, Steven C; Ahn, Soohyoun

2018-04-01

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Fluorescence detection of single molecules using pulsed near-field optical excitation and time correlated photon counting

International Nuclear Information System (INIS)

Ambrose, W.P.; Goodwin, P.M.; Martin, J.C.; Keller, R.A.

1994-01-01

Pulsed excitation, time correlated single photon counting and time gated detection are used in near-field optical microscopy to enhance fluorescence images and measure the fluorescence lifetimes of single molecules of Rhodamine 6G on silica surfaces. Time gated detection is used to reject prompt scattered background and to improve the image signal to noise ratio. The excited state lifetime of a single Rhodamine 6G molecule is found to depend on the position of the near-field probe. We attribute the lifetime variations to spontaneous emission rate alterations by the fluorescence reflected from and quenching by the aluminum coated probe

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.

Multiplex gas chromatography

Science.gov (United States)

Valentin, Jose R.

1990-01-01

The principles of the multiplex gas chromatography (GC) technique, which is a possible candidate for chemical analysis of planetary atmospheres, are discussed. Particular attention is given to the chemical modulators developed by present investigators for multiplex GC, namely, the thermal-desorption, thermal-decomposition, and catalytic modulators, as well as to mechanical modulators. The basic technique of multiplex GC using chemical modulators and a mechanical modulator is demonstrated. It is shown that, with the chemical modulators, only one gas stream consisting of the carrier in combination with the components is being analyzed, resulting in a simplified instrument that requires relatively few consumables. The mechanical modulator demonstrated a direct application of multiplex GC for the analysis of gases in atmosphere of Titan at very low pressures.

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise.

Science.gov (United States)

Sanchez, J J; Børsting, C; Balogh, K; Berger, B; Bogus, M; Butler, J M; Carracedo, A; Court, D Syndercombe; Dixon, L A; Filipović, B; Fondevila, M; Gill, P; Harrison, C D; Hohoff, C; Huel, R; Ludes, B; Parson, W; Parsons, T J; Petkovski, E; Phillips, C; Schmitter, H; Schneider, P M; Vallone, P M; Morling, N

2008-06-01

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.

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.

Phosphorescence and delayed fluorescence properties of fluorone dyes in bio-related films

International Nuclear Information System (INIS)

Penzkofer, A.; Tyagi, A.; Slyusareva, E.; Sizykh, A.

2010-01-01

Graphical abstract: The spectral and temporal phosphorescence and delayed fluorescence behaviour of five fluorescein dyes in gelatine, starch, and chitosan is studied and basic parameters are determined. Research highlights: → Phosphorescence quantum yields of fluorone dyes in bio-related films are measured at room temperature. → Delayed fluorescence quantum yields of fluorone dyes in bio-related films are measured at room temperature. → Phosphorescence lifetimes of fluorone dyes in bio-related films are measured at room temperature. → Delayed fluorescence lifetimes of fluorone dyes in bio-related films are measured at room temperature. → General theory of short-pulse excited phosphorescence and delayed fluorescence is presented and relevant parameters are extracted. - Abstract: The phosphorescence and delayed fluorescence behaviour of the fluorone dyes disodium fluorescein (FL, uranine), 4,5-dibromofluorescein (DBF), eosin Y (EO), erythrosine B (ER), and rose bengal (RB) in bio-films of gelatine, starch, and chitosan at room temperature is studied. Phosphorescence and delayed fluorescence quantum yields and lifetimes were measured. The singlet-triplet dynamics is described and applied to the fluorone dyes for parameter extraction. For uranine films at room temperature no phosphorescence could be resolved. The efficiency of singlet-triplet intersystem crossing increased in the order φ ISC (DBF) ISC (EO) ISC (ER) ISC (RB) due to the heavy atom effect on spin-orbit coupling. The phosphorescence quantum yields increased in the order φ P (DBF) P (EO) P (RB) P (ER). The phosphorescence lifetimes followed the order τ P (DBF) > τ P (EO) > τ P (ER) > τ P (RB).

Distinct roles of the photosystem II protein PsbS and zeaxanthin in the regulation of light harvesting in plants revealed by fluorescence lifetime snapshots.

Science.gov (United States)

Sylak-Glassman, Emily J; Malnoë, Alizée; De Re, Eleonora; Brooks, Matthew D; Fischer, Alexandra Lee; Niyogi, Krishna K; Fleming, Graham R

2014-12-09

The photosystem II (PSII) protein PsbS and the enzyme violaxanthin deepoxidase (VDE) are known to influence the dynamics of energy-dependent quenching (qE), the component of nonphotochemical quenching (NPQ) that allows plants to respond to fast fluctuations in light intensity. Although the absence of PsbS and VDE has been shown to change the amount of quenching, there have not been any measurements that can detect whether the presence of these proteins alters the type of quenching that occurs. The chlorophyll fluorescence lifetime probes the excited-state chlorophyll relaxation dynamics and can be used to determine the amount of quenching as well as whether two different genotypes with the same amount of NPQ have similar dynamics of excited-state chlorophyll relaxation. We measured the fluorescence lifetimes on whole leaves of Arabidopsis thaliana throughout the induction and relaxation of NPQ for wild type and the qE mutants, npq4, which lacks PsbS; npq1, which lacks VDE and cannot convert violaxanthin to zeaxanthin; and npq1 npq4, which lacks both VDE and PsbS. These measurements show that although PsbS changes the amount of quenching and the rate at which quenching turns on, it does not affect the relaxation dynamics of excited chlorophyll during quenching. In addition, the data suggest that PsbS responds not only to ΔpH but also to the Δψ across the thylakoid membrane. In contrast, the presence of VDE, which is necessary for the accumulation of zeaxanthin, affects the excited-state chlorophyll relaxation dynamics.

Consideration for wavelength multiplexing versus time multiplexing in optical transport network

DEFF Research Database (Denmark)

Limal, Emmanuel; Stubkjær, Kristian Elmholdt

1999-01-01

We compare optical wavelength multiplexing and time multiplexing techniquesfor optical transport network by studying the space switch sizes of OXCs andtheir interfaces as a function of the fraction of add/drop traffic....

Determination of absolute Ba densities during dimming operation of fluorescent lamps by laser-induced fluorescence measurements

International Nuclear Information System (INIS)

Hadrath, S; Beck, M; Garner, R C; Lieder, G; Ehlbeck, J

2007-01-01

Investigations of fluorescent lamps (FL) are often focused on the electrodes, since the lifetime of the lamps is typically limited by the electrode lifetime and durability. During steady state operation, the work function lowering emitter material, in particular, barium, is lost. Greater barium losses occur under dimming conditions, in which reduced discharge currents lead to increased cathode falls, the result of the otherwise diminished heating of the electrode by the bombarding plasma ions. In this work the barium density near the electrodes of (FL), operating in high frequency dimming mode is investigated using the high-sensitivity method of laser-induced fluorescence. From these measurements we infer barium loss for a range of discharge currents and auxiliary coil heating currents. We show that the Ba loss can very easily be reduced by moderate auxiliary coil heating

Dual fluorescence of single LH2 antenna nanorings

International Nuclear Information System (INIS)

Freiberg, A.; Raetsep, M.; Timpmann, K.; Trinkunas, G.

2004-01-01

A dual nature of fluorescence from LH2 pigment-protein complexes, which is a part of the light harvesting system of purple bacteria, is confirmed by fluorescence-lifetime dependence on recording wavelength and spectrally selective spectroscopy. An analysis based on the Holstein molecular crystal model, modified by allowing diagonal disorder, suggests coexistence of large- and small-radius self-trapped excitons, which serve as the origin of the dual fluorescence

Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

Science.gov (United States)

Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

2017-01-01

Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

Extracting information from multiplex networks

Science.gov (United States)

Iacovacci, Jacopo; Bianconi, Ginestra

2016-06-01

Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ ˜ S for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science.

Improving fluorescence diagnosis of cancer by SLIM

Science.gov (United States)

Rück, Angelika; Dolp, Frank; Kinzler, Ingrid; Hauser, Carmen; Scalfi-Happ, Claudia

2006-02-01

Although during the last years, significant progress was made in cancer diagnosis, using either intrinsic or specially designed fluorophores, still problems exist, due to difficulties in spectral separation of highly overlapping probes or in lack of specificity. Many of the problems could be circumvented by focusing on time-resolved methods. In combination with spectral resolved detection (spectral fluorescence lifetime imaging, SLIM) highly sophisticated fluorescence lifetime imaging can be performed which might improve specificity of cell diagnosis. To record lifetime images (τ-mapping) with spectral resolution a setup was realized consisting of a laser scanning microscope equipped with a 16 channel array for time-correlated single photon counting (TCSPC) and a spectrograph in front of the array. A Ti:Saphir laser can be used for excitation or alternatively ps diode lasers. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in solution and in cell culture. As an example, not only the mitochondria staining dye rhodamine 123 could be easily distinguished from DAPI, which intercalates into nucleic acids, but also different binding sites of DAPI. This was proved by the appearance of different lifetime components within different spectral channels. Another example is Photofrin, a photosensitizer which is approved for bladder cancer and for palliative lung and esophageal cancer in 20 countries, including the United States, Canada and many European countries. Photofrin is a complex mixture of different monomeric and aggregated porphyrins. The phototoxic efficiency during photodynamic therapy (PDT) seems to be correlated with the relative amounts of monomers and aggregates. With SLIM different lifetimes could be attributed to various, spectrally highly overlapping compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during

Automatic segmentation of fluorescence lifetime microscopy images of cells using multiresolution community detection--a first study.

Science.gov (United States)

Hu, D; Sarder, P; Ronhovde, P; Orthaus, S; Achilefu, S; Nussinov, Z

2014-01-01

Inspired by a multiresolution community detection based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Furthermore, using the proposed method, the mean-square error in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The multiresolution community detection method appeared to perform better than a popular spectral clustering-based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in mean-square error with increasing resolution. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

Science.gov (United States)

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors.

Science.gov (United States)

Bereza-Malcolm, Lara; Aracic, Sanja; Kannan, Ruban; Mann, Gülay; Franks, Ashley E

2017-08-15

Widespread presence of cadmium in soil and water systems is a consequence of industrial and agricultural processes. Subsequent accumulation of cadmium in food and drinking water can result in accidental consumption of dangerous concentrations. As such, cadmium environmental contamination poses a significant threat to human health. Development of microbial biosensors, as a novel alternative method for in situ cadmium detection, may reduce human exposure by complementing traditional analytical methods. In this study, a multiplex cadmium biosensing construct was assembled by cloning a single-output cadmium biosensor element, cadRgfp, and a constitutively expressed mrfp1 onto a broad-host range vector. Incorporation of the duplex fluorescent output [green and red fluorescence proteins] allowed measurement of biosensor functionality and viability. The biosensor construct was tested in several Gram-negative bacteria including Pseudomonas, Shewanella and Enterobacter. The multiplex cadmium biosensors were responsive to cadmium concentrations ranging from 0.01 to 10µgml -1 , as well as several other heavy metals, including arsenic, mercury and lead at similar concentrations. The biosensors were also responsive within 20-40min following exposure to 3µgml -1 cadmium. This study highlights the importance of testing biosensor constructs, developed using synthetic biology principles, in different bacterial genera. Copyright © 2017 Elsevier B.V. All rights reserved.

Riboflavin enhanced fluorescence of highly reduced graphene oxide

Science.gov (United States)

Iliut, Maria; Gabudean, Ana-Maria; Leordean, Cosmin; Simon, Timea; Teodorescu, Cristian-Mihail; Astilean, Simion

2013-10-01

The improvement of graphene derivates' fluorescence properties is a challenging topic and very few ways were reported up to now. In this Letter we propose an easy method to enhance the fluorescence of highly reduced graphene oxide (rGO) through non-covalent binding to a molecular fluorophore, namely the riboflavin (Rb). While the fluorescence of Rb is quenched, the Rb - decorated rGO exhibits strong blue fluorescence and significantly increased fluorescence lifetime, as compared to its pristine form. The data reported here represent a promising start towards tailoring the optical properties of rGOs, having utmost importance in optical applications.

Metal Stable Isotope Tagging: Renaissance of Radioimmunoassay for Multiplex and Absolute Quantification of Biomolecules.

Science.gov (United States)

Liu, Rui; Zhang, Shixi; Wei, Chao; Xing, Zhi; Zhang, Sichun; Zhang, Xinrong

2016-05-17

The unambiguous quantification of biomolecules is of great significance in fundamental biological research as well as practical clinical diagnosis. Due to the lack of a detectable moiety, the direct and highly sensitive quantification of biomolecules is often a "mission impossible". Consequently, tagging strategies to introduce detectable moieties for labeling target biomolecules were invented, which had a long and significant impact on studies of biomolecules in the past decades. For instance, immunoassays have been developed with radioisotope tagging by Yalow and Berson in the late 1950s. The later languishment of this technology can be almost exclusively ascribed to the use of radioactive isotopes, which led to the development of nonradioactive tagging strategy-based assays such as enzyme-linked immunosorbent assay, fluorescent immunoassay, and chemiluminescent and electrochemiluminescent immunoassay. Despite great success, these strategies suffered from drawbacks such as limited spectral window capacity for multiplex detection and inability to provide absolute quantification of biomolecules. After recalling the sequences of tagging strategies, an apparent question is why not use stable isotopes from the start? A reasonable explanation is the lack of reliable means for accurate and precise quantification of stable isotopes at that time. The situation has changed greatly at present, since several atomic mass spectrometric measures for metal stable isotopes have been developed. Among the newly developed techniques, inductively coupled plasma mass spectrometry is an ideal technique to determine metal stable isotope-tagged biomolecules, for its high sensitivity, wide dynamic linear range, and more importantly multiplex and absolute quantification ability. Since the first published report by our group, metal stable isotope tagging has become a revolutionary technique and gained great success in biomolecule quantification. An exciting research highlight in this area

Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research

Czech Academy of Sciences Publication Activity Database

Benda, Aleš; Fagulová, Veronika; Deyneka, Alexander; Enderlain, J.; Hof, Martin

2006-01-01

Roč. 22, č. 23 (2006), s. 9580-9585 ISSN 0743-7463 R&D Projects: GA ČR GA203/05/2308; GA MŠk LC06063 Institutional research plan: CEZ:AV0Z40400503; CEZ:AV0Z10100522 Keywords : spectroscopy * fluorescence * FLCS Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.902, year: 2006

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.

Super-multiplex vibrational imaging

Science.gov (United States)

Wei, Lu; Chen, Zhixing; Shi, Lixue; Long, Rong; Anzalone, Andrew V.; Zhang, Luyuan; Hu, Fanghao; Yuste, Rafael; Cornish, Virginia W.; Min, Wei

2017-04-01

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a ‘colour barrier’, owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the

Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

Science.gov (United States)

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

Fluorescence lifetime studies of MeV erbium implanted silica glass

International Nuclear Information System (INIS)

Lidgard, A.; Polman, A.; Jacobsen, D.C.; Blonder, G.E.; Kistler, R.; Poate, J.M.; Becker, P.C.

1991-01-01

MeV erbium ion implantation into various SiO 2 glasses has been studied with the aim of incorporating the rare-earth dopant as an optically active ion in the silica network. The lifetime of the excited state ranges from 1.6 to 12.8 ms, depending on base material and implantation fluence. These results have positive implications for silica-based integrated optical technology. (Author)

Fluorescence lifetime studies of MeV erbium implanted silica glass

Energy Technology Data Exchange (ETDEWEB)

Lidgard, A.; Polman, A.; Jacobsen, D.C.; Blonder, G.E.; Kistler, R.; Poate, J.M.; Becker, P.C. (AT and T Bell Labs., Murray Hill, NJ (USA))

1991-05-23

MeV erbium ion implantation into various SiO{sub 2} glasses has been studied with the aim of incorporating the rare-earth dopant as an optically active ion in the silica network. The lifetime of the excited state ranges from 1.6 to 12.8 ms, depending on base material and implantation fluence. These results have positive implications for silica-based integrated optical technology. (Author).

Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model

OpenAIRE

Klemm, Matthias; Blum, Johannes; Link, Dietmar; Hammer, Martin; Haueisen, Jens; Schweitzer, Dietrich

2016-01-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes d...

Detection of radiation-induced brain necrosis in live rats using label-free time-resolved fluorescence spectroscopy (TRFS) (Conference Presentation)

Science.gov (United States)

Hartl, Brad A.; Ma, Htet S. W.; Sridharan, Shamira; Hansen, Katherine; Klich, Melanie; Perks, Julian; Kent, Michael; Kim, Kyoungmi; Fragoso, Ruben; Marcu, Laura

2017-02-01

Differentiating radiation-induced necrosis from recurrent tumor in the brain remains a significant challenge to the neurosurgeon. Clinical imaging modalities are not able to reliably discriminate the two tissue types, making biopsy location selection and surgical management difficult. Label-free fluorescence lifetime techniques have previously been shown to be able to delineate human brain tumor from healthy tissues. Thus, fluorescence lifetime techniques represent a potential means to discriminate the two tissues in real-time during surgery. This study aims to characterize the endogenous fluorescence lifetime signatures from radiation induced brain necrosis in a tumor-free rat model. Fischer rats received a single fraction of 60 Gy of radiation to the right hemisphere using a linear accelerator. Animals underwent a terminal live surgery after gross necrosis had developed, as verified with MRI. During surgery, healthy and necrotic brain tissue was measured with a fiber optic needle connected to a multispectral fluorescence lifetime system. Measurements of the necrotic tissue showed a 48% decrease in intensity and 20% increase in lifetimes relative to healthy tissue. Using a support vector machine classifier and leave-one-out validation technique, the necrotic tissue was correctly classified with 94% sensitivity and 97% specificity. Spectral contribution analysis also confirmed that the primary source of fluorescence contrast lies within the redox and bound-unbound population shifts of nicotinamide adenine dinucleotide. A clinical trial is presently underway to measure these tissue types in humans. These results show for the first time that radiation-induced necrotic tissue in the brain contains significantly different metabolic signatures that are detectable with label-free fluorescence lifetime techniques.

Gated Detection Measurements of Phosphorescence Lifetimes

Directory of Open Access Journals (Sweden)

Yordan Kostov

2004-10-01

Full Text Available A low-cost, gated system for measurements of phosphorescence lifetimes is presented. An extensive description of the system operating principles and metrological characteristics is given. Remarkably, the system operates without optical filtering of the LED excitation source. A description of a practical system is also given and its performance is discussed. Because the device effectively suppresses high-level background fluorescence and scattered light, it is expected to find wide-spread application in bioprocess, environmental and biomedical fields.

Confocal fluorescence techniques in industrial application

Science.gov (United States)

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

2003-06-01

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

A New Theoretical Approach to Single-Molecule Fluorescence Optical Studies of RNA Dynamics

International Nuclear Information System (INIS)

Zhao Xinghai; Shan Guangcun; Bao Shuying

2011-01-01

Single-molecule fluorescence spectroscopy in condensed phases has many important chemical and biological applications. The single-molecule fluorescence measurements contain information about conformational dynamics on a vast range of time scales. Based on the data analysis protocols methodology proposed by X. Sunney Xie, the theoretical study here mainly focuses on the single-molecule studies of single RNA with interconversions among different conformational states, to with a single FRET pair attached. We obtain analytical expressions for fluorescence lifetime correlation functions that relate changes in fluorescence lifetime to the distance-dependent FRET mechanism within the context of the Smoluchowski diffusion model. The present work establishes useful guideline for the single-molecule studies of biomolecules to reveal the complicated folding dynamics of single RNA molecules at nanometer scale.

Facile and Scalable Preparation of Fluorescent Carbon Dots for Multifunctional Applications

Directory of Open Access Journals (Sweden)

Dan Wang

2017-06-01

Full Text Available The synthesis of fluorescent nanomaterials has received considerable attention due to the great potential of these materials for a wide range of applications, from chemical sensing through bioimaging to optoelectronics. Herein, we report a facile and scalable approach to prepare fluorescent carbon dots (FCDs via a one-pot reaction of citric acid with ethylenediamine at 150 °C under ambient air pressure. The resultant FCDs possess an optical bandgap of 3.4 eV and exhibit strong excitation-wavelength-independent blue emission (λEm = 450 nm under either one- or two-photon excitation. Owing to their low cytotoxicity and long fluorescence lifetime, these FCDs were successfully used as internalized fluorescent probes in human cancer cell lines (HeLa cells for two-photon excited imaging of cells by fluorescence lifetime imaging microscopy with a high-contrast resolution. They were also homogenously mixed with commercial inks and used to draw fluorescent patterns on normal papers and on many other substrates (e.g., certain flexible plastic films, textiles, and clothes. Thus, these nanomaterials are promising for use in solid-state fluorescent sensing, security labeling, and wearable optoelectronics.

Time-Resolved Fluorescence of Water-Soluble Pyridinium Salt: Sensitive Detection of the Conformational Changes of Bovine Serum Albumin.

Science.gov (United States)

Li, Lei; Yi, Hua; Jia, Menghui; Chang, Mengfang; Zhou, Zhongneng; Zhang, Sanjun; Pan, Haifeng; Chen, Yan; Chen, Jinquan; Xu, Jianhua

2016-06-20

In this paper, we report a pyridinium salt "turn-on" fluorescent probe, 4-[2-(4-Dimethylamino-phenyl)-vinyl]-1-methylpyridinium iodide (p-DASPMI), and applied its time-resolved fluorescence (TRF) to monitor the protein conformational changes. Both the fluorescence lifetime and quantum yield (QY) of p-DASPMI were increased about two orders of magnitude after binding to the protein bovine serum albumin (BSA). The free p-DASPMI in solution presents an ultrashort fluorescence lifetime (12.4 ps), thus it does not interfere the detection of bound p-DASPMI which has nanosecond fluorescence lifetime. Decay-associated spectra (DAS) show that p-DASPMI molecules bind to subdomains IIA and IIIA of BSA. The TRF decay profiles of p-DASPMI can be described by multi-exponential decay function ([Formula: see text]), and the obtained parameters, such as lifetimes ([Formula: see text]), fractional amplitudes ([Formula: see text]), and fractional intensities ([Formula: see text]), may be used to deduce the conformational changes of BSA. The pH and Cu 2+ induced conformational changes of BSA were investigated through the TRF of p-DASPMI. The results show that the p-DASPMI is a candidate fluorescent probe in studying the conformational changes of proteins through TRF spectroscopy and microscopy in the visible range. © The Author(s) 2016.

Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system.

Science.gov (United States)

Luo, Yuan; Gelsinger-Austin, Paul J; Watson, Jonathan M; Barbastathis, George; Barton, Jennifer K; Kostuk, Raymond K

2008-09-15

A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral transmittance filtering properties that enable obtaining spatial-spectral information within an object. We demonstrate this imaging system's ability to obtain multiple depth-resolved fluorescence images simultaneously.

Time resolved fluorescence of cow and goat milk powder

Science.gov (United States)

Brandao, Mariana P.; de Carvalho dos Anjos, Virgílio; Bell., Maria José V.

2017-01-01

Milk powder is an international dairy commodity. Goat and cow milk powders are significant sources of nutrients and the investigation of the authenticity and classification of milk powder is particularly important. The use of time-resolved fluorescence techniques to distinguish chemical composition and structure modifications could assist develop a portable and non-destructive methodology to perform milk powder classification and determine composition. This study goal is to differentiate milk powder samples from cows and goats using fluorescence lifetimes. The samples were excited at 315 nm and the fluorescence intensity decay registered at 468 nm. We observed fluorescence lifetimes of 1.5 ± 0.3, 6.4 ± 0.4 and 18.7 ± 2.5 ns for goat milk powder; and 1.7 ± 0.3, 6.9 ± 0.2 and 29.9 ± 1.6 ns for cow's milk powder. We discriminate goat and cow powder milk by analysis of variance using Fisher's method. In addition, we employed quadratic discriminant analysis to differentiate the milk samples with accuracy of 100%. Our results suggest that time-resolved fluorescence can provide a new method to the analysis of powder milk and its composition.

Nanostructure induced changes in lifetime and enhanced second-harmonic response of organic-plasmonic hybrids

Energy Technology Data Exchange (ETDEWEB)

Leißner, Till [NanoSYD, Mads Clausen Institute, University of Southern Denmark, Alsion 2, 6400 Sønderborg (Denmark); Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense (Denmark); Kostiučenko, Oksana; Rubahn, Horst-Günter; Fiutowski, Jacek, E-mail: fiutowski@mci.sdu.dk [NanoSYD, Mads Clausen Institute, University of Southern Denmark, Alsion 2, 6400 Sønderborg (Denmark); Brewer, Jonathan R. [Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense (Denmark)

2015-12-21

In this letter we show that the optical response of organic nanofibers, grown from functionalized para-quaterphenylene molecules, can be controlled by forming organic-plasmonic hybrid systems. The interaction between nanofibers and supporting regular arrays of nanostructures leads to a strongly enhanced second harmonic response. At the same time, the fluorescence lifetime of the nanofibers is reduced from 0.32 ns for unstructured gold films to 0.22 ns for gold nanosquare arrays, demonstrating efficient organic–plasmonic interaction. To study the origin of these effects, we applied two-photon laser scanning microscopy and fluorescence lifetime imaging microscopy. These findings provide an effective approach for plasmon-enhanced second-harmonic generation at the nanoscale, which is attractive for nanophotonic circuitry.

Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

Science.gov (United States)

Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

2011-07-01

Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

Recombinant Helicobacter bilis Protein P167 for Mouse Serodiagnosis in a Multiplex Microbead Assay

OpenAIRE

Feng, Sunlian; Kendall, Lon V.; Hodzic, Emir; Wong, Scott; Lorenzana, Edward; Freet, Kimberly; Ku, Karin S.; Luciw, Paul A.; Barthold, Stephen W.; Khan, Imran H.

2004-01-01

Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated t...

Synthesis of water-soluble, ring-substituted squaraine dyes and their evaluation as fluorescent probes and labels

Energy Technology Data Exchange (ETDEWEB)

Tatarets, Anatoliy L. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); Fedyunyayeva, Irina A. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); Dyubko, Tatyana S. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); Povrozin, Yevgeniy A. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); Doroshenko, Andrey O. [Institute of Chemistry, V.N. Karazin National University, 4 Svobody Sq., Kharkov 61077 (Ukraine); Terpetschnig, Ewald A. [SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801 (United States) and ISS, Inc., 1602 Newton Drive, Champaign, IL 61822 (United States)]. E-mail: ewaldte@juno.com; Patsenker, Leonid D. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801 (United States)

2006-06-16

A series of ring-substituted squaraines absorbing and emitting in the red and NIR spectral region was synthesized and their spectral and photophysical properties (quantum yields, fluorescence lifetimes) and photostabilities were measured and compared to Cy5, a commonly used fluorescent label. The absorption maxima in aqueous media were found to be between 628 and 667 nm and the emission maxima are between 642 and 685 nm. Squaraine dyes exhibit high extinction coefficients (163,000-265,000 M{sup -1} cm{sup -1}) and lower quantum yields (2-7%) in aqueous buffer but high quantum yields (up to 45%) and long fluorescence lifetimes (up to 3.3 ns) in presence of BSA. Dicyanomethylene- and thio-substituted squaraines exhibit an additional absorption around 400 nm with extinction coefficients between 21,500 and 44,500 M{sup -1} cm{sup -1}. These dyes are excitable not only with red but also with blue diode lasers or light emitting diodes. Due to the favourable spectral and photophysical properties these dyes can be used as fluorescent probes and labels for intensity- and fluorescence lifetime-based biomedical applications.

Synthesis of water-soluble, ring-substituted squaraine dyes and their evaluation as fluorescent probes and labels

International Nuclear Information System (INIS)

Tatarets, Anatoliy L.; Fedyunyayeva, Irina A.; Dyubko, Tatyana S.; Povrozin, Yevgeniy A.; Doroshenko, Andrey O.; Terpetschnig, Ewald A.; Patsenker, Leonid D.

2006-01-01

A series of ring-substituted squaraines absorbing and emitting in the red and NIR spectral region was synthesized and their spectral and photophysical properties (quantum yields, fluorescence lifetimes) and photostabilities were measured and compared to Cy5, a commonly used fluorescent label. The absorption maxima in aqueous media were found to be between 628 and 667 nm and the emission maxima are between 642 and 685 nm. Squaraine dyes exhibit high extinction coefficients (163,000-265,000 M -1 cm -1 ) and lower quantum yields (2-7%) in aqueous buffer but high quantum yields (up to 45%) and long fluorescence lifetimes (up to 3.3 ns) in presence of BSA. Dicyanomethylene- and thio-substituted squaraines exhibit an additional absorption around 400 nm with extinction coefficients between 21,500 and 44,500 M -1 cm -1 . These dyes are excitable not only with red but also with blue diode lasers or light emitting diodes. Due to the favourable spectral and photophysical properties these dyes can be used as fluorescent probes and labels for intensity- and fluorescence lifetime-based biomedical applications

Multiplexed Engineering in Biology.

Science.gov (United States)

Rogers, Jameson K; Church, George M

2016-03-01

Biotechnology is the manufacturing technology of the future. However, engineering biology is complex, and many possible genetic designs must be evaluated to find cells that produce high levels of a desired drug or chemical. Recent advances have enabled the design and construction of billions of genetic variants per day, but evaluation capacity remains limited to thousands of variants per day. Here we evaluate biological engineering through the lens of the design–build–test cycle framework and highlight the role that multiplexing has had in transforming the design and build steps. We describe a multiplexed solution to the ‘test’ step that is enabled by new research. Achieving a multiplexed test step will permit a fully multiplexed engineering cycle and boost the throughput of biobased product development by up to a millionfold.

Automatic Segmentation of Fluorescence Lifetime Microscopy Images of Cells Using Multi-Resolution Community Detection -A First Study

Science.gov (United States)

Hu, Dandan; Sarder, Pinaki; Ronhovde, Peter; Orthaus, Sandra; Achilefu, Samuel; Nussinov, Zohar

2014-01-01

Inspired by a multi-resolution community detection (MCD) based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Further, using the proposed method, the mean-square error (MSE) in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The MCD method appeared to perform better than a popular spectral clustering based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in MSE with increasing resolution. PMID:24251410

Silicon Chip-to-Chip Mode-Division Multiplexing

DEFF Research Database (Denmark)

Baumann, Jan Markus; Porto da Silva, Edson; Ding, Yunhong

2018-01-01

A chip-to-chip mode-division multiplexing connection is demonstrated using a pair of multiplexers/demultiplexers fabricated on the silicon-on-insulator platform. Successful mode multiplexing and demultiplexing is experimentally demonstrated, using the LP01, LP11a and LP11b modes.......A chip-to-chip mode-division multiplexing connection is demonstrated using a pair of multiplexers/demultiplexers fabricated on the silicon-on-insulator platform. Successful mode multiplexing and demultiplexing is experimentally demonstrated, using the LP01, LP11a and LP11b modes....

Multiplex measuring systems in physics

International Nuclear Information System (INIS)

Soroko, L.M.

1980-01-01

The principles of operation of multiplex devices used in different spheres of physics are discussed. The ''multiplex'' notion means that the data output of the device is an integral image of the functional dependence under investigation, but not its readings as in usual instruments. The analysis of the present state of developments of the multiplex systems in optics, nuclear magnetic resonance spectroscopy, in time-of-flight spectrometers for slow and fast neutrons, as well as elementary particle detectors, is given. The construction algorithms for the digital codes are presented, the history of development of the multiplex measuring principle is given [ru

Design of a Modular DNA Triangular-Prism Sensor Enabling Ratiometric and Multiplexed Biomolecule Detection on a Single Microbead.

Science.gov (United States)

Liu, Yu; Chen, Qiaoshu; Liu, Jianbo; Yang, Xiaohai; Guo, Qiuping; Li, Li; Liu, Wei; Wang, Kemin

2017-03-21

DNA nanostructures have emerged as powerful and versatile building blocks for the construction of programmable nanoscale structures and functional sensors for biomarker detection, disease diagnostics, and therapy. Here we integrated multiple sensing modules into a single DNA three-dimensional (3D) nanoarchitecture with a triangular-prism (TP) structure for ratiometric and multiplexed biomolecule detection on a single microbead. In our design, the complementary hybridization of three clip sequences formed TP nanoassemblies in which the six single-strand regions in the top and bottom faces act as binding sites for different sensing modules, including an anchor module, reference sequence module, and capture sequence module. The multifunctional modular TP nanostructures were thus exploited for ratiometric and multiplexed biomolecule detection on microbeads. Microbead imaging demonstrated that, after ratiometric self-calibration analysis, the imaging deviations resulting from uneven fluorescence intensity distribution and differing probe concentrations were greatly reduced. The rigid nanostructure also conferred the TP as a framework for geometric positioning of different capture sequences. The inclusion of multiple targets led to the formation of sandwich hybridization structures that gave a readily detectable optical response at different fluorescence channels and distinct fingerprint-like pattern arrays. This approach allowed us to discriminate multiplexed biomolecule targets in a simple and efficient fashion. In this module-designed strategy, the diversity of the controlled DNA assembly coupled with the geometrically well-defined rigid nanostructures of the TP assembly provides a flexible and reliable biosensing approach that shows great promise for biomedical applications.

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

Science.gov (United States)

Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

2016-02-01

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescence decay data analysis correcting for detector pulse pile-up at very high count rates

Science.gov (United States)

Patting, Matthias; Reisch, Paja; Sackrow, Marcus; Dowler, Rhys; Koenig, Marcelle; Wahl, Michael

2018-03-01

Using time-correlated single photon counting for the purpose of fluorescence lifetime measurements is usually limited in speed due to pile-up. With modern instrumentation, this limitation can be lifted significantly, but some artifacts due to frequent merging of closely spaced detector pulses (detector pulse pile-up) remain an issue to be addressed. We propose a data analysis method correcting for this type of artifact and the resulting systematic errors. It physically models the photon losses due to detector pulse pile-up and incorporates the loss in the decay fit model employed to obtain fluorescence lifetimes and relative amplitudes of the decay components. Comparison of results with and without this correction shows a significant reduction of systematic errors at count rates approaching the excitation rate. This allows quantitatively accurate fluorescence lifetime imaging at very high frame rates.

Integrated photonics : compact multiplexing

NARCIS (Netherlands)

Pile, D.; Chen, H.; Uden, van R.G.H.; Okonkwo, C.M.; Koonen, A.M.J.

2015-01-01

Spatial multiplexers (SMUXs) for mode division multiplexing often involve multiple strategies for mode-selective excitation and the minimization of insertion and other losses. Haoshuo Chen, Roy van Uden, Chigo Okonkwo and Ton Koonen, working at the COBRA Institute at the Eindhoven University of

Distribution of diffusion times determined by fluorescence (lifetime) correlation spectroscopy

Czech Academy of Sciences Publication Activity Database

Pánek, Jiří; Loukotová, Lenka; Hrubý, Martin; Štěpánek, Petr

2018-01-01

Roč. 51, č. 8 (2018), s. 2796-2804 ISSN 0024-9297 R&D Projects: GA MŠk(CZ) LO1507 Institutional support: RVO:61389013 Keywords : polymer solution * fluorescence correlation spectroscopy * diffusion time distribution Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 5.835, year: 2016

Fluorescent metal nanoshell and CK19 detection on single cell image

International Nuclear Information System (INIS)

Zhang, Jian; Fu, Yi; Li, Ge; Lakowicz, Joseph R.; Zhao, Richard Y.

2011-01-01

Highlights: → Novel metal nanoshell as fluorescence imaging agent. → Fluorescent mAb-metal complex with enhanced intensity and shortened lifetime. → Immuno-interactions of mAb-metal complexes with CK19 molecules on CNCAP and HeLa cell surfaces. → Isolation of conjugated mAb-metal complexes from cellular autofluorescence on cell image. -- Abstract: In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.

Dynamic Optically Multiplexed Imaging

Science.gov (United States)

2015-07-29

Dynamic Optically Multiplexed Imaging Yaron Rachlin, Vinay Shah, R. Hamilton Shepard, and Tina Shih Lincoln Laboratory, Massachusetts Institute of...V. Shah, and T. Shih “Design Architectures for Optically Multiplexed Imaging,” in submission 9 R. Gupta , P. Indyk, E. Price, and Y. Rachlin

A novel multiplex bead-based platform highlights the diversity of extracellular vesicles.

Science.gov (United States)

Koliha, Nina; Wiencek, Yvonne; Heider, Ute; Jüngst, Christian; Kladt, Nikolay; Krauthäuser, Susanne; Johnston, Ian C D; Bosio, Andreas; Schauss, Astrid; Wild, Stefan

2016-01-01

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell-derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.

Polarization-multiplexing ghost imaging

Science.gov (United States)

Dongfeng, Shi; Jiamin, Zhang; Jian, Huang; Yingjian, Wang; Kee, Yuan; Kaifa, Cao; Chenbo, Xie; Dong, Liu; Wenyue, Zhu

2018-03-01

A novel technique for polarization-multiplexing ghost imaging is proposed to simultaneously obtain multiple polarimetric information by a single detector. Here, polarization-division multiplexing speckles are employed for object illumination. The light reflected from the objects is detected by a single-pixel detector. An iterative reconstruction method is used to restore the fused image containing the different polarimetric information by using the weighted sum of the multiplexed speckles based on the correlation coefficients obtained from the detected intensities. Next, clear images of the different polarimetric information are recovered by demultiplexing the fused image. The results clearly demonstrate that the proposed method is effective.

Origins of fluorescence in evolved bacteriophytochromes.

Science.gov (United States)

Bhattacharya, Shyamosree; Auldridge, Michele E; Lehtivuori, Heli; Ihalainen, Janne A; Forest, Katrina T

2014-11-14

Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) biomarkers that exploit the ability of light in this range to penetrate tissue. Bacteriophytochromes (BphPs) are photoreceptors that couple absorbance of NIR light to photoisomerization, protein conformational changes, and signal transduction. BphPs have been engineered to form NIR fluorophores, including IFP1.4, Wi-Phy, and the iRFP series, initially by replacement of Asp-207 by His. This position was suggestive because its main chain carbonyl is within hydrogen-bonding distance to pyrrole ring nitrogens of the biliverdin chromophore, thus potentially functioning as a crucial transient proton sink during photoconversion. To explain the origin of fluorescence in these phytofluors, we solved the crystal structures of IFP1.4 and a comparison non-fluorescent monomeric phytochrome DrCBDmon. Met-186 and Val-288 in IFP1.4 are responsible for the formation of a tightly packed hydrophobic hub around the biliverdin D ring. Met-186 is also largely responsible for the blue-shifted IFP1.4 excitation maximum relative to the parent BphP. The structure of IFP1.4 revealed decreased structural heterogeneity and a contraction of two surface regions as direct consequences of side chain substitutions. Unexpectedly, IFP1.4 with Asp-207 reinstalled (IFPrev) has a higher fluorescence quantum yield (∼9%) than most NIR phytofluors published to date. In agreement, fluorescence lifetime measurements confirm the exceptionally long excited state lifetimes, up to 815 ps, in IFP1.4 and IFPrev. Our research helps delineate the origin of fluorescence in engineered BphPs and will facilitate the wide-spread adoption of phytofluors as biomarkers. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

An ion quencher operated lamp for multiplexed fluorescent bioassays.

Science.gov (United States)

Qing, Taiping; Sun, Huanhuan; He, Xiaoxiao; Huang, Xiaoqin; He, Dinggeng; Bu, Hongchang; Qiao, Zhenzhen; Wang, Kemin

2018-02-01

A novel and adjustable lamp based on competitive interaction among dsDNA-SYBR Green I (SGI), ion quencher, and analyte was designed for bioanalysis. The "filament" and switch of the lamp could be customized by employing different dsDNA and ion quencher. The poly(AT/TA) dsDNA was successfully screened as the most effective filament of the lamp. Two common ions, Hg 2+ and Fe 3+ , were selected as the model switch, and the corresponding ligand molecules cysteine (Cys) and pyrophosphate ions (PPi) were selected as the targets. When the fluorescence-quenched dsDNA/SGI-ion complex was introduced into a target-containing system, ions could be bound by competitive molecules and separate from the complex, thereby lighting the lamp. However, no light was observed if the biomolecule could not snatch the metal ions from the complex. Under the optimal conditions, sensitive and selective detection of Cys and PPi was achieved by the lamp, with practical applications in fetal bovine serum and human urine. This ion quencher regulated lamp for fluorescent bioassays is simple in design, fast in operation, and is more convenient than other methods. Significantly, as many molecules could form stable complexes with metal ions selectively, this ion quencher operated lamp has potential for the detection of a wide spectrum of analytes. Graphical abstract A novel and adjustable lamp on the basis of competitive interaction among dsDNA-SYBR Green I, ions quencher and analyte was designed for bioanalysis. The filament and switch of lamp could be customized by employing different dsDNA and ions quencher.

Lifetime-vibrational interference effects in resonantly excited x-ray emission spectra of CO

Energy Technology Data Exchange (ETDEWEB)

Skytt, P.; Glans, P.; Gunnelin, K. [Uppsala Univ. (Sweden)] [and others

1997-04-01

The parity selection rule for resonant X-ray emission as demonstrated for O{sub 2} and N{sub 2} can be seen as an effect of interference between coherently excited degenerate localized core states. One system where the core state degeneracy is not exact but somewhat lifted was previously studied at ALS, namely the resonant X-ray emission of amino-substituted benzene (aniline). It was shown that the X-ray fluorescence spectrum resulting from excitation of the C1s at the site of the {open_quotes}aminocarbon{close_quotes} could be described in a picture separating the excitation and the emission processes, whereas the spectrum corresponding to the quasi-degenerate carbons could not. Thus, in this case it was necessary to take interference effects between the quasi-degenerate intermediate core excited states into account in order to obtain agreement between calculations and experiment. The different vibrational levels of core excited states in molecules have energy splittings which are of the same order of magnitude as the natural lifetime broadening of core excitations in the soft X-ray range. Therefore, lifetime-vibrational interference effects are likely to appear and influence the band shapes in resonant X-ray emission spectra. Lifetime-vibrational interference has been studied in non-resonant X-ray emission, and in Auger spectra. In this report the authors discuss results of selectively excited soft X-ray fluorescence spectra of molecules, where they focus on lifetime-interference effects appearing in the band shapes.

Fluorescent imaging of cancerous tissues for targeted surgery

Science.gov (United States)

Bu, Lihong; Shen, Baozhong; Cheng, Zhen

2014-01-01

To maximize tumor excision and minimize collateral damage is the primary goal of cancer surgery. Emerging molecular imaging techniques have to “image-guided surgery” developing into “molecular imaging-guided surgery”, which is termed “targeted surgery” in this review. Consequently, the precision of surgery can be advanced from tissue-scale to molecule-scale, enabling “targeted surgery” to be a component of “targeted therapy”. Evidence from numerous experimental and clinical studies has demonstrated significant benefits of fluorescent imaging in targeted surgery with preoperative molecular diagnostic screening. Fluorescent imaging can help to improve intraoperative staging and enable more radical cytoreduction, detect obscure tumor lesions in special organs, highlight tumor margins, better map lymph node metastases, and identify important normal structures intraoperatively. Though limited tissue penetration of fluorescent imaging and tumor heterogeneity are two major hurdles for current targeted surgery, multimodality imaging and multiplex imaging may provide potential solutions to overcome these issues, respectively. Moreover, though many fluorescent imaging techniques and probes have been investigated, targeted surgery remains at a proof-of-principle stage. The impact of fluorescent imaging on cancer surgery will likely be realized through persistent interdisciplinary amalgamation of research in diverse fields. PMID:25064553

On-chip mode division multiplexing technologies

DEFF Research Database (Denmark)

Ding, Yunhong; Frellsen, Louise Floor; Guan, Xiaowei

2016-01-01

Space division multiplexing (SDM) is currently widely investigated in order to provide enhanced capacity thanks to the utilization of space as a new degree of multiplexing freedom in both optical fiber communication and on-chip interconnects. Basic components allowing the processing of spatial...... photonic integrated circuit mode (de) multiplexer for few-mode fibers (FMFs)....

The fluorescence behaviour of methyl and phenyl salicylate

Science.gov (United States)

Ford, D.; Thistlethwaite, P. J.; Woolfe, G. J.

1980-01-01

Fluorcsccnce lifetimes tor the 450 nm emission of methyl and phenyl salicylate in various solvents have been measured. Qucnching studics on the 340 nm fluorescence of these molecules point to the existence of three distinct ground state conformers.

Fluorescent Nanodiamonds in Biomedical Applications.

Science.gov (United States)

Mitura, Katarzyna Anna; Włodarczyk, Elżbieta

2018-04-18

Nanoparticles have an extended surface and a large surface area, which is the ratio of the size of the surfacearea to the volume. A functionalized surface can give rise to more modifications and therefore allows this nanomaterial to have new properties. Fluorescent molecules contain fluorophore, which is capable of being excited via the absorption of light energy at a specific wavelength and subsequently emitting radiation energy of a longer wavelength. A chemically modified surface of nanodiamond (ND; by carboxylation) demonstrated biocompatibility with DNA, cytochrome C, and antigens. In turn, fluorescent nanodiamonds (FNDs) belong to a group of new nanomaterials. Their surface can be modified by joining functional groups such as carboxyl, hydroxyl, or amino, after which they can be employed as a fluorescence agent. Their fluorescent properties result from defects in the crystal lattice. FNDs reach dimensions of 4-100 nm, have attributes such as photostability, long fluorescence lifetimes (10 ns), and fluorescence emission between 600 and 700 nm. They are also nontoxic, chemically inert, biocompatible, and environmentally harmless. The main purpose of this article was to present the medical applications of various types of modified NDs.

A new front-face optical cell for measuring weak fluorescent emissions with time resolution in the picosecond time scale.

Science.gov (United States)

Gryczynski, Z; Bucci, E

1993-11-01

Recent developments of ultrafast fluorimeters allow measuring time-resolved fluorescence on the picosecond time scale. This implies one is able to monitor lifetimes and anisotropy decays of highly quenched systems and of systems that contain fluorophores having lifetimes in the subnanosecond range; both systems that emit weak signals. The combination of weak signals and very short lifetimes makes the measurements prone to distortions which are negligible in standard fluorescence experiments. To cope with these difficulties, we have designed a new optical cell for front-face optics which offers to the excitation beam a horizontal free liquid surface in the absence of interactions with optical windows. The new cell has been tested with probes of known lifetimes and anisotropies. It proved very useful in detecting tryptophan fluorescence in hemoglobin. If only diluted samples are available, which cannot be used in front-face optics, regular square geometry can still be utilized by inserting light absorbers into a cuvette of 1 cm path length.

Time-resolved laser-induced fluorescence system

Science.gov (United States)

Bautista, F. J.; De la Rosa, J.; Gallegos, F. J.

2006-02-01

Fluorescence methods are being used increasingly in the measurement of species concentrations in gases, liquids and solids. Laser induced fluorescence is spontaneous emission from atoms or molecules that have been excited by laser radiation. Here we present a time resolved fluorescence instrument that consists of a 5 μJ Nitrogen laser (337.1 nm), a sample holder, a quartz optical fiber, a spectrometer, a PMT and a PC that allows the measurement of visible fluorescence spectra (350-750 nm). Time response of the system is approximately 5 ns. The instrument has been used in the measurement of colored bond paper, antifreeze, diesel, cochineal pigment and malignant tissues. The data acquisition was achieved through computer control of a digital oscilloscope (using General Purpose Interface Bus GPIB) and the spectrometer via serial (RS232). The instrument software provides a graphic interface that lets make some data acquisition tasks like finding fluorescence spectra, and fluorescence lifetimes. The software was developed using the Lab-View 6i graphic programming package and can be easily managed in order to add more functions to it.

Steatocystoma multiplex hos 39-årig kvinde

DEFF Research Database (Denmark)

Duffy, Jonas Raymond; Siersen, Hans Erik; Bonde, Christian T

2011-01-01

-coloured cystic lesions on the chest, abdomen, axillae and back. The patient's clinical presentations and history were compatible with steatocystoma multiplex. Various treatment options for steatocystoma multiplex and steatocystoma multiplex suppurativum have been published and include oral antibiotics...

Explaining HIV Risk Multiplexity: A Social Network Analysis.

Science.gov (United States)

Felsher, Marisa; Koku, Emmanuel

2018-04-21

Risk multiplexity (i.e., overlap in drug-use, needle exchange and sexual relations) is a known risk factor for HIV. However, little is known about predictors of multiplexity. This study uses egocentric data from the Colorado Springs study to examine how individual, behavioral and social network factors influence engagement in multiplex risk behavior. Analyses revealed that compared to Whites, Hispanics were significantly more likely to engage in risk multiplexity and Blacks less so. Respondents who were similar to each other (e.g., in terms of race) had significantly higher odds of being in risk multiplex relationships, and respondents' risk perceptions and network size were significantly associated with engaging in multiplex risk behaviors. Findings from interaction analysis showed the effect of knowing someone with HIV on the odds of multiplexity depends partly on whether respondents' know their HIV status. Findings suggest that demographics, HIV behaviors and network factors impact engagement in multiplex risk behaviors, highlighting the need for multi-level interventions aimed at reducing HIV risk behavior.

Fluorescein Derivatives in Intravital Fluorescence Imaging

Directory of Open Access Journals (Sweden)

Michael S. Roberts

2013-08-01

Full Text Available Intravital fluorescence microscopy enables the direct imaging of fluorophores in vivo and advanced techniques such as fluorescence lifetime imaging (FLIM enable the simultaneous detection of multiple fluorophores. Consequently, it is now possible to record distribution and metabolism of a chemical in vivo and to optimise the delivery of fluorophores in vivo. Recent clinical applications with fluorescein and other intravital fluorescent stains have occurred in neurosurgery, dermatology [including photodynamic therapy (PDT] and endomicroscopy. Potential uses have been identified in periodontal disease, skin graft and cancer surgery. Animal studies have demonstrated that diseased tissue can be specifically stained with fluorophore conjugates. This review focuses on the fluorescein derived fluorophores in common clinical use and provides examples of novel applications from studies in tissue samples.

Percolation in real multiplex networks

Science.gov (United States)

Bianconi, Ginestra; Radicchi, Filippo

2016-12-01

We present an exact mathematical framework able to describe site-percolation transitions in real multiplex networks. Specifically, we consider the average percolation diagram valid over an infinite number of random configurations where nodes are present in the system with given probability. The approach relies on the locally treelike ansatz, so that it is expected to accurately reproduce the true percolation diagram of sparse multiplex networks with negligible number of short loops. The performance of our theory is tested in social, biological, and transportation multiplex graphs. When compared against previously introduced methods, we observe improvements in the prediction of the percolation diagrams in all networks analyzed. Results from our method confirm previous claims about the robustness of real multiplex networks, in the sense that the average connectedness of the system does not exhibit any significant abrupt change as its individual components are randomly destroyed.

Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

Directory of Open Access Journals (Sweden)

Yogini P. Bhavsar

2011-01-01

Full Text Available Topological variants of single-strand DNA (ssDNA structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC and 1,3-diaza-2-oxophenoxazine (tC∘, can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

Photoluminescence decay lifetime measurements of hemicyanine derivatives of different alkyl chain lengths

International Nuclear Information System (INIS)

Shim, Taekyu; Lee, Myounghee; Kim, Sungho; Sung, Jaeho; Rhee, Bum Ku; Kim, Doseok; Kim, Hyunsung; Yoon, Kyung Byung

2004-01-01

The fluorescence upconversion setup for the detection of photoluminescence (PL) decay lifetime with subpicosecond time resolution was constructed, and the photoluminescence phenomena of several hemicyanine dyes with alkyl chains of different chain lengths tethered to the N atom of the pyridine moiety (HC-n, n=6, 15, 22) in methanol were investigated. The average decay lifetimes of the solutions determined from the measured data by multi-order exponential decay curve fitting were ∼27 ps at the PL peak wavelength. It was found that the PL decay properties did not depend on the alkyl chain length in the molecule, implying that the twist of the alkylpyridinium ring of the molecule is not possible as a nonfluorescing relaxation pathway. The time-dependent PL spectra constructed from the PL lifetime data showed the dynamic Stokes shift of ∼1000 cm -1

Spatial analysis of various multiplex cinema types

Directory of Open Access Journals (Sweden)

Young-Seo Park

2016-03-01

Full Text Available This study identifies the spatial characteristics and relationships of each used space according to the multiplex type. In this study, multiplexes are classified according to screen rooms and circulation systems, and each used space is quantitatively analyzed. The multiplex type based on screen rooms and moving line systems influences the relationship and characteristics of each used space in various ways. In particular, the structure of the used space of multiplexes has a significant effect on profit generation and audience convenience.

Fluorescence enhancement by Au nanostructures: nanoshells and nanorods.

Science.gov (United States)

Bardhan, Rizia; Grady, Nathaniel K; Cole, Joseph R; Joshi, Amit; Halas, Naomi J

2009-03-24

Metallic nanoparticles influence the quantum yield and lifetime of adjacent fluorophores in a manner dependent on the properties of the nanostructure. Here we directly compare the fluorescence enhancement of the near-infrared fluorophore IR800 by Au nanoshells (NSs) and Au nanorods (NRs), where human serum albumin (HSA) serves as a spacer layer between the nanoparticle and the fluorophore. Our measurements reveal that the quantum yield of IR800 is enhanced from approximately 7% as an isolated fluorophore to 86% in a NSs-HSA-IR800 complex and 74% in a NRs-HSA-IR800 complex. This dramatic increase in fluorescence shows tremendous potential for contrast enhancement in fluorescence-based bioimaging.

A novel IPTV program multiplex access system to EPON

Science.gov (United States)

Xu, Xian; Liu, Deming; He, Wei; Lu, Xi

2007-11-01

With the rapid development of high speed networks, such as Ethernet Passive Optical Network (EPON), traffic patterns in access networks have evolved from traditional text-oriented service to the mixed text-, voice- and video- based services, leading to so called "Triple Play". For supporting IPTV service in EPON access network infrastructure, in this article we propose a novel IPTV program multiplex access system to EPON, which enables multiple IPTV program source servers to seamlessly access to IPTV service access port of optical line terminal (OLT) in EPON. There are two multiplex schemes, namely static multiplex scheme and dynamic multiplex scheme, in implementing the program multiplexing. Static multiplex scheme is to multiplex all the IPTV programs and forward them to the OLT, regardless of the need of end-users. While dynamic multiplex scheme can dynamically multiplex and forward IPTV programs according to what the end-users actually demand and those watched by no end-user would not be multiplexed. By comparing these two schemes, a reduced traffic of EPON can be achieved by using dynamic multiplex scheme, especially when most end-users are watching the same few IPTV programs. Both schemes are implemented in our system, with their hardware and software designs described.

Bilevel alarm monitoring multiplexer

International Nuclear Information System (INIS)

Johnson, C.S.

1977-06-01

This report describes the operation of the Bilevel Alarm Monitoring Multiplexer used in the Adaptive Intrusion Data System (AIDS) to transfer and control alarm signals being sent to the Nova 2 computer, the Memory Controlled Data Processor, and its own integral Display Panel. The multiplexer can handle 48 alarm channels and format the alarms into binary formats compatible with the destination of the alarm data

5f state interaction with inner coordination sphere ligands: einsteinium 3+ ion fluorescence in aqueous and organic phases

International Nuclear Information System (INIS)

Beitz, J.V.; Wester, D.W.; Williams, C.W.

1983-01-01

The interaction between 5f electron states of einsteinium 3+ ion and coordinated ligands in solution has been probed using laser-induced fluorescence. Aquo einsteinium 3+ ion was observed to fluoresce from its first excited J = 5 state in a broad-band peaking at 9260 wavenumbers. The observed fluorescence lifetimes were 1.05 microseconds and 2.78 microseconds in H 2 O and D 2 O (99+ % D atom), respectively. The non-radiative decay rates derived from the lifetime data are compared with previously reported data for Cm, Sm, Eu, Tb, and Dy aquo 3+ ions. The 5f actinide states exhibit substantially greater non-radiative decay rates than do lanthanide 4f states of similar energy gap. This provides evidence that actinide 5f electrons interact more strongly with their inner coordination sphere than do lanthanide ion 4f electrons. The fluorescence lifetime of einsteinium 3+ ion complexed with 1 formal di(2-ethylhexyl)orthophosphoric acid in h-heptane was 2.34 microseconds. 3 figures, 1 table

Rapid Simultaneous Amplification and Detection of the MBR/JH Chromosomal Translocation by Fluorescence Melting Curve Analysis

Science.gov (United States)

Bohling, Sandra D.; King, Thomas C.; Wittwer, Carl T.; Elenitoba-Johnson, Kojo S. J.

1999-01-01

Polymerase chain reaction (PCR) amplification and product analysis for the detection of chromosomal translocations, such as the t(14;18), has traditionally been a two-step process. PCR product detection has generally entailed gel electrophoresis and/or hybridization or sequencing for confirmation of assay specificity. Using a microvolume fluorimeter integrated with a thermal cycler and a PCR-compatible double-stranded DNA (dsDNA) binding fluorescent dye (SYBR Green I), we investigated the feasibility of simultaneous thermal amplification and detection of MBR/JH translocation products by fluorescence melting curve analysis. We analyzed DNA from 30 cases of lymphoproliferative disorders comprising 19 cases of previously documented MBR/JH-positive follicle center lymphoma and 11 reactive lymphadenopathies. The samples were coded and analyzed blindly for the presence of MBR/JH translocations by fluorescence melting curve analysis. We also performed dilutional assays using the MBR/JH-positive cell line SUDHL-6. Multiplex PCR for MBR/JH and β-globin was used to simultaneously assess sample adequacy. All (100%) of the 19 cases previously determined to be MBR/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at ∼90°C by melting curve analysis after amplification. Fluorescence melting peaks obtained by plotting the negative derivative of fluorescence over temperature (−dF/dT) versus temperature (T) showed melting temperatures (Tm) at 88.85 ± 1.15°C. In addition, multiplex assays using both MBR/JH and β-globin primers yielded easily distinguishable fluorescence melting peaks at ∼90°C and 81.2°C, respectively. Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 100-fold. Simultaneous amplification and fluorescence melting curve analysis is a simple, reliable, and sensitive method

Quantitative frequency-domain fluorescence spectroscopy in tissues and tissue-like media

Science.gov (United States)

Cerussi, Albert Edward

1999-09-01

In the never-ending quest for improved medical technology at lower cost, modern near-infrared optical spectroscopy offers the possibility of inexpensive technology for quantitative and non-invasive diagnoses. Hemoglobin is the dominant chromophore in the 700-900 nm spectral region and as such it allows for the optical assessment of hemoglobin concentration and tissue oxygenation by absorption spectroscopy. However, there are many other important physiologically relevant compounds or physiological states that cannot be effectively sensed via optical methods because of poor optical contrast. In such cases, contrast enhancements are required. Fluorescence spectroscopy is an attractive component of optical tissue spectroscopy. Exogenous fluorophores, as well as some endogenous ones, may furnish the desperately needed sensitivity and specificity that is lacking in near-infrared optical tissue spectroscopy. The main focus of this thesis was to investigate the generation and propagation of fluorescence photons inside tissues and tissue-like media (i.e., scattering dominated media). The standard concepts of fluorescence spectroscopy have been incorporated into a diffusion-based picture that is sometimes referred to as photon migration. The novelty of this work lies in the successful quantitative recovery of fluorescence lifetimes, absolute fluorescence quantum yields, fluorophore concentrations, emission spectra, and both scattering and absorption coefficients at the emission wavelength from a tissue-like medium. All of these parameters are sensitive to the fluorophore local environment and hence are indicators of the tissue's physiological state. One application demonstrating the capabilities of frequency-domain lifetime spectroscopy in tissue-like media is a study of the binding of ethidium bromide to bovine leukocytes in fresh milk. Ethidium bromide is a fluorescent dye that is commonly used to label DNA, and hence visualize chromosomes in cells. The lifetime of

Frequency multiplexing for readout of spin qubits

Energy Technology Data Exchange (ETDEWEB)

Hornibrook, J. M.; Colless, J. I.; Mahoney, A. C.; Croot, X. G.; Blanvillain, S.; Reilly, D. J., E-mail: david.reilly@sydney.edu.au [ARC Centre of Excellence for Engineered Quantum Systems, School of Physics, University of Sydney, Sydney, NSW 2006 (Australia); Lu, H.; Gossard, A. C. [Materials Department, University of California, Santa Barbara, California 93106 (United States)

2014-03-10

We demonstrate a low loss, chip-level frequency multiplexing scheme for readout of scaled-up spin qubit devices. By integrating separate bias tees and resonator circuits on-chip for each readout channel, we realise dispersive gate-sensing in combination with charge detection based on two radio frequency quantum point contacts. We apply this approach to perform multiplexed readout of a double quantum dot in the few-electron regime and further demonstrate operation of a 10-channel multiplexing device. Limitations for scaling spin qubit readout to large numbers of multiplexed channels are discussed.

Preliminary study of visual effect of multiplex hologram

Science.gov (United States)

Fu, Huaiping; Xiong, Bingheng; Yang, Hong; Zhang, Xueguo

2004-06-01

The process of any movement of real object can be recorded and displayed by a multiplex holographic stereogram. An embossing multiplex holographic stereogram and a multiplex rainbow holographic stereogram have been made by us, the multiplex rainbow holographic stereogram reconstructs the dynamic 2D line drawing of speech organs, the embossing multiplex holographic stereogram reconstructs the process of an old man drinking water. In this paper, we studied the visual result of an embossing multiplex holographic stereogram made with 80 films of 2-D pictures. Forty-eight persons of aged from 13 to 67 were asked to see the hologram and then to answer some questions about the feeling of viewing. The results indicate that this kind of holograms could be accepted by human visual sense organ without any problem. This paper also discusses visual effect of the multiplex holography stereograms base on visual perceptual psychology. It is open out that the planar multiplex holograms can be recorded and present the movement of real animal and object. Not only have the human visual perceptual constancy for shape, just as that size, color, etc... but also have visual perceptual constancy for binocular parallax.

Development of a GeXP-multiplex PCR assay for the simultaneous detection and differentiation of six cattle viruses.

Directory of Open Access Journals (Sweden)

Qing Fan

Full Text Available Foot-and-mouth disease virus (FMDV, Bluetongue virus (BTV, Vesicular stomatitis Virus (VSV, Bovine viral diarrheal (BVDV, Bovine rotavirus (BRV, and Bovine herpesvirus 1 (IBRV are common cattle infectious viruses that cause a great economic loss every year in many parts of the world. A rapid and high-throughput GenomeLab Gene Expression Profiler (GeXP analyzer-based multiplex PCR assay was developed for the simultaneous detection and differentiation of these six cattle viruses. Six pairs of chimeric primers consisting of both the gene-specific primer and a universal primer were designed and used for amplification. Then capillary electrophoresis was used to separate the fluorescent labeled PCR products according to the amplicons size. The specificity of GeXP-multiplex PCR assay was examined with samples of the single template and mixed template of six viruses. The sensitivity was evaluated using the GeXP-multiplex PCR assay on serial 10-fold dilutions of ssRNAs obtained via in vitro transcription. To further evaluate the reliability, 305 clinical samples were tested by the GeXP-multiplex PCR assay. The results showed that the corresponding virus specific fragments of genes were amplified. The detection limit of the GeXP-multiplex PCR assay was 100 copies/μL in a mixed sample of ssRNAs containing target genes of six different cattle viruses, whereas the detection limit for the Gexp-mono PCR assay for a single target gene was 10 copies/μL. In detection of viruses in 305 clinical samples, the results of GeXP were consistent with simplex real-time PCR. Analysis of positive samples by sequencing demonstrated that the GeXP-multiplex PCR assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP-multiplex PCR assay is a high throughput, specific, sensitive, rapid and simple method for the detection and differentiation of six cattle viruses. It is an effective tool that can be applied for the rapid differential diagnosis

Demonstration of hybrid orbital angular momentum multiplexing and time-division multiplexing passive optical network.

Science.gov (United States)

Wang, Andong; Zhu, Long; Liu, Jun; Du, Cheng; Mo, Qi; Wang, Jian

2015-11-16

Mode-division multiplexing passive optical network (MDM-PON) is a promising scheme for next-generation access networks to further increase fiber transmission capacity. In this paper, we demonstrate the proof-of-concept experiment of hybrid mode-division multiplexing (MDM) and time-division multiplexing (TDM) PON architecture by exploiting orbital angular momentum (OAM) modes. Bidirectional transmissions with 2.5-Gbaud 4-level pulse amplitude modulation (PAM-4) downstream and 2-Gbaud on-off keying (OOK) upstream are demonstrated in the experiment. The observed optical signal-to-noise ratio (OSNR) penalties for downstream and upstream transmissions at a bit-error rate (BER) of 2 × 10(-3) are less than 2.0 dB and 3.0 dB, respectively.

Developing a novel fiber optic fluorescence device for multiplexed high-throughput cytotoxic screening.

Science.gov (United States)

Lee, Dennis; Barnes, Stephen

2010-01-01

The need for new pharmacological agents is unending. Yet the drug discovery process has changed substantially over the past decade and continues to evolve in response to new technologies. There is presently a high demand to reduce discovery time by improving specific lab disciplines and developing new technology platforms in the area of cell-based assay screening. Here we present the developmental concept and early stage testing of the Ab-Sniffer, a novel fiber optic fluorescence device for high-throughput cytotoxicity screening using an immobilized whole cell approach. The fused silica fibers are chemically functionalized with biotin to provide interaction with fluorescently labeled, streptavidin functionalized alginate-chitosan microspheres. The microspheres are also functionalized with Concanavalin A to facilitate binding to living cells. By using lymphoma cells and rituximab in an adaptation of a well-known cytotoxicity protocol we demonstrate the utility of the Ab-Sniffer for functional screening of potential drug compounds rather than indirect, non-functional screening via binding assay. The platform can be extended to any assay capable of being tied to a fluorescence response including multiple target cells in each well of a multi-well plate for high-throughput screening.

Signal multiplexing scheme for LINAC

International Nuclear Information System (INIS)

Sujo, C.I.; Mohan, Shyam; Joshi, Gopal; Singh, S.K.; Karande, Jitendra

2004-01-01

For the proper operation of the LINAC some signals, RF (radio frequency) as well as LF (low frequency) have to be available at the Master Control Station (MCS). These signals are needed to control, calibrate and characterize the RF fields in the resonators. This can be achieved by proper multiplexing of various signals locally and then routing the selected signals to the MCS. A multiplexing scheme has been designed and implemented, which will allow the signals from the selected cavity to the MCS. High isolation between channels and low insertion loss for a given signal are important issues while selecting the multiplexing scheme. (author)

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

Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA

Science.gov (United States)

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-10-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ˜40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

Thermally multiplexed polymerase chain reaction.

Science.gov (United States)

Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

2015-07-01

Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

Science.gov (United States)

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Sensitive determination of nucleic acids using organic nanoparticle fluorescence probes

Science.gov (United States)

Zhou, Yunyou; Bian, Guirong; Wang, Leyu; Dong, Ling; Wang, Lun; Kan, Jian

2005-06-01

This paper describes the preparation of organic nanoparticles by reprecipitation method under sonication and vigorous stirring. Transmission electron microscopy (TEM) was used to characterize the size and size distribution of the luminescent nanoparticles. Their average diameter was about 25 nm with a size variation of ±18%. The fluorescence decay lifetime of the nanoparticles also was determined on a self-equipped fluorospectrometer with laser light source. The lifetime (˜0.09 μs) of nanoparticles is about three times long as that of the monomer. The nanoparticles were in abundant of hydrophilic groups, which increased their miscibility in aqueous solution. These organic nanoparticles have high photochemical stability, excellent resistance to chemical degradation and photodegradation, and a good fluorescence quantum yield (25%). The fluorescence can be efficiently quenched by nucleic acids. Based on the fluorescence quenching of nanoparticles, a fluorescence quenching method was developed for determination of microamounts of nucleic acids by using the nanoparticles as a new fluorescent probe. Under optimal conditions, maximum fluorescence quenching is produced, with maximum excitation and emission wavelengths of 345 and 402 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.4-19.0 μg ml -1 for calf thymus DNA (ct-DNA) and 0.3-19.0 μg ml -1 for fish sperm DNA (fs-DNA). The corresponding detection limits are 0.25 μg ml -1 for ct-DNA and 0.17 μg ml -1 for fs-DNA. The relative standard deviation of six replicate measurements is 1.3-2.1%. The method is simple, rapid and sensitive with wide linear range. The recovery and relative standard deviation are very satisfactory.

Long term optical stability of fluorescent solar concentrator plates

NARCIS (Netherlands)

Slooff, L.H.; Bakker, N.J.; Sommeling, P.M.; Büchtemann, A.; Wedel, A.; Sark, W.G.J.H.M. van

2014-01-01

Fluorescent solar concentrators offer an alternative approach for low-cost photovoltaic energy conversion. For successful application, not only the power conversion efficiency and cost are important, but also lifetime or stability of the devices. As today’s concentrator is made of polymer sheets

Long-term optical stability of fluorescent solar concentrator plates

NARCIS (Netherlands)

Slooff, Lenneke H.; Bakker, Nicolaas J.; Sommeling, Paul M.; Büchtemann, Andreas; Wedel, Armin; Van Sark, Wilfried G J H M

2014-01-01

Fluorescent solar concentrators offer an alternative approach for low-cost photovoltaic energy conversion. For successful application, not only the power conversion efficiency and cost are important, but also lifetime or stability of the devices. As today's concentrator is made of polymer sheets

Fluorescence lifetime spectroscopy: potential for in-vivo estimation of skin fluorophores changes after low power laser treatment

Science.gov (United States)

Ferulova, Inesa; Lihachev, Alexey; Spigulis, Janis

2013-11-01

The impact of visible cwlaser irradiation on skin autofluorescence lifetimes was investigated in spectral range from 450 nm to 600 nm. Skin optical provocations were performed during 1 min by 405 nm low power cw laser with power density up to 20 mW/cm2. Autofluorescence lifetimes were measured before and immediately after the optical provocation.

Excited-state lifetime measurement of silicon vacancy centers in diamond by single-photon frequency upconversion

Science.gov (United States)

Rong, Youying; Ma, Jianhui; Chen, Lingxiao; Liu, Yan; Siyushev, Petr; Wu, Botao; Pan, Haifeng; Jelezko, Fedor; Wu, E.; Zeng, Heping

2018-05-01

We report a method with high time resolution to measure the excited-state lifetime of silicon vacancy centers in bulk diamond avoiding timing jitter from the single-photon detectors. Frequency upconversion of the fluorescence emitted from silicon vacancy centers was achieved from 738 nm to 436 nm via sum frequency generation with a short pump pulse. The excited-state lifetime can be obtained by measuring the intensity of upconverted light while the pump delay changes. As a probe, a pump laser with pulse duration of 11 ps provided a high temporal resolution of the measurement. The lifetime extracted from the pump–probe curve was 0.755 ns, which was comparable to the timing jitter of the single-photon detectors.

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

Time-resolved fluorescence monitoring of cholesterol in peripheral blood mononuclear cells

Science.gov (United States)

Martinakova, Z.; Horilova, J.; Lajdova, I.; Marcek Chorvatova, A.

2014-12-01

Precise evaluation of intracellular cholesterol distribution is crucial for improving diagnostics of diseased states associated with cholesterol alteration. Time-resolved fluorescence techniques are tested for non-invasive investigation of cholesterol in living cells. Fluorescent probe NBD attached to cholesterol was employed to evaluate cholesterol distribution in peripheral blood mononuclear cells (PBMC) isolated from the human blood. Fluorescence Lifetime Imaging Microscopy (FLIM) was successfully applied to simultaneously monitor the spatial distribution and the timeresolved characteristics of the NBD-cholesterol fluorescence in PBMC. Gathered data are the first step in the development of a new perspective non-invasive diagnostic method for evaluation of cholesterol modifications in diseases associated with disorders of lipid metabolism.

Laguerre Gaussian beam multiplexing through turbulence

CSIR Research Space (South Africa)

Trichili, A

2014-08-17

Full Text Available We analyze the effect of atmospheric turbulence on the propagation of multiplexed Laguerre Gaussian modes. We present a method to multiplex Laguerre Gaussian modes using digital holograms and decompose the resulting field after encountering a...

Fluorescent nanoparticles for intracellular sensing: a review.

Science.gov (United States)

Ruedas-Rama, Maria J; Walters, Jamie D; Orte, Angel; Hall, Elizabeth A H

2012-11-02

Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy. Copyright © 2012 Elsevier B.V. All rights reserved.

The photoluminescence of a fluorescent lamp: didactic experiments on the exponential decay

Science.gov (United States)

Onorato, Pasquale; Gratton, Luigi; Malgieri, Massimiliano; Oss, Stefano

2017-01-01

The lifetimes of the photoluminescent compounds contained in the coating of fluorescent compact lamps are usually measured using specialised instruments, including pulsed lasers and/or spectrofluorometers. Here we discuss how some low cost apparatuses, based on the use of either sensors for the educational lab or commercial digital photo cameras, can be employed to the same aim. The experiments do not require that luminescent phosphors are hazardously extracted from the compact fluorescent lamp, that also contains mercury. We obtain lifetime measurements for specific fluorescent elements of the bulb coating, in good agreement with the known values. We also address the physical mechanisms on which fluorescence lamps are based in a simplified way, suitable for undergraduate students; and we discuss in detail the physics of the lamp switch-off by analysing the time dependent spectrum, measured through a commercial fiber-optic spectrometer. Since the experiment is not hazardous in any way, requires a simple setup up with instruments which are commonly found in educational labs, and focuses on the typical features of the exponential decay, it is suitable for being performed in the undergraduate laboratory.

Advanced combinational microfluidic multiplexer for fuel cell reactors

International Nuclear Information System (INIS)

Lee, D W; Kim, Y; Cho, Y-H; Doh, I

2013-01-01

An advanced combinational microfluidic multiplexer capable to address multiple fluidic channels for fuel cell reactors is proposed. Using only 4 control lines and two different levels of control pressures, the proposed multiplexer addresses up to 19 fluidic channels, at least two times larger than the previous microfluidic multiplexers. The present multiplexer providing high control efficiency and simple structure for channel addressing would be used in the application areas of the integrated microfluidic systems such as fuel cell reactors and dynamic pressure generators

Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR.

Science.gov (United States)

Woo, Nain; Kim, Su-Kang; Sun, Yucheng; Kang, Seong Ho

2018-01-01

Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer's disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer's. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120-180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4-62.0pM, which were approximately 100-100,000 times more sensitive than previous Alzheimer's diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer's patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer's with high sensitivity and short analysis time even with direct use of whole blood. Copyright © 2017 Elsevier B.V. All rights reserved.

Platform for Quantitative Evaluation of Spatial Intratumoral Heterogeneity in Multiplexed Fluorescence Images.

Science.gov (United States)

Spagnolo, Daniel M; Al-Kofahi, Yousef; Zhu, Peihong; Lezon, Timothy R; Gough, Albert; Stern, Andrew M; Lee, Adrian V; Ginty, Fiona; Sarachan, Brion; Taylor, D Lansing; Chennubhotla, S Chakra

2017-11-01

We introduce THRIVE (Tumor Heterogeneity Research Interactive Visualization Environment), an open-source tool developed to assist cancer researchers in interactive hypothesis testing. The focus of this tool is to quantify spatial intratumoral heterogeneity (ITH), and the interactions between different cell phenotypes and noncellular constituents. Specifically, we foresee applications in phenotyping cells within tumor microenvironments, recognizing tumor boundaries, identifying degrees of immune infiltration and epithelial/stromal separation, and identification of heterotypic signaling networks underlying microdomains. The THRIVE platform provides an integrated workflow for analyzing whole-slide immunofluorescence images and tissue microarrays, including algorithms for segmentation, quantification, and heterogeneity analysis. THRIVE promotes flexible deployment, a maintainable code base using open-source libraries, and an extensible framework for customizing algorithms with ease. THRIVE was designed with highly multiplexed immunofluorescence images in mind, and, by providing a platform to efficiently analyze high-dimensional immunofluorescence signals, we hope to advance these data toward mainstream adoption in cancer research. Cancer Res; 77(21); e71-74. ©2017 AACR . ©2017 American Association for Cancer Research.

Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

Directory of Open Access Journals (Sweden)

Kumar Sukhdeo

Full Text Available Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116. Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

Multiplexed image storage by electromagnetically induced transparency in a solid

Science.gov (United States)

Heinze, G.; Rentzsch, N.; Halfmann, T.

2012-11-01

We report on frequency- and angle-multiplexed image storage by electromagnetically induced transparency (EIT) in a Pr3+:Y2SiO5 crystal. Frequency multiplexing by EIT relies on simultaneous storage of light pulses in atomic coherences, driven in different frequency ensembles of the inhomogeneously broadened solid medium. Angular multiplexing by EIT relies on phase matching of the driving laser beams, which permits simultaneous storage of light pulses propagating under different angles into the crystal. We apply the multiplexing techniques to increase the storage capacity of the EIT-driven optical memory, in particular to implement multiplexed storage of larger two-dimensional amounts of data (images). We demonstrate selective storage and readout of images by frequency-multiplexed EIT and angular-multiplexed EIT, as well as the potential to combine both multiplexing approaches towards further enhanced storage capacities.

Frequency-domain readout multiplexing of transition-edge sensor arrays

Energy Technology Data Exchange (ETDEWEB)

Lanting, T.M. [Physics Department, University of California, Berkeley, CA 94720 (United States)]. E-mail: tlanting@berkeley.edu; Arnold, K. [Physics Department, University of California, Berkeley, CA 94720 (United States); Cho, Hsiao-Mei [Physics Department, University of California, Berkeley, CA 94720 (United States); Clarke, John [Physics Department, University of California, Berkeley, CA 94720 (United States); Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Dobbs, Matt [Physics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Holzapfel, William [Physics Department, University of California, Berkeley, CA 94720 (United States); Lee, Adrian T. [Physics Department, University of California, Berkeley, CA 94720 (United States); Physics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Lueker, M. [Physics Department, University of California, Berkeley, CA 94720 (United States); Richards, P.L. [Physics Department, University of California, Berkeley, CA 94720 (United States); Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Space Sciences Laboratory, University of California, Berkeley, CA 94720 (United States); Smith, A.D. [Northrop-Grumman, Redondo Beach, CA 94278 (United States); Spieler, H.G. [Physics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)

2006-04-15

We have demonstrated frequency-domain readout multiplexing of eight channels for superconducting transition-edge sensor bolometer arrays. The multiplexed readout noise is 6.5 pA/{radical}Hz, well below the bolometer dark noise of 15-20 pA/{radical}Hz. We measure an upper limit on crosstalk of 0.004 between channels adjacent in frequency which meets our design requirement of 0.01. We have observed vibration insensitivity in our frequency-domain multiplexed transition-edge sensors, making this system very attractive for telescope and satellite observations. We also discuss extensions to our multiplexed readout. In particular, we are developing a SQUID flux-locked loop that is entirely cold and collaborating on digital multiplexer technology in order to scale up the number of multiplexed channels.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

Science.gov (United States)

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Immunization of Epidemics in Multiplex Networks

Science.gov (United States)

Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

2014-01-01

Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks. PMID:25401755

Immunization of epidemics in multiplex networks.

Science.gov (United States)

Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

2014-01-01

Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks.

Multiwavelength FLIM: new concept for fluorescence diagnosis

Science.gov (United States)

Rück, Angelika; Lorenz, S.; Hauser, Carmen; Mosch, S.; Kalinina, S.

2012-03-01

Fluorescence guided tumor resection is very well accepted in the case of bladder cancer and brain tumor, respectively. However, false positive results are one of the major problems, which will make the discrimination between tumor tissue and inflammation difficult. In contrast fluorescence lifetime imaging (FLIM) and especially spectral resolved FLIM (SLIM) can significantly improve the analysis. The fluorescence decay of a fluorophore in many cases does not show a simple monoexponential profile. A very complex situation arises, when more than one compound has to be analyzed. This could be the case when endogenous fluorophores of living cells and tissues have to be discriminated to identify oxidative metabolic changes. Other examples are PDT, when different photosensitizer metabolites are observed simultaneously. In those cases a considerable improvement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated. Within this presentation the principles of spectral and time-resolved fluorescence imaging will be discussed. Successful applications as autofluorescence and 5-ALA induced porphyrin fluorescence will be described in more detail.

Multiplexed interfacial transduction of nucleic acid hybridization using a single color of immobilized quantum dot donor and two acceptors in fluorescence resonance energy transfer.

Science.gov (United States)

Algar, W Russ; Krull, Ulrich J

2010-01-01

A multiplexed solid-phase assay for the detection of nucleic acid hybridization was developed on the basis of a single color of immobilized CdSe/ZnS quantum dot (QD) as a donor in fluorescence resonance energy transfer (FRET). This work demonstrated that two channels of detection did not necessitate two different QD donors. Two probe oligonucleotides were coimmobilized on optical fibers modified with QDs, and a sandwich assay was used to associate the acceptor dyes with interfacial hybridization events without target labeling. FRET-sensitized acceptor emission provided an analytical signal that was concentration dependent down to 10 nM. Changes in the ratio of coimmobilized probe oligonucleotides were found to yield linear changes in the relative amounts of acceptor emission. These changes were compared to previous studies that used mixed films of two QD donors for two detection channels. The analysis indicated that probe dilution effects were primarily driven by changes in acceptor number density and that QD dilution effects or changes in mean donor-acceptor distance were secondary. Hybridization kinetics were found to be consistent between different ratios of coimmobilized probes, suggesting that hybridization in this type of system occurred via the accepted model for solid-phase hybridization, where adsorption and then diffusion at the solid interface drove hybridization.

Multiplexed detection of mycotoxins in foods with a regenerable array.

Science.gov (United States)

Ngundi, Miriam M; Shriver-Lake, Lisa C; Moore, Martin H; Ligler, Frances S; Taitt, Chris R

2006-12-01

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

Science.gov (United States)

Petryayeva, Eleonora; Algar, W Russ

2014-03-18

Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

Science.gov (United States)

Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

2018-04-03

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

Proximal Sensing of Plant-Pathogen Interactions in Spring Barley with Three Fluorescence Techniques

Directory of Open Access Journals (Sweden)

Georg Leufen

2014-06-01

Full Text Available In the last years fluorescence spectroscopy has come to be viewed as an essential approach in key research fields of applied plant sciences. However, the quantity and particularly the quality of information produced by different equipment might vary considerably. In this study we investigate the potential of three optical devices for the proximal sensing of plant-pathogen interactions in four genotypes of spring barley. For this purpose, the fluorescence lifetime, the image-resolved multispectral fluorescence and selected indices of a portable multiparametric fluorescence device were recorded at 3, 6, and 9 days after inoculation (dai from healthy leaves as well as from leaves inoculated with powdery mildew (Blumeria graminis or leaf rust (Puccinia hordei. Genotype-specific responses to pathogen infections were revealed already at 3 dai by higher fluorescence mean lifetimes in the spectral range from 410 to 560 nm in the less susceptible varieties. Noticeable pathogen-induced modifications were also revealed by the ‘Blue-to-Far-Red Fluorescence Ratio’ and the ‘Simple Fluorescence Ratio’. Particularly in the susceptible varieties the differences became more evident in the time-course of the experiment i.e., following the pathogen development. The relevance of the blue and green fluorescence to exploit the plant-pathogen interaction was demonstrated by the multispectral fluorescence imaging system. As shown, mildewed leaves were characterized by exceptionally high blue fluorescence, contrasting the values observed in rust inoculated leaves. Further, we confirm that the intensity of green fluorescence depends on the pathogen infection and the stage of disease development; this information might allow a differentiation of both diseases. Moreover, our results demonstrate that the detection area might influence the quality of the information, although it had a minor impact only in the current study. Finally, we highlight the relevance of

Prototype data terminal-multiplexer/demultiplexer

Science.gov (United States)

Leck, D. E.; Goodwin, J. E.

1972-01-01

The design and operation of a quad redundant data terminal and a multiplexer/demultiplexer (MDU) is described. The most unique feature is the design of the quad redundant data terminal. This is one of the few designs where the unit is fail/op, fail/op, fail/safe. Laboratory tests confirm that the unit will operate satisfactorily with the failure of three out of four channels. Although the design utilizes state-of-the-art technology, the waveform error checks, the voting techniques, and the parity bit checks are believed to be used in unique configurations. Correct word selection routines are also novel. The MDU design, while not redundant, utilizes, the latest state-of-the-art advantages of light coupler and interested amplifiers. Much of the technology employed was an evolution of prior NASA contracts related to the Addressable Time Division Data System. A good example of the earlier technology development was the development of a low level analog multiplexer, a high level analog multiplexer, and a digital multiplexer. A list of all drawings is included for reference and all schematic, block and timing diagrams are incorporated.

Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

Science.gov (United States)

Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael

2009-01-01

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

Steady state and time resolved fluorescence studies of azadioxatriangulenium (ADOTA) fluorophore in silica and PVA thin films

DEFF Research Database (Denmark)

Chib, Rahul; Raut, Sangram; Shah, Sunil

2015-01-01

A cationic azadioxatriangulenium dye was entrapped in silica thin films obtained by the sol-gel process and in poly (vinyl) alcohol (PVA) thin films. Azadioxatriangulenium is a red emitting fluorophore with a long fluorescence lifetime of ∼20 ns. The fluorescent properties of azadioxatriangulenium...

Immunization of epidemics in multiplex networks.

Directory of Open Access Journals (Sweden)

Dawei Zhao

Full Text Available Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted immunization and layer node-based random (targeted immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF networks.

FastFLIM, the all-in-one engine for measuring photoluminescence lifetime of 100 picoseconds to 100 milliseconds

Science.gov (United States)

Sun, Yuansheng; Coskun, Ulas; Liao, Shih-Chu Jeff; Barbieri, Beniamino

2018-02-01

Photoluminescence (PL) refers to light emission initiated by any form of photon excitation. PL spectroscopy and microscopy imaging has been widely applied in material, chemical and life sciences. Measuring its lifetime yields a new dimension of the PL imaging and opens new opportunities for many PL applications. In solar cell research, quantification of the PL lifetime has become an important evaluation for the characteristics of the Perovskite thin film. Depending upon the PL process (fluorescence, phosphorescence, photon upconversion, etc.), the PL lifetimes to be measured can vary in a wide timescale range (e.g. from sub-nanoseconds to microseconds or even milliseconds) - it is challenging to cover this wide range of lifetime measurements by a single technique efficiently. Here, we present a novel digital frequency domain (DFD) technique named FastFLIM, capable of measuring the PL lifetime from 100 ps to 100 ms at the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear leastsquare fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the PL lifetime microscopy imaging.

Optimizing diffusion in multiplexes by maximizing layer dissimilarity

Science.gov (United States)

Serrano, Alfredo B.; Gómez-Gardeñes, Jesús; Andrade, Roberto F. S.

2017-05-01

Diffusion in a multiplex depends on the specific link distribution between the nodes in each layer, but also on the set of the intralayer and interlayer diffusion coefficients. In this work we investigate, in a quantitative way, the efficiency of multiplex diffusion as a function of the topological similarity among multiplex layers. This similarity is measured by the distance between layers, taken among the pairs of layers. Results are presented for a simple two-layer multiplex, where one of the layers is held fixed, while the other one can be rewired in a controlled way in order to increase or decrease the interlayer distance. The results indicate that, for fixed values of all intra- and interlayer diffusion coefficients, a large interlayer distance generally enhances the global multiplex diffusion, providing a topological mechanism to control the global diffusive process. For some sets of networks, we develop an algorithm to identify the most sensitive nodes in the rewirable layer, so that changes in a small set of connections produce a drastic enhancement of the global diffusion of the whole multiplex system.

Synthesis and characterization of titania-based monodisperse fluorescent europium nanoparticles for biolabeling

International Nuclear Information System (INIS)

Tan Mingqian; Wang Guilan; Ye Zhiqiang; Yuan Jingli

2006-01-01

Inorganic-organic hybrid titania-based nanoparticles covalently bound to a fluorescent Eu 3+ chelate of 4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedion-6''-yl) chlorosulfo-o-terphenyl (BHHCT-Eu 3+ ) were synthesized by a sol-gel technique. A conjugate of BHHCT with 3-[2-(2-aminoethylamino) ethylamino]propyl-trimethoxysilane (APTS) was used as a precursor for the nanoparticle preparation and monodisperse nanoparticles consisting of titania network and silica sub-network covalently bound to the Eu 3+ chelate were prepared by the copolymerization of APTS-BHHCT conjugate, titanium tetraisopropoxide (TTIP) and free APTS in EuCl 3 water-alcohol solution. The effects of reaction conditions on size and fluorescence lifetime of the nanoparticles were investigated. The characterizations by transmission electron microscopy and fluorometric methods indicate that the nanoparticles are near spherical and strongly fluorescent having a fluorescence quantum yield of 11.6% and a long fluorescence lifetime of ∼0.4 ms. The direct-introduced amino groups on the nanoparticle's surface by using free APTS in nanoparticle preparation facilitated the biolabeling process of the nanoparticles. The nanoparticle-labeled streptavidin (SA) was prepared and used in a sandwich-type time-resolved fluoroimmunoassay (TR-FIA) of human prostate-specific antigen (PSA) by using a 96-well microtiter plate as the solid phase carrier. The method gives a detection limit of 66 pg/ml for the PSA assay

Available number of multiplexed holograms based on signal-to-noise ratio analysis in reflection-type holographic memory using three-dimensional speckle-shift multiplexing.

Science.gov (United States)

Nishizaki, Tatsuya; Matoba, Osamu; Nitta, Kouichi

2014-09-01

The recording properties of three-dimensional speckle-shift multiplexing in reflection-type holographic memory are analyzed numerically. Three-dimensional recording can increase the number of multiplexed holograms by suppressing the cross-talk noise from adjacent holograms by using depth-direction multiplexing rather than in-plane multiplexing. Numerical results indicate that the number of multiplexed holograms in three-layer recording can be increased by 1.44 times as large as that of a single-layer recording when an acceptable signal-to-noise ratio is set to be 2 when NA=0.43 and the thickness of the recording medium is 0.5 mm.

Strain measurement using multiplexed fiber optic sensors

International Nuclear Information System (INIS)

Kwon, Il Bum; Kim, Chi Yeop; Yoon, Dong Jin; Lee, Seung Seok

2003-01-01

FBG(Fiber Bragg grating) sensor, which is one of the fiber optic sensors for the application of smart structures, can not only measure one specific point but also multiple points by multiplexing techniques. We have proposed a novel multiplexing technique of FBG sensor by the intensity modulation of light source. This technique is applicable to WDM(Wavelength Division Multiplexing) technique and number of sensors in this system can be increased by using this technique with WDM technique.

Fluorescence spectral studies of Gum Arabic: Multi-emission of Gum Arabic in aqueous solution

Energy Technology Data Exchange (ETDEWEB)

Dhenadhayalan, Namasivayam, E-mail: ndhena@gmail.com [Department of Chemistry, National Taiwan University, Taipei, Taiwan (China); Mythily, Rajan, E-mail: rajanmythily@gmail.com [Department of Chemistry, Dwaraka Doss Goverdhan Doss Vaishnav College (Autonomous), 833, Gokul Bagh, E.V.R. Periyar Road, Arumbakkam, Chennai 600 106 (India); Kumaran, Rajendran, E-mail: kumaranwau@rediffmail.com [Department of Chemistry, Dwaraka Doss Goverdhan Doss Vaishnav College (Autonomous), 833, Gokul Bagh, E.V.R. Periyar Road, Arumbakkam, Chennai 600 106 (India)

2014-11-15

Gum Arabic (GA), a food hydrocolloid is a natural composite obtained from the stems and branches of Acacia Senegal and Acacia Seyal trees. GA structure is made up of highly branched arabinogalactan polysaccharides. Steady-state absorption, fluorescence, and time-resolved fluorescence spectral studies of acid hydrolyzed GA solutions were carried out at various pH conditions. The fluorescence in GA is predominantly attributed to the presence of tyrosine and phenylalanine amino acids. The presence of multi-emissive peaks at different pH condition is attributed to the exposure of the fluorescing amino acids to the aqueous phase, which contains several sugar units, hydrophilic and hydrophobic moieties. Time-resolved fluorescence studies of GA exhibits a multi-exponential decay with different fluorescence lifetime of varying amplitude which confirms that tyrosine is confined to a heterogeneous microenvironment. The existence of multi-emissive peaks with large variation in the fluorescence intensities were established by 3D emission contour spectral studies. The probable location of the fluorophore in a heterogeneous environment was further ascertained by constructing a time-resolved emission spectrum (TRES) and time-resolved area normalized emission spectrum (TRANES) plots. Fluorescence spectral technique is used as an analytical tool in understanding the photophysical properties of a water soluble complex food hydrocolloid containing an intrinsic fluorophore located in a multiple environment is illustrated. - Highlights: • The Manuscript deals with the steady state absorption, emission, fluorescence lifetime and time-resolved emission spectrum studies of Gum Arabic in aqueous medium at various pH conditions. • The fluorescence emanates from the tyrosine amino acid present in GA. • Change in pH results in marked variation in the fluorescence spectral properties of tyrosine. • Fluorescence spectral techniques are employed as a tool in establishing the

Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

Science.gov (United States)

Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

2014-05-01

The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

Study of lifetimes of fluorescence levels of tetravalent uranium in the incommensurate phase of thorium tetrabromide and tetrachloride

International Nuclear Information System (INIS)

Milicic, A.

1989-01-01

The lifetimes of radiative levels of tetravalent uranium in the incommensurate phase of thorium tetrahalides have been measured as a function of different parameters: site symmetry, temperature and concentration. The incommensurate phase of thorium tetrabromide and tetrachloride is characterized by a continuous distribution of site symmetries induced by a continuous and weak displacement of the halides around the thorium (uranium) ions. At low temperature, 4.2 K, the lifetime variation as a function of excited classes of symmetry is governed by the radiative process probability as well as the energy transfer between uranium ions in different sites. At higher temperature, a model based on a Boltzmann equilibrium between closed energy levels is able to reproduce the experimental lifetime variation as a function of the temperature, for a given class of symmetry. For the variation of lifetime as a function of uranium ion concentrations, at high dilution and in the case of U 4+ : ThBr 4 , there is a competition between the energy transfer and thermal population of excited states [fr

Lifetimes of partial charge transfer exciplexes of 9-cyanophenanthrene and 9-cyanoanthracene

OpenAIRE

Dolotova, Elena; Dogadkin, Denis; Soboleva, Irina; Kuzmin, Michael; Nicolet, Olivier; Vauthey, Eric

2003-01-01

The fluorescence decays of several exciplexes with partial charge transfer have been investigated in solvents of various polarity. The measured lifetimes are found to be in reasonable agreement with the activation enthalpy and entropy of exciplex decay obtained earlier from the temperature dependence of the exciplex emission quantum yields. For exciplexes with 9-cyanophenanthrene substantial contribution of the higher local excited state into the exciplex electronic structure is found and bor...

Light-Addressed Electrodeposition of Enzyme-Entrapped Chitosan Membranes for Multiplexed Enzyme-Based Bioassays Using a Digital Micromirror Device

Directory of Open Access Journals (Sweden)

Yeu-Long Jiang

2013-08-01

Full Text Available This paper describes a light-addressed electrolytic system used to perform an electrodeposition of enzyme-entrapped chitosan membranes for multiplexed enzyme-based bioassays using a digital micromirror device (DMD. In this system, a patterned light illumination is projected onto a photoconductive substrate serving as a photo-cathode to electrolytically produce hydroxide ions, which leads to an increased pH gradient. The high pH generated at the cathode can cause a local gelation of chitosan through sol-gel transition. By controlling the illumination pattern on the DMD, a light-addressed electrodeposition of chitosan membranes with different shapes and sizes, as well as multiplexed micropatterning, was performed. The effect of the illumination time of the light pattern on the dimensional resolution of chitosan membrane formation was examined experimentally. Moreover, multiplexed enzyme-based bioassay of enzyme-entrapped chitosan membranes was also successfully demonstrated through the electrodeposition of the chitosan membranes with various shapes/sizes and entrapping different enzymes. As a model experiment, glucose and ethanol were simultaneously detected in a single detection chamber without cross-talk using shape-coded chitosan membranes entrapped with glucose oxidase (GOX, peroxidase (POD, and Amplex Red (AmR or alcohol oxidase (AOX, POD, and AmR by using same fluorescence indicator (AmR.

Nuclear lifetimes

International Nuclear Information System (INIS)

Caraca, J.M.G.

1976-01-01

The importance of the results obtained in experiments of measurement of lifetimes for a detailed knowledge of nuclear structure is referred. Direct methods of measurement of nuclear lifetimes are described, namely, electronic methods, recoil-distance method, doppler shift atenuation method and blocking-method. A brief reference is made to indirect methods for measurement of life-times

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.

Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

Science.gov (United States)

Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-01-15

Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

Time-resolved fluorescence microscopy (FLIM) as an analytical tool in skin nanomedicine.

Science.gov (United States)

Alexiev, Ulrike; Volz, Pierre; Boreham, Alexander; Brodwolf, Robert

2017-07-01

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief, and for monitoring of disease progression. Topical application of drug-loaded nanoparticles for the treatment of skin disorders is a promising strategy to overcome the stratum corneum, the upper layer of the skin, which represents an effective physical and biochemical barrier. The understanding of drug penetration into skin and enhanced penetration into skin facilitated by nanocarriers requires analytical tools that ideally allow to visualize the skin, its morphology, the drug carriers, drugs, their transport across the skin and possible interactions, as well as effects of the nanocarriers within the different skin layers. Here, we review some recent developments in the field of fluorescence microscopy, namely Fluorescence Lifetime Imaging Microscopy (FLIM)), for improved characterization of nanocarriers, their interactions and penetration into skin. In particular, FLIM allows for the discrimination of target molecules, e.g. fluorescently tagged nanocarriers, against the autofluorescent tissue background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle and its interactions with other biomolecules. Thus, FLIM shows the potential to overcome several limits of intensity based microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

Potential of Fluorescence Imaging Techniques To Monitor Mutagenic PAH Uptake by Microalga

Science.gov (United States)

2015-01-01

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is one of the major environmental pollutants that causes mutagenesis and cancer. BaP has been shown to accumulate in phytoplankton and zooplankton. We have studied the localization and aggregation of BaP in Chlorella sp., a microalga that is one of the primary producers in the food chain, using fluorescence confocal microscopy and fluorescence lifetime imaging microscopy with the phasor approach to characterize the location and the aggregation of BaP in the cell. Our results show that BaP accumulates in the lipid bodies of Chlorella sp. and that there is Förster resonance energy transfer between BaP and photosystems of Chlorella sp., indicating the close proximity of the two molecular systems. The lifetime of BaP fluorescence was measured to be 14 ns in N,N-dimethylformamide, an average of 7 ns in Bold’s basal medium, and 8 ns in Chlorella cells. Number and brightness analysis suggests that BaP does not aggregate inside Chlorella sp. (average brightness = 5.330), while it aggregates in the supernatant. In Chlorella grown in sediments spiked with BaP, in 12 h the BaP uptake could be visualized using fluorescence microscopy. PMID:25020149

ConA-based glucose sensing using the long-lifetime azadioxatriangulenium fluorophore

Science.gov (United States)

Cummins, Brian; Simpson, Jonathan; Gryczynski, Zygmunt; Sørensen, Thomas Just; Laursen, Bo W.; Graham, Duncan; Birch, David; Coté, Gerard

2014-02-01

Fluorescent glucose sensing technologies have been identified as possible alternatives to current continuous glucose monitoring approaches. We have recently introduced a new, smart fluorescent ligand to overcome the traditional problems of ConA-based glucose sensors. For this assay to be translated into a continuous glucose monitoring device where both components are free in solution, the molecular weight of the smart fluorescent ligand must be increased. We have identified ovalbumin as a naturally-occurring glycoprotein that could serve as the core-component of a 2nd generation smart fluorescent ligand. It has a single asparagine residue that is capable of displaying an N-linked glycan and a similar isoelectric point to ConA. Thus, binding between ConA and ovalbumin can potentially be monovalent and sugar specific. This work is the preliminary implementation of fluorescently-labeled ovalbumin in the ConA-based assay. We conjugate the red-emitting, long-lifetime azadioxatriangulenium (ADOTA+) dye to ovalbumin, as ADOTA have many advantageous properties to track the equilibrium binding of the assay. The ADOTA-labeled ovalbumin is paired with Alexa Fluor 647-labeled ConA to create a Förster Resonance Energy Transfer (FRET) assay that is glucose dependent. The assay responds across the physiologically relevant glucose range (0-500 mg/dL) with increasing intensity from the ADOTA-ovalbumin, showing that the strategy may allow for the translation of the smart fluorescent ligand concept into a continuous glucose monitoring device.

Surface plasmon-enhanced molecular fluorescence induced by gold nanostructures

International Nuclear Information System (INIS)

Teng, Y.; Ueno, K.; Shi, X.; Aoyo, D.; Misawa, H.; Qiu, J.

2012-01-01

The authors report on surface plasmon-enhanced fluorescence of Eosin Y molecules induced by gold nanostructures. Al 2 O 3 films deposited by atomic layer deposition with sub-nanometer resolution were used as the spacer layer to control the distance between molecules and the gold surface. As the thickness of the Al 2 O 3 film increased, the fluorescence intensity first increased and then decreased. The highest enhancement factor is achieved with a 1 nm Al 2 O 3 film. However, the trend for the fluorescence lifetime is the opposite. It first decreased and then increased. The changes in the fluorescence quantum yield were also calculated. The yield shows a similar trend to the fluorescence intensity. The competition between the surface plasmon-induced increase in the radiative decay rate and the gold-induced fluorescence quenching is responsible for the observed phenomenon. In addition, this competition strongly depends on the thickness of the spacer layer between Eosin Y molecules and the gold surface. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

Cavity enhanced eigenmode multiplexing for volume holographic data storage

Science.gov (United States)

Miller, Bo E.; Takashima, Yuzuru

2017-08-01

Previously, we proposed and experimentally demonstrated enhanced recording speeds by using a resonant optical cavity to semi-passively increase the reference beam power while recording image bearing holograms. In addition to enhancing the reference beam power the cavity supports the orthogonal reference beam families of its eigenmodes, which can be used as a degree of freedom to multiplex data pages and increase storage densities for volume Holographic Data Storage Systems (HDSS). While keeping the increased recording speed of a cavity enhanced reference arm, image bearing holograms are multiplexed by orthogonal phase code multiplexing via Hermite-Gaussian eigenmodes in a Fe:LiNbO3 medium with a 532 nm laser at two Bragg angles for expedited recording of four multiplexed holograms. We experimentally confirmed write rates are enhanced by an average factor of 1.1, and page crosstalk is about 2.5%. This hybrid multiplexing opens up a pathway to increase storage density while minimizing modifications to current angular multiplexing HDSS.

In vivo detection of oral epithelial cancer using endogenous fluorescence lifetime imaging: a pilot human study (Conference Presentation)

Science.gov (United States)

Jo, Javier A.; Hwang, Dae Yon; Palma, Jorge; Cheng, Shuna; Cuenca, Rodrigo; Malik, Bilal; Jabbour, Joey; Cheng, Lisa; Wright, John; Maitland, Kristen

2016-03-01

Endogenous fluorescence lifetime imaging (FLIM) provides direct access to the concomitant functional and biochemical changes accompanying tissue transition from benign to precancerous and cancerous. Since FLIM can noninvasively measure different and complementary biomarkers of precancer and cancer, we hypothesize that it will aid in clinically detecting early oral epithelial cancer. Our group has recently demonstrated the detection of benign from premalignant and malignant lesions based on endogenous multispectral FLIM in the hamster cheek-pouch model. Encouraged by these positive preliminary results, we have developed a handheld endoscope capable of acquiring multispectral FLIM images in real time from the oral mucosa. This novel FLIM endoscope is being used for imaging clinically suspicious pre-malignant and malignant lesions from patients before undergoing tissue biopsy for histopathological diagnosis of oral epithelial cancer. Our preliminary results thus far are already suggesting the potential of endogenous FLIM for distinguishing a variety of benign lesions from advanced dysplasia and squamous cell carcinoma (SCC). To the best of out knowledge, this is the first in vivo human study aiming to demonstrate the ability to predict the true malignancy of clinically suspicious lesions using endogenous FLIM. If successful, the resulting clinical tool will allow noninvasive real-time detection of epithelial precancerous and cancerous lesions in the oral mucosa and could potentially be used to assist at every step involved on the clinical management of oral cancer patients, from early screening and diagnosis, to treatment and monitoring of recurrence.

Fluorescence properties of 3-amino phenylboronic acid and its interaction with glucose and ZnS:Cu quantum dots.

Science.gov (United States)

Kur-Kowalska, Karolina; Przybyt, Małgorzata; Ziółczyk, Paulina; Sowiński, Przemysław; Miller, Ewa

2014-08-14

Preliminary results of a study of the interaction between 3-amino phenylboronic acid and glucose or ZnS:Cu quantum dots are presented in this paper. ZnS:Cu quantum dots with mercaptopropionic acid as a capping agent were obtained and characterized. Quenching of 3-amino phenylboronic acid fluorescence was studied by steady-state and timeresolved measurements. For fluorescence quenching with glucose the results of steady-state measurements fulfill Stern-Volmer equation. The quenching constants are increasing with growing pH. The decay of fluorescence is monoexponential with lifetime about 8.4 ns, which does not depend on pH and glucose concentration indicating static quenching. The quenching constant can be interpreted as apparent equilibrium constant of estrification of boronic group with diol. Quantum dots are also quenching 3-amino phenylboronic acid fluorescence. Fluorescence lifetime, in this case, is slightly decreasing with increasing concentration of quantum dots. The quenching constants are increasing slightly with pH's growth. Quenching mechanism of 3-amino phenylboronic acid fluorescence by quantum dots needs further experiments to be fully explained. Copyright © 2014 Elsevier B.V. All rights reserved.

Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model.

Science.gov (United States)

Klemm, Matthias; Blum, Johannes; Link, Dietmar; Hammer, Martin; Haueisen, Jens; Schweitzer, Dietrich

2016-09-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes determined from the intensity decays. Here, we present a new technique to suppress the autofluorescence of the crystalline lens by introducing an annular stop into the detection light path, which we call Schweitzer's principle. The efficacy of annular stops with an outer diameter of 7 mm and inner diameters of 1 to 5 mm are analyzed in an experimental setup using a model eye based on fluorescent dyes. Compared to the confocal principle, Schweitzer's principle with an inner diameter of 3 mm is able to reduce the simulated crystalline lens fluorescence to 4%, while 42% of the simulated retina fluorescence is preserved. Thus, we recommend the implementation of Schweitzer's principle in scanning laser ophthalmoscopes used for fundus autofluorescence measurements, especially the FLIO device, for improved image quality.

Nanoscale Test Strips for Multiplexed Blood Analysis

Science.gov (United States)

Chan, Eugene

2015-01-01

A critical component of the DNA Medicine Institute's Reusable Handheld Electrolyte and Lab Technology for Humans (rHEALTH) sensor are nanoscale test strips, or nanostrips, that enable multiplexed blood analysis. Nanostrips are conceptually similar to the standard urinalysis test strip, but the strips are shrunk down a billionfold to the microscale. Each nanostrip can have several sensor pads that fluoresce in response to different targets in a sample. The strips carry identification tags that permit differentiation of a specific panel from hundreds of other nanostrip panels during a single measurement session. In Phase I of the project, the company fabricated, tested, and demonstrated functional parathyroid hormone and vitamin D nanostrips for bone metabolism, and thrombin aptamer and immunoglobulin G antibody nanostrips. In Phase II, numerous nanostrips were developed to address key space flight-based medical needs: assessment of bone metabolism, immune response, cardiac status, liver metabolism, and lipid profiles. This unique approach holds genuine promise for space-based portable biodiagnostics and for point-of-care (POC) health monitoring and diagnostics here on Earth.

Recommendations for fluorescence instrument qualification: the new ASTM Standard Guide.

Science.gov (United States)

DeRose, Paul C; Resch-Genger, Ute

2010-03-01

Aimed at improving quality assurance and quantitation for modern fluorescence techniques, ASTM International (ASTM) is about to release a Standard Guide for Fluorescence, reviewed here. The guide's main focus is on steady state fluorometry, for which available standards and instrument characterization procedures are discussed along with their purpose, suitability, and general instructions for use. These include the most relevant instrument properties needing qualification, such as linearity and spectral responsivity of the detection system, spectral irradiance reaching the sample, wavelength accuracy, sensitivity or limit of detection for an analyte, and day-to-day performance verification. With proper consideration of method-inherent requirements and limitations, many of these procedures and standards can be adapted to other fluorescence techniques. In addition, procedures for the determination of other relevant fluorometric quantities including fluorescence quantum yields and fluorescence lifetimes are briefly introduced. The guide is a clear and concise reference geared for users of fluorescence instrumentation at all levels of experience and is intended to aid in the ongoing standardization of fluorescence measurements.

Theoretical and experimental investigation of atomic radiative lifetimes and hyperfine structures

International Nuclear Information System (INIS)

Joensson, Per.

1992-01-01

Atomic radiative lifetimes and hyperfine structures as well as other properties, such as total energy and specific mass shift, have been studied theoretically and experimentally. Computer programs to calculate hyperfine structure constants from non-relativistic multiconfiguration Hartree-Fock (MCHF) and relativistic multi-configuration Dirac-Fock (MCDF) wavefunctions have been written. Using these programs large-scale calculations of hyperfine structures in lithium and sodium have been performed. It is shown, that the MCHF method is able to predict hyperfine structures to an accuracy of a few per mille in lithium, whereas for the more complex hyperfine structures to an accuracy of a few per mille in lithium, whereas for the more complex sodium atom an accuracy of a few per cent is obtainable. For lithium convergence of the total energy, ionization energy, specific mass shift and hyperfine parameters has been studied with the MCHF method. Radiative lifetimes and hyperfine structures of excited states in sodium and silver have been experimentally determined using time-resolved laser spectroscopy. By recording the fluorescence light decay curves following VUV excitation, the radiative lifetimes and hyperfine structures of the 7p 2 P states in silver were measured. The delayed-coincidence technique has been used to make very accurate measurements of the radiative lifetimes and hyperfine structures of the lowest P states in sodium and silver

Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection.

Science.gov (United States)

Sahoo, Harekrushna; Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-12-26

The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

Lifetime measurements

International Nuclear Information System (INIS)

Fossan, D.B.; Warburton, E.K.

1974-01-01

Lifetime measurements are discussed, concentrating on the electronic technique, the recoil distance method (RDM), and the Doppler shift attenuation method (DSAM). A brief review of several indirect timing techniques is given, and their specific advantages and applicability are considered. The relationship between lifetimes of nuclear states and the nuclear structure information obtained from them is examined. A short discussion of channeling and microwave methods of lifetime measurement is presented. (23 figures, 171 references) (U.S.)

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

Science.gov (United States)

Nyström, Sofie; Bäck, Marcus; Nilsson, K Peter R; Hammarström, Per

2017-10-20

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Mobility-lifetime product in epitaxial GaAs X-ray detectors

Energy Technology Data Exchange (ETDEWEB)

Sun, G.C. [GESEC R and D, Universite Pierre et Marie Curie, Bat.11, 140 rue de Lourmel, 75015 Paris (France)]. E-mail: guocsun@ccr.jussieu.fr; Zazoui, M. [LPMC, Faculte des Sciences et Techniques-Mohammedia, B.P. 146 Bd Hassan II, Mohammedia, Maroc (Morocco); Talbi, N. [Faculte des Sciences, Universite de Gabes, Route de Medenine, 6029 Gabes (Tunisia); Khirouni, K. [Faculte des Sciences, Universite de Gabes, Route de Medenine, 6029 Gabes (Tunisia); Bourgoin, J.C. [GESEC R and D, Universite Pierre et Marie Curie, Bat.11, 140 rue de Lourmel, 75015 Paris (France)

2007-04-01

Self-supported thick (200-500 {mu}m), non-intentionally doped, epitaxial GaAs layers are good candidates for X-ray imaging for the following reasons. Their electronic properties are homogeneous over large areas, they can be grown at low cost, the technology to realize pixel detectors of various size is standard, the defect concentration is low and the fluorescence yield is small. Here, we characterize the defects present in the material and evaluate the mobility-lifetime product, using Deep Level Transient Spectroscopy combined with current-voltage and charge collection measurements.

Interpretation of measurements of dynamic fluorescence of the eye

Science.gov (United States)

Schweitzer, Dietrich; Hammer, Martin; Jentsch, Susanne; Schenke, Stefan

2007-09-01

First pathological alterations occur at cellular level, most in metabolism. An indirect estimation of metabolic activity in cells is measurement of microcirculation. Measurements of tissue autofluorescence are potentially suited for direct investigation of cellular metabolism. Besides redox pairs of co-enzymes (NADH-NAD, FADH2-FAD) several other fluorophores are excited in tissue. In addition, a number of anatomical structures are simultaneously excited, when investigating the eye-ground. In this study, spectral and time resolved comparison was performed between purified substances, single ocular structures and in vivo measurements of the time-resolved autofluorescence at the human eye. In human eyes, the ageing pigment lipofuscin covers other fluorophores at the fundus in long - wave visible range. Applying lifetime measurements, weakly emitting fluorophores can be detected, when the lifetimes are different from the strongly emitting fluorophore. For this, the autofluorescence was excited at 468 nm and detected in two spectral ranges (500 nm-560 nm, 560 nm-700 nm). In tri-exponential fitting, the short lifetime corresponds to retinal pigment epithelium, the mean lifetime corresponds probably to neural retina and the long lifetime is caused by fluorescence of connective tissue.

The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae, Lamiaceae, Rosaceae.

Science.gov (United States)

Sytar, Oksana; Bruckova, Klaudia; Hunkova, Elena; Zivcak, Marek; Konate, Kiessoun; Brestic, Marian

2015-01-16

The aim of our research work was to quantify total flavonoid contents in the leaves of 13 plant species family Asteraceae, 8 representatives of family Lamiaceae and 9 plant species belonging to family Rosaceae, using the multiplex fluorimetric sensor. Fluorescence was measured using optical fluorescence apparatus Multiplex(R) 3 (Force-A, France) for non-destructive flavonoids estimation. The content of total flavonoids was estimated by FLAV index (expressed in relative units), that is deduced from flavonoids UV absorbing properties. Among observed plant species, the highest amount of total flavonoids has been found in leaves of Helianthus multiflorus (1.65 RU) and Echinops ritro (1.27 RU), Rudbeckia fulgida (1.13 RU) belonging to the family Asteraceae. Lowest flavonoid content has been observed in the leaves of marigold (Calendula officinalis) (0.14 RU) also belonging to family Asteraceae. The highest content of flavonoids among experimental plants of family Rosaceae has been estimated in the leaves of Rosa canina (1.18 RU) and among plant species of family Lamiaceae in the leaves of Coleus blumei (0.90 RU). This research work was done as pre-screening of flavonoids content in the leaves of plant species belonging to family Asteraceae, Lamiaceae and Rosaceae. Results indicated that statistically significant differences (P > 0.05) in flavonoids content were observed not only between families, but also among individual plant species within one family.

Radiative-lifetime measurements and calculations of odd-parity highly excited levels in Ba i

International Nuclear Information System (INIS)

Zhang Wei; Du Shan; Palmeri, Patrick; Quinet, Pascal; Biemont, Emile; Dai Zhenwen

2010-01-01

Natural radiative lifetime measurements have been performed for 70 odd-parity highly excited levels of neutral barium in the energy range from 308 15.512 to 417 59.93 cm -1 by a time-resolved laser-induced fluorescence technique in a laser-produced plasma. The lifetime values measured in this paper are in the range from 11.3 to 901 ns. They are compared with the published lifetimes of four levels. Two of them are in good agreement, whereas for the other two our measurements are slightly longer than the published data. The reasons for the discrepancies are discussed. Comparisons with theoretical results of the Hartree-Fock method with relativistic corrections illustrate the difficulties associated with the use of Cowan's codes for obtaining accurate branching fractions for transitions depopulating highly excited levels along the Rydberg series of heavy neutral elements. This work will be useful to extend the set of oscillator strengths available in Ba i.

Helicity multiplexed broadband metasurface holograms.

Science.gov (United States)

Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Pun, Edwin Yue Bun; Zhang, Shuang; Chen, Xianzhong

2015-09-10

Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices.

User Multiplexing in Relay Enhanced LTE-Advanced Networks

DEFF Research Database (Denmark)

Teyeb, Oumer Mohammed; Frederiksen, Frank; Redana, Simone

2010-01-01

is radio relaying. This uses relay nodes that act as surrogate base stations for mobile users whose radio links with the base stations are not experiencing good enough conditions. In the downlink, the data that is destined for the relayed users may first have to be multiplexed by the base station, sent...... over the wireless backhaul link towards the relay node, and de-multiplexed and forwarded to the individual users by the relay node. The reverse process also has to be undertaken in the uplink. In this paper, we present a novel multiplexing scheme which is able to adapt the addressing and bitmapping...... of user identification to the actual number of users being served by the relay nodes, and thus greatly reduce the multiplexing overhead....

Multiple routes transmitted epidemics on multiplex networks

International Nuclear Information System (INIS)

Zhao, Dawei; Li, Lixiang; Peng, Haipeng; Luo, Qun; Yang, Yixian

2014-01-01

This letter investigates the multiple routes transmitted epidemic process on multiplex networks. We propose detailed theoretical analysis that allows us to accurately calculate the epidemic threshold and outbreak size. It is found that the epidemic can spread across the multiplex network even if all the network layers are well below their respective epidemic thresholds. Strong positive degree–degree correlation of nodes in multiplex network could lead to a much lower epidemic threshold and a relatively smaller outbreak size. However, the average similarity of neighbors from different layers of nodes has no obvious effect on the epidemic threshold and outbreak size. -- Highlights: •We studies multiple routes transmitted epidemic process on multiplex networks. •SIR model and bond percolation theory are used to analyze the epidemic processes. •We derive equations to accurately calculate the epidemic threshold and outbreak size. •ASN has no effect on the epidemic threshold and outbreak size. •Strong positive DDC leads to a lower epidemic threshold and a smaller outbreak size.

Multiple routes transmitted epidemics on multiplex networks

Energy Technology Data Exchange (ETDEWEB)

Zhao, Dawei [Information Security Center, State Key Laboratory of Networking and Switching Technology, Beijing University of Posts and Telecommunications, P.O. Box 145, Beijing 100876 (China); National Engineering Laboratory for Disaster Backup and Recovery, Beijing University of Posts and Telecommunications, Beijing 100876 (China); Shandong Provincial Key Laboratory of Computer Network, Shandong Computer Science Center, Jinan 250014 (China); Li, Lixiang [Information Security Center, State Key Laboratory of Networking and Switching Technology, Beijing University of Posts and Telecommunications, P.O. Box 145, Beijing 100876 (China); National Engineering Laboratory for Disaster Backup and Recovery, Beijing University of Posts and Telecommunications, Beijing 100876 (China); Peng, Haipeng, E-mail: penghaipeng@bupt.edu.cn [Information Security Center, State Key Laboratory of Networking and Switching Technology, Beijing University of Posts and Telecommunications, P.O. Box 145, Beijing 100876 (China); National Engineering Laboratory for Disaster Backup and Recovery, Beijing University of Posts and Telecommunications, Beijing 100876 (China); Luo, Qun; Yang, Yixian [Information Security Center, State Key Laboratory of Networking and Switching Technology, Beijing University of Posts and Telecommunications, P.O. Box 145, Beijing 100876 (China); National Engineering Laboratory for Disaster Backup and Recovery, Beijing University of Posts and Telecommunications, Beijing 100876 (China)

2014-02-01

This letter investigates the multiple routes transmitted epidemic process on multiplex networks. We propose detailed theoretical analysis that allows us to accurately calculate the epidemic threshold and outbreak size. It is found that the epidemic can spread across the multiplex network even if all the network layers are well below their respective epidemic thresholds. Strong positive degree–degree correlation of nodes in multiplex network could lead to a much lower epidemic threshold and a relatively smaller outbreak size. However, the average similarity of neighbors from different layers of nodes has no obvious effect on the epidemic threshold and outbreak size. -- Highlights: •We studies multiple routes transmitted epidemic process on multiplex networks. •SIR model and bond percolation theory are used to analyze the epidemic processes. •We derive equations to accurately calculate the epidemic threshold and outbreak size. •ASN has no effect on the epidemic threshold and outbreak size. •Strong positive DDC leads to a lower epidemic threshold and a smaller outbreak size.

TR-LIF LIFETIME MEASUREMENTS AND HFR+CPOL CALCULATIONS OF RADIATIVE PARAMETERS IN VANADIUM ATOM (V I)

International Nuclear Information System (INIS)

Wang, Q.; Jiang, L. Y.; Shang, X.; Tian, Y. S.; Dai, Z. W.; Quinet, P.; Palmeri, P.; Zhang, W.

2014-01-01

Radiative lifetimes of 79 levels belonging to the 3d 3 4s4p, 3d 4 4p, 3d 3 4s5p, 3d 4 5p, and 3d 3 4s4d configurations of V I with energy from 26,604.807 to 46,862.786 cm –1 have been measured using time-resolved laser-induced fluorescence (TR-LIF) spectroscopy in laser-produced plasma. The lifetime values reported in this paper are in the range of 3.3-494 ns, and the uncertainties of these measurements are within ±10%. A good agreement was obtained with previous data. HFR+CPOL calculations have been performed and used to combine the calculated branching fractions with the available experimental lifetimes to determine semi-empirical transition probabilities for 784 V I transitions

An image fiber based fluorescent probe with associated signal processing scheme for biomedical diagnostics

International Nuclear Information System (INIS)

Vaishakh, M; Murukeshan, V M; Seah, L K

2008-01-01

A dual-modality image fiber based fluorescent probe that can be used for depth sensitive imaging and suppression of fluorescent emissions with nanosecond lifetime difference is proposed and illustrated in this paper. The system can give high optical sectioning and employs an algorithm for obtaining phase sensitive images. The system can find main application in in vivo biomedical diagnostics for detecting biochemical changes for distinguishing malignant tissue from healthy tissue

Novel Fluorescent Microemulsion: Probing Properties, Investigating Mechanism, and Unveiling Potential Application.

Science.gov (United States)

Hou, Mengna; Dang, Leping; Liu, Tiankuo; Guo, Yun; Wang, Zhanzhong

2017-08-09

Nanoscale microemulsions have been utilized as delivery carriers for nutraceuticals and active biological drugs. Herein, we designed and synthesized a novel oil in water (O/W) fluorescent microemulsion based on isoamyl acetate, polyoxyethylene castor oil EL (CrEL), and water. The microemulsion emitted bright blue fluorescence, thus exhibiting its potential for active drug detection with label-free strategy. The microemulsion exhibited excitation-dependent emission and distinct red shift with longer excitation wavelengths. Lifetime and quantum yield of fluorescent microemulsion were 2.831 ns and 5.0%, respectively. An excellent fluorescent stability of the microemulsion was confirmed by altering pH, ionic strength, temperature, and time. Moreover, we proposed a probable mechanism of fluorochromic phenomenon, in connection with the aromatic ring structure of polyoxyethylene ether substituent in CrEL. Based on our findings, we concluded that this new fluorescent microemulsion is a promising drug carrier that can facilitate active drug detection with a label-free strategy. Although further research is required to understand the exact mechanism behind its fluorescence property, this work provided valuable guidance to develop new biosensors based on fluorescent microemulsion.

Lifetime measurements in Crsub(I) by laser excitation from the metastable states

International Nuclear Information System (INIS)

Kwiatkowski, M.; Micali, G.; Werner, K.; Zimmermann, P.

1981-01-01

A combination of collisional and laser excitation was used to measure radiative lifetimes in Cr I. By a discharge an atomic beam of metastable atoms in the 3d 5 4sa 5 S, a 5 G, b 5 D, a 3 I, b 1 I and 3d 4 4s 2 a 5 D terms was produced. Spatially separated from the place of collisional excitation laser radiation selectively populated levels belonging to the 3d 5 4p z 5 P, y 5 P, u 5 F, u 5 D, x 3 I, y 1 I, 3d 5 5pz 5 G and 3d 4 4s4px 5 G terms. Time-resolved observation of the reemitted resonance fluorescence yielded the lifetimes of 28 levels. The values are compared with other experimental and theoretical results. (orig.)

Miniaturized high-temperature superconducting multiplexer with cascaded quadruplet structure

Science.gov (United States)

Xu, Zhang; Jingping, Liu; Shaolin, Yan; Lan, Fang; Bo, Zhang; Xinjie, Zhao

2015-06-01

In this paper, compact high temperature superconducting (HTS) multiplexers are presented for satellite communication applications. The first multiplexer consists of an input coupling node and three high-order bandpass filters, which is named triplexer. The node is realized by a loop microstrip line instead of conventional T-junction to eliminate the redundant susceptance due to combination of three filters. There are two eight-pole band-pass filters and one ten-pole band-pass filter with cascaded quadruplet structure for realizing high isolation. Moreover, the triplexer is extended to a multiplexer with six channels so as to verify the expansibility of the suggested approach. The triplexer is fabricated using double-sided YBa2Cu3O7 thin films on a 38 × 25 mm2 LaAlO3 substrate. The experimental results, when compared with those ones from the T-junction multiplexer, show that our multiplexer has lower insertion loss, smaller sizes and higher isolation between any two channels. Also, good agreement has been achieved between simulations and measurements, which illustrate the effectiveness of our methods for the design of high performance HTS multiplexers.

Enzyme entrapment in polyaniline films observed via fluorescence anisotropy and antiquenching

Science.gov (United States)

Nemzer, Louis R.; McCaffrey, Marisa; Epstein, Arthur J.

2014-05-01

The facile entrapment of oxidoreductase enzymes within polyaniline polymer films by inducing hydrophobic collapse using phosphate buffered saline (PBS) has been shown to be a cost-effective method for fabricating organic biosensors. Here, we use fluorescence anisotropy measurements to verify enzyme immobilization and subsequent electron donation to the polymer matrix, both prerequisites for an effective biosensor. Specifically, we measure a three order of magnitude decrease in the ratio of the fluorescence to rotational lifetimes. In addition, the observed fluorescence antiquenching supports the previously proposed model that the polymer chain assumes a severely coiled conformation when exposed to PBS. These results help to empirically reinforce the theoretical basis previously laid out for this biosensing platform.

The role of defects in fluorescent silicon carbide layers grown by sublimation epitaxy

DEFF Research Database (Denmark)

Schimmel, Saskia; Kaiser, Michl; Jokubavicius, Valdas

2014-01-01

Donor-acceptor co-doped SiC is a promising light converter for novel monolithic all-semiconductor white LEDs due to its broad-band donor-acceptor pair luminescence and potentially high internal quantum efficiency. Besides sufficiently high doping concentrations in an appropriate ratio yielding...... short radiative lifetimes, long nonradiative lifetimes are crucial for efficient light conversion. The impact of different types of defects is studied by characterizing fluorescent silicon carbide layers with regard to photoluminescence intensity, homogeneity and efficiency taking into account...

Effects of short term changes in the blood glucose level on the autofluorescence lifetime of the human retina in healthy volunteers

Science.gov (United States)

Klemm, Matthias; Nagel, Edgar; Schweitzer, Dietrich; Schramm, Stefan; Haueisen, Jens

2016-03-01

Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO) provides in vivo metabolic mapping of the ocular fundus. Changes in FLIO have been found in e.g. diabetes patients. The influence of short term metabolic changes caused by blood glucose level changes on is unknown. Aim of this work is the detection of short-term changes in fundus autofluorescence lifetime during an oral glucose tolerance test. Methods: FLIO was performed in 10 healthy volunteers (29+/-4 years, fasting for 12h) using a scanning laser ophthalmoscope (30° fundus, 34μm resolution, excitation with 473nm diode laser with 70 ps pulses at 80 MHz repetition rate, detection in two spectral channels 500-560nm (ch1) and 560-720nm (ch2) using the timecorrelated single photon counting method). The blood glucose level (BGL) was measured by an Accu-Chek® Aviva self-monitoring device. Before and after a glucose drink (300ml solution, containing 75g of glucose (Accu-Chek® Dextrose O.G.T.), BGL and FLIO were measured every 15min. The FLIMX software package was applied to compute the average fluorescence lifetime τ on the inner ring of the ETDRS grid using a modified 3-exponential approach. Results: The results are given as mean +/- standard deviation over all volunteers in ch1. Baseline measurement: BGL: 5.3+/-0.4 mmol/l, τ1: 49+/-6ps. A significant reduction (α=5% Wilcoxon rank-sum test) in τ1 is detected after 15min (BGL: 8.4+/-1.1 mmol/l, τ1: 44+/-5ps) and after 90min (BGL: 6.3+/-1.4 mmol/l, τ1: 41+/-5ps). Results of ch2 show smaller reductions in the fluorescence lifetimes over time.

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

Science.gov (United States)

Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

SQUID readout multiplexers for transition-edge sensor arrays

Energy Technology Data Exchange (ETDEWEB)

Lee, Adrian T. [Physics Department, University of California, Berkeley, CA 94720 (United States) and Physics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)]. E-mail: atl@physics.berkeley.edu

2006-04-15

Two classes of SQUID multiplexer are being developed for large arrays of cryogenic sensors, distinguished by their operation in either the time domain or frequency domain. Several systems optimized for use with Transition-Edge Sensors (TES) are reaching a high level of maturity, and will be deployed on funded astrophysics experiments in the next several years. A useful technical figure of merit is the product of the number of detectors multplexed multipled by the bandwidth of the detectors, which can be termed the 'total signal bandwidth' of a multiplexer system. This figure of merit is comparable within a factor of two for the mature systems. Several new concepts for increasing the total bandwidth are being developed in the broad class of frequency domain multiplexers. Another notable area of progress is in the level of integration of muliplexer and detector array. The time domain system for SCUBA-II is a sophisticated bump-bonded sandwich structure, and the Jena/MPI group is integrating detectors and a time domain multiplexer on one substrate. Finally, the Kinetic Inductance Detectors (KID)/HEMT (non-SQUID) detector/multiplexer system, will be discussed briefly.

Multiplexing schemes for an achromatic programmable diffractive lens

Energy Technology Data Exchange (ETDEWEB)

Millan, M S; Perez-Cabre, E; Oton, J [Technical University of Catalonia, Dep. Optics and Optometry, Terrassa-Barcelona, 08222 (Spain)], E-mail: millan@oo.upc.edu

2008-11-01

A multiplexed programmable diffractive lens, displayed on a pixelated liquid crystal device under broadband illumination, is proposed to compensate for the severe chromatic aberration that affects diffractive elements. The proposed lens is based on multiplexing a set of sublenses with a common focal length for different wavelengths. We consider different types of integration of the optical information (spatial only, temporal only and hybrid spatial-temporal) combined with a proper selection of the spectral bandwidth. The properties and limits of the achromatic programmable multiplexed lens are described. Experimental results are presented and discussed.

Multiplexing schemes for an achromatic programmable diffractive lens

International Nuclear Information System (INIS)

Millan, M S; Perez-Cabre, E; Oton, J

2008-01-01

A multiplexed programmable diffractive lens, displayed on a pixelated liquid crystal device under broadband illumination, is proposed to compensate for the severe chromatic aberration that affects diffractive elements. The proposed lens is based on multiplexing a set of sublenses with a common focal length for different wavelengths. We consider different types of integration of the optical information (spatial only, temporal only and hybrid spatial-temporal) combined with a proper selection of the spectral bandwidth. The properties and limits of the achromatic programmable multiplexed lens are described. Experimental results are presented and discussed.

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

Science.gov (United States)

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Reduced γ–γ time walk to below 50 ps using the multiplexed-start and multiplexed-stop fast-timing technique with LaBr_3(Ce) detectors

International Nuclear Information System (INIS)

Régis, J.-M.; Saed-Samii, N.; Rudigier, M.; Ansari, S.; Dannhoff, M.; Esmaylzadeh, A.; Fransen, C.; Gerst, R.-B.; Jolie, J.; Karayonchev, V.; Müller-Gatermann, C.; Stegemann, S.

2016-01-01

The electronic γ–γ fast-timing technique using arrays consisting of many LaBr_3(Ce) detectors is a powerful method to determine lifetimes of nuclear excited states with a lower limit of about 5 ps. This method requires the determination of the energy-dependent time walk of the zero time which is represented by the centroid of a prompt γ–γ time distribution. The full-energy peak versus full-energy peak prompt response difference which represents the linearly combined mean γ–γ time walk of a fast-timing array consisting of 8 LaBr_3(Ce) detectors was measured using a standard "1"5"2Eu γ-ray source for the energy region of 40–1408 keV. The data were acquired using a “multiplexed-start and multiplexed-stop” analogue electronics circuitry and analysed by employing the generalized centroid difference method. Concerning the cylindrical 1.5 in.×1.5 in. LaBr_3(Ce) crystals which are coupled to the Hamamatsu R9779 photomultiplier tubes, the best fast-timing array time resolution of 202(3) ps is obtained for the two prompt γ lines of "6"0Co by using the leading-edge timing principle. When using the zero-crossover timing principle the time resolution is degraded by up to 30%, dependent on the energy and the shaping delay time of the constant fraction discriminator model Ortec 935. The smallest γ–γ time walk to below 50 ps is obtained by using a shaping delay time of about 17 ns and an optimum “time-walk adjustment” needed for detector output pulses with amplitudes smaller than 400 mV.

Autofluorescence lifetime imaging during transoral robotic surgery: a clinical validation study of tumor detection (Conference Presentation)

Science.gov (United States)

Lagarto, João. L.; Phipps, Jennifer E.; Unger, Jakob; Faller, Leta M.; Gorpas, Dimitris; Ma, Dinglong M.; Bec, Julien; Moore, Michael G.; Bewley, Arnaud F.; Yankelevich, Diego R.; Sorger, Jonathan M.; Farwell, Gregory D.; Marcu, Laura

2017-02-01

Autofluorescence lifetime spectroscopy is a promising non-invasive label-free tool for characterization of biological tissues and shows potential to report structural and biochemical alterations in tissue owing to pathological transformations. In particular, when combined with fiber-optic based instruments, autofluorescence lifetime measurements can enhance intraoperative diagnosis and provide guidance in surgical procedures. We investigate the potential of a fiber-optic based multi-spectral time-resolved fluorescence spectroscopy instrument to characterize the autofluorescence fingerprint associated with histologic, morphologic and metabolic changes in tissue that can provide real-time contrast between healthy and tumor regions in vivo and guide clinicians during resection of diseased areas during transoral robotic surgery. To provide immediate feedback to the surgeons, we employ tracking of an aiming beam that co-registers our point measurements with the robot camera images and allows visualization of the surgical area augmented with autofluorescence lifetime data in the surgeon's console in real-time. For each patient, autofluorescence lifetime measurements were acquired from normal, diseased and surgically altered tissue, both in vivo (pre- and post-resection) and ex vivo. Initial results indicate tumor and normal regions can be distinguished based on changes in lifetime parameters measured in vivo, when the tumor is located superficially. In particular, results show that autofluorescence lifetime of tumor is shorter than that of normal tissue (p robot assisted cancer removal interventions.

Eigenmode multiplexing with SLM for volume holographic data storage

Science.gov (United States)

Chen, Guanghao; Miller, Bo E.; Takashima, Yuzuru

2017-08-01

The cavity supports the orthogonal reference beam families as its eigenmodes while enhancing the reference beam power. Such orthogonal eigenmodes are used as additional degree of freedom to multiplex data pages, consequently increase storage densities for volume Holographic Data Storage Systems (HDSS) when the maximum number of multiplexed data page is limited by geometrical factor. Image bearing holograms are multiplexed by orthogonal phase code multiplexing via Hermite-Gaussian eigenmodes in a Fe:LiNbO3 medium with a 532 nm laser at multiple Bragg angles by using Liquid Crystal on Silicon (LCOS) spatial light modulators (SLMs) in reference arms. Total of nine holograms are recorded with three angular and three eigenmode.

Laser diagnostics in combustion. Elastic scattering and picosecond laser-induced fluorescence

Energy Technology Data Exchange (ETDEWEB)

Ossler, Frederik

1999-05-01

Elastic scattering and the Lorenz-Mie (LM) theory in particular is used for the characterization of sub-micron- and micron-sized droplets of organic fuels in sprays and aerosols. Calculations on the Lorenz-Mie theory show that backward-sideward scattered visible radiation can be used for unambiguous detection of ensembles of homogeneous droplets of organic substances with diameters around 1 micrometer (size parameter between 2 and 6). A backward feature in the polarization ratio appears with a value considerably higher than one, on the opposite to the case of the rainbow observed for larger droplets. A comparison between measurements and LM calculations showed that a large amount of droplets in aerosols and well-atomized sprays were smaller than one micrometer in diameter. The LM theory was also used to characterize different size groups in a burning spray. A 3 - D technique based on a picosecond laser and a streak camera was demonstrated for measurements of fast and turbulent biphase flows. The entire 3 - D information was obtained within a time-span of less than 15 nanoseconds. A 2 - D technique for lifetime measurements based on a picosecond laser and a streak camera has been demonstrated on static objects. An analysis indicates that the technique may be applied to measurements of lifetimes around or below one picosecond employing femtosecond lasers and femtosecond streak-cameras. The technique may in principle be used to study dynamic systems when two detectors are used. Fluorescence lifetime measurements on hydrogen and oxygen atoms in flames at atmospheric pressure demonstrate the need of lasers with suiting spectral properties such as jitter and linewidth and the need of detectors with high sensitivity in the near IR in the case of oxygen atoms. The fluorescence lifetimes of gas phase acetone and 3-pentanone at 266 nm excitation wavelength have been measured for mixtures with nitrogen and air at temperatures between 323 and 723 K and pressures between 0

Time-resolved measurements of the external electric field effects on fluorescence in electron donor and acceptor pairs of N-ethylcarbazole and dimethyl terephthalate doped in a polymer film

International Nuclear Information System (INIS)

Iimori, Toshifumi; Yoshizawa, Tomokazu; Nakabayashi, Takakazu; Ohta, Nobuhiro

2005-01-01

Electric-field-induced change in fluorescence decay has been measured for electron donor and acceptor pairs of N-ethylcarbazole (ECZ) and dimethyl terephthalate (DMTP) doped in a polymer film. Field-induced change in lifetime of the fluorescence emitted from the locally excited state of ECZ clearly shows that the electron transfer from the excited state of ECZ to DMTP is enhanced by an external electric field ( F ). A comparison is made between the experimental results of the field effect on decay profile of the ECZ fluorescence and the simulated results. Time-resolved electrofluorescence spectra as well as the field-induced change in decay profile of exciplex fluorescence show that exciplex fluorescence is quenched by F at the early stage of time following photoexcitation, but enhanced by F at a later stage of time. Both the decrease in the initial population of the fluorescent exciplex and the lengthening of the exciplex fluorescence in lifetime are shown to be induced by F

Time-resolved measurements of the external electric field effects on fluorescence in electron donor and acceptor pairs of N-ethylcarbazole and dimethyl terephthalate doped in a polymer film

Energy Technology Data Exchange (ETDEWEB)

Iimori, Toshifumi [Research Institute for Electronic Science (RIES), Hokkaido University, Sapporo 060-0812 (Japan); Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810 (Japan); Yoshizawa, Tomokazu [Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810 (Japan); Nakabayashi, Takakazu [Research Institute for Electronic Science (RIES), Hokkaido University, Sapporo 060-0812 (Japan); Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810 (Japan); Ohta, Nobuhiro [Research Institute for Electronic Science (RIES), Hokkaido University, Sapporo 060-0812 (Japan); Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810 (Japan)], E-mail: nohta@es.hokudai.ac.jp

2005-12-07

Electric-field-induced change in fluorescence decay has been measured for electron donor and acceptor pairs of N-ethylcarbazole (ECZ) and dimethyl terephthalate (DMTP) doped in a polymer film. Field-induced change in lifetime of the fluorescence emitted from the locally excited state of ECZ clearly shows that the electron transfer from the excited state of ECZ to DMTP is enhanced by an external electric field ( F ). A comparison is made between the experimental results of the field effect on decay profile of the ECZ fluorescence and the simulated results. Time-resolved electrofluorescence spectra as well as the field-induced change in decay profile of exciplex fluorescence show that exciplex fluorescence is quenched by F at the early stage of time following photoexcitation, but enhanced by F at a later stage of time. Both the decrease in the initial population of the fluorescent exciplex and the lengthening of the exciplex fluorescence in lifetime are shown to be induced by F.

Characterization of a new series of fluorescent probes for imaging membrane order.

Directory of Open Access Journals (Sweden)

Joanna M Kwiatek

Full Text Available Visualization and quantification of lipid order is an important tool in membrane biophysics and cell biology, but the availability of environmentally sensitive fluorescent membrane probes is limited. Here, we present the characterization of the novel fluorescent dyes PY3304, PY3174 and PY3184, whose fluorescence properties are sensitive to membrane lipid order. In artificial bilayers, the fluorescence emission spectra are red-shifted between the liquid-ordered and liquid-disordered phases. Using ratiometric imaging we demonstrate that the degree of membrane order can be quantitatively determined in artificial liposomes as well as live cells and intact, live zebrafish embryos. Finally, we show that the fluorescence lifetime of the dyes is also dependent on bilayer order. These probes expand the current palate of lipid order-sensing fluorophores affording greater flexibility in the excitation/emission wavelengths and possibly new opportunities in membrane biology.

Social contagions on correlated multiplex networks

Science.gov (United States)

Wang, Wei; Cai, Meng; Zheng, Muhua

2018-06-01

The existence of interlayer degree correlations has been disclosed by abundant multiplex network analysis. However, how they impose on the dynamics of social contagions are remain largely unknown. In this paper, we propose a non-Markovian social contagion model in multiplex networks with inter-layer degree correlations to delineate the behavior spreading, and develop an edge-based compartmental (EBC) theory to describe the model. We find that multiplex networks promote the final behavior adoption size. Remarkably, it can be observed that the growth pattern of the final behavior adoption size, versus the behavioral information transmission probability, changes from discontinuous to continuous once decreasing the behavior adoption threshold in one layer. We finally unravel that the inter-layer degree correlations play a role on the final behavior adoption size but have no effects on the growth pattern, which is coincidence with our prediction by using the suggested theory.

Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset

Directory of Open Access Journals (Sweden)

Nayereh Nouri

2014-01-01

Full Text Available Background: The Duchenne muscular dystrophy (DMD gene is located in the short arm of the X chromosome (Xp21. It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA over multiplex polymerase chain reaction (PCR assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae, Lamiaceae, Rosaceae

Directory of Open Access Journals (Sweden)

Oksana Sytar

2015-01-01

Full Text Available BACKGROUND: The aim of our research work was to quantify total flavonoid contents in the leaves of 13 plant species family Asteraceae, 8 representatives of family Lamiaceae and 9 plant species belonging to familyRosaceae, using the multiplex fluorimetric sensor. Fluorescence was measured using optical fluorescence apparatus Multiplex(R 3 (Force-A, France for non-destructive flavonoids estimation. The content of total flavonoids was estimated by FLAV index (expressed in relative units, that is deduced from flavonoids UV absorbing properties. RESULTS: Among observed plant species, the highest amount of total flavonoids has been found in leaves ofHelianthus multiflorus (1.65 RU and Echinops ritro (1.27 RU, Rudbeckia fulgida (1.13 RU belonging to the family Asteraceae. Lowest flavonoid content has been observed in the leaves of marigold (Calendula officinalis (0.14 RU also belonging to family Asteraceae. The highest content of flavonoids among experimental plants of family Rosaceae has been estimated in the leaves of Rosa canina (1.18 RU and among plant species of family Lamiaceae in the leaves of Coleus blumei (0.90 RU. CONCLUSIONS: This research work was done as pre-screening of flavonoids content in the leaves of plant species belonging to family Asteraceae, Lamiaceae and Rosaceae. Results indicated that statistically significant differences (P > 0.05 in flavonoids content were observed not only between families, but also among individual plant species within one family.

Nuclear lifetime measurement

International Nuclear Information System (INIS)

Guillaume, Georges

Three direct techniques of lifetime measurement are emphasized: electronic methods and two methods based on the Doppler effect (the recoil distance methods or RDM, the Doppler shift attenuation methods or DSAM). Said direct methods are concerned with the direct measurement of the radioactive decay constants of nuclear excited states. They allow lifetimes of nucleus bound states whose deexcitations occur by electromagnetic transitions, to be determined. Other methods for measuring lifetimes are also examined: microwave techniques and those involving the blocking effect in crystals (direct methods) and also various indirect methods of obtaining lifetimes (γ resonance scattering, capture reactions, inelastic electron and nucleus scattering, and Coulomb deexcitation) [fr

Moving through a multiplex holographic scene

Science.gov (United States)

Mrongovius, Martina

2013-02-01

This paper explores how movement can be used as a compositional element in installations of multiplex holograms. My holographic images are created from montages of hand-held video and photo-sequences. These spatially dynamic compositions are visually complex but anchored to landmarks and hints of the capturing process - such as the appearance of the photographer's shadow - to establish a sense of connection to the holographic scene. Moving around in front of the hologram, the viewer animates the holographic scene. A perception of motion then results from the viewer's bodily awareness of physical motion and the visual reading of dynamics within the scene or movement of perspective through a virtual suggestion of space. By linking and transforming the physical motion of the viewer with the visual animation, the viewer's bodily awareness - including proprioception, balance and orientation - play into the holographic composition. How multiplex holography can be a tool for exploring coupled, cross-referenced and transformed perceptions of movement is demonstrated with a number of holographic image installations. Through this process I expanded my creative composition practice to consider how dynamic and spatial scenes can be conveyed through the fragmented view of a multiplex hologram. This body of work was developed through an installation art practice and was the basis of my recently completed doctoral thesis: 'The Emergent Holographic Scene — compositions of movement and affect using multiplex holographic images'.

Laser induced fluorescence and phosphorescence of matrix isolated glyoxal - Evidence for exciplex formation in the A 1Au and a 3Au states

Science.gov (United States)

Van Ijzendoorn, L. J.; Baas, F.; Koernig, S.; Greenberg, J. M.; Allamandola, L. J.

1986-01-01

Laser-induced fluorescence and phosphorescence as well as infrared and visible absorption spectra of glyoxal in Ar, N2, and CO matrices are presented and analyzed. Glyoxal in its first excited electronic state is shown to form an exciplex with its nearest neighbors in all three matrices, and transitions normally forbidden dominate the emission spectra. The spectral characteristics of these complexes are similar to those of the Ar-glyoxal complex found in supersonic beam experiments. Due to the matrix cage effect, no vibrational predissociation is observed. The phosphorescence lifetime is determined and an upper limit is given for the fluorescence lifetime. This, in combination with the relative intensities of fluorescence and phosphorescence, can be used to place limits on the quantum yields of the various relaxation processes.

Reduced γ–γ time walk to below 50 ps using the multiplexed-start and multiplexed-stop fast-timing technique with LaBr{sub 3}(Ce) detectors

Energy Technology Data Exchange (ETDEWEB)

Régis, J.-M., E-mail: regis@ikp.uni-koeln.de [Institut für Kernphysik der Universität zu Köln, Zülpicher Str. 77, 50937 Köln (Germany); Saed-Samii, N., E-mail: nima@ikp.uni-koeln.de [Institut für Kernphysik der Universität zu Köln, Zülpicher Str. 77, 50937 Köln (Germany); Rudigier, M. [Institut für Kernphysik der Universität zu Köln, Zülpicher Str. 77, 50937 Köln (Germany); Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom); Ansari, S.; Dannhoff, M.; Esmaylzadeh, A.; Fransen, C.; Gerst, R.-B.; Jolie, J.; Karayonchev, V.; Müller-Gatermann, C.; Stegemann, S. [Institut für Kernphysik der Universität zu Köln, Zülpicher Str. 77, 50937 Köln (Germany)

2016-07-01

The electronic γ–γ fast-timing technique using arrays consisting of many LaBr{sub 3}(Ce) detectors is a powerful method to determine lifetimes of nuclear excited states with a lower limit of about 5 ps. This method requires the determination of the energy-dependent time walk of the zero time which is represented by the centroid of a prompt γ–γ time distribution. The full-energy peak versus full-energy peak prompt response difference which represents the linearly combined mean γ–γ time walk of a fast-timing array consisting of 8 LaBr{sub 3}(Ce) detectors was measured using a standard {sup 152}Eu γ-ray source for the energy region of 40–1408 keV. The data were acquired using a “multiplexed-start and multiplexed-stop” analogue electronics circuitry and analysed by employing the generalized centroid difference method. Concerning the cylindrical 1.5 in.×1.5 in. LaBr{sub 3}(Ce) crystals which are coupled to the Hamamatsu R9779 photomultiplier tubes, the best fast-timing array time resolution of 202(3) ps is obtained for the two prompt γ lines of {sup 60}Co by using the leading-edge timing principle. When using the zero-crossover timing principle the time resolution is degraded by up to 30%, dependent on the energy and the shaping delay time of the constant fraction discriminator model Ortec 935. The smallest γ–γ time walk to below 50 ps is obtained by using a shaping delay time of about 17 ns and an optimum “time-walk adjustment” needed for detector output pulses with amplitudes smaller than 400 mV.

Radiometric and signal-to-noise ratio properties of multiplex dispersive spectrometry

International Nuclear Information System (INIS)

Barducci, Alessandro; Guzzi, Donatella; Lastri, Cinzia; Nardino, Vanni; Marcoionni, Paolo; Pippi, Ivan

2010-01-01

Recent theoretical investigations have shown important radiometric disadvantages of interferential multiplexing in Fourier transform spectrometry that apparently can be applied even to coded aperture spectrometers. We have reexamined the methods of noninterferential multiplexing in order to assess their signal-to-noise ratio (SNR) performance, relying on a theoretical modeling of the multiplexed signals. We are able to show that quite similar SNR and radiometric disadvantages affect multiplex dispersive spectrometry. The effect of noise on spectral estimations is discussed.

Topology-optimized silicon photonic wire mode (de)multiplexer

DEFF Research Database (Denmark)

Frellsen, Louise Floor; Frandsen, Lars Hagedorn; Ding, Yunhong

2015-01-01

We have designed and for the first time experimentally verified a topology optimized mode (de)multiplexer, which demultiplexes the fundamental and the first order mode of a double mode photonic wire to two separate single mode waveguides (and multiplexes vice versa). The device has a footprint...

Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

International Nuclear Information System (INIS)

Gartia, Manas Ranjan; Hsiao, Austin; Logan Liu, G; Sivaguru, Mayandi; Chen Yi

2011-01-01

We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.

Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

Energy Technology Data Exchange (ETDEWEB)

Gartia, Manas Ranjan [Department of Nuclear, Plasma and Radiological Engineering, University of Illinois, Urbana, IL 61801 (United States); Hsiao, Austin; Logan Liu, G [Department of Bioengineering, University of Illinois, Urbana, IL 61801 (United States); Sivaguru, Mayandi [Institute for Genomic Biology, University of Illinois, Urbana, IL 61801 (United States); Chen Yi, E-mail: loganliu@illinois.edu [Department of Electrical and Computer Engineering, University of Illinois, Urbana, IL 61801 (United States)

2011-09-07

We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.

Link overlap, viability, and mutual percolation in multiplex networks

International Nuclear Information System (INIS)

Min, Byungjoon; Lee, Sangchul; Lee, Kyu-Min; Goh, K.-I.

2015-01-01

Many real-world complex systems are best modeled by multiplex networks. The multiplexity has proved to have broad impact on the system’s structure and function. Most theoretical studies on multiplex networks to date, however, have largely ignored the effect of the link overlap across layers despite strong empirical evidences for its significance. In this article, we investigate the effect of the link overlap in the viability of multiplex networks, both analytically and numerically. After a short recap of the original multiplex viability study, the distinctive role of overlapping links in viability and mutual connectivity is emphasized and exploited for setting up a proper analytic framework. A rich phase diagram for viability is obtained and greatly diversified patterns of hysteretic behavior in viability are observed in the presence of link overlap. Mutual percolation with link overlap is revisited as a limit of multiplex viability problem, and the controversy between existing results is clarified. The distinctive role of overlapping links is further demonstrated by the different responses of networks under random removals of overlapping and non-overlapping links, respectively, as well as under several link-removal strategies. Our results show that the link overlap facilitates the viability and mutual percolation; at the same time, the presence of link overlap poses a challenge in analytical approaches to the problem

Fluorescence of sanguinarine: fundamental characteristics and analysis of interconversion between various forms.

Science.gov (United States)

Janovská, Marika; Kubala, Martin; Simánek, Vilím; Ulrichová, Jitka

2009-09-01

The quaternary isoquinoline alkaloid, sanguinarine (SG) plays an important role in both traditional and modern medicine, exhibiting a wide range of biological activities. Under physiological conditions, there is an equilibrium between the quaternary cation (SG+) and a pseudobase (SGOH) forms of SG. In the gastrointestinal tract, SG is converted to dihydrosanguinarine (DHSG). All forms exhibit bright fluorescence. However, their spectra overlap, which limited the use of powerful techniques based on fluorescence spectroscopy/microscopy. Our experiments using a combination of steady-state and time-resolved techniques enabled the separation of individual components. The results revealed that (a) the equilibrium constant between SG+ and SGOH is pKa = 8.06, while fluorescence of DHSG exhibited no changes in the pH range 5-12, (b) the SGOH has excitation/emission spectra with maxima at 327/418 nm and excited-state lifetime 3.2 ns, the spectra of the SG+ have maxima at 475/590 nm and excited-state lifetime 2.4 ns. The DHSG spectra have maxima at 327/446 nm and 2-exponential decay with components 4.2 and 2.0 ns, (c) NADH is able to convert SG to DHSG, while there is no apparent interaction between NADH and DHSG. These techniques are applicable for monitoring the SG to DHSG conversion in hepatocytes.

Simultaneous Expression of GUS and Actin Genes by Using the Multiplex RT-PCR and Multiplex Gold Nanoparticle Probes.

Science.gov (United States)

Ghazi, Yaser; Vaseghi, Akbar; Ahmadi, Sepideh; Haddadi, Fatemeh

2018-04-23

Gene expression analysis is considered to be extremely important in many different biological researches. DNA-based diagnostic test, which contributes to DNA identification, has higher specificity, cost, and speed than some biochemical and molecular methods. In this study, we try to use the novel nano technology approach with Multiplex RT-PCR and Gold nano particular probes (GNPs-probes) in order to get gene expression in Curcumas melons. We used Agrobacterium tumefactions for gene transfer and GUS reporter gene as a reporter. After cDNA synthesis, Multiplex PCR and Multiplex RT-PCR techniques were used. Finally, probes were designed for RNA of GUS and Actin genes, and then the analysis of the gene expression using the probes attached to GNPs was carried out and the color changes in the GNPs were applied. In the following, probes hybridization was checked with DNA between 400 to 700 nm wavelengths and the highest rate was observed in the 550 to 650 nm. The results show that the simultaneous use of GNP-attached detectors and Multiplex RT-PCRcan reduce time and costmore considerably than somelaboratory methods for gene expiration investigation. Additionally, it can be seen thatthere is an increase in sensitivity and specificity of our investigation. Based on our findings, this can bea novel study doneusingMultiplex RT-PCRand unmodified AuNPs for gene transfer and expression detection to plants. We can claim that this assay has a remarkable advantage including rapid, cost-effectiveness, specificity and accuracy to detect transfer and expression genes in plants. Also,we can use this technique from other gene expressionsin many different biology samples.

Investigation of the inclusion behavior between p-sulfoniccalix[8]arene and norfloxacin by fluorescence spectroscopy

International Nuclear Information System (INIS)

Wang Xueying; Luo Chuannan; Lv Zhen; Lu Fuguang

2011-01-01

The host-guest complexation between p-sulfoniccalix[8]arene (SC 8 A) and norfloxacin (NFLX) in aqueous solution was investigated by fluorescence spectroscopy. Strong fluorescence intensity of the NFLX aqueous solution alone and obvious fluorescence quenching of NFLX solution in the presence of SC 8 A were observed. The fluorescence lifetimes of NFLX and SC 8 A-NFLX inclusion complex were determined and the effect of temperature on SC 8 A-NFLX inclusion complex was studied. The static quenching of the inclusion was obtained, that is the SC 8 A can form a nonfluorescent ground-state inclusion complex with NFLX. As the results show, the combined ratio (n) was 1:1 and association constant K was 1.17x10 5 L/mol. Based on the experimental results, the mechanism of the inclusion complex was explored. The space matching, electrostatic force and hydrogen bond play important effects in the inclusion process. Subsequently, the addition of bovine serum albumin (BSA) solution led to the recovery of fluorescence intensity. It is indicated that BSA can liberate the NFLX into the solution by destructing the SC 8 A-NFLX inclusion complex. Hence SC 8 A may be used for controlled-release drug delivery in the pharmaceutical industry. - Highlights: → Fluorescence lifetimes of NFLX and SC8A-NFLX inclusion complex were determined. → Mechanism of the SC8A-NFLX inclusion complex was explored. → It is proved that SC8A can form a nonfluorescent ground-state inclusion complex with NFLX.

Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes

Science.gov (United States)

Liu, Jianbo; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Wei; Wang, Dong

2013-10-01

The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies.The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to

Shift-Peristrophic Multiplexing for High Density Holographic Data Storage

Directory of Open Access Journals (Sweden)

Zenta Ushiyama

2014-03-01

Full Text Available Holographic data storage is a promising technology that provides very large data storage capacity, and the multiplexing method plays a significant role in increasing this capacity. Various multiplexing methods have been previously researched. In the present study, we propose a shift-peristrophic multiplexing technique that uses spherical reference waves, and experimentally verify that this method efficiently increases the data capacity. In the proposed method, a series of holograms is recorded with shift multiplexing, in which the recording material is rotated with its axis perpendicular to the material’s surface. By iterating this procedure, multiplicity is shown to improve. This method achieves more than 1 Tbits/inch2 data density recording. Furthermore, a capacity increase of several TB per disk is expected by maximizing the recording medium performance.

Fluorescence properties of europium and samarium. beta. -diketonates and their use in fluorometry

Energy Technology Data Exchange (ETDEWEB)

Huang, H; Hiraki, K; Nishikawa, Y [Kinki Univ., Higashi-Osaka, Osaka (Japan). Faculty of Science and Technology

1981-01-01

Several europium and samarium ..beta..-diketonates (tta, ntfa, bfa) complexed with 1, 10-phenanthroline, or with trioctylphosphine oxide (topo) were synthesized. The fluorescence properties of these compounds in benzene or hexane have been studied. Absorption and fluorescence spectra, fluorescence quantum yield, fluorescence sensitivity index (F.S.I.), and fluorescence lifetime were measured. From the measurement of fluorescence lifetime of the ..beta..-diketonates, the velocity of radiative process (k sub(f)/phi sub(f)) has almost the same value for benzene and hexane solvent. The red fluorescence (Em. max. : 619 nm) of Eu(III) in these chelates is attributed to transitions from /sup 5/D/sub 0/ ..-->.. /sup 7/F/sub 2/ levels of this ion, and the three-band spectrum (Em. max. : 569 nm, 606 nm, 650 nm) indicates the transitions from the /sup 4/G sub(5/2) ..-->.. /sup 6/H sub(5/2), /sup 4/G sub(5/2) ..-->.. /sup 6/H sub(7/2), and /sup 4/G sub(5/2) ..-->.. /sup 6/H sub(9/2) levels of Sm(III), respectively. These spectra are not changed by any solvents and ligands. From the results, the fluorescence of the ..beta..-diketonates in organic solvent has been attributed to m* ..-->.. m luminescence transition. The complexes of Eu(III) and Sm(III) show radiative transition within orbitals, composed exclusively of 4f orbitals of rare earth ions (m* ..-->.. m radiative transition). Fluorinated ligands show better sensitivity than unfluorinated ligands, and the best sensitivity is obtained with TTA-phen system, and/or TTA-topo system for the spectrofluorometric determination of the two metals. In the case of Eu determination, 619 nm emission wavelength is used (the determinable range : 0.2 -- 10 ppb Eu), and in the case of Sm determination, 650 nm emission wavelength is adopted (the determinable range : 0.1 -- 1 ppm Sm), because of much higher sensitivity than the other two peaks (569, 606 nm) without interference from europium complex.

Hadronization, spin and lifetimes

International Nuclear Information System (INIS)

Grossman, Yuval; Nachshon, Itay

2008-01-01

Measurements of lifetimes can be done in two ways. For very short lived particles, the width can be measured. For long lived ones, the lifetime can be directly measured, for example, using a displaced vertex. Practically, the lifetime cannot be extracted for particles with intermediate lifetimes. We show that for such cases information about the lifetime can be extracted for heavy colored particles that can be produced with known polarization. For example, a t-like particle with intermediate lifetime hadronizes into a superposition of the lowest two hadronic states, T* and T (the equivalent of B* and B). Depolarization effects are governed by time scales that are much longer than the hadronization time scale, Λ QCD -1 . After a time of order 1/Δm, with Δm≡m(T*)-m(T), half of the initial polarization is lost. The polarization is totally lost after a time of order 1/Γ γ , with Γ γ = Γ(T* → Tγ). Thus, by comparing the initial and final polarization, we get information on the particle's lifetime.

Digital holograms for laser mode multiplexing

CSIR Research Space (South Africa)

Mhlanga, T

2014-10-02

Full Text Available multiplexing Thandeka Mhlangaa, b, Abderrahmen Trichilic, Angela Dudleya, Darryl Naidooa, b, Mourad Zghalc and Andrew Forbesa, b aCSIR National Laser Centre, P.O. Box 395, Pretoria 0001, South Africa bSchool of Physics, University of KwaZulu-Natal, Private Bag... problems. In this context, we demonstrate a method of multiplexing laser modes using spatial light modulators (SLMs). In our proposed technique, we use Laguerre Gaussian (LG) modes, which form a complete basis set; hence multi-mode masks can be created...

Simple Multiplexing Hand-Held Control Unit

Science.gov (United States)

Hannaford, Blake

1989-01-01

Multiplexer consists of series of resistors, each shunted by single-pole, single-throw switch. User operates switches by pressing buttons or squeezing triggers. Prototype includes three switches operated successfully in over 200 hours of system operations. Number of switches accommodated determined by signal-to-noise ratio of current source, noise induced in control unit and cable, and number of bits in output of analog-to-digital converter. Because many computer-contolled robots have extra analog-to-digital channels, such multiplexer added at little extra cost.

Two-photon-excited fluorescence spectroscopy of atomic fluorine at 170 nm

Science.gov (United States)

Herring, G. C.; Dyer, Mark J.; Jusinski, Leonard E.; Bischel, William K.

1988-01-01

Two-photon-excited fluorescence spectroscopy of atomic fluorine is reported. A doubled dye laser at 286-nm is Raman shifted in H2 to 170 nm (sixth anti-Stokes order) to excite ground-state 2P(0)J fluorine atoms to the 2D(0)J level. The fluorine atoms are detected by one of two methods: observing the fluorescence decay to the 2PJ level or observing F(+) production through the absorption of an additional photon by the excited atoms. Relative two-photon absorption cross sections to and the radiative lifetimes of the 2D(0)J states are measured.

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...

Instantaneous imaging of ozone in a gliding arc discharge using photofragmentation laser-induced fluorescence

Science.gov (United States)

Larsson, Kajsa; Hot, Dina; Gao, Jinlong; Kong, Chengdong; Li, Zhongshan; Aldén, Marcus; Bood, Joakim; Ehn, Andreas

2018-04-01

Ozone vapor, O3, is here visualized in a gliding arc discharge using photofragmentation laser-induced fluorescence. Ozone is imaged by first photodissociating the O3 molecule into an O radical and a vibrationally hot O2 fragment by a pump photon. Thereafter, the vibrationally excited O2 molecule absorbs a second (probe) photon that further transits the O2-molecule to an excited electronic state, and hence, fluorescence from the deexcitation process in the molecule can be detected. Both the photodissociation and excitation processes are achieved within one 248 nm KrF excimer laser pulse that is formed into a laser sheet and the fluorescence is imaged using an intensified CCD camera. The laser-induced signal in the vicinity of the plasma column formed by the gliding arc is confirmed to stem from O3 rather than plasma produced vibrationally hot O2. While both these products can be produced in plasmas a second laser pulse at 266 nm was utilized to separate the pump- from the probe-processes. Such arrangement allowed lifetime studies of vibrationally hot O2, which under these conditions were several orders of magnitude shorter than the lifetime of plasma-produced ozone.

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

Science.gov (United States)

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

2005-08-01

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

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

International Nuclear Information System (INIS)

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

2005-01-01

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

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

Science.gov (United States)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

Light extraction efficiency enhancement for fluorescent SiC based white light-emitting diodes

DEFF Research Database (Denmark)

Ou, Haiyan; Ou, Yiyu; Argyraki, Aikaterini

Fluorescent SiC based white light-emitting diodes(LEDs) light source, as an innovative energy-efficient light source, would even have longer lifetime, better light quality and eliminated blue-tone effect, compared to the current phosphor based white LED light source. In this paper, the yellow...

Lifetime-management and lifetime-extension at PAKS nuclear power plant

International Nuclear Information System (INIS)

Katona, Tamas; Ratkai, Sandor; Janosi, Agnes Biro

2002-01-01

Paks Nuclear Power Plant provides 38-40% of domestic generation at lowest price. It has an important energy-policy role in Hungary. NPP Paks shall be a decisive and perspectively permanent element of the domestic electricity generation during the next two decades, which shall be ensured by plant safe operation, the lifetime extension and power uprating. Paks Nuclear Power Plant investigated the nuclear power plant's lifetime extension possibilities and alternatives, as well as technical and business feasibility of such alternatives. The feasibility study is based on the evaluation of a representative set of systems, structures and components, operational, test, in-service inspection and maintenance practice, experience and findings of the Periodic Safety Review. The most important results of this study showing the feasibility of 20 years lifetime extension is summarised in the paper. It was found that there are no technical or safety issues or limits, which may inhibit the operation of the Nuclear Power Plant Paks up to 50 years. In case of most systems and equipment the recent monitoring, maintenance and regular reconstruction practice of the NPP Paks allows the lifetime extension without outstanding cost. Replacement or reconstruction of a few equipment and systems requires significant investment costs. Material of reactor vessels of VVER/213 incorporated at Paks, compared to vessels of the similar units, is less sensitive to the embrittlement. At units 3-4 reactor vessels do not require any measure, consequently, any additional cost, even in case of a lifetime of 50 years. At unit 2 to extend the lifetime of the reactor vessel, only heating-up of emergency core cooling tanks is needed in order to decrease thermal stress levels caused by pressure thermal shock (PST) transients. For this purpose cost-effective technical solutions are available. At unit 1, beside the heating-up of the emergency core cooling tanks annealing of the welded joint No. 5/6 close to the

Multiplexed salivary protein profiling for patients with respiratory diseases using fiber-optic bundles and fluorescent antibody-based microarrays.

Science.gov (United States)

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-01

Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody-based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. On the basis of a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin-1 beta (IL-1β) salivary biomarkers in the subpicomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p < 0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis.

Microprocessorized message multiplexer

International Nuclear Information System (INIS)

Ejzman, S.; Guglielmi, L.; Jaeger, J.J.

1980-07-01

The 'Microprocessorized Message Multiplexer' is an elementary development tool used to create and debug the software of a target microprocessor (User Module: UM). It connects together four devices: a terminal, a cassette recorder, the target microprocessor and a host computer where macro and editor for the M 6800 microprocessor are resident [fr