two-dimensional fluorescence difference: Topics by Science.gov

Sample records for two-dimensional fluorescence difference

Characterization of humic acids by two-dimensional correlation fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

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

2008-07-01

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

PubMed

Serpengüzel, A; Swindal, J C; Chang, R K; Acker, W P

1992-06-20

Two-dimensional fluorescence, lasing, and stimulated Raman scattering images of a hollow-cone nozzle spray are observed. The various constituents of the spray, such as vapor, liquid ligaments, small droplets, and large droplets, are distinguished by selectively imaging different colors associated with the inelastic light-scattering processes.
Measurement of Zeta-Potential at Microchannel Wall by a Nanoscale Laser Induced Fluorescence Imaging

NASA Astrophysics Data System (ADS)

Kazoe, Yutaka; Sato, Yohei

A nanoscale laser induced fluorescence imaging was proposed by using fluorescent dye and the evanescent wave with total internal reflection of a laser beam. The present study focused on the two-dimensional measurement of zeta-potential at the microchannel wall, which is an electrostatic potential at the wall surface and a dominant parameter of electroosmotic flow. The evanescent wave, which decays exponentially from the wall, was used as an excitation light of the fluorescent dye. The fluorescent intensity detected by a CCD camera is closely related to the zeta-potential. Two kinds of fluorescent dye solution at different ionic concentrations were injected into a T-shaped microchannel, and formed a mixing flow field in the junction area. The two-dimensional distribution of zeta-potential at the microchannel wall in the pressure-driven flow field was measured. The obtained zeta-potential distribution has a transverse gradient toward the mixing flow field and was changed by the difference in the averaged velocity of pressure-driven flow. To understand the ion motion in the mixing flow field, the three-dimensional flow structure was analyzed by the velocity measurement using micron-resolution particle image velocimetry and the numerical simulation. It is concluded that the two-dimensional distribution of zeta-potential at the microchannel wall was dependent on the ion motion in the flow field, which was governed by the convection and molecular diffusion.
2D fluorescence spectra measurement of six kinds of bioagents simulants by short range Lidar

NASA Astrophysics Data System (ADS)

Sanpedro, Man

2018-02-01

Pantoea agglomerans (Pan), Staphylococcus aureus (Sta), Bacillus globigii (BG) and Escherichia coli (EH), these four kinds of bioagents simulants of were cultured and then their growth curves were measured, the generation time was 0.99h, 0.835h, 1.07h and 1.909h, respectively. A small short range fluorescence lidar working at wavelengths of 266nm and 355nm was designed and used to measure the two-dimensional fluorescence spectra of bioagents simulants in the amino acid segment and NADH segment, respectively. In a controllable fluorescence measurement chamber, the two-dimensional fluorescence spectra of vegetative liquid bacterial aerosols as well as BSA and OVA, two protein toxinic simulants were measured with a resolution of 4nm. The two-dimensional fluorescence spectral shape of Pan, Sta, EH and BG, BSA and OVA were consistent with the standard fluorescent component tryptophan in the amino acid band with FWHM of 60nm, but the central wavelength of the fluorescence spectra of these simulants blue/purple shifted obviously as affected by the external biochemical environment, concentration and ratio of different bacterial internal fluorophores, so the energy level between the excited state and the ground state of the fluorescence molecule increased. Differently, weak NADH fluorescence spectra with 100nm FWHM inside the four vegetative bacteria aerosols were detected, but Rayleigh scattering, Raman scattering contribution of water, nitrogen in the fluorescence spectra could not be effectively extracted. The second - order derivative fluorescence spectra of four simulants showed that the high - order processing and recognition of the fluorescence spectra was feasible.
Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

PubMed

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

2013-07-29

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

NASA Astrophysics Data System (ADS)

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

2014-04-01

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

PubMed

Wang, Zhiwei; Wu, Zhichao; Tang, Shujuan

2009-04-01

Three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy was employed to characterize dissolved organic matter (DOM) in a submerged membrane bioreactor (MBR). Three fluorescence peaks could be identified from the EEM fluorescence spectra of the DOM samples in the MBR. Two peaks were associated with the protein-like fluorophores, and the third was related to the visible humic acid-like fluorophores. Only two main peaks were observed in the EEM fluorescence spectra of the extracellular polymeric substance (EPS) samples, which were due to the fluorescence of protein-like and humic acid-like matters, respectively. However, the EEM fluorescence spectra of membrane foulants were observed to have three peaks. It was also found that the dominant fluorescence substances in membrane foulants were protein-like substances, which might be due to the retention of proteins in the DOM and/or EPS in the MBR by the fine pores of the membrane. Quantitative analysis of the fluorescence spectra including peak locations, fluorescence intensity, and different peak intensity ratios and the fluorescence regional integration (FRI) analysis were also carried out in order to better understand the similarities and differences among the EEM spectra of the DOM, EPS, and membrane foulant samples and to further provide an insight into membrane fouling caused by the fluorescence substances in the DOM in submerged MBRs.
Two-dimensional fluorescence lifetime correlation spectroscopy. 2. Application.

PubMed

Ishii, Kunihiko; Tahara, Tahei

2013-10-03

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

NASA Astrophysics Data System (ADS)

Gascooke, Jason R.; Lawrance, Warren D.

2017-11-01

Two dimensional laser induced fluorescence (2D-LIF) extends the usual laser induced fluorescence technique by adding a second dimension, the wavelength at which excited states emit, thereby significantly enhancing the information that can be extracted. It allows overlapping absorption features, whether they arise from within the same molecule or from different molecules in a mixture, to be associated with their appropriate "parent" state and/or molecule. While the first gas phase version of the technique was published a decade ago, the technique is in its infancy, having been exploited by only a few groups to date. However, its potential in gas phase spectroscopy and dynamics is significant. In this article we provide an overview of the technique and illustrate its potential with examples, with a focus on those utilising high resolution in the dispersed fluorescence dimension.
Resonance fluorescence based two- and three-dimensional atom localization

NASA Astrophysics Data System (ADS)

Wahab, Abdul; Rahmatullah; Qamar, Sajid

2016-06-01

Two- and three-dimensional atom localization in a two-level atom-field system via resonance fluorescence is suggested. For the two-dimensional localization, the atom interacts with two orthogonal standing-wave fields, whereas for the three-dimensional atom localization, the atom interacts with three orthogonal standing-wave fields. The effect of the detuning and phase shifts associated with the corresponding standing-wave fields is investigated. A precision enhancement in position measurement of the single atom can be noticed via the control of the detuning and phase shifts.
Comparison of Mars Science Laboratory Reaction Control System Jet Computations With Flow Visualization and Velocimetry

NASA Technical Reports Server (NTRS)

Bathel, Brett F.; Danehy, Paul M.; Johansen, Craig T.; Ashcraft, Scott W.; Novak, Luke A.

2013-01-01

Numerical predictions of the Mars Science Laboratory reaction control system jets interacting with a Mach 10 hypersonic flow are compared to experimental nitric oxide planar laser-induced fluorescence data. The steady Reynolds Averaged Navier Stokes equations using the Baldwin-Barth one-equation turbulence model were solved using the OVERFLOW code. The experimental fluorescence data used for comparison consists of qualitative two-dimensional visualization images, qualitative reconstructed three-dimensional flow structures, and quantitative two-dimensional distributions of streamwise velocity. Through modeling of the fluorescence signal equation, computational flow images were produced and directly compared to the qualitative fluorescence data.
Recent Advances in Silicon Nanomaterial-Based Fluorescent Sensors.

PubMed

Wang, Houyu; He, Yao

2017-02-03

During the past decades, owing to silicon nanomaterials' unique optical properties, benign biocompatibility, and abundant surface chemistry, different dimensional silicon nanostructures have been widely employed for rationally designing and fabricating high-performance fluorescent sensors for the detection of various chemical and biological species. Among of these, zero-dimensional silicon nanoparticles (SiNPs) and one-dimensional silicon nanowires (SiNWs) are of particular interest. Herein, we focus on reviewing recent advances in silicon nanomaterials-based fluorescent sensors from a broad perspective and discuss possible future directions. Firstly, we introduce the latest achievement of zero-dimensional SiNP-based fluorescent sensors. Next, we present recent advances of one-dimensional SiNW-based fluorescent sensors. Finally, we discuss the major challenges and prospects for the development of silicon-based fluorescent sensors.
Recent Advances in Silicon Nanomaterial-Based Fluorescent Sensors

PubMed Central

Wang, Houyu; He, Yao

2017-01-01

During the past decades, owing to silicon nanomaterials’ unique optical properties, benign biocompatibility, and abundant surface chemistry, different dimensional silicon nanostructures have been widely employed for rationally designing and fabricating high-performance fluorescent sensors for the detection of various chemical and biological species. Among of these, zero-dimensional silicon nanoparticles (SiNPs) and one-dimensional silicon nanowires (SiNWs) are of particular interest. Herein, we focus on reviewing recent advances in silicon nanomaterials-based fluorescent sensors from a broad perspective and discuss possible future directions. Firstly, we introduce the latest achievement of zero-dimensional SiNP-based fluorescent sensors. Next, we present recent advances of one-dimensional SiNW-based fluorescent sensors. Finally, we discuss the major challenges and prospects for the development of silicon-based fluorescent sensors. PMID:28165357
Lateral resolution testing of a novel developed confocal microscopic imaging system

NASA Astrophysics Data System (ADS)

Zhang, Xin; Zhang, Yunhai; Chang, Jian; Huang, Wei; Xue, Xiaojun; Xiao, Yun

2015-10-01

Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths 405nm 488nm 561nm and 638nm which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.
Ultrafast fluorescent decay induced by metal-mediated dipole–dipole interaction in two-dimensional molecular aggregates

DOE PAGES

Hu, Qing; Jin, Dafei; Xiao, Jun; ...

2017-09-05

Two-dimensional molecular aggregate (2DMA), a thin sheet of strongly interacting dipole molecules self-assembled at close distance on an ordered lattice, is a fascinating fluorescent material. It is distinctively different from the conventional (single or colloidal) dye molecules and quantum dots. Here, in this paper, we verify that when a 2DMA is placed at a nanometric distance from a metallic substrate, the strong and coherent interaction between the dipoles inside the 2DMA dominates its fluorescent decay at a picosecond timescale. Our streak-camera lifetime measurement and interacting lattice–dipole calculation reveal that the metal-mediated dipole–dipole interaction shortens the fluorescent lifetime to about one-halfmore » and increases the energy dissipation rate by 10 times that expected from the noninteracting single-dipole picture. In conclusion, our finding can enrich our understanding of nanoscale energy transfer in molecular excitonic systems and may designate a unique direction for developing fast and efficient optoelectronic devices.« less
Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin.

PubMed

Nakashima, Kenichi; Yuda, Kazuki; Ozaki, Yukihiro; Noda, Isao

2003-11-01

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.
Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis.

PubMed

Gutiérrez-Sánchez, Gerardo; Atwood, James; Kolli, V S Kumar; Roussos, Sévastianos; Augur, Christopher

2012-04-01

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.
Temporal focusing microscopy combined with three-dimensional structured illumination

NASA Astrophysics Data System (ADS)

Isobe, Keisuke; Toda, Keisuke; Song, Qiyuan; Kannari, Fumihiko; Kawano, Hiroyuki; Miyawaki, Atsushi; Midorikawa, Katsumi

2017-05-01

Temporal focusing microscopy provides the optical sectioning capability in wide-field two-photon fluorescence imaging. Here, we demonstrate temporal focusing microscopy combined with three-dimensional structured illumination, which enables us to enhance the three-dimensional spatial resolution and reject the background fluorescence. Experimentally, the periodic pattern of the illumination was produced not only in the lateral direction but also in the axial direction by the interference between three temporal focusing pulses, which were easily generated using a digital micromirror device. The lateral resolution and optical sectioning capability were successfully enhanced by factors of 1.6 and 3.6, respectively, compared with those of temporal focusing microscopy. In the two-photon fluorescence imaging of a tissue-like phantom, the out-of-focus background fluorescence and the scattered background fluorescence could also be rejected.
Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS.

PubMed

Lanzanò, Luca; Scipioni, Lorenzo; Di Bona, Melody; Bianchini, Paolo; Bizzarri, Ranieri; Cardarelli, Francesco; Diaspro, Alberto; Vicidomini, Giuseppe

2017-07-06

The observation of molecular diffusion at different spatial scales, and in particular below the optical diffraction limit (<200 nm), can reveal details of the subcellular topology and its functional organization. Stimulated-emission depletion microscopy (STED) has been previously combined with fluorescence correlation spectroscopy (FCS) to investigate nanoscale diffusion (STED-FCS). However, stimulated-emission depletion fluorescence correlation spectroscopy has only been used successfully to reveal functional organization in two-dimensional space, such as the plasma membrane, while, an efficient implementation for measurements in three-dimensional space, such as the cellular interior, is still lacking. Here we integrate the STED-FCS method with two analytical approaches, the recent separation of photons by lifetime tuning and the fluorescence lifetime correlation spectroscopy, to simultaneously probe diffusion in three dimensions at different sub-diffraction scales. We demonstrate that this method efficiently provides measurement of the diffusion of EGFP at spatial scales tunable from the diffraction size down to ∼80 nm in the cytoplasm of living cells.The measurement of molecular diffusion at sub-diffraction scales has been achieved in 2D space using STED-FCS, but an implementation for 3D diffusion is lacking. Here the authors present an analytical approach to probe diffusion in 3D space using STED-FCS and measure the diffusion of EGFP at different spatial scales.
Two-Dimensional Fluorescence Difference Spectroscopy of ZnO and Mg Composites in the Detection of Physiological Protein and RNA Interactions

PubMed Central

Hoffman, Amanda; Wu, Xiaotong; Wang, Jianjie; Brodeur, Amanda; Thomas, Rintu; Thakkar, Ravindra; Hadi, Halena; Glaspell, Garry P.; Duszynski, Molly; Wanekaya, Adam; DeLong, Robert K.

2017-01-01

Two-dimensional fluorescence difference spectroscopy (2-D FDS) was used to determine the unique spectral signatures of zinc oxide (ZnO), magnesium oxide (MgO), and 5% magnesium zinc oxide nanocomposite (5% Mg/ZnO) and was then used to demonstrate the change in spectral signature that occurs when physiologically important proteins, such as angiotensin-converting enzyme (ACE) and ribonuclease A (RNase A), interact with ZnO nanoparticles (NPs). When RNase A is bound to 5% Mg/ZnO, the intensity is quenched, while the intensity is magnified and a significant shift is seen when torula yeast RNA (TYRNA) is bound to RNase A and 5% Mg/ZnO. The intensity of 5% Mg/ZnO is quenched also when thrombin and thrombin aptamer are bound to the nanocomposite. These data indicate that RNA–protein interaction can occur unimpeded on the surface of NPs, which was confirmed by gel electrophoresis, and importantly that the change in fluorescence excitation, emission, and intensity shown by 2-D FDS may indicate specificity of biomolecular interactions. PMID:29244716

On-line analysis of algae in water by discrete three-dimensional fluorescence spectroscopy.

PubMed

Zhao, Nanjing; Zhang, Xiaoling; Yin, Gaofang; Yang, Ruifang; Hu, Li; Chen, Shuang; Liu, Jianguo; Liu, Wenqing

2018-03-19

In view of the problem of the on-line measurement of algae classification, a method of algae classification and concentration determination based on the discrete three-dimensional fluorescence spectra was studied in this work. The discrete three-dimensional fluorescence spectra of twelve common species of algae belonging to five categories were analyzed, the discrete three-dimensional standard spectra of five categories were built, and the recognition, classification and concentration prediction of algae categories were realized by the discrete three-dimensional fluorescence spectra coupled with non-negative weighted least squares linear regression analysis. The results show that similarities between discrete three-dimensional standard spectra of different categories were reduced and the accuracies of recognition, classification and concentration prediction of the algae categories were significantly improved. By comparing with that of the chlorophyll a fluorescence excitation spectra method, the recognition accuracy rate in pure samples by discrete three-dimensional fluorescence spectra is improved 1.38%, and the recovery rate and classification accuracy in pure diatom samples 34.1% and 46.8%, respectively; the recognition accuracy rate of mixed samples by discrete-three dimensional fluorescence spectra is enhanced by 26.1%, the recovery rate of mixed samples with Chlorophyta 37.8%, and the classification accuracy of mixed samples with diatoms 54.6%.
Characterization of binding site heterogeneity for copper within dissolved organic matter fractions using two-dimensional correlation fluorescence spectroscopy.

PubMed

Hur, Jin; Lee, Bo-Mi

2011-06-01

The heterogeneity of copper binding characteristics for dissolved organic matter (DOM) fractions was investigated based on the fluorescence quenching of the synchronous fluorescence spectra upon the addition of copper and two-dimensional correlation spectroscopy (2D-COS). Hydrophobic acid (HoA) and hydrophilic (Hi) fractions of two different DOM (algal and leaf litter DOM) were used for this study. For both DOM, fluorescence quenching occurred at a wider range of wavelengths for the HoA fractions compared to the Hi fractions. The combined information of the synchronous and asynchronous maps derived from 2D-COS provided a clear picture of the heterogeneous distribution of the copper binding sites within each DOM fraction, which was not readily recognized by a simple comparison of the changes in the synchronous fluorescence spectra upon the addition of copper. For the algal DOM, higher stability constants were exhibited for the HoA versus the Hi fractions. The logarithms of the stability constants ranged from 4.8 to 6.1 and from 4.5 to 5.0 for the HoA and the Hi fractions of the algal DOM, respectively, depending on the associated wavelength and the fitted models. In contrast, no distinctive difference in the binding characteristics was found between the two fractions of the leaf litter DOM. This suggests that influences of the structural and chemical properties of DOM on copper binding may differ for DOM from different sources. The relative difference of the calculated stability constants within the DOM fractions were consistent with the sequential orders interpreted from the asynchronous 2D-COS. It is expected that 2D-COS will be widely applied to other DOM studies requiring detailed information on the heterogeneous nature and subsequent effects under a range of environmental conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.
Two-dimensional fluorescence-detected coherent spectroscopy with absolute phasing by confocal imaging of a dynamic grating and 27-step phase-cycling

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

De, Arijit K., E-mail: akde@lbl.gov; Fleming, Graham R., E-mail: grfleming@lbl.gov; Department of Chemistry, University of California at Berkeley, Berkeley, California 94702

2014-05-21

We present a novel experimental scheme for two-dimensional fluorescence-detected coherent spectroscopy (2D-FDCS) using a non-collinear beam geometry with the aid of “confocal imaging” of dynamic (population) grating and 27-step phase-cycling to extract the signal. This arrangement obviates the need for distinct experimental designs for previously developed transmission detected non-collinear two-dimensional coherent spectroscopy (2D-CS) and collinear 2D-FDCS. We also describe a novel method for absolute phasing of the 2D spectrum. We apply this method to record 2D spectra of a fluorescent dye in solution at room temperature and observe “spectral diffusion.”.
Navigating surgical fluorescence cameras using near-infrared optical tracking.

PubMed

van Oosterom, Matthias; den Houting, David; van de Velde, Cornelis; van Leeuwen, Fijs

2018-05-01

Fluorescence guidance facilitates real-time intraoperative visualization of the tissue of interest. However, due to attenuation, the application of fluorescence guidance is restricted to superficial lesions. To overcome this shortcoming, we have previously applied three-dimensional surgical navigation to position the fluorescence camera in reach of the superficial fluorescent signal. Unfortunately, in open surgery, the near-infrared (NIR) optical tracking system (OTS) used for navigation also induced an interference during NIR fluorescence imaging. In an attempt to support future implementation of navigated fluorescence cameras, different aspects of this interference were characterized and solutions were sought after. Two commercial fluorescence cameras for open surgery were studied in (surgical) phantom and human tissue setups using two different NIR OTSs and one OTS simulating light-emitting diode setup. Following the outcome of these measurements, OTS settings were optimized. Measurements indicated the OTS interference was caused by: (1) spectral overlap between the OTS light and camera, (2) OTS light intensity, (3) OTS duty cycle, (4) OTS frequency, (5) fluorescence camera frequency, and (6) fluorescence camera sensitivity. By optimizing points 2 to 4, navigation of fluorescence cameras during open surgery could be facilitated. Optimization of the OTS and camera compatibility can be used to support navigated fluorescence guidance concepts. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).
Enhanced fluorescence from CdSe/ZnS quantum dot nanophosphors embedded in a one-dimensional photonic crystal backbone structure.

PubMed

Min, Kyungtaek; Choi, Serok; Choi, Yunkyoung; Jeon, Heonsu

2014-11-06

A nano-engineered phosphor structure that produces enhanced fluorescence is reported. Two kinds of polymer materials with different refractive indices are spin-coated alternately to realize a one-dimensional (1D) photonic crystal (PC) phosphor platform, in which CdSe/ZnS core-shell quantum dots (QDs) were embedded as a fluorescence agent. The 1D PC phosphor structure is designed to match the pump photon energy with one of the photonic band-edges (PBEs), where the photon group velocity becomes zero, and thus the interaction between pump photons and fluorescent centres strengthened. A reference phosphor structure is also designed and fabricated; however, it has no PBE and exhibited bulk-like photonic properties. The fluorescence intensity from the 1D PC phosphors is examined during the pump photon energy scanning across the PBE. It is found that fluorescence from the 1D PC phosphor reaches its maximum when the pump photon energy coincides with the PBE, which is consistent with the theoretical prediction. In comparison with the reference phosphor, the fluorescence from the 1D PC phosphor is measured to be enhanced by a factor of 1.36.
Two-dimensional nanosecond electric field mapping based on cell electropermeabilization.

PubMed

Chen, Meng-Tse; Jiang, Chunqi; Vernier, P Thomas; Wu, Yu-Hsuan; Gundersen, Martin A

2009-11-11

Nanosecond, megavolt-per-meter electric pulses cause permeabilization of cells to small molecules, programmed cell death (apoptosis) in tumor cells, and are under evaluation as a treatment for skin cancer. We use nanoelectroporation and fluorescence imaging to construct two-dimensional maps of the electric field associated with delivery of 15 ns, 10 kV pulses to monolayers of the human prostate cancer cell line PC3 from three different electrode configurations: single-needle, five-needle, and flat-cut coaxial cable. Influx of the normally impermeant fluorescent dye YO-PRO-1 serves as a sensitive indicator of membrane permeabilization. The level of fluorescence emission after pulse exposure is proportional to the applied electric field strength. Spatial electric field distributions were compared in a plane normal to the center axis and 15-20 mum from the tip of the center electrode. Measurement results agree well with models for the three electrode arrangements evaluated in this study. This live-cell method for measuring a nanosecond pulsed electric field distribution provides an operationally meaningful calibration of electrode designs for biological applications and permits visualization of the relative sensitivities of different cell types to nanoelectropulse stimulation. PACS Codes: 87.85.M-
Three-dimensional optical-transfer-function analysis of fiber-optical two-photon fluorescence microscopy.

PubMed

Gu, Min; Bird, Damian

2003-05-01

The three-dimensional optical transfer function is derived for analyzing the imaging performance in fiber-optical two-photon fluorescence microscopy. Two types of fiber-optical geometry are considered: The first involves a single-mode fiber for delivering a laser beam for illumination, and the second is based on the use of a single-mode fiber coupler for both illumination delivery and signal collection. It is found that in the former case the transverse and axial cutoff spatial frequencies of the three-dimensional optical transfer function are the same as those in conventional two-photon fluorescence microscopy without the use of a pinhole.However, the transverse and axial cutoff spatial frequencies in the latter case are 1.7 times as large as those in the former case. Accordingly, this feature leads to an enhanced optical sectioning effect when a fiber coupler is used, which is consistent with our recent experimental observation.
A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes

PubMed Central

Kang, Minchul; Day, Charles A.; Drake, Kimberly; Kenworthy, Anne K.; DiBenedetto, Emmanuele

2009-01-01

Abstract Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells. PMID:19720039
8-Anilino-1-naphthalenesulfonate/Layered Double Hydroxide Ultrathin Films: Small Anion Assembly and Its Potential Application as a Fluorescent Biosensor.

PubMed

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

2016-09-06

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

PubMed

Shih, Wenting; Yamada, Soichiro

2011-12-22

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network. By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.
Imaging of molecular hydrogen and oxygen by single and two-photon fluorescence using laser and flashlamp sources

NASA Technical Reports Server (NTRS)

Diskin, Glenn S.; Lempert, Walter R.; Miles, Richard B.; Kumar, Vinod; Glesk, Ivan

1991-01-01

Two flow visualization techniques, i.e., simultaneous two-dimensional fluorescence imaging of H2 and O2 in a diffusion flame, and quasi-linear fluorescence imaging of O2, are presented. The first uses an injection-locked argon-fluoride excimer laser and a partial overlap of a two-photon ground state absorption in H2 with a single photon absorption from a vibrational level in O2. The second uses a simple, high-intensity ultraviolet flashlamp which provides a flux of photons in the 180-195 nm range, sufficient to produce a quasi-one-dimensional fluorescence image of hot/room temperature oxygen. Both techniques do not require that a seed material be introduced into the flow, they can image major flow constituents, and provide an instantaneous snapshot of the flow.
The application of laser induced predissociation fluorescence to the measurement of vibrational temperatures in a shock layer flow

NASA Technical Reports Server (NTRS)

Sutton, D. J.; Houwing, A. F. P.; Palma, P. C.; Boyce, R. R.; Sandeman, R. J.; Mundt, CH.

1993-01-01

Single shot spatially and spectrally resolved laser induced predissociation fluorescence measurements in a shock layer around a cylinder in a pulsed supersonic free stream are presented. Fluorescence signals were produced using the tuned output of an argon fluoride excimer laser to excite a mixture of rovibrational transitions in molecular oxygen. The signals produced along a line inside the shock layer were focussed onto a two dimensional detector coupled to a spectrometer, thus allowing spectral and spatial resolution of the fluorescence. In this way, it was possible to detect two fluorescence signals from two different transitions simultaneously, allowing the determination of vibrational temperatures without the need for calibration. However, to minimize problems associated with low signal to noise ratios, background subtraction and spatial averaging was required. The experimental measurements are compared with theoretical inviscid shock layer calculations for nonequilibrium air. A description of the strategies employed in these calculations is also provided.
Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

PubMed Central

Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R.

2013-01-01

Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821
A novel quantitative analysis method of three-dimensional fluorescence spectra for vegetable oils contents in edible blend oil

NASA Astrophysics Data System (ADS)

Xu, Jing; Wang, Yu-Tian; Liu, Xiao-Fei

2015-04-01

Edible blend oil is a mixture of vegetable oils. Eligible blend oil can meet the daily need of two essential fatty acids for human to achieve the balanced nutrition. Each vegetable oil has its different composition, so vegetable oils contents in edible blend oil determine nutritional components in blend oil. A high-precision quantitative analysis method to detect the vegetable oils contents in blend oil is necessary to ensure balanced nutrition for human being. Three-dimensional fluorescence technique is high selectivity, high sensitivity, and high-efficiency. Efficiency extraction and full use of information in tree-dimensional fluorescence spectra will improve the accuracy of the measurement. A novel quantitative analysis is proposed based on Quasi-Monte-Carlo integral to improve the measurement sensitivity and reduce the random error. Partial least squares method is used to solve nonlinear equations to avoid the effect of multicollinearity. The recovery rates of blend oil mixed by peanut oil, soybean oil and sunflower are calculated to verify the accuracy of the method, which are increased, compared the linear method used commonly for component concentration measurement.
Coherent fluorescence emission by using hybrid photonic–plasmonic crystals

PubMed Central

Shi, Lei; Yuan, Xiaowen; Zhang, Yafeng; Hakala, Tommi; Yin, Shaoyu; Han, Dezhuan; Zhu, Xiaolong; Zhang, Bo; Liu, Xiaohan; Törmä, Päivi; Lu, Wei; Zi, Jian

2014-01-01

The spatial and temporal coherence of the fluorescence emission controlled by a quasi-two-dimensional hybrid photonic–plasmonic crystal structure covered with a thin fluorescent-molecular-doped dielectric film is investigated experimentally. A simple theoretical model to describe how a confined quasi-two-dimensional optical mode may induce coherent fluorescence emission is also presented. Concerning the spatial coherence, it is experimentally observed that the coherence area in the plane of the light source is in excess of 49 μm2, which results in enhanced directional fluorescence emission. Concerning temporal coherence, the obtained coherence time is 4 times longer than that of the normal fluorescence emission in vacuum. Moreover, a Young's double-slit interference experiment is performed to directly confirm the spatially coherent emission. This smoking gun proof of spatial coherence is reported here for the first time for the optical-mode-modified emission. PMID:25793015
Instantaneous three-dimensional visualization of concentration distributions in turbulent flows with crossed-plane laser-induced fluorescence imaging

NASA Astrophysics Data System (ADS)

Hoffmann, A.; Zimmermann, F.; Scharr, H.; Krömker, S.; Schulz, C.

2005-01-01

A laser-based technique for measuring instantaneous three-dimensional species concentration distributions in turbulent flows is presented. The laser beam from a single laser is formed into two crossed light sheets that illuminate the area of interest. The laser-induced fluorescence (LIF) signal emitted from excited species within both planes is detected with a single camera via a mirror arrangement. Image processing enables the reconstruction of the three-dimensional data set in close proximity to the cutting line of the two light sheets. Three-dimensional intensity gradients are computed and compared to the two-dimensional projections obtained from the two directly observed planes. Volume visualization by digital image processing gives unique insight into the three-dimensional structures within the turbulent processes. We apply this technique to measurements of toluene-LIF in a turbulent, non-reactive mixing process of toluene and air and to hydroxyl (OH) LIF in a turbulent methane-air flame upon excitation at 248 nm with a tunable KrF excimer laser.
Characterizing the Interaction between tartrazine and two serum albumins by a hybrid spectroscopic approach.

PubMed

Pan, Xingren; Qin, Pengfei; Liu, Rutao; Wang, Jing

2011-06-22

Tartrazine is an artificial azo dye commonly used in food products. The present study evaluated the interaction of tartrazine with two serum albumins (SAs), human serum albumin (HSA) and bovine serum albumin (BSA), under physiological conditions by means of fluorescence, three-dimensional fluorescence, UV-vis absorption, and circular dichroism (CD) techniques. The fluorescence data showed that tartrazine could bind to the two SAs to form a complex. The binding process was a spontaneous molecular interaction procedure, in which van der Waals and hydrogen bond interactions played a major role. Additionally, as shown by the UV-vis absorption, three-dimensional fluorescence, and CD results, tartrazine could lead to conformational and some microenvironmental changes of both SAs, which may affect the physiological functions of SAs. The work provides important insight into the mechanism of toxicity of tartrazine in vivo.
Simultaneous three-dimensional velocity and mixing measurements by use of laser Doppler velocimetry and fluorescence probes in a water tunnel

NASA Technical Reports Server (NTRS)

Neuhart, Dan H.; Wing, David J.; Henderson, Uleses C., Jr.

1994-01-01

A water tunnel investigation was conducted to demonstrate the capabilities of a laser-based instrument that can measure velocity and fluorescence intensity simultaneously. Fluorescence intensity of an excited fluorescent dye is directly related to concentration level and is used to indicate the extent of mixing in flow. This instrument is a three-dimensional laser Doppler velocimeter (LDV) in combination with a fluorometer for measuring fluorescence intensity variations. This capability allows simultaneous flow measurements of the three orthogonal velocity components and mixing within the same region. Two different flows which were generated by two models were studied: a generic nonaxisymmetric nozzle propulsion simulation model with an auxiliary internal water source that generated a jet flow and an axisymmetric forebody model with a circular sector strake that generated a vortex flow. The off-body flow fields around these models were investigated in the Langley 16- by 24-Inch Water Tunnel. The experimental results were used to calculate 17 quantities that included mean and fluctuating velocities, Reynolds stresses, mean and fluctuating dye fluorescence intensities (proportional to concentration), and fluctuating velocity and dye concentration correlations. An uncertainty analysis was performed to establish confidence levels in the experimental results. In general, uncertainties in mean velocities varied between 1 and 7 percent of free-stream velocity; uncertainties in fluctuating velocities varied between 1 and 5 percent of reference values. The results show characteristics that are unique to each type of flow.
Indirect determination of the electric field in plasma discharges using laser-induced fluorescence spectroscopy

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

Vaudolon, J., E-mail: julien.vaudolon@cnrs-orleans.fr; Mazouffre, S., E-mail: stephane.mazouffre@cnrs-orleans.fr

2014-09-15

The evaluation of electric fields is of prime interest for the description of plasma characteristics. In this work, different methods for determining the electric field profile in low-pressure discharges using one- and two-dimensional Laser-Induced Fluorescence (LIF) measurements are presented and discussed. The energy conservation, fluid, and kinetic approaches appear to be well-suited for the electric field evaluation in this region of the plasma flow. However, the numerical complexity of a two-dimensional kinetic model is penalizing due to the limited signal-to-noise ratio that can be achieved, making the computation of the electric field subject to large error bars. The ionization contributionmore » which appears in the fluid model makes it unattractive on an experimental viewpoint. The energy conservation and 1D1V kinetic approaches should therefore be preferred for the determination of the electric field when LIF data are used.« less
DIGE Analysis of Human Tissues.

PubMed

Gelfi, Cecilia; Capitanio, Daniele

2018-01-01

Two-dimensional difference gel electrophoresis (2-D DIGE) is an advanced and elegant gel electrophoretic analytical tool for comparative protein assessment. It is based on two-dimensional gel electrophoresis (2-DE) separation of fluorescently labeled protein extracts. The tagging procedures are designed to not interfere with the chemical properties of proteins with respect to their pI and electrophoretic mobility, once a proper labeling protocol is followed. The two-dye or three-dye systems can be adopted and their choice depends on specific applications. Furthermore, the use of an internal pooled standard makes 2-D DIGE a highly accurate quantitative method enabling multiple protein samples to be separated on the same two-dimensional gel. The image matching and cross-gel statistical analysis generates robust quantitative results making data validation by independent technologies successful.

Fluorescence enhancement through the formation of a single-layer two-dimensional supramolecular organic framework and its application in highly selective recognition of picric acid.

PubMed

Zhang, Ying; Zhan, Tian-Guang; Zhou, Tian-You; Qi, Qiao-Yan; Xu, Xiao-Na; Zhao, Xin

2016-06-18

A two-dimensional (2D) supramolecular organic framework (SOF) has been constructed through the co-assembly of a triphenylamine-based building block and cucurbit[8]uril (CB[8]). Fluorescence turn-on of the non-emissive building block was observed upon the formation of the 2D SOF, which displayed highly selective and sensitive recognition of picric acid over a variety of nitroaromatics.
Two-dimensional fluorescence spectroscopy of uranium isotopes in femtosecond laser ablation plumes

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

Phillips, Mark C.; Brumfield, Brian E.; LaHaye, Nicole

Here, we demonstrate measurement of uranium isotopes in femtosecond laser ablation plumes using two-dimensional fluorescence spectroscopy (2DFS). The high-resolution, tunable CW-laser spectroscopy technique clearly distinguishes atomic absorption from 235U and 238U in natural and highly enriched uranium metal samples. We present analysis of spectral resolution and analytical performance of 2DFS as a function of ambient pressure. Simultaneous measurement using time-resolved absorption spectroscopy provides information on temporal dynamics of the laser ablation plume and saturation behavior of fluorescence signals. The rapid, non-contact measurement is promising for in-field, standoff measurements of uranium enrichment for nuclear safety and security.
Two-dimensional fluorescence spectroscopy of uranium isotopes in femtosecond laser ablation plumes

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

Phillips, Mark C.; Brumfield, Brian E.; LaHaye, Nicole L.

We demonstrate measurement of uranium isotopes in femtosecond laser ablation plumes using two-dimensional fluorescence spectroscopy (2DFS). The high-resolution, tunable CW-laser spectroscopy technique clearly distinguishes atomic absorption from 235U and 238U in natural and highly enriched uranium metal samples. We present analysis of spectral resolution and analytical performance of 2DFS as a function of ambient pressure. Simultaneous measurement using time-resolved absorption spectroscopy provides information on temporal dynamics of the laser ablation plume and saturation behavior of fluorescence signals. The rapid, non-contact measurement is promising for in-field, standoff measurements of uranium enrichment for nuclear safety and security applications.
Two-dimensional fluorescence spectroscopy of uranium isotopes in femtosecond laser ablation plumes

DOE PAGES

Phillips, Mark C.; Brumfield, Brian E.; LaHaye, Nicole; ...

2017-06-19

Here, we demonstrate measurement of uranium isotopes in femtosecond laser ablation plumes using two-dimensional fluorescence spectroscopy (2DFS). The high-resolution, tunable CW-laser spectroscopy technique clearly distinguishes atomic absorption from 235U and 238U in natural and highly enriched uranium metal samples. We present analysis of spectral resolution and analytical performance of 2DFS as a function of ambient pressure. Simultaneous measurement using time-resolved absorption spectroscopy provides information on temporal dynamics of the laser ablation plume and saturation behavior of fluorescence signals. The rapid, non-contact measurement is promising for in-field, standoff measurements of uranium enrichment for nuclear safety and security.
Visualization of Two-Phase Fluid Distribution Using Laser Induced Exciplex Fluorescence

NASA Astrophysics Data System (ADS)

Kim, J. U.; Darrow, J.; Schock, H.; Golding, B.; Nocera, D.; Keller, P.

1998-03-01

Laser-induced exciplex (excited state complex) fluorescence has been used to generate two-dimensional images of dispersed liquid and vapor phases with spectrally resolved two-color emissions. In this method, the vapor phase is tagged by the monomer fluorescence while the liquid phase is tracked by the exciplex fluorescence. A new exciplex visualization system consisting of DMA and 1,4,6-TMN in an isooctane solvent was developed.(J.U. Kim et al., Chem. Phys. Lett. 267, 323-328 (1997)) The direct ca
Compact three-dimensional super-resolution system based on fluorescence emission difference microscopy

NASA Astrophysics Data System (ADS)

Zhu, Dazhao; Chen, Youhua; Fang, Yue; Hussain, Anwar; Kuang, Cuifang; Zhou, Xiaoxu; Xu, Yingke; Liu, Xu

2017-12-01

A compact microscope system for three-dimensional (3-D) super-resolution imaging is presented. The super-resolution capability of the system is based on a size-reduced effective 3-D point spread function generated through the fluorescence emission difference (FED) method. The appropriate polarization direction distribution and manipulation allows the panel active area of the spatial light modulator to be fully utilized. This allows simultaneous modulation of the incident light by two kinds of phase masks to be performed with a single spatial light modulator in order to generate a 3-D negative spot. The system is more compact than standard 3-D FED systems while maintaining all the advantages of 3-D FED microscopy. The experimental results demonstrated the improvement in 3-D resolution by nearly 1.7 times and 1.6 times compared to the classic confocal resolution in the lateral and axial directions, respectively.
Synthesis, structure and photoluminescence properties of amine-templated open-framework bismuth sulfates

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

Marri, Subba R.; Behera, J.N., E-mail: jnbehera@niser.ac.in

2014-02-15

Two organically-templated bismuth sulfates of the compositions, [C{sub 6}N{sub 2}H{sub 14}] [Bi(SO{sub 4}){sub 2}(NO{sub 3})], (1) and [C{sub 4}N{sub 2}H{sub 12}]{sub 4}[Bi{sub 4}(SO{sub 4}){sub 10}(H{sub 2}O){sub 4}], (2), with open architecture have been synthesized and their structures determined by single crystal X-ray diffraction. 1 has a corrugated layered structure with 8-membered aperture wherein the SO{sub 4} tetrahedra and the BiO{sub 8} polyhedra join together to form (4, 4) net sheets of the metal centers while 2 has a three-dimensional structure possessing 8- and 12-membered channels. Both the compounds show good fluorescence properties exhibiting blue luminescence. Time-resolved fluorescence behavior of 1more » and 2 shows mean fluorescence life time of 0.9 and 1.0 ns, respectively. - Graphical abstract: Two open-framework bismuth sulfates with the layered and three-dimensional structures have been synthesized and characterized. Both the compounds show good fluorescence properties exhibiting blue luminescence. Display Omitted - Highlights: • Two organically-templated bismuth sulfates with open architecture have been synthesized and characterized. • One has a corrugated layered structure while the other one has a three-dimensional structure possessing channels. • They are novel in that open-framework three-dimensional main group metal sulfates are first to be reported. • They show good fluorescence properties exhibiting blue luminescence.« less
Towards pH-sensitive imaging of small animals with photon-counting difference diffuse fluorescence tomography

NASA Astrophysics Data System (ADS)

Li, Jiao; Wang, Xin; Yi, Xi; Zhang, Limin; Zhou, Zhongxing; Zhao, Huijuan; Gao, Feng

2012-09-01

The importance of cellular pH has been shown clearly in the study of cell activity, pathological feature, and drug metabolism. Monitoring pH changes of living cells and imaging the regions with abnormal pH-values, in vivo, could provide invaluable physiological and pathological information for the research of the cell biology, pharmacokinetics, diagnostics, and therapeutics of certain diseases such as cancer. Naturally, pH-sensitive fluorescence imaging of bulk tissues has been attracting great attentions from the realm of near infrared diffuse fluorescence tomography (DFT). Herein, the feasibility of quantifying pH-induced fluorescence changes in turbid medium is investigated using a continuous-wave difference-DFT technique that is based on the specifically designed computed tomography-analogous photon counting system and the Born normalized difference image reconstruction scheme. We have validated the methodology using two-dimensional imaging experiments on a small-animal-sized phantom, embedding an inclusion with varying pH-values. The results show that the proposed approach can accurately localize the target with a quantitative resolution to pH-sensitive variation of the fluorescent yield, and might provide a promising alternative method of pH-sensitive fluorescence imaging in addition to the fluorescence-lifetime imaging.
Improvement of antigen detection efficiency with the use of two-dimensional photonic crystal as a substrate

NASA Astrophysics Data System (ADS)

Dovzhenko, Dmitriy; Terekhin, Vladimir; Vokhmincev, Kirill; Sukhanova, Alyona; Nabiev, Igor

2017-01-01

Multiplex detection of different antigens in human serum in order to reveal diseases at the early stage is of interest nowadays. There are a lot of biosensors, which use the fluorescent labels for specific detection of analytes. For instance, common method for detection of antigens in human serum samples is enzyme-linked immunosorbent assay (ELISA). One of the most effective ways to improve the sensitivity of this detection method is the use of a substrate that could enhance the fluorescent signal and make it easier to collect. Two-dimensional (2D) photonic crystals are very suitable structures for these purposes because of the ability to enhance the luminescent signal, control the light propagation and perform the analysis directly on its surface. In our study we have calculated optimal parameters for 2D-dimensional photonic crystal consisting of the array of silicon nano-rods, fabricated such photonic crystal on a silicon substrate using reactive ion etching and showed the possibility of its efficient application as a substrate for ELISA detection of human cancer antigens.
[Three dimensional fluorescent characteristics of soil dissolved organic matter (DOM) in Jiaozhou Bay coastal wetlands, China].

PubMed

Zi, Yuan Yuan; Kong, Fan Long; Xi, Min; Li, Yue; Yang, Ling

2016-12-01

In order to elucidate the structure characteristics of soil dissolved organic matter (DOM) and analyze the sources in Jiaozhou Bay coastal wetlands, four typical types of wetlands around Jiaozhou Bay were chosen, including Spartina anglica wetland, the barren wetland, Suaeda glauca wetland and Phragmites australis wetland. The soil samples were collected in January 2014. The contents of soil DOM were determined and the spectral analysis was made by three-dimensional fluorescent technology. The results showed that the contents of soil dissolved organic carbon (DOC) in four types of wetlands all decreased with the increasing soil depth, and S. anglica wetland ranked the first in the contents of soil DOC, followed by the barren wetland, S. glauca wetland and P. australis wetland. Five fluorescence peaks including B, T, A, D and C were found in the three-dimensional fluorescence spectrum (3DEEMs), indicating tyrosine-like, tryptophan-like, phenol-like, soluble microbial byproduct-like and humic acid-like- substances, respectively. Fluorescence integration (FRI) was applied in the qualitative analysis of five components. The results showed that tryptophan-like, phenol-like and tyrosine-like substances ranked in top three in content, followed by soluble microbial byproduct-like and humic acid-like substances which were not significantly different. Pearson correlation analysis demonstrated that a positive correlation existed between any two of the five components of DOM, and they were all positively related to DOC content. In addition, there existed different correlations between the five components of DOM and total phosphorus (TP), available phosphorus (AP) and total nitrogen (TN). The soil DOM in the four types of wetlands was mainly produced by biotic interactions, and the degree of humification was relatively low.
Two-dimensional fruit ripeness estimation using thermal imaging

NASA Astrophysics Data System (ADS)

Sumriddetchkajorn, Sarun; Intaravanne, Yuttana

2013-06-01

Some green fruits do not change their color from green to yellow when being ripe. As a result, ripeness estimation via color and fluorescent analytical approaches cannot be applied. In this article, we propose and show for the first time how a thermal imaging camera can be used to two-dimensionally classify fruits into different ripeness levels. Our key idea relies on the fact that the mature fruits have higher heat capacity than the immature ones and therefore the change in surface temperature overtime is slower. Our experimental proof of concept using a thermal imaging camera shows a promising result in non-destructively identifying three different ripeness levels of mangoes Mangifera indica L.
Preparation, one- and two-photon properties of carbazole derivatives containing nitrogen heterocyclic ring

NASA Astrophysics Data System (ADS)

Zhang, Yichi; Wang, Ping; Li, Liang; Chen, Zhimin; He, Chunying; Wu, Yiqun

Preparation of recording materials with high two-photon absorption activities is one of the important issues to superhigh- density two-photon absorption (TPA) three-dimensional (3D) optical data storage. In this paper, three new carbazole derivatives containing nitrogen heterocyclic ring with symmetric and asymmetric structures are prepared using ethylene as the π bridge between the carbazole unit and nitrogen heterocyclic ring, namely, 9-butyl-3-(2-(1,8- naphthyridin)vinyl)-carbazole (material 1), 9-butyl-3,6-bis(2-(1,8-naphthyl)vinyl)-carbazole (material 2) and 9-butyl-3,6- bis(2-(quinolin)vinyl)-carbazole (material 3). Their one photon properties including linear absorption spectra, fluorescence emission spectra, and fluorescence quantum yields are studied. The fluorescence excited by 120 fs pulse at 800 nm Ti: sapphire laser operating at 1 kHz repetition rate with different incident powers of 9-butyl-3-(2-(quinolin) vinyl)-carbazole (material 3) was investigated, and two-photon absorption cross-sections has been obtained. It is shown that material 3 containing quinoline rings as electron acceptor with symmetric structure exhibit high two-photon absorption activity. The result implies that material 3 (9-butyl-3-(2-(quinolin) vinyl)-carbazole) is a good candidate as a promising recording material for super-high-density two-photon absorption (TPA) three-dimensional (3D) optical data storage. The influence of chemical structure of the materials on the optical properties is discussed.
Dual-color three-dimensional STED microscopy with a single high-repetition-rate laser

PubMed Central

Han, Kyu Young; Ha, Taekjip

2016-01-01

We describe a dual-color three-dimensional stimulated emission depletion (3D-STED) microscopy employing a single laser source with a repetition rate of 80 MHz. Multiple excitation pulses synchronized with a STED pulse were generated by a photonic crystal fiber and the desired wavelengths were selected by an acousto-optic tunable filter with high spectral purity. Selective excitation at different wavelengths permits simultaneous imaging of two fluorescent markers at a nanoscale resolution in three dimensions. PMID:26030581
3D fluorescence anisotropy imaging using selective plane illumination microscopy.

PubMed

Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

2015-08-24

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.
Ultrafast optical excitations in supramolecular metallacycles with charge transfer properties.

PubMed

Flynn, Daniel C; Ramakrishna, Guda; Yang, Hai-Bo; Northrop, Brian H; Stang, Peter J; Goodson, Theodore

2010-02-03

New organometallic materials such as two-dimensional metallacycles and three-dimensional metallacages are important for the development of novel optical, electronic, and energy related applications. In this article, the ultrafast dynamics of two different platinum-containing metallacycles have been investigated by femtosecond fluorescence upconversion and transient absorption. These measurements were carried out in an effort to probe the charge transfer dynamics and the rate of intersystem crossing in metallacycles of different geometries and dimensions. The processes of ultrafast intersystem crossing and charge transfer vary between the two different classes of metallacyclic systems studied. For rectangular anthracene-containing metallacycles, the electronic coupling between adjacent ligands was relatively weak, whereas for the triangular phenanthrene-containing structures, there was a clear interaction between the conjugated ligand and the metal complex center. The transient lifetimes increased with increasing conjugation in that case. The results show that differences in the dimensionality and structure of metallacycles result in different optical properties, which may be utilized in the design of nonlinear optical materials and potential new, longer-lived excited state materials for further electronic applications.
Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy.

PubMed

Indovina, Paola; Collini, Maddalena; Chirico, Giuseppe; Santini, Maria Teresa

2007-02-20

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1alpha (hypoxia-inducible factor-1alpha) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (<100 microm) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.
Motor Oil Classification Based on Time-Resolved Fluorescence

PubMed Central

Mu, Taotao; Chen, Siying; Zhang, Yinchao; Guo, Pan; Chen, He; Meng, Fandong

2014-01-01

A time-resolved fluorescence (TRF) technique is presented for classifying motor oils. The system is constructed with a third harmonic Nd:YAG laser, a spectrometer, and an intensified charge coupled device (ICCD) camera. Steady-state and time-resolved fluorescence (TRF) measurements are reported for several motor oils. It is found that steady-state fluorescence is insufficient to distinguish the motor oil samples. Then contour diagrams of TRF intensities (CDTRFIs) are acquired to serve as unique fingerprints to identify motor oils by using the distinct TRF of motor oils. CDTRFIs are preferable to steady-state fluorescence spectra for classifying different motor oils, making CDTRFIs a particularly choice for the development of fluorescence-based methods for the discrimination and characterization of motor oils. The two-dimensional fluorescence contour diagrams contain more information, not only the changing shapes of the LIF spectra but also the relative intensity. The results indicate that motor oils can be differentiated based on the new proposed method, which provides reliable methods for analyzing and classifying motor oils. PMID:24988439
Three-Dimensional Imaging of the Mouse Organ of Corti Cytoarchitecture for Mechanical Modeling

NASA Astrophysics Data System (ADS)

Puria, Sunil; Hartman, Byron; Kim, Jichul; Oghalai, John S.; Ricci, Anthony J.; Liberman, M. Charles

2011-11-01

Cochlear models typically use continuous anatomical descriptions and homogenized parameters based on two-dimensional images for describing the organ of Corti. To produce refined models based more closely on the actual cochlear cytoarchitecture, three-dimensional morphometric parameters of key mechanical structures are required. Towards this goal, we developed and compared three different imaging methods: (1) A fixed cochlear whole-mount preparation using the fluorescent dye Cellmask®, which is a molecule taken up by cell membranes and clearly delineates Deiters' cells, outer hair cells, and the phalangeal process, imaged using confocal microscopy; (2) An in situ fixed preparation with hair cells labeled using anti-prestin and supporting structures labeled using phalloidin, imaged using two-photon microscopy; and (3) A membrane-tomato (mT) mouse with fluorescent proteins expressed in all cell membranes, which enables two-photon imaging of an in situ live preparation with excellent visualization of the organ of Corti. Morphometric parameters including lengths, diameters, and angles, were extracted from 3D cellular surface reconstructions of the resulting images. Preliminary results indicate that the length of the phalangeal processes decreases from the first (inner most) to third (outer most) row of outer hair cells, and that their length also likely varies from base to apex and across species.
A brief review of other notable protein detection methods on acrylamide gels.

PubMed

Kurien, Biji T; Scofield, R Hal

2012-01-01

Several methods have been described to stain proteins analyzed on acrylamide gels. These include ultrasensitive protein detection in one-dimensional and two-dimensional gel electrophoresis using a fluorescent product from the fungus Epicoccum nigrum; a fluorescence-based Coomassie Blue protein staining; visualization of proteins in acrylamide gels using ultraviolet illumination; fluorescence visualization of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution; and increasing the sensitivity four- to sixfold for detecting trace proteins in dye or silver stained polyacrylamide gels using polyethylene glycol 6000. All these methods are reviewed briefly in this chapter.
Investigating axial diffusion in cylindrical pores using confocal single-particle fluorescence correlation spectroscopy.

PubMed

Chen, Fang; Neupane, Bhanu; Li, Peiyuan; Su, Wei; Wang, Gufeng

2016-08-01

We explored the feasibility of using confocal fluorescence correlation spectroscopy to study small nanoparticle diffusion in hundred-nanometer-sized cylindrical pores. By modeling single particle diffusion in tube-like confined three-dimensional space aligned parallel to the confocal optical axis, we showed that two diffusion dynamics can be observed in both original intensity traces and the autocorrelation functions (ACFs): the confined two-dimensional lateral diffusion and the unconfined one-dimensional (1D) axial diffusion. The separation of the axial and confined lateral diffusion dynamics provides an opportunity to study diffusions in different dimensions separately. We further experimentally studied 45 nm carboxylated polystyrene particles diffusing in 300 nm alumina pores. The experimental data showed consistency with the simulation. To extract the accurate axial diffusion coefficient, we found that a 1D diffusion model with a Lorentzian axial collection profile needs to be used to analyze the experimental ACFs. The diffusion of the 45 nm nanoparticles in polyethyleneglycol-passivated 300 nm pores slowed down by a factor of ∼2, which can be satisfactorily explained by hydrodynamic frictions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Three-dimensional fluorescence analysis of chernozem humic acids and their electrophoretic fractions

NASA Astrophysics Data System (ADS)

Trubetskoi, O. A.; Trubetskaya, O. E.

2017-09-01

Polyacrylamide gel electrophoresis in combination with size-exclusion chromatography (SEC-PAGE) has been used to obtain stable electrophoretic fractions of different molecular size (MS) from chernozem humic acids (HAs). Three-dimensional fluorescence charts of chernozem HAs and their fractions have been obtained for the first time, and all fluorescence excitation-emission maxima have been identified in the excitation wavelength range of 250-500 nm. It has been found that fractionation by the SEC-PAGE method results in a nonuniform distribution of protein- and humin-like fluorescence of the original HA preparation among the electrophoretic fractions. The electrophoretic fractions of the highest and medium MSs have only the main protein-like fluorescence maximum and traces of humin-like fluorescence. In the electrophoretic fraction of the lowest MS, the intensity of protein-like fluorescence is low, but the major part of humin-like fluorescence is localized there. Relationships between the intensity of protein-like fluorescence and the weight distribution of amino acids have been revealed, as well as between the degree of aromaticity and the intensity of humin-like fluorescence in electrophoretic fractions of different MSs. The obtained relationships can be useful in the interpretation of the spatial structural organization and ecological functions of soil HAs.
Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

PubMed Central

Scolaro, Loretta; Lorenser, Dirk; Madore, Wendy-Julie; Kirk, Rodney W.; Kramer, Anne S.; Yeoh, George C.; Godbout, Nicolas; Sampson, David D.; Boudoux, Caroline; McLaughlin, Robert A.

2015-01-01

Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue. PMID:26137379
Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

PubMed

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.
[Studies on the action features between cefuroxime axetil and bovine serum albumin].

PubMed

Wu, Gang-ke; Yan, Cheng-nong; Liu, Yi

2008-09-01

Under different temperatures and physiological conditions, with cefuroxime axetil concentrations in the range of 1.959 X 10(-6) to 13.71 X 10(-6) mol x L(-1), and bovine serum albumin (BSA) concentrations at 2.0 X 10(-6) mol x L(-1), the interaction between cefuroxime axetil and BSA was studied by fluorescence spectroscopy, three-dimensional fluorescence spectrum, synchronous fluorescence spectrum and UV-Vis absorption spectroscopy. After analyzing and processing the fluorescence quenching data at different temperatures according to Sterm-Volmer equation, Lineweaver-Burk equation and thermodynamic equation, the average value of the apparent binding constant (K(LB): 3.907 X 10(6) L x mol(-1)), and thermodynamics parameters (enthalpy change delta H: -13.43 kJ x mol(-1), entropy change delta S: 81.90 J x K(-1) and standard Gibbs free energy change delta G0: -38.34 kJ x mol(-1)) were calculated, and the amounts of binding sites (n: 1.042)were measured. The fluorescence quenching mechanism of BSA after cefuroxime axetil was added was discussed. BSA was bound with cefuroxime axetil and formed a new compound. The quenching belonged to static fluorescence quenching. The thermodynamic parameters agree with delta H approximately 0, delta S > 0 and delta G0 < 0, and the binding reaction is mainly entropy-driven and electro-static interaction force plays a major role in the reaction. The maximum emission wavelength of Tyr and Trp had an obvious red shift in the synchronous fluorescence spectra, the fluorescence emission wavelength of two peaks had a blue shift in the three-dimensional fluorescence spectrum of BSA in the presence of cefuroxime axetil and the maximum absorbtion wavelenghs of three systems in the UV-Vis absorption spectra were obviously different. These showed that the changes in the micro-environment of Tyr and Trp and demonstrated that the conformation of BSA changed as cefuroxime axetil had been added. This provides important information for discussing the configuration modification of BSA because of the added cefuroxime axetil, and for elucidating the pharmacological effects of cefuroxime axetil and biological effects in the organism.
Two-Dimensional Imaging of OH in a Lean Burning High Pressure Combustor

NASA Technical Reports Server (NTRS)

Locke, R. J.; Hicks, Y. R.; Anderson, R. C.; Ockunzzi, K. A.; North, G. L.

1995-01-01

Planar laser-induced fluorescence (PLIF) images of OH have been obtained from an optically accessible, lean burning high pressure combustor burning Jet-A fuel. These images were obtained using various laser excitation lines of the OH A (reverse arrow) X (1,0) band for several fuel injector configurations with pressures ranging from 1013 kPa (10 atm) to 1419 kPa (14 atm). Non-uniformities in the combusting flow, attributed to differences in fuel injector configuration, are revealed by these images. Contributions attributable to fluorescent aromatic hydrocarbons and complex fuel chemistries are also not evident.
Discrimination of Rhizoma Gastrodiae (Tianma) using 3D synchronous fluorescence spectroscopy coupled with principal component analysis

NASA Astrophysics Data System (ADS)

Fan, Qimeng; Chen, Chaoyin; Huang, Zaiqiang; Zhang, Chunmei; Liang, Pengjuan; Zhao, Shenglan

2015-02-01

Rhizoma Gastrodiae (Tianma) of different variants and different geographical origins has vital difference in quality and physiological efficacy. This paper focused on the classification and identification of Tianma of six types (two variants from three different geographical origins) using three dimensional synchronous fluorescence spectroscopy (3D-SFS) coupled with principal component analysis (PCA). 3D-SF spectra of aqueous extracts, which were obtained from Tianma of the six types, were measured by a LS-50B luminescence spectrofluorometer. The experimental results showed that the characteristic fluorescent spectral regions of the 3D-SF spectra were similar, while the intensities of characteristic regions are different significantly. Coupled these differences in peak intensities with PCA, Tianma of six types could be discriminated successfully. In conclusion, 3D-SFS coupled with PCA, which has such advantages as effective, specific, rapid, non-polluting, has an edge for discrimination of the similar Chinese herbal medicine. And the proposed methodology is a useful tool to classify and identify Tianma of different variants and different geographical origins.
Image scanning fluorescence emission difference microscopy based on a detector array.

PubMed

Li, Y; Liu, S; Liu, D; Sun, S; Kuang, C; Ding, Z; Liu, X

2017-06-01

We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Bie Haiying; Lu Jing; Yu Jiehui

Three novel thiocyanate supramolecular compounds have been synthesized and characterized by X-ray diffraction and fluorescent spectra. Compound [pipH]{sub 2}[Co(NCS){sub 4}] (pip=piperazine) 1 possesses a two-dimensional layer connected by the combination of N-H...N hydrogen bonds and weak S...S contacts. Under the same conditions, using nickel salt instead of cobalt salt as a starting material, we obtained a different two-dimensional supramolecular layer [pipH]{sub 2}[Ni(NCS){sub 4}] 2 connected by unusual N-H...S hydrogen bonds and weak S...S contacts. In order to observe the influence of the dimension of ligand on the self-assembly structure, dabco was used for substituting pip, and compound [dabcoH]{sub 2}[Ni(NCS){sub 4}]more » (dabco=1,4-Diazabicyclo[2.2.2] octane) 3 was gained, which constructed two-dimensional, highly wavy network with hourglass-shaped cavities only through N-H...S hydrogen bonds.« less
Assessment of microbial products in the biosorption process of Cu(II) onto aerobic granular sludge: Extracellular polymeric substances contribution and soluble microbial products release.

PubMed

Huang, Linxian; Li, Meilin; Si, Guangchao; Wei, Jinglin; Ngo, Huu Hao; Guo, Wenshan; Xu, Weiying; Du, Bin; Wei, Qin; Wei, Dong

2018-05-18

In the present study, the responses of microbial products in the biosorption process of Cu(II) onto aerobic granular sludge were evaluated by using batch and spectroscopic approaches. Batch experimental data showed that extracellular polymeric substances (EPSs) contributed to Cu(II) removal from an aqueous solution, especially when treating low metal concentrations, whereas soluble microbial products (SMPs) were released under the metal stress during biosorption process. A three-dimensional excitation-emission matrix (3D-EEM) identified four main fluorescence peaks in the EPS, i.e., tryptophan protein-like, aromatic protein-like, humic-like and fulvic acid-like substances, and their fluorescence intensities decreased gradually in the presence of Cu(II) during the sorption process. Particularly, tryptophan protein-like substances quenched the Cu(II) binding to a much higher extent through a static quenching process with less than one class of binding sites. According to the synchronous fluorescence spectra, the whole fluorescence intensity of released SMP samples expressed an increased trend with different degrees along with contact time. Two-dimensional correlation spectroscopy (2D-COS) suggested that the fulvic-like fluorescence fraction might be more susceptible to metal exposure than other fractions. The result of molecular weight distribution demonstrated that the SMPs released from the biosorption process differed significantly according to contact time. The result obtained could provide new insights into the responses of microbial products from aerobic granular sludge with heavy metal treatment. Copyright © 2018. Published by Elsevier Inc.
Ethanolic extraction, purification, and partial characterization of a fluorescent toxin from the coral reef crab, Lophozozymus pictor.

PubMed

Lau, C O; Tan, C H; Khoo, H E; Li, Q T; Yuen, R

1995-01-01

A purification procedure for Lophozozymus pictor toxin (LPTX) following ethanolic extraction of whole crab homogenate is described. The ethanol-extracted toxin (LPTX-E) had higher yield and specific activity than the hot aqueous-extracted one (LPTX-H). It was found that LPTX-E was fluorescent and cochromatographed with LPTX-H on two-dimensional thin-layer chromatography. Although LPTX-E, LPTX-H, and palytoxin (P. caribaeorum, PTX) had similar migration and retention times when analysed on high performance capillary electrophoresis and gel permeation-high performance liquid chromatography respectively, LPTX-E and LPTX-H were both fluorescent in contrast to PTX. In addition, LPTX-E had a different retention time compared with PTX when chromatographed on reversed phase high performance liquid chromatography in the solvent system 80% acetonitrile and 0.02 M Tris-HCl, pH 7.2, at a 4:1 ratio, respectively, indicating some differences in their chemical structures.
Simultaneous two-dimensional laser-induced-fluorescence measurements of argon ions.

PubMed

Hansen, A K; Galante, Matthew; McCarren, Dustin; Sears, Stephanie; Scime, E E

2010-10-01

Recent laser upgrades on the Hot Helicon Experiment at West Virginia University have enabled multiplexed simultaneous measurements of the ion velocity distribution function at a single location, expanding our capabilities in laser-induced fluorescence diagnostics. The laser output is split into two beams, each modulated with an optical chopper and injected perpendicular and parallel to the magnetic field. Light from the crossing point of the beams is transported to a narrow-band photomultiplier tube filtered at the fluorescence wavelength and monitored by two lock-in amplifiers, each referenced to one of the two chopper frequencies.
Syntheses, structures and luminescent properties of zero-/two-dimensional Cd(II) and Eu(III) complexes

NASA Astrophysics Data System (ADS)

Fan, Rui-Qing; Wang, Li-Yuan; Wang, Ping; Chen, Hong; Sun, Cun-fa; Yang, Yu-Lin; Su, Qing

2012-12-01

Three metal-organic complexes Cd(HBIDC)(phen)2·4H2O (1), [Cd(BIC)(phen)]n (2) and {[Eu(HBIDC)(H2BIDC)(H2O)]·H2O}n (3) (H3BIDC=benzimidazole-5,6-dicarboxylic acid, H2BIC=benzimidazole-6-carboxylic acid, phen=1,10-phenanthroline) have been synthesized under hydro(solvo)thermal conditions and structurally characterized by elemental analysis, IR spectrum, and single-crystal X-ray diffraction. With similar reaction conditions, reactions of the same ligand with different metal cations selected from different blocks (d-block and f-block) result in different coordination modes of carboxylate groups and final frameworks of complexes 1 and 3. The decarboxylation was observed in complex 2 and resulted in the formation of BIC2- ligand. Complexes 1-3 have intense fluorescent emissions at room temperature in dimethylsulfoxide (DMSO) solution and in the solid-state, which indicate they are potential fluorescence materials. The quantum yields and fluorescence lifetimes of these three complexes were systematically studied.
Multimodal fluorescence molecular imaging for in vivo characterization of skin cancer using endogenous and exogenous fluorophores

NASA Astrophysics Data System (ADS)

Miller, Jessica P.; Habimana-Griffin, LeMoyne; Edwards, Tracy S.; Achilefu, Samuel

2017-06-01

Similarity of skin cancer with many benign skin pathologies requires reliable methods to detect and differentiate the different types of these lesions. Previous studies have explored the use of disparate optical techniques to identify and estimate the invasive nature of melanoma and basal cell carcinoma with varying outcomes. Here, we used a concerted approach that provides complementary information for rapid screening and characterization of tumors, focusing on squamous cell carcinoma (SCC) of the skin. Assessment of in vivo autofluorescence lifetime (FLT) imaging of endogenous fluorophores that are excitable at longer wavelengths (480 nm) than conventional NADH and FAD revealed a decrease in the short FLT component for SCC compared to normal skin, with mean values of 0.57±0.026 ns and 0.61±0.021 ns, respectively (p=0.004). Subsequent systemic administration of a near-infrared fluorescent molecular probe in SCC bearing mice, followed by the implementation of image processing methods on data acquired from two-dimensional and three-dimensional fluorescence molecular imaging, allowed us to estimate the tumor volume and depth, as well as quantify the fluorescent probe in the tumor. The result suggests the involvement of lipofuscin-like lipopigments and riboflavin in SCC metabolism and serves as a model for staging SCC.
Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

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

PubMed

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

2017-04-03

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

PubMed Central

Jones, Christopher A. R.; Groves, Nicholas Scott; Sun, Bo

2016-01-01

Cells in three-dimensional (3D) environments exhibit very different biochemical and biophysical phenotypes compared to the behavior of cells in two-dimensional (2D) environments. As an important biomechanical measurement, 2D traction force microscopy can not be directly extended into 3D cases. In order to quantitatively characterize the contraction field, we have developed 3D reflectance traction microscopy which combines confocal reflection imaging and partial volume correlation postprocessing. We have measured the deformation field of collagen gel under controlled mechanical stress. We have also characterized the deformation field generated by invasive breast cancer cells of different morphologies in 3D collagen matrix. In contrast to employ dispersed tracing particles or fluorescently-tagged matrix proteins, our methods provide a label-free, computationally effective strategy to study the cell mechanics in native 3D extracellular matrix. PMID:27304456
Two-Dimensional Algal Collection and Assembly by Combining AC-Dielectrophoresis with Fluorescence Detection for Contaminant-Induced Oxidative Stress Sensing.

PubMed

Siebman, Coralie; Velev, Orlin D; Slaveykova, Vera I

2015-06-15

An alternative current (AC) dielectrophoretic lab-on-chip setup was evaluated as a rapid tool of capture and assembly of microalga Chlamydomonas reinhardtii in two-dimensional (2D) close-packed arrays. An electric field of 100 V·cm⁻¹, 100 Hz applied for 30 min was found optimal to collect and assemble the algae into single-layer structures of closely packed cells without inducing cellular oxidative stress. Combined with oxidative stress specific staining and fluorescence microscopy detection, the capability of using the 2D whole-cell assembly on-chip to follow the reactive oxygen species (ROS) production and oxidative stress during short-term exposure to several environmental contaminants, including mercury, methylmercury, copper, copper oxide nanoparticles (CuO-NPs), and diuron was explored. The results showed significant increase of the cellular ROS when C. reinhardtii was exposed to high concentrations of methylmercury, CuO-NPs, and 10⁻⁵ M Cu. Overall, this study demonstrates the potential of combining AC-dielectrophoretically assembled two-dimensional algal structures with cell metabolic analysis using fluorescence staining, as a rapid analytical tool for probing the effect of contaminants in highly impacted environment.
Singlet oxygen luminescence kinetics under PDI relevant conditions of pathogenic dermatophytes and molds.

PubMed

Bornhütter, Tobias; Shamali, Nedaa; Saltsman, Irena; Mahammed, Atif; Gross, Zeev; Däschlein, Georg; Röder, Beate

2018-01-01

A treatment of onychomycosis using the photodynamic effect would be a favorable alternative to currently used antimycotic drugs. This study should be considered as a first step towards development and control of an efficient photodynamic inactivation of onychomycosis causative pathogens. Here, we evaluate the usage of time-resolved 2D singlet oxygen luminescence detection in combination with 2D fluorescence scanning as a tool to understand the behavior of the photosensitizer when applied to fungi on Petri dishes. To investigate the interaction of photosensitizer with fungi in various concentrations and in different stages of live, a photodynamic inactivation was avoided by keeping the samples in darkness. Scans of singlet oxygen luminescence and photosensitizer fluorescence were performed over a period of 24days. Two different photosensitizer, a cationic porphyrin and cationic corrole and two fungi strains, the dermatophyte Trichophyton rubrum and the mold Scopulariopsis brevicaulis, were investigated in this study. The two-dimensional correlation of photosensitizer fluorescence and singlet oxygen luminescence revealed differences in the diffusion of both photosensitizer. Even though the singlet oxygen luminescence was quenched with increasing growth of fungi, it was found that the kinetics of singlet oxygen luminescence could be detected on Petri dishes for both photosensitizers and both fungi strains for up to seven days. Copyright © 2017 Elsevier B.V. All rights reserved.
UV plasmonic enhancement through three dimensional nano-cavity antenna array in aluminum

NASA Astrophysics Data System (ADS)

Mao, Jieying; Stevenson, Peter; Montanaric, Danielle; Wang, Yunshan; Shumaker-Parry, Jennifer S.; Harris, Joel M.; Blair, Steve

2017-08-01

Metallic nanostructure can enhance fluorescence through excited surface plasmons which increase the local field as well as improve its quantum efficiency. When coupling to cavity resonance with proper gap dimension, gap hot spots can be generated to interact with fluorescence at their excitation/emission region in UV. A 3D nano-cavity antenna array in Aluminum has been conducted to generate local hot spot resonant at fluorescence emission resonance. Giant field enhancement has been achieved through coupling fundamental resonance modes of nanocavity into surface plasmons polaritons (SPPs). In this work, two distinct plasmonic structure of 3D resonant cavity nanoantenna has been studied and its plasmonic response has been scaled down to the UV regime through finite-difference-time-domain (FDTD) method. Two different strategies for antenna fabrication will be conducted to obtain D-coupled Dots-on-Pillar Antenna array (D2PA) through Focus Ion Beam (FIB) and Cap- Hole Pair Antenna array (CHPA) through nanosphere template lithography (NTL). With proper optimization of the structures, D2PA and CHPA square array with 280nm pitch have achieved distinct enhancement at fluorophore emission wavelength 350nm and excitation wavelength 280nm simultaneously. Maximum field enhancement can reach 20 and 65 fold in the gap of D2PA and CHPA when light incident from substrate, which is expected to greatly enhance fluorescent quantum efficiency that will be confirmed in fluorescence lifetime measurement.
Two-photon excitation based photochemistry and neural imaging

NASA Astrophysics Data System (ADS)

Hatch, Kevin Andrew

Two-photon microscopy is a fluorescence imaging technique which provides distinct advantages in three-dimensional cellular and molecular imaging. The benefits of this technology may extend beyond imaging capabilities through exploitation of the quantum processes responsible for fluorescent events. This study utilized a two-photon microscope to investigate a synthetic photoreactive collagen peptidomimetic, which may serve as a potential material for tissue engineering using the techniques of two-photon photolysis and two-photon polymerization. The combination of these techniques could potentially be used to produce a scaffold for the vascularization of engineered three-dimensional tissues in vitro to address the current limitations of tissue engineering. Additionally, two-photon microscopy was used to observe the effects of the application of the neurotransmitter dopamine to the mushroom body neural structures of Drosophila melanogaster to investigate dopamine's connection to cognitive degeneration.

Stereoscopic Planar Laser-Induced Fluorescence Imaging at 500 kHz

NASA Technical Reports Server (NTRS)

Medford, Taylor L.; Danehy, Paul M.; Jones, Stephen B.; Jiang, N.; Webster, M.; Lempert, Walter; Miller, J.; Meyer, T.

2011-01-01

A new measurement technique for obtaining time- and spatially-resolved image sequences in hypersonic flows is developed. Nitric-oxide planar laser-induced fluorescence (NO PLIF) has previously been used to investigate transition from laminar to turbulent flow in hypersonic boundary layers using both planar and volumetric imaging capabilities. Low flow rates of NO were typically seeded into the flow, minimally perturbing the flow. The volumetric imaging was performed at a measurement rate of 10 Hz using a thick planar laser sheet that excited NO fluorescence. The fluorescence was captured by a pair of cameras having slightly different views of the flow. Subsequent stereoscopic reconstruction of these images allowed the three-dimensional flow structures to be viewed. In the current paper, this approach has been extended to 50,000 times higher repetition rates. A laser operating at 500 kHz excites the seeded NO molecules, and a camera, synchronized with the laser and fitted with a beam-splitting assembly, acquires two separate images of the flow. The resulting stereoscopic images provide three-dimensional flow visualizations at 500 kHz for the first time. The 200 ns exposure time in each frame is fast enough to freeze the flow while the 500 kHz repetition rate is fast enough to time-resolve changes in the flow being studied. This method is applied to visualize the evolving hypersonic flow structures that propagate downstream of a discrete protuberance attached to a flat plate. The technique was demonstrated in the NASA Langley Research Center s 31-Inch Mach 10 Air Tunnel facility. Different tunnel Reynolds number conditions, NO flow rates and two different cylindrical protuberance heights were investigated. The location of the onset of flow unsteadiness, an indicator of transition, was observed to move downstream during the tunnel runs, coinciding with an increase in the model temperature.
Confocal Imaging of porous media

NASA Astrophysics Data System (ADS)

Shah, S.; Crawshaw, D.; Boek, D.

2012-12-01

Carbonate rocks, which hold approximately 50% of the world's oil and gas reserves, have a very complicated and heterogeneous structure in comparison with sandstone reservoir rock. We present advances with different techniques to image, reconstruct, and characterize statistically the micro-geometry of carbonate pores. The main goal here is to develop a technique to obtain two dimensional and three dimensional images using Confocal Laser Scanning Microscopy. CLSM is used in epi-fluorescent imaging mode, allowing for the very high optical resolution of features well below 1μm size. Images of pore structures were captured using CLSM imaging where spaces in the carbonate samples were impregnated with a fluorescent, dyed epoxy-resin, and scanned in the x-y plane by a laser probe. We discuss the sample preparation in detail for Confocal Imaging to obtain sub-micron resolution images of heterogeneous carbonate rocks. We also discuss the technical and practical aspects of this imaging technique, including its advantages and limitation. We present several examples of this application, including studying pore geometry in carbonates, characterizing sub-resolution porosity in two dimensional images. We then describe approaches to extract statistical information about porosity using image processing and spatial correlation function. We have managed to obtain very low depth information in z -axis (~ 50μm) to develop three dimensional images of carbonate rocks with the current capabilities and limitation of CLSM technique. Hence, we have planned a novel technique to obtain higher depth information to obtain high three dimensional images with sub-micron resolution possible in the lateral and axial planes.
Emission wavelength tuning of fluorescence by fine structural control of optical metamaterials with Fano resonance

PubMed Central

Moritake, Y.; Kanamori, Y.; Hane, K.

2016-01-01

We demonstrated fine emission wavelength tuning of quantum dot (QD) fluorescence by fine structural control of optical metamaterials with Fano resonance. An asymmetric-double-bar (ADB), which was composed of only two bars with slightly different bar lengths, was used to obtain Fano resonance in the optical region. By changing the short bar length of ADB structures with high dimensional accuracy in the order of 10 nm, resonant wavelengths of Fano resonance were controlled from 1296 to 1416 nm. Fluorescence of QDs embedded in a polymer layer on ADB metamaterials were modified due to coupling to Fano resonance and fine tuning from 1350 to 1376 nm was observed. Wavelength tuning of modified fluorescence was reproduced by analysis using absorption peaks of Fano resonance. Tuning range of modified fluorescence became narrow, which was interpreted by a simple Gaussian model and resulted from comparable FWHM in QD fluorescence and Fano resonant peaks. The results will help the design and fabrication of metamaterial devices with fluorophores such as light sources and biomarkers. PMID:27622503
DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Lingyun; Lin, Zekai; Shi, Wenjie

The dimensionality dependency of resonance energy transfer is of great interest due to its importance in understanding energy transfer on cell membranes and in low-dimension nanostructures. Light harvesting two-dimensional metal–organic layers (2D-MOLs) and three-dimensional metal–organic frameworks (3D-MOFs) provide comparative models to study such dimensionality dependence with molecular accuracy. Here we report the construction of 2D-MOLs and 3D-MOFs from a donor ligand 4,4',4''-(benzene-1,3,5-triyl-tris(ethyne-2,1-diyl))tribenzoate (BTE) and a doped acceptor ligand 3,3',3''-nitro-4,4',4''-(benzene-1,3,5-triyl-tris(ethyne-2,1-diyl))tribenzoate (BTE-NO2). These 2D-MOLs and 3D-MOFs are connected by similar hafnium clusters, with key differences in the topology and dimensionality of the metal–ligand connection. Energy transfer from donors to acceptors through themore » 2D-MOL or 3D-MOF skeletons is revealed by measuring and modeling the fluorescence quenching of the donors. We found that energy transfer in 3D-MOFs is more efficient than that in 2D-MOLs, but excitons on 2D-MOLs are more accessible to external quenchers as compared with those in 3D-MOFs. These results not only provide support to theoretical analysis of energy transfer in low dimensions, but also present opportunities to use efficient exciton migration in 2D materials for light-harvesting and fluorescence sensing.« less
Further Insights into Metal-DOM Interaction: Consideration of Both Fluorescent and Non-Fluorescent Substances

PubMed Central

Xu, Huacheng; Zhong, Jicheng; Yu, Guanghui; Wu, Jun; Jiang, Helong; Yang, Liuyan

2014-01-01

Information on metal binding with fluorescent substances has been widely studied. By contrast, information on metal binding with non-fluorescent substances remains lacking despite the dominance of these substances in aquatic systems. In this study, the metal binding properties of both fluorescent and non-fluorescent substances were investigated by using metal titration combined with two-dimensional correlation spectroscopy (2D–COS) analysis. The organic matters in the eutrophic algae-rich lake, including natural organic matters (NOM) and algae-induced extracellular polymeric substances (EPS), both contained fluorescent and non-fluorescent substances. The peaks in the one-dimensional spectra strongly overlapped, while 2D–COS can decompose the overlapped peaks and thus enhanced the spectral resolution. Moreover, 2D FTIR COS demonstrated that the binding susceptibility of organic ligands in both NOM and algal EPS matrices followed the order: 3400>1380>1650 cm−1, indicative the significant contribution of non-fluorescent ligands in metal binding. The modified Stern-Volmer equation also revealed a substantial metal binding potential for the non-fluorescent substances (logKM: 3.57∼4.92). As for the effects of organic ligands on metal binding, EPS was characterized with higher binding ability than NOM for both fluorescent and non-fluorescent ligands. Algae-induced EPS and the non-fluorescent substances in eutrophic algae-rich lakes should not be overlooked because of their high metal binding potential. PMID:25380246
High-efficiency in situ resonant inelastic x-ray scattering (iRIXS) endstation at the Advanced Light Source

NASA Astrophysics Data System (ADS)

Qiao, Ruimin; Li, Qinghao; Zhuo, Zengqing; Sallis, Shawn; Fuchs, Oliver; Blum, Monika; Weinhardt, Lothar; Heske, Clemens; Pepper, John; Jones, Michael; Brown, Adam; Spucces, Adrian; Chow, Ken; Smith, Brian; Glans, Per-Anders; Chen, Yanxue; Yan, Shishen; Pan, Feng; Piper, Louis F. J.; Denlinger, Jonathan; Guo, Jinghua; Hussain, Zahid; Chuang, Yi-De; Yang, Wanli

2017-03-01

An endstation with two high-efficiency soft x-ray spectrographs was developed at Beamline 8.0.1 of the Advanced Light Source, Lawrence Berkeley National Laboratory. The endstation is capable of performing soft x-ray absorption spectroscopy, emission spectroscopy, and, in particular, resonant inelastic soft x-ray scattering (RIXS). Two slit-less variable line-spacing grating spectrographs are installed at different detection geometries. The endstation covers the photon energy range from 80 to 1500 eV. For studying transition-metal oxides, the large detection energy window allows a simultaneous collection of x-ray emission spectra with energies ranging from the O K-edge to the Ni L-edge without moving any mechanical components. The record-high efficiency enables the recording of comprehensive two-dimensional RIXS maps with good statistics within a short acquisition time. By virtue of the large energy window and high throughput of the spectrographs, partial fluorescence yield and inverse partial fluorescence yield signals could be obtained for all transition metal L-edges including Mn. Moreover, the different geometries of these two spectrographs (parallel and perpendicular to the horizontal polarization of the beamline) provide contrasts in RIXS features with two different momentum transfers.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiao, Ruimin; Li, Qinghao; Zhuo, Zengqing

In this paper, an endstation with two high-efficiency soft x-ray spectrographs was developed at Beamline 8.0.1 of the Advanced Light Source, Lawrence Berkeley National Laboratory. The endstation is capable of performing soft x-ray absorption spectroscopy, emission spectroscopy, and, in particular, resonant inelastic soft x-ray scattering (RIXS). Two slit-less variable line-spacing grating spectrographs are installed at different detection geometries. The endstation covers the photon energy range from 80 to 1500 eV. For studying transition-metal oxides, the large detection energy window allows a simultaneous collection of x-ray emission spectra with energies ranging from the O K-edge to the Ni L-edge without movingmore » any mechanical components. The record-high efficiency enables the recording of comprehensive two-dimensional RIXS maps with good statistics within a short acquisition time. By virtue of the large energy window and high throughput of the spectrographs, partial fluorescence yield and inverse partial fluorescence yield signals could be obtained for all transition metal L-edges including Mn. Finally and moreover, the different geometries of these two spectrographs (parallel and perpendicular to the horizontal polarization of the beamline) provide contrasts in RIXS features with two different momentum transfers.« less
Feasibility of the simultaneous determination of polycyclic aromatic hydrocarbons based on two-dimensional fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Ren, Yixia, E-mail: renyixia1@163.com; Chai, Hongmei; Tang, Long

Three novel coordination polymers, namely, [Cd(2-Hstp)(1,4-bbi)(H{sub 2}O){sub 2}]·3H{sub 2}O (1), [Cd{sub 1.5}(2-stp)(1,4-bbi)(H{sub 2}O){sub 2}]·H{sub 2}O (2) and [Zn{sub 2}(2-stp)(μ{sub 2}-OH)(1,4-bbi){sub 1.5}(H{sub 2}O)]·6H{sub 2}O (3) (2-H{sub 3}stp is equal to 2-sulfoterephthalate and 1,4-bisbenzimidazole is equal to 1,4-bbi), have been synthesized by hydrothermal reaction. The structural analyses show that 1 and 2 possess different structural features despite the same raw materials, which are 1D chain structure featuring 6-member-water H-bonds cluster and 3D bbi-pillared wavy-like layer framework, respectively. As changing the metal ion to zinc ion, 3 exhibits 3D stp-pillared layer architecture, which discovers the effect of the central metal ions on themore » formation of metal–organic frameworks. The fluorescence studies show that the emissions of the coordination polymers are attributed to the ligand π–π* transition, which means they could be potential fluorescence materials. - Graphical abstract: Three new Cd{sup II}/Zn{sup II} 2-sulfoterephthalate (2-H{sub 3}stp) complexes with 1,4-bisbenzimidazole (1,4-bbi) are described. Complex 1 exhibits one-dimensional chain-like structure, 2 is a three-dimensional bbi-pillared wavy-like layer framework, while 3 is a three-dimensional stp-pillared layer architecture. Fluorescence spectra exhibit the π–π* transition of two organic ligands. - Highlights: • Three Cd{sup II}/Mn{sup II} 2-sulfoterephthalate complexes containing 1,4-bisbenzimidazole. • Different structural features despite the same raw materials for 1 and 2. • Fluorescence spectra exhibit the π–π* transition of organic ligands.« less
Portable Fluorescence Imaging System for Hypersonic Flow Facilities

NASA Technical Reports Server (NTRS)

Wilkes, J. A.; Alderfer, D. W.; Jones, S. B.; Danehy, P. M.

2003-01-01

A portable fluorescence imaging system has been developed for use in NASA Langley s hypersonic wind tunnels. The system has been applied to a small-scale free jet flow. Two-dimensional images were taken of the flow out of a nozzle into a low-pressure test section using the portable planar laser-induced fluorescence system. Images were taken from the center of the jet at various test section pressures, showing the formation of a barrel shock at low pressures, transitioning to a turbulent jet at high pressures. A spanwise scan through the jet at constant pressure reveals the three-dimensional structure of the flow. Future capabilities of the system for making measurements in large-scale hypersonic wind tunnel facilities are discussed.
Fluorescence intermittency originates from reclustering in two-dimensional organic semiconductors

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

PubMed Central

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

2014-01-01

Abstract. Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures. PMID:24390370
Nonlinear Spectroscopy of Multicomponent Droplets and Two- and Three Dimensional Measurements in Flames.

DTIC Science & Technology

1994-03-31

fluorescence intensity with temperature , which allows the fuel cn ce to be found directly from the acetaldehyde fluorescence. An alternative means of measuring... oxidizer . The measured quantities are used to form 17 a conserved scalar from which the mixtur fraction is determined in an iterative process. We have...turbulent nonpemIixed acetaklehyde flame. Acetaldehyde (CH3CHO) was chosen for its relatively high fluorescence yield and small variation of
Sample holder for axial rotation of specimens in 3D microscopy.

PubMed

Bruns, T; Schickinger, S; Schneckenburger, H

2015-10-01

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.
All Fiber-Coupled OH Planar Laser-Induced-Fluorescence (OH-PLIF)-Based Two-Dimensional Thermometry.

PubMed

Hsu, Paul S; Jiang, Naibo; Patnaik, Anil K; Katta, Vish; Roy, Sukesh; Gord, James R

2018-04-01

Two-color, planar laser-induced fluorescence (PLIF)-based two-dimensional (2D) thermometry techniques for reacting flows, which are typically developed in the laboratory conditions, face a stiff challenge in their practical implementation in harsh environments such as combustion rigs. In addition to limited optical access, the critical experimental conditions (i.e., uncontrolled humidity, vibration, and large thermal gradients) often restrict sensitive laser system operation and cause difficulties maintaining beam-overlap. Thus, an all fiber-coupled, two-color OH-PLIF system has been developed, employing two long optical fibers allowing isolation of the laser and signal-collection systems. Two OH-excitation laser beams (∼283 nm and ∼286 nm) are delivered through a common 6 m long, 400 µm core, deep ultraviolet (UV)-enhanced multimode fiber. The fluorescence signal (∼310 nm) is collected by a 3 m long, UV-grade imaging fiber. Proof-of-principle temperature measurements are demonstrated in atmospheric pressure, near adiabatic, CH 4 /O 2 /N 2 jet flames. The effects of the excitation pulse interval on fiber transmission are investigated. The proof-of-principle measurements show significant promise for thermometry in harsh environments such as gas turbine engine tests.
Hyper-spectrum scanning laser optical tomography

NASA Astrophysics Data System (ADS)

Chen, Lingling; Li, Guiye; Li, Yingchao; Liu, Lina; Liu, Ang; Hu, Xuejuan; Ruan, Shuangchen

2018-02-01

We describe a quantitative fluorescence projection tomography technique which measures the three-dimensional fluorescence spectrum in biomedical samples with size up to several millimeters. This is achieved by acquiring a series of hyperspectral images, by using laser scanning scheme, at different projection angles. We demonstrate that this technique provide a quantitative measure of the fluorescence signal by comparing the spectrum and intensity profile of a fluorescent bead phantom and also demonstrate its application to differentiating the extrinsic label and the autofluorescence in a mouse embryo.
Single-Molecule Resolution of Antimicrobial Peptide Interactions with Supported Lipid A Bilayers.

PubMed

Nelson, Nathaniel; Schwartz, Daniel K

2018-06-05

The molecular interactions between antimicrobial peptides (AMPs) and lipid A-containing supported lipid bilayers were probed using single-molecule total internal reflection fluorescence microscopy. Hybrid supported lipid bilayers with lipid A outer leaflets and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) inner leaflets were prepared and characterized, and the spatiotemporal trajectories of individual fluorescently labeled LL37 and Melittin AMPs were determined as they interacted with the bilayer surfaces comprising either monophosphoryl or diphosphoryl lipid A (from Escherichia coli) to determine the impact of electrostatic interactions. Large numbers of trajectories were obtained and analyzed to obtain the distributions of surface residence times and the statistics of the spatial trajectories. Interestingly, the AMP species were sensitive to subtle differences in the charge of the lipid, with both peptides diffusing more slowly and residing longer on the diphosphoryl lipid A. Furthermore, the single-molecule dynamics indicated a qualitative difference between the behavior of AMPs on hybrid Lipid A bilayers and on those composed entirely of DOPE. Whereas AMPs interacting with a DOPE bilayer exhibited two-dimensional Brownian diffusion with a diffusion coefficient of ∼1.7 μm 2 /s, AMPs adsorbed to the lipid A surface exhibited much slower apparent diffusion (on the order of ∼0.1 μm 2 /s) and executed intermittent trajectories that alternated between two-dimensional Brownian diffusion and desorption-mediated three-dimensional flights. Overall, these findings suggested that bilayers with lipid A in the outer leaflet, as it is in bacterial outer membranes, are valuable model systems for the study of the initial stage of AMP-bacterium interactions. Furthermore, single-molecule dynamics was sensitive to subtle differences in electrostatic interactions between cationic AMPs and monovalent or divalent anionic lipid A moieties. Copyright © 2018 Biophysical Society. All rights reserved.
Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation

NASA Astrophysics Data System (ADS)

Lien, Chi-Hsiang; Abrigo, Gerald; Chen, Pei-Hsuan; Chien, Fan-Ching

2017-02-01

Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore-labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830 lines/mm. TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0±0.7 μm and the shifting distance of the temporal focal plane was less than 0.95 μm within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements.
Comparison of three-dimensional fluorescence analysis methods for predicting formation of trihalomethanes and haloacetic acids.

PubMed

Peleato, Nicolás M; Andrews, Robert C

2015-01-01

This work investigated the application of several fluorescence excitation-emission matrix analysis methods as natural organic matter (NOM) indicators for use in predicting the formation of trihalomethanes (THMs) and haloacetic acids (HAAs). Waters from four different sources (two rivers and two lakes) were subjected to jar testing followed by 24hr disinfection by-product formation tests using chlorine. NOM was quantified using three common measures: dissolved organic carbon, ultraviolet absorbance at 254 nm, and specific ultraviolet absorbance as well as by principal component analysis, peak picking, and parallel factor analysis of fluorescence spectra. Based on multi-linear modeling of THMs and HAAs, principle component (PC) scores resulted in the lowest mean squared prediction error of cross-folded test sets (THMs: 43.7 (μg/L)(2), HAAs: 233.3 (μg/L)(2)). Inclusion of principle components representative of protein-like material significantly decreased prediction error for both THMs and HAAs. Parallel factor analysis did not identify a protein-like component and resulted in prediction errors similar to traditional NOM surrogates as well as fluorescence peak picking. These results support the value of fluorescence excitation-emission matrix-principal component analysis as a suitable NOM indicator in predicting the formation of THMs and HAAs for the water sources studied. Copyright © 2014. Published by Elsevier B.V.
[2D correlation spectral study of a coordination polymer [Eu(PCPOA)3 (H2O)]n].

PubMed

Sun, Rui-qing; Zhang, Han-hui; Cao, Yan-ning; Chen, Yi-ping; Yang, Qi-yu; Wang, Zhi-yang

2007-05-01

A novel two dimensional coordination polymer [Eu(PCPOA)3 (H2O)], was synthesized under hydrothermal condition. Based on the determination of the structure, the 2D correlation FTIR spectra with the perturbation of magnetism and the 2D correlation fluorescence spectra with the perturbation of temperature were investigated. The energy bonds were calculated using CASTEP Program of Material studio. The Europium ions are nine-coordinated and the ligands adopted two different modes to connect the Eu3+ ions to 2D layer structure. The study of the 2D-FTIR reveals that the carboxylates coordinate with the center ions not only as monodentate, but also as bidentate chelate. The 2D fluorescence spectra indicates that the transition of (5)D0-->(7)F2 is influenced intensively by the perturbation of temperature.

Integrated three-dimensional optical MEMS for chip-based fluorescence detection

NASA Astrophysics Data System (ADS)

Hung, Kuo-Yung; Tseng, Fan-Gang; Khoo, Hwa-Seng

2009-04-01

This paper presents a novel fluorescence sensing chip for parallel protein microarray detection in the context of a 3-in-1 protein chip system. This portable microchip consists of a monolithic integration of CMOS-based avalanche photo diodes (APDs) combined with a polymer micro-lens, a set of three-dimensional (3D) inclined mirrors for separating adjacent light signals and a low-noise transformer-free dc-dc boost mini-circuit to power the APDs (ripple below 1.28 mV, 0-5 V input, 142 V and 12 mA output). We fabricated our APDs using the planar CMOS process so as to facilitate the post-CMOS integration of optical MEMS components such as the lenses. The APD arrays were arranged in unique circular patterns appropriate for detecting the specific fluorescently labelled protein spots in our study. The array-type APDs were designed so as to compensate for any alignment error as detected by a positional error signal algorithm. The condenser lens was used as a structure for light collection to enhance the fluorescent signals by about 25%. This element also helped to reduce the light loss due to surface absorption. We fabricated an inclined mirror to separate two adjacent fluorescent signals from different specimens. Excitation using evanescent waves helped reduce the interference of the excitation light source. This approach also reduced the number of required optical lenses and minimized the complexity of the structural design. We achieved detection floors for anti-rabbit IgG and Cy5 fluorescent dye as low as 0.5 ng/µl (~3.268 nM). We argue that the intrinsic nature of point-to-point and batch-detection methods as showcased in our chip offers advantages over the serial-scanning approach used in traditional scanner systems. In addition, our system is low cost and lightweight.
Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance

NASA Astrophysics Data System (ADS)

Li, Shihui; Li, Daojin

2011-11-01

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC ( CBZC/ CLys < 9) and a combined quenching process at higher concentration of BZC ( CBZC/ CLys > 9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn 2+ on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied.
Performance Enhancement of Pharmacokinetic Diffuse Fluorescence Tomography by Use of Adaptive Extended Kalman Filtering.

PubMed

Wang, Xin; Wu, Linhui; Yi, Xi; Zhang, Yanqi; Zhang, Limin; Zhao, Huijuan; Gao, Feng

2015-01-01

Due to both the physiological and morphological differences in the vascularization between healthy and diseased tissues, pharmacokinetic diffuse fluorescence tomography (DFT) can provide contrast-enhanced and comprehensive information for tumor diagnosis and staging. In this regime, the extended Kalman filtering (EKF) based method shows numerous advantages including accurate modeling, online estimation of multiparameters, and universal applicability to any optical fluorophore. Nevertheless the performance of the conventional EKF highly hinges on the exact and inaccessible prior knowledge about the initial values. To address the above issues, an adaptive-EKF scheme is proposed based on a two-compartmental model for the enhancement, which utilizes a variable forgetting-factor to compensate the inaccuracy of the initial states and emphasize the effect of the current data. It is demonstrated using two-dimensional simulative investigations on a circular domain that the proposed adaptive-EKF can obtain preferable estimation of the pharmacokinetic-rates to the conventional-EKF and the enhanced-EKF in terms of quantitativeness, noise robustness, and initialization independence. Further three-dimensional numerical experiments on a digital mouse model validate the efficacy of the method as applied in realistic biological systems.
Characterization of organic membrane foulants in a submerged membrane bioreactor with pre-ozonation using three-dimensional excitation-emission matrix fluorescence spectroscopy.

PubMed

Liu, Ting; Chen, Zhong-lin; Yu, Wen-zheng; You, Shi-jie

2011-02-01

This study focuses on organic membrane foulants in a submerged membrane bioreactor (MBR) process with pre-ozonation compared to an individual MBR using three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy. While the influent was continuously ozonated at a normal dosage, preferable organic matter removal was achieved in subsequent MBR, and trans-membrane pressure increased at a much lower rate than that of the individual MBR. EEM fluorescence spectroscopy was employed to characterize the dissolved organic matter (DOM) samples, extracellular polymeric substance (EPS) samples and membrane foulants. Four main peaks could be identified from the EEM fluorescence spectra of the DOM samples in both MBRs. Two peaks were associated with the protein-like fluorophores, and the other ones were related to the humic-like fluorophores. The results indicated that pre-ozonation decreased fluorescence intensities of all peaks in the EEM spectra of influent DOM especially for protein-like substances and caused red shifts of all fluorescence peaks to different extents. The peak intensities of the protein-like substances represented by Peak T(1) and T(2) in EPS spectra were obviously decreased as a result of pre-ozonation. Both external and internal fouling could be effectively mitigated by the pre-ozonation. The most primary component of external foulants was humic acid-like substance (Peak C) in the MBR with pre-ozonation and protein-like substance (Peak T(1)) in the individual MBR, respectively. The content decrease of protein-like substances and structural change of humic-like substances were observed in external foulants from EEM fluorescence spectra due to pre-ozonation. However, it could be seen that ozonation resulted in significant reduction of intensities but little location shift of all peaks in EEM fluorescence spectra of internal foulants. Copyright © 2010 Elsevier Ltd. All rights reserved.
Longitudinal in vivo two-photon fluorescence imaging

PubMed Central

Crowe, Sarah E.; Ellis-Davies, Graham C.R.

2014-01-01

Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in 1980s, that enabled imaging both fixed and living biological tissue with three-dimensional precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to two years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. PMID:24214350
DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Qing; Jin, Dafei; Xiao, Jun

Two-dimensional molecular aggregate (2DMA), a thin sheet of strongly interacting dipole molecules self-assembled at close distance on an ordered lattice, is a fascinating fluorescent material. It is distinctively different from the conventional (single or colloidal) dye molecules and quantum dots. Here, in this paper, we verify that when a 2DMA is placed at a nanometric distance from a metallic substrate, the strong and coherent interaction between the dipoles inside the 2DMA dominates its fluorescent decay at a picosecond timescale. Our streak-camera lifetime measurement and interacting lattice–dipole calculation reveal that the metal-mediated dipole–dipole interaction shortens the fluorescent lifetime to about one-halfmore » and increases the energy dissipation rate by 10 times that expected from the noninteracting single-dipole picture. In conclusion, our finding can enrich our understanding of nanoscale energy transfer in molecular excitonic systems and may designate a unique direction for developing fast and efficient optoelectronic devices.« less
An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

PubMed

Gregoretti, Francesco; Cesarini, Elisa; Lanzuolo, Chiara; Oliva, Gennaro; Antonelli, Laura

2016-01-01

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Harilal, Sivanandan S.; LaHaye, Nicole L.; Phillips, Mark C.

We use a two-dimensional laser-induced fluorescence spectroscopy technique to measure the coupled absorption and emission properties of atomic species in plasmas produced via laser ablation of solid aluminum targets at atmospheric pressure. Emission spectra from the Al I 394.4 nm and Al I 396.15 nm transitions are measured while a frequency-doubled, continuous-wave, Ti:Sapphire laser is tuned across the Al I 396.15 nm transition. The resulting two-dimensional spectra show the energy coupling between the two transitions via increased emission intensity for both transitions during resonant absorption of the continuous-wave laser at one transition. Time-delayed and gated detection of the emission spectrummore » is used to isolate the resonantly-excited fluorescence emission from the thermally-excited emission from the plasma. In addition, the tunable continuous-wave laser measures the absorption spectrum of the Al transition with ultra-high resolution after the plasma has cooled, resulting in narrower spectral linewidths than observed in emission spectra. Our results highlight that fluorescence spectroscopy employing continuous-wave laser re-excitation after pulsed laser ablation combines benefits of both traditional emission and absorption spectroscopic methods.« less
Use of intrinsic fluorescent signals for characterizing tissue metabolic states in health and disease

NASA Astrophysics Data System (ADS)

Chance, Britton

1996-04-01

The large content of mitochondria in metabolizing cells, coupled with intrinsic NADH and flavoprotein signals makes these signals ideal for characterizing tissue metabolic states in health and disease. The first few millimeters of tissue are reached by the fluorescence excitation in the exposed surfaces of the cervix, bladder, rectum and esophagus, etc. Thus, extensive use has been made of fluorescent signals by a large number of investigators for tumor diagnosis from an empirical standpoint where the fluorescent signals are generally diminished in precancerous and cancerous tissue. This article reviews the biochemical basis for the fluorescent signals and points to a 'gold standard' for fluorescent signal examination involving freeze trapping and low temperature two- or three-dimensional high resolution fluorescence spectroscopy.
Electron beam dispersion measurements in nitrogen using two-dimensional imaging of N2(+) fluorescence

NASA Technical Reports Server (NTRS)

Clapp, L. H.; Twiss, R. G.; Cattolica, R. J.

1991-01-01

Experimental results are presented related to the radial spread of fluorescence excited by 10 and 20 KeV electron beams passing through nonflowing rarefied nitrogen at 293 K. An imaging technique for obtaining species distributions from measured beam-excited fluorescence is described, based on a signal inversion scheme mathematically equivalent to the inversion of the Abel integral equation. From fluorescence image data, measurements of beam radius, integrated signal intensity, and spatially resolved distributions of N2(+) first-negative-band fluorescence-emitting species have been made. Data are compared with earlier measurements and with an heuristic beam spread model.
[Vermicomposting of different organic materials and three-dimensional excitation emission matrix fluorescence spectroscopic characterization of their dissolved organic matter].

PubMed

Yang, Wei; Wang, Dong-sheng; Liu, Man-qiang; Hu, Feng; Li, Hui-xin; Huang, Zhong-yang; Chang, Yi-jun; Jiao, Jia-guo

2015-10-01

In this experiment, different proportions of the cattle manure, tea-leaf, herb and mushroom residues, were used as food for earthworm (Eisenia fetida) to study the growth of the earth-worm. Then the characteristics and transformation of nutrient content and three-dimensional excitation emission matrix fluorescence (3DEEM) of dissolved organic matter (DOM) during vermistabilization were investigated by means of chemical and spectroscopic methods. The result showed that the mixture of different ratios of cattle manure with herb residue, and cattle manure with tea-leaf were conducive to the growth of earthworm, while the materials compounded with mushroom residue inhibited the growth of earthworm. With the increasing time of verimcomposting, the pH in vermicompost tended to be circumneutral and weakly acidic, and there were increases in electrical conductivity, and the contents of total nitrogen, total phosphorus, available nitrogen, and available phosphorus, while the total potassium and available potassium increased first and then decreased, and the organic matter content decreased. 3DEEM and fluorescence regional integration results indicated that, the fluorescence of protein-like fluorescence peaks declined significantly, while the intensity of humic-like fluorescence peak increased significantly in DOM. Vermicomposting process might change the compositions of DOM with elevated concentrations of humic acid and fulvic acid in the organics. In all, this study suggested the suitability of 3DEEM for monitoring the organics transformation and assessing the maturity in the vermicomposting.
Analytical Applications Of High-Resolution Molecular Fluorescence Spectroscopy In Low Temperature Solid Matrices

NASA Astrophysics Data System (ADS)

Hofstraat, Johannes W.; van Zeijl, W. J.; Smedes, F.; Ariese, Freek; Gooijer, Cees; Velthorst, Nel H.; Locher, R.; Renn, Alois; Wild, Urs P.

1989-05-01

High-resolution fluorescence spectroscopy may be used to obtain highly specific, vibrationally resolved spectral signatures of molecules. Two techniques are presented that both make use of low temperature, solid matrices. In Shpol'skii spectroscopy highly resolved spectra are obtained by employing n-alkanes as solvents that form neat crystalline matrices at low temperatures in which the guest molecules occupy well defined substitutional sites. Fluorescence line-narrowing spectroscopy is based on the application of selective (mostly laser-) excitation of the guest molecules. Principles and analytical applications of both techniques will be discussed. Specific attention will be paid to the determination of pyrene in bird meat by means of Shpol'skii spectroscopy and to the possibilities of applying two-dimensional fluorescence line-narrowing spectroscopy.
Two-dimensional correlation spectroscopic analysis on the interaction between humic acids and aluminum coagulant.

PubMed

Jin, Pengkang; Song, Jina; Wang, Xiaochang C; Jin, Xin

2018-02-01

In this study, two-dimensional correlation spectroscopy integrated with synchronous fluorescence and infrared absorption spectroscopy was employed to investigate the interaction between humic acids and aluminum coagulant at slightly acidic and neutral pH. Higher fluorescence quenching was produced for fulvic-like and humic-like fractions at pH5. At pH5, the humic-like fractions originating from the carboxylic acid, carboxyl and polysaccharide compounds were bound to aluminum first, followed by the fulvic-like fractions originating from the carboxyl and polysaccharide compounds. This finding also demonstrated that the activated functional groups of HA were involved in forming the Al-HA complex, which was accompanied by the removal of other groups by co-precipitation. Meanwhile, at pH7, almost no fluorescence quenching occurred, and surface complexation was observed to occur, in which the activated functional groups were absorbed on the amorphous Al(OH) 3 . Two-dimensional FT-IR correlation spectroscopy indicated the sequence of HA structural change during coagulation with aluminum, with IR bands affected in the order of COOH>COO - >NH deformation of amide II>aliphatic hydroxyl COH at pH5, and COO - >aliphatic hydroxyl COH at pH7. This study provides a promising pathway for analysis and insight into the priority of functional groups in the interaction between organic matters and metal coagulants. Copyright © 2017. Published by Elsevier B.V.
Fluorescence imaging in the last two decades

PubMed Central

Miyawaki, Atsushi

2013-01-01

In commemoration of the 20th anniversary of the molecular cloning of the gene for the green fluorescent protein from the jellyfish Aequorea victoria, I would like to reflect on the development of new fluorescence imaging technology in the last two decades. As this technology has become increasingly diversified, it has become more and more of a challenge to come up with a comprehensive and exhaustive review of it. Here I will focus on optogenetics and large-scale, three-dimensional reconstruction. Those two technological innovations have been achieved in the neuroscience community owing to the combined efforts of molecular biologists and light microscopists. In addition, modern fluorescence imaging has indeed improved our understanding of the spatiotemporal regulation of fundamental biological functions at cellular level. As an example, I will introduce some findings we made regarding the movement of biomolecules across the nuclear membrane. The above-mentioned imaging approaches are possible today but were impossible two decades ago. PMID:23393311
Fast and background-free three-dimensional (3D) live-cell imaging with lanthanide-doped upconverting nanoparticles.

PubMed

Jo, Hong Li; Song, Yo Han; Park, Jinho; Jo, Eun-Jung; Goh, Yeongchang; Shin, Kyujin; Kim, Min-Gon; Lee, Kang Taek

2015-12-14

We report on the development of a three-dimensional (3D) live-cell imaging technique with high spatiotemporal resolution using lanthanide-doped upconverting nanoparticles (UCNPs). It employs the sectioning capability of confocal microscopy except that the two-dimensional (2D) section images are acquired by wide-field epi-fluorescence microscopy. Although epi-fluorescence images are contaminated with the out-of-focus background in general, the near-infrared (NIR) excitation used for the excitation of UCNPs does not generate any autofluorescence, which helps to lower the background. Moreover, the image blurring due to defocusing was naturally eliminated in the image reconstruction process. The 3D images were used to investigate the cellular dynamics such as nuclear uptake and single-particle tracking that require 3D description.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, Mark C.; Brumfield, Brian E.; Harilal, Sivanandan S.

We present the first two-dimensional fluorescence spectroscopy measurements of uranium isotopes in femtosecond laser ablation plasmas. A new method of signal normalization is presented to reduce noise in absorption-based measurements of laser ablation.
Targeted nanosensor aided three-dimensional pH mapping in tumor spheroids using two-photon microscopy

NASA Astrophysics Data System (ADS)

Ray, Aniruddha; Lee, Yong-Eun Koo; Elbez, Remy; Kopelman, Raoul

2012-03-01

Tumors are generally characterized by a pH lower than the surrounding tissues. The mapping of tumor pH is of great importance as it plays a critical role in drug delivery and its effectiveness. Here we present a pH mapping technique in tumor spheroids, using targeted, ratiometric, fluorescent, pH nano-sensor that is based on two-photon excitation. Spheroids are micro-tumors that are widely used as an in-vitro three dimensional tumor model to study the different properties of the tumor for the purpose of drug delivery, therapy etc. The nanosensor consists of 8-Hydroxypyrene- 1,3,6-trisulfonic acid (HPTS), a pH sensitive dye, encapsulated in polyacrylamide hydrogel nanoparticle matrix and F3 peptide, conjugated to the nanoparticle's surface. The nanosensor has an average size of 68nm and contains approximately 0.5% dye by weight. The fluorescence intensity ratio, at the two-photon excitation wavelengths of 900nm and 750nm, increases linearly in the pH range from 6.0 to 8.0 and is used to determine the pH of the local environment. Our study reveals the pH distribution inside human cervix cancer spheroids (of different sizes) during the various stages of their formation. This information can be used to develop more efficient drug delivery mechanisms. The two-photon excitation used for this purpose is especially useful as it drastically minimizes both photobleaching and autofluorescence, thus leading to an increase in the signal-to-noise ratio. It also enables deep tissue imaging due to higher photon penetration depth.
High-efficiency in situ resonant inelastic x-ray scattering (iRIXS) endstation at the Advanced Light Source

DOE PAGES

Qiao, Ruimin; Li, Qinghao; Zhuo, Zengqing; ...

2017-03-17

In this paper, an endstation with two high-efficiency soft x-ray spectrographs was developed at Beamline 8.0.1 of the Advanced Light Source, Lawrence Berkeley National Laboratory. The endstation is capable of performing soft x-ray absorption spectroscopy, emission spectroscopy, and, in particular, resonant inelastic soft x-ray scattering (RIXS). Two slit-less variable line-spacing grating spectrographs are installed at different detection geometries. The endstation covers the photon energy range from 80 to 1500 eV. For studying transition-metal oxides, the large detection energy window allows a simultaneous collection of x-ray emission spectra with energies ranging from the O K-edge to the Ni L-edge without movingmore » any mechanical components. The record-high efficiency enables the recording of comprehensive two-dimensional RIXS maps with good statistics within a short acquisition time. By virtue of the large energy window and high throughput of the spectrographs, partial fluorescence yield and inverse partial fluorescence yield signals could be obtained for all transition metal L-edges including Mn. Finally and moreover, the different geometries of these two spectrographs (parallel and perpendicular to the horizontal polarization of the beamline) provide contrasts in RIXS features with two different momentum transfers.« less
[Spectroscopic Diagnosis of Two-Dimensional Distribution of OH Radicals in Wire-Plate Pulsed Corona Discharge Reactor].

PubMed

Jiang, Jian-ping; Luo, Zhong-yang; Xuan, Jian-yong; Zhao, Lei; Fang, Meng-xiang; Gao, Xiang

2015-10-01

Pulsed corona discharge in atmosphere has been widely regarded as an efficient flue gas treatment technology for the generation of active radical species, such as the OH radicals. The spatial distribution of OH radicals generated by pulsed corona discharge plays an important role in decomposing pollutants. The two-dimensional (2-D) distribution of OH radicals of positive wire--plate pulsed corona discharge was detected using laser-induced fluorescence (LIF). The influence of relative humidity (RH) and oxygen concentration on the 2-D distribution of OH radicals were investigated. The results indicated that the 2-D distribution of OH radicals was characterized by a fan-shaped distribution from the wire electrode to plate electrode, and both the maximum values of vertical length and horizontal width of the fan area was less than 1 cm. The 2-D distribution area of OH radicals increased significantly with increasing the RH and the optimum condition was 65% RH. The optimal level of the oxygen concentration for the 2-D distribution area of OH radicals was 2%. The process of OH radical generation and 2-D distribution area of OH radicals were significantly interfered when the oxygen concentration was larger than 15%. The total quenching rate coefficients for different RH values and oxygen concentration in this study were used to calculate the fluorescence yield of OH radical. The fluorescence yield, which is the ratio between the emission rate (Einstein coefficient) and the sum of the emission rate and quenching rate, was used to normalize the 2-D distribution area of OH radicals. The fluorescence yield of OH radical decreased with increasing the RH and oxygen concentration linearly and rapidly. It was also found that compared with the RH, the influence of the oxygen concentration had more notable effect on the fluorescence yield of OH radical and 2-D distribution area of OH radicals.
Four-dimensional data coupled to alternating weighted residue constraint quadrilinear decomposition model applied to environmental analysis: Determination of polycyclic aromatic hydrocarbons

NASA Astrophysics Data System (ADS)

Liu, Tingting; Zhang, Ling; Wang, Shutao; Cui, Yaoyao; Wang, Yutian; Liu, Lingfei; Yang, Zhe

2018-03-01

Qualitative and quantitative analysis of polycyclic aromatic hydrocarbons (PAHs) was carried out by three-dimensional fluorescence spectroscopy combining with Alternating Weighted Residue Constraint Quadrilinear Decomposition (AWRCQLD). The experimental subjects were acenaphthene (ANA) and naphthalene (NAP). Firstly, in order to solve the redundant information of the three-dimensional fluorescence spectral data, the wavelet transform was used to compress data in preprocessing. Then, the four-dimensional data was constructed by using the excitation-emission fluorescence spectra of different concentration PAHs. The sample data was obtained from three solvents that are methanol, ethanol and Ultra-pure water. The four-dimensional spectral data was analyzed by AWRCQLD, then the recovery rate of PAHs was obtained from the three solvents and compared respectively. On one hand, the results showed that PAHs can be measured more accurately by the high-order data, and the recovery rate was higher. On the other hand, the results presented that AWRCQLD can better reflect the superiority of four-dimensional algorithm than the second-order calibration and other third-order calibration algorithms. The recovery rate of ANA was 96.5% 103.3% and the root mean square error of prediction was 0.04 μgL- 1. The recovery rate of NAP was 96.7% 115.7% and the root mean square error of prediction was 0.06 μgL- 1.

Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE™ lightning red.

PubMed

Griebel, Anja; Obermaier, Christian; Westermeier, Reiner; Moche, Martin; Büttner, Knut

2013-07-01

A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins.
Fluorescence intermittency originates from reclustering in two-dimensional organic semiconductors

DOE PAGES

Ruth, Anthony; Hayashi, Michitoshi; Zapol, Peter; ...

2017-02-22

Fluorescence intermittency or blinking is observed in nearly all nanoscale fluorophores. It is characterized by universal power-law distributions in on- and off-times as well as 1/f behaviour in corresponding emission power spectral densities. Blinking, previously seen in confined zero- and one-dimensional systems has recently been documented in two-dimensional reduced graphene oxide. Here we show that unexpected blinking during graphene oxide-to-reduced graphene oxide photoreduction is attributed, in large part, to the redistribution of carbon sp 2 domains. This reclustering generates fluctuations in the number/size of emissive graphenic nanoclusters wherein multiscale modelling captures essential experimental aspects of reduced graphene oxide’s absorption/emission trajectories,more » while simultaneously connecting them to the underlying photochemistry responsible for graphene oxide’s reduction. These simulations thus establish causality between currently unexplained, long timescale emission intermittency in a quantum mechanical fluorophore and identifiable chemical reactions that ultimately lead to switching between on and off states.« less
Multimodal imaging system for dental caries detection

NASA Astrophysics Data System (ADS)

Liang, Rongguang; Wong, Victor; Marcus, Michael; Burns, Peter; McLaughlin, Paul

2007-02-01

Dental caries is a disease in which minerals of the tooth are dissolved by surrounding bacterial plaques. A caries process present for some time may result in a caries lesion. However, if it is detected early enough, the dentist and dental professionals can implement measures to reverse and control caries. Several optical, nonionized methods have been investigated and used to detect dental caries in early stages. However, there is not a method that can singly detect the caries process with both high sensitivity and high specificity. In this paper, we present a multimodal imaging system that combines visible reflectance, fluorescence, and Optical Coherence Tomography (OCT) imaging. This imaging system is designed to obtain one or more two-dimensional images of the tooth (reflectance and fluorescence images) and a three-dimensional OCT image providing depth and size information of the caries. The combination of two- and three-dimensional images of the tooth has the potential for highly sensitive and specific detection of dental caries.
Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

NASA Astrophysics Data System (ADS)

Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

2017-03-01

Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work.
Super resolution imaging of HER2 gene amplification

NASA Astrophysics Data System (ADS)

Okada, Masaya; Kubo, Takuya; Masumoto, Kanako; Iwanaga, Shigeki

2016-02-01

HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.
Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance.

PubMed

Li, Shihui; Li, Daojin

2011-11-01

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (C(BZC)/C(Lys)<9) and a combined quenching process at higher concentration of BZC (C(BZC)/C(Lys)>9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn(2+) on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied. Copyright © 2011 Elsevier B.V. All rights reserved.
Temperature-Dependent Conformations of a Membrane Supported ‘Zinc Porphyrin Tweezer’ by 2D Fluorescence Spectroscopy

PubMed Central

Widom, Julia R.; Lee, Wonbae; Perdomo-Ortiz, Alejandro; Rappoport, Dmitrij; Molinski, Tadeusz F.; Aspuru-Guzik, Alán; Marcus, Andrew H.

2013-01-01

We studied the equilibrium conformations of a ‘zinc porphyrin tweezer’ composed of two carboxylphenyl-functionalized zinc tetraphenyl porphyrin subunits connected by a 1,4 butyndiol spacer, which was suspended inside the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) liposomes. By combining phase-modulation two-dimensional fluorescence spectroscopy (2D FS) with linear absorbance and fluorimetry, we determined that the zinc porphyrin tweezer adopts a mixture of ‘folded’ and ‘extended’ conformations in the membrane. By fitting an exciton-coupling model to a series of data sets recorded over a range of temperatures (17 – 85 °C) and at different laser center wavelengths, we determined that the folded form of the tweezer is stabilized by a favorable change in the entropy of the local membrane environment. Our results provide insights toward understanding the balance of thermodynamic factors that govern molecular assembly in membranes. PMID:23480874
Excitation-resolved multispectral method for imaging pharmacokinetic parameters in dynamic fluorescent molecular tomography

NASA Astrophysics Data System (ADS)

Chen, Maomao; Zhou, Yuan; Su, Han; Zhang, Dong; Luo, Jianwen

2017-04-01

Imaging of the pharmacokinetic parameters in dynamic fluorescence molecular tomography (DFMT) can provide three-dimensional metabolic information for biological studies and drug development. However, owing to the ill-posed nature of the FMT inverse problem, the relatively low quality of the parametric images makes it difficult to investigate the different metabolic processes of the fluorescent targets with small distances. An excitation-resolved multispectral DFMT method is proposed; it is based on the fact that the fluorescent targets with different concentrations show different variations in the excitation spectral domain and can be considered independent signal sources. With an independent component analysis method, the spatial locations of different fluorescent targets can be decomposed, and the fluorescent yields of the targets at different time points can be recovered. Therefore, the metabolic process of each component can be independently investigated. Simulations and phantom experiments are carried out to evaluate the performance of the proposed method. The results demonstrated that the proposed excitation-resolved multispectral method can effectively improve the reconstruction accuracy of the parametric images in DFMT.
Light sheet-based fluorescence microscopy (LSFM) reduces phototoxic effects and provides new means for the modern life sciences

NASA Astrophysics Data System (ADS)

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

2011-07-01

Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack). The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed cell movements during gastrulation, revealed the development during cell migration processes and showed that an LSFM exposes an embryo to 200 times less energy than a conventional and 5,000 times less energy than a confocal fluorescence microscope. Most recently, we implemented incoherent structured illumination in our DSLM. The intensity modulated light sheets can be generated with dynamic frequencies and allow us to estimate the effect of the specimen on the image formation process at various depths in objects of different age.
Direct observation of vibrational energy dispersal via methyl torsions.

PubMed

Gardner, Adrian M; Tuttle, William D; Whalley, Laura E; Wright, Timothy G

2018-02-28

Explicit evidence for the role of methyl rotor levels in promoting energy dispersal is reported. A set of coupled zero-order vibration/vibration-torsion (vibtor) levels in the S 1 state of para -fluorotoluene ( p FT) are investigated. Two-dimensional laser-induced fluorescence (2D-LIF) and two-dimensional zero-kinetic-energy (2D-ZEKE) spectra are reported, and the assignment of the main features in both sets of spectra reveals that the methyl torsion is instrumental in providing a route for coupling between vibrational levels of different symmetry classes. We find that there is very localized, and selective, dissipation of energy via doorway states, and that, in addition to an increase in the density of states, a critical role of the methyl group is a relaxation of symmetry constraints compared to direct vibrational coupling.
Stereoscopic Imaging in Hypersonics Boundary Layers using Planar Laser-Induced Fluorescence

NASA Technical Reports Server (NTRS)

Danehy, Paul M.; Bathel, Brett; Inman, Jennifer A.; Alderfer, David W.; Jones, Stephen B.

2008-01-01

Stereoscopic time-resolved visualization of three-dimensional structures in a hypersonic flow has been performed for the first time. Nitric Oxide (NO) was seeded into hypersonic boundary layer flows that were designed to transition from laminar to turbulent. A thick laser sheet illuminated and excited the NO, causing spatially-varying fluorescence. Two cameras in a stereoscopic configuration were used to image the fluorescence. The images were processed in a computer visualization environment to provide stereoscopic image pairs. Two methods were used to display these image pairs: a cross-eyed viewing method which can be viewed by naked eyes, and red/blue anaglyphs, which require viewing through red/blue glasses. The images visualized three-dimensional information that would be lost if conventional planar laser-induced fluorescence imaging had been used. Two model configurations were studied in NASA Langley Research Center's 31-Inch Mach 10 Air Wind tunnel. One model was a 10 degree half-angle wedge containing a small protuberance to force the flow to transition. The other model was a 1/3-scale, truncated Hyper-X forebody model with blowing through a series of holes to force the boundary layer flow to transition to turbulence. In the former case, low flowrates of pure NO seeded and marked the boundary layer fluid. In the latter, a trace concentration of NO was seeded into the injected N2 gas. The three-dimensional visualizations have an effective time resolution of about 500 ns, which is fast enough to freeze this hypersonic flow. The 512x512 resolution of the resulting images is much higher than high-speed laser-sheet scanning systems with similar time response, which typically measure 10-20 planes.
Fluorescence Intrinsic Characterization of Excitation-Emission Matrix Using Multi-Dimensional Ensemble Empirical Mode Decomposition

PubMed Central

Chang, Chi-Ying; Chang, Chia-Chi; Hsiao, Tzu-Chien

2013-01-01

Excitation-emission matrix (EEM) fluorescence spectroscopy is a noninvasive method for tissue diagnosis and has become important in clinical use. However, the intrinsic characterization of EEM fluorescence remains unclear. Photobleaching and the complexity of the chemical compounds make it difficult to distinguish individual compounds due to overlapping features. Conventional studies use principal component analysis (PCA) for EEM fluorescence analysis, and the relationship between the EEM features extracted by PCA and diseases has been examined. The spectral features of different tissue constituents are not fully separable or clearly defined. Recently, a non-stationary method called multi-dimensional ensemble empirical mode decomposition (MEEMD) was introduced; this method can extract the intrinsic oscillations on multiple spatial scales without loss of information. The aim of this study was to propose a fluorescence spectroscopy system for EEM measurements and to describe a method for extracting the intrinsic characteristics of EEM by MEEMD. The results indicate that, although PCA provides the principal factor for the spectral features associated with chemical compounds, MEEMD can provide additional intrinsic features with more reliable mapping of the chemical compounds. MEEMD has the potential to extract intrinsic fluorescence features and improve the detection of biochemical changes. PMID:24240806
Multiphoton-Excited Fluorescence of Silicon-Vacancy Color Centers in Diamond

NASA Astrophysics Data System (ADS)

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

2017-05-01

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

NASA Technical Reports Server (NTRS)

Ryan, Robert E.; O'Neal, S. Duane; Lanoue, Mark; Russell, Jeffrey

2008-01-01

The system is a single hyperspectral imaging instrument that has the unique capability to acquire both fluorescence and reflectance high-spatial-resolution data that is inherently spatially and spectrally registered. Potential uses of this instrument include plant stress monitoring, counterfeit document detection, biomedical imaging, forensic imaging, and general materials identification. Until now, reflectance and fluorescence spectral imaging have been performed by separate instruments. Neither a reflectance spectral image nor a fluorescence spectral image alone yields as much information about a target surface as does a combination of the two modalities. Before this system was developed, to benefit from this combination, analysts needed to perform time-consuming post-processing efforts to co-register the reflective and fluorescence information. With this instrument, the inherent spatial and spectral registration of the reflectance and fluorescence images minimizes the need for this post-processing step. The main challenge for this technology is to detect the fluorescence signal in the presence of a much stronger reflectance signal. To meet this challenge, the instrument modulates artificial light sources from ultraviolet through the visible to the near-infrared part of the spectrum; in this way, both the reflective and fluorescence signals can be measured through differencing processes to optimize fluorescence and reflectance spectra as needed. The main functional components of the instrument are a hyperspectral imager, an illumination system, and an image-plane scanner. The hyperspectral imager is a one-dimensional (line) imaging spectrometer that includes a spectrally dispersive element and a two-dimensional focal plane detector array. The spectral range of the current imaging spectrometer is between 400 to 1,000 nm, and the wavelength resolution is approximately 3 nm. The illumination system consists of narrowband blue, ultraviolet, and other discrete wavelength light-emitting-diode (LED) sources and white-light LED sources designed to produce consistently spatially stable light. White LEDs provide illumination for the measurement of reflectance spectra, while narrowband blue and UV LEDs are used to excite fluorescence. Each spectral type of LED can be turned on or off depending on the specific remote-sensing process being performed. Uniformity of illumination is achieved by using an array of LEDs and/or an integrating sphere or other diffusing surface. The image plane scanner uses a fore optic with a field of view large enough to provide an entire scan line on the image plane. It builds up a two-dimensional image in pushbroom fashion as the target is scanned across the image plane either by moving the object or moving the fore optic. For fluorescence detection, spectral filtering of a narrowband light illumination source is sometimes necessary to minimize the interference of the source spectrum wings with the fluorescence signal. Spectral filtering is achieved with optical interference filters and absorption glasses. This dual spectral imaging capability will enable the optimization of reflective, fluorescence, and fused datasets as well as a cost-effective design for multispectral imaging solutions. This system has been used in plant stress detection studies and in currency analysis.
Endophytic colonization of olive roots by the biocontrol strain Pseudomonas fluorescens PICF7.

PubMed

Prieto, Pilar; Mercado-Blanco, Jesús

2008-05-01

Confocal microscopy combined with three-dimensional olive root tissue sectioning was used to provide evidence of the endophytic behaviour of Pseudomonas fluorescens PICF7, an effective biocontrol strain against Verticillium wilt of olive. Two derivatives of the green fluorescent protein (GFP), the enhanced green and the red fluorescent proteins, have been used to visualize simultaneously two differently fluorescently tagged populations of P. fluorescens PICF7 within olive root tissues at the single cell level. The time-course of colonization events of olive roots cv. Arbequina by strain PICF7 and the localization of tagged bacteria within olive root tissues are described. First, bacteria rapidly colonized root surfaces and were predominantly found in the differentiation zone. Thereafter, microscopy observations showed that PICF7-tagged populations eventually disappeared from the root surface, and increasingly colonized inner root tissues. Localized and limited endophytic colonization by the introduced bacteria was observed over time. Fluorescent-tagged bacteria were always visualized in the intercellular spaces of the cortex region, and no colonization of the root xylem vessels was detected at any time. To the best of our knowledge, this is the first time this approach has been used to demonstrate endophytism of a biocontrol Pseudomonas spp. strain in a woody host such as olive using a nongnotobiotic system.
Hydrothermal synthesis of zinc(II)-phosphonate coordination polymers with different dimensionality (0D, 2D, 3D) and dimensionality change in the solid phase (0D→3D) induced by temperature

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

Fernández-Zapico, Eva; Montejo-Bernardo, Jose; Fernández-González, Alfonso

2015-05-15

Three new zinc(II) coordination polymers, [Zn(HO{sub 3}PCH{sub 2}CH{sub 2}COO)(C{sub 12}H{sub 8}N{sub 2})(H{sub 2}O)] (1), [Zn{sub 3}(O{sub 3}PCH{sub 2}CH{sub 2}COO){sub 2}(C{sub 12}H{sub 8}N{sub 2})](H{sub 2}O){sub 3.40} (2) and [Zn{sub 5}(HO{sub 3}PCH{sub 2}CH{sub 2}COO){sub 2}(O{sub 3}PCH{sub 2}CH{sub 2}COO){sub 2}(C{sub 12}H{sub 8}N{sub 2}){sub 4}](H{sub 2}O){sub 0.32} (3), with different structural dimensionality (0D, 2D and 3D, respectively) have been prepared by hydrothermal synthesis, and their structures were determined by single-crystal X-ray diffraction. Compound 1 crystallizes in the monoclinic system (P2{sub 1}/c) forming discrete dimeric units bonded through H-bonds, while compounds 2 and 3 crystallize in the triclinic (P−1) and the monoclinic (C2/c) systems, respectively.more » Compound 3, showing three different coordination numbers (4, 5 and 6) for the zinc atoms, has also been obtained by thermal treatment of 1 (probed by high-temperature XRPD experiments). The crystalline features of these compounds, related to the coordination environments for the zinc atoms in each structure, provoke the increase of the relative fluorescence for 2 and 3, compared to the free phenanthroline. Thermal analysis (TG and DSC) and XPS studies have been also carried out for all compounds. - Graphical abstract: Three new coordination compounds of zinc with 2-carboxyethylphosphonic acid (H{sub 2}PPA) and phenanthroline have been obtained by hydrothermal synthesis. The crystalline structure depends on the different coordination environments of the zinc atoms (see two comparative Zn{sub 6}-moieties). The influence of the different coordination modes of H{sub 2}PPA with the central atom in all structures have been studied, being found new coordination modes for this ligand. Several compounds show a significant increase in relative fluorescence with respect to the free phenanthroline. - Highlights: • Compounds have been obtained modifying the reaction time and the rate of reagents. • Dimensionality and crystalline structure is a function of the zinc environments. • New coordination modes for 2-carboxyethylphosphonic acid are reported. • 3D-compound presents three different coordination environments for the zinc atoms. • Fluorescence properties are related to the structural dimensionality.« less
Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery.

PubMed

Stock, Kristin; Estrada, Marta F; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vítor E; Barbier, Michaël; Blom, Sami; Arundkar, Sharath C; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph

2016-07-01

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.
Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery

PubMed Central

Stock, Kristin; Estrada, Marta F.; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vítor E.; Barbier, Michaël; Blom, Sami; Arundkar, Sharath C.; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph

2016-01-01

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models. PMID:27364600
Four-dimensional data coupled to alternating weighted residue constraint quadrilinear decomposition model applied to environmental analysis: Determination of polycyclic aromatic hydrocarbons.

PubMed

Liu, Tingting; Zhang, Ling; Wang, Shutao; Cui, Yaoyao; Wang, Yutian; Liu, Lingfei; Yang, Zhe

2018-03-15

Qualitative and quantitative analysis of polycyclic aromatic hydrocarbons (PAHs) was carried out by three-dimensional fluorescence spectroscopy combining with Alternating Weighted Residue Constraint Quadrilinear Decomposition (AWRCQLD). The experimental subjects were acenaphthene (ANA) and naphthalene (NAP). Firstly, in order to solve the redundant information of the three-dimensional fluorescence spectral data, the wavelet transform was used to compress data in preprocessing. Then, the four-dimensional data was constructed by using the excitation-emission fluorescence spectra of different concentration PAHs. The sample data was obtained from three solvents that are methanol, ethanol and Ultra-pure water. The four-dimensional spectral data was analyzed by AWRCQLD, then the recovery rate of PAHs was obtained from the three solvents and compared respectively. On one hand, the results showed that PAHs can be measured more accurately by the high-order data, and the recovery rate was higher. On the other hand, the results presented that AWRCQLD can better reflect the superiority of four-dimensional algorithm than the second-order calibration and other third-order calibration algorithms. The recovery rate of ANA was 96.5%~103.3% and the root mean square error of prediction was 0.04μgL -1 . The recovery rate of NAP was 96.7%~115.7% and the root mean square error of prediction was 0.06μgL -1 . Copyright © 2017 Elsevier B.V. All rights reserved.
Three-dimensional image formation in fiber-optical second-harmonic-generation microscopy.

PubMed

Gu, Min; Fu, Ling

2006-02-06

Three-dimensional (3-D) image formation in fiber-optical second-harmonic-generation microscopy is revealed to be purely coherent and therefore can be described by a 3-D coherent transfer function (CTF) that exhibits the same spatial frequency passband as that of fiber-optical reflection-mode non-fluorescence microscopy. When the numerical aperture of the fiber is much larger than the angle of convergence of the illumination on the fiber aperture, the performance of fiber-optical second-harmonic-generation microscopy behaves as confocal second-harmonic-generation microscopy. The dependence of axial resolution on fiber coupling parameters shows an improvement of approximately 7%, compared with that in fiber-optical two-photon fluorescence microscopy.

Derivation of a closed form analytical expression for fluorescence recovery after photo bleaching in the case of continuous bleaching during read out

NASA Astrophysics Data System (ADS)

Endress, E.; Weigelt, S.; Reents, G.; Bayerl, T. M.

2005-01-01

Measurements of very slow diffusive processes in membranes, like the diffusion of integral membrane proteins, by fluorescence recovery after photo bleaching (FRAP) are hampered by bleaching of the probe during the read out of the fluorescence recovery. In the limit of long observation time (very slow diffusion as in the case of large membrane proteins), this bleaching may cause errors to the recovery function and thus provides error-prone diffusion coefficients. In this work we present a new approach to a two-dimensional closed form analytical solution of the reaction-diffusion equation, based on the addition of a dissipative term to the conventional diffusion equation. The calculation was done assuming (i) a Gaussian laser beam profile for bleaching the spot and (ii) that the fluorescence intensity profile emerging from the spot can be approximated by a two-dimensional Gaussian. The detection scheme derived from the analytical solution allows for diffusion measurements without the constraint of observation bleaching. Recovery curves of experimental FRAP data obtained under non-negligible read-out bleaching for native membranes (rabbit endoplasmic reticulum) on a planar solid support showed excellent agreement with the analytical solution and allowed the calculation of the lipid diffusion coefficient.
A one-dimensional free energy surface does not account for two-probe folding kinetics of protein alpha(3)D.

PubMed

Liu, Feng; Dumont, Charles; Zhu, Yongjin; DeGrado, William F; Gai, Feng; Gruebele, Martin

2009-02-14

We present fluorescence-detected measurements of the temperature-jump relaxation kinetics of the designed three-helix bundle protein alpha(3)D taken under solvent conditions identical to previous infrared-detected kinetics. The fluorescence-detected rate is similar to the IR-detected rate only at the lowest temperature where we could measure it (326 K). The fluorescence-detected rate decreases by a factor of 3 over the 326-344 K temperature range, whereas the IR-detected rate remains nearly constant over the same range. To investigate this probe dependence, we tested an extensive set of physically reasonable one-dimensional (1D) free energy surfaces by Langevin dynamics simulation. The simulations included coordinate- and temperature-dependent roughness, diffusion coefficients, and IR/fluorescence spectroscopic signatures. None of these can reproduce the IR and fluorescence data simultaneously, forcing us to the conclusion that a 1D free energy surface cannot accurately describe the folding of alpha(3)D. This supports the hypothesis that alpha(3)D has a multidimensional free energy surface conducive to downhill folding at 326 K, and that it is already an incipient downhill folder with probe-dependent kinetics near its melting point.
Quantitative fluorescence microscopy and image deconvolution.

PubMed

Swedlow, Jason R

2013-01-01

Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used to remove blurred signal from an image. There are two major types of deconvolution approaches, deblurring and restoration algorithms. Deblurring algorithms remove blur, but treat a series of optical sections as individual two-dimensional entities, and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed. Copyright © 1998 Elsevier Inc. All rights reserved.
Contextual analysis of immunological response through whole-organ fluorescent imaging.

PubMed

Woodruff, Matthew C; Herndon, Caroline N; Heesters, B A; Carroll, Michael C

2013-09-01

As fluorescent microscopy has developed, significant insights have been gained into the establishment of immune response within secondary lymphoid organs, particularly in draining lymph nodes. While established techniques such as confocal imaging and intravital multi-photon microscopy have proven invaluable, they provide limited insight into the architectural and structural context in which these responses occur. To interrogate the role of the lymph node environment in immune response effectively, a new set of imaging tools taking into account broader architectural context must be implemented into emerging immunological questions. Using two different methods of whole-organ imaging, optical clearing and three-dimensional reconstruction of serially sectioned lymph nodes, fluorescent representations of whole lymph nodes can be acquired at cellular resolution. Using freely available post-processing tools, images of unlimited size and depth can be assembled into cohesive, contextual snapshots of immunological response. Through the implementation of robust iterative analysis techniques, these highly complex three-dimensional images can be objectified into sortable object data sets. These data can then be used to interrogate complex questions at the cellular level within the broader context of lymph node biology. By combining existing imaging technology with complex methods of sample preparation and capture, we have developed efficient systems for contextualizing immunological phenomena within lymphatic architecture. In combination with robust approaches to image analysis, these advances provide a path to integrating scientific understanding of basic lymphatic biology into the complex nature of immunological response.
[Applications of three-dimensional fluorescence spectrum of dissolved organic matter to identification of red tide algae].

PubMed

Lü, Gui-Cai; Zhao, Wei-Hong; Wang, Jiang-Tao

2011-01-01

The identification techniques for 10 species of red tide algae often found in the coastal areas of China were developed by combining the three-dimensional fluorescence spectra of fluorescence dissolved organic matter (FDOM) from the cultured red tide algae with principal component analysis. Based on the results of principal component analysis, the first principal component loading spectrum of three-dimensional fluorescence spectrum was chosen as the identification characteristic spectrum for red tide algae, and the phytoplankton fluorescence characteristic spectrum band was established. Then the 10 algae species were tested using Bayesian discriminant analysis with a correct identification rate of more than 92% for Pyrrophyta on the level of species, and that of more than 75% for Bacillariophyta on the level of genus in which the correct identification rates were more than 90% for the phaeodactylum and chaetoceros. The results showed that the identification techniques for 10 species of red tide algae based on the three-dimensional fluorescence spectra of FDOM from the cultured red tide algae and principal component analysis could work well.
Extraction and characterization of bound extracellular polymeric substances from cultured pure cyanobacterium (Microcystis wesenbergii).

PubMed

Liu, Lizhen; Qin, Boqiang; Zhang, Yunlin; Zhu, Guangwei; Gao, Guang; Huang, Qi; Yao, Xin

2014-08-01

Preliminary characterization of bound extracellular polymeric substances (bEPS) of cyanobacteria is crucial to obtain a better understanding of the formation mechanism of cyanobacterial bloom. However, the characterization of bEPS can be affected by extraction methods. Five sets (including the control) of bEPS from Microcystis extracted by different methods were characterized using three-dimensional excitation and emission matrix (3DEEM) fluorescence spectroscopy combined chemical spectrophotometry; and the characterization results of bEPS samples were further compared. The agents used for extraction were NaOH, pure water and phosphate buffered saline (PBS) containing cationic exchange resins, and hot water. Extraction methods affected the fluorescence signals and intensities in the bEPS. Five fluorescence peaks were observed in the excitation and emission matrix fluorescence spectra of bEPS samples. Two peaks (peaks T₁ and T₂) present in all extractions were identified as protein-like fluorophores, two (peaks A and C) as humic-like fluorophores, and one (peak E) as a fulvic-like substance. Among these substances, the humic-like and fulvic-like fluorescences were only seen in the bEPS extracted with hot water. Also, NaOH solution extraction could result in strong fluorescence intensities compared to the other extraction methods. It was suggested that NaOH at pH10.0 was the most appropriate method to extract bEPS from Microcystis. In addition, dialysis could affect the yields and characteristics of extracted bEPS during the determination process. These results will help us to explore the issues of cyanobacterial blooms. Copyright © 2014. Published by Elsevier B.V.
Snapshot Hyperspectral Volumetric Microscopy

NASA Astrophysics Data System (ADS)

Wu, Jiamin; Xiong, Bo; Lin, Xing; He, Jijun; Suo, Jinli; Dai, Qionghai

2016-04-01

The comprehensive analysis of biological specimens brings about the demand for capturing the spatial, temporal and spectral dimensions of visual information together. However, such high-dimensional video acquisition faces major challenges in developing large data throughput and effective multiplexing techniques. Here, we report the snapshot hyperspectral volumetric microscopy that computationally reconstructs hyperspectral profiles for high-resolution volumes of ~1000 μm × 1000 μm × 500 μm at video rate by a novel four-dimensional (4D) deconvolution algorithm. We validated the proposed approach with both numerical simulations for quantitative evaluation and various real experimental results on the prototype system. Different applications such as biological component analysis in bright field and spectral unmixing of multiple fluorescence are demonstrated. The experiments on moving fluorescent beads and GFP labelled drosophila larvae indicate the great potential of our method for observing multiple fluorescent markers in dynamic specimens.
Construction and optical properties of infinite Cd and finite Cu molecules stairs

NASA Astrophysics Data System (ADS)

Zhao, Qiang; Mao, Wutao; Shen, Zhi; Wang, Qinghong; Zhou, Qian

2017-02-01

Two coordination complexes, namely [(hpdq)(pta)Cd]n (1) and [(pptp)(pta)Cu2Cl] (2) have been synthesized by solvothermal method based on two polypyridyl ligands, 2,3,6,7,10,11-hexakis- (2-pyridyl)dipyrazino[2,3-f:2‧,3‧-h]quinoxaline) (hpdq), 4‧-(4- (3H-pyrrol-3-yl)phenyl)- 2,2‧:6‧,2″- terpyridine (pptp) and auxiliary ligand p-phthalic acid (pta), respectively. Single crystal x-ray diffraction analyses reveal that complexes 1 and 2 assembled based on distinct asymmetric unit comprising one and two respective polypyridyl ligands but one Cd(II) and two Cu(I)ions, respectively. Among them, The asymmetric units in 1 was extended to one dimensional chain via the link of auxiliary ligand pta, just like infinite layers of stairs that connected by cadmium ions as the node. While that in 2 to Zero dimensional tetranuclear structure via the link of auxiliary ligand pta, just like finite four layers of stairs that Copper ion as the node connection. Furthermore, solid fluorescence spectra properties of two complexes were also investigated, and the result shows the fluorescence intensity of complex 1 is stronger than that of the hpdq ligand, but the fluorescence intensity of complex 2 is weaker than that of the pptp ligand. CCDC number of 1and 2 are 1483301 and 1483302.
Kinetics of intercalation of fluorescent probes in magnesium–aluminium layered double hydroxide within a multiscale reaction–diffusion framework

PubMed Central

Saliba, Daniel

2016-01-01

We report the synthesis of magnesium–aluminium layered double hydroxide (LDH) using a reaction–diffusion framework (RDF) that exploits the multiscale coupling of molecular diffusion with chemical reactions, nucleation and growth of crystals. In an RDF, the hydroxide anions are allowed to diffuse into an organic gel matrix containing the salt mixture needed for the precipitation of the LDH. The chemical structure and composition of the synthesized magnesium–aluminium LDHs are determined using powder X-ray diffraction (PXRD), thermo-gravimetric analysis, differential scanning calorimetry, solid-state nuclear magnetic resonance (SSNMR), Fourier transform infrared and energy dispersive X-ray spectroscopy. This novel technique also allows the investigation of the mechanism of intercalation of some fluorescent probes, such as the neutral three-dimensional rhodamine B (RhB) and the negatively charged two-dimensional 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), using in situ steady-state fluorescence spectroscopy. The incorporation of these organic dyes inside the interlayer region of the LDH is confirmed via fluorescence microscopy, solid-state lifetime, SSNMR and PXRD. The activation energies of intercalation of the corresponding molecules (RhB and HPTS) are computed and exhibit dependence on the geometry of the involved probe (two or three dimensions), the charge of the fluorescent molecule (anionic, cationic or neutral) and the cationic ratio of the corresponding LDH. This article is part of the themed issue ‘Multiscale modelling at the physics–chemistry–biology interface’. PMID:27698034
Kinetics of intercalation of fluorescent probes in magnesium-aluminium layered double hydroxide within a multiscale reaction-diffusion framework

NASA Astrophysics Data System (ADS)

Saliba, Daniel; Al-Ghoul, Mazen

2016-11-01

We report the synthesis of magnesium-aluminium layered double hydroxide (LDH) using a reaction-diffusion framework (RDF) that exploits the multiscale coupling of molecular diffusion with chemical reactions, nucleation and growth of crystals. In an RDF, the hydroxide anions are allowed to diffuse into an organic gel matrix containing the salt mixture needed for the precipitation of the LDH. The chemical structure and composition of the synthesized magnesium-aluminium LDHs are determined using powder X-ray diffraction (PXRD), thermo-gravimetric analysis, differential scanning calorimetry, solid-state nuclear magnetic resonance (SSNMR), Fourier transform infrared and energy dispersive X-ray spectroscopy. This novel technique also allows the investigation of the mechanism of intercalation of some fluorescent probes, such as the neutral three-dimensional rhodamine B (RhB) and the negatively charged two-dimensional 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), using in situ steady-state fluorescence spectroscopy. The incorporation of these organic dyes inside the interlayer region of the LDH is confirmed via fluorescence microscopy, solid-state lifetime, SSNMR and PXRD. The activation energies of intercalation of the corresponding molecules (RhB and HPTS) are computed and exhibit dependence on the geometry of the involved probe (two or three dimensions), the charge of the fluorescent molecule (anionic, cationic or neutral) and the cationic ratio of the corresponding LDH. This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'.
[Study of Determination of Oil Mixture Components Content Based on Quasi-Monte Carlo Method].

PubMed

Wang, Yu-tian; Xu, Jing; Liu, Xiao-fei; Chen, Meng-han; Wang, Shi-tao

2015-05-01

Gasoline, kerosene, diesel is processed by crude oil with different distillation range. The boiling range of gasoline is 35 ~205 °C. The boiling range of kerosene is 140~250 °C. And the boiling range of diesel is 180~370 °C. At the same time, the carbon chain length of differentmineral oil is different. The carbon chain-length of gasoline is within the scope of C7 to C11. The carbon chain length of kerosene is within the scope of C12 to C15. And the carbon chain length of diesel is within the scope of C15 to C18. The recognition and quantitative measurement of three kinds of mineral oil is based on different fluorescence spectrum formed in their different carbon number distribution characteristics. Mineral oil pollution occurs frequently, so monitoring mineral oil content in the ocean is very important. A new method of components content determination of spectra overlapping mineral oil mixture is proposed, with calculation of characteristic peak power integrationof three-dimensional fluorescence spectrum by using Quasi-Monte Carlo Method, combined with optimal algorithm solving optimum number of characteristic peak and range of integral region, solving nonlinear equations by using BFGS(a rank to two update method named after its inventor surname first letter, Boyden, Fletcher, Goldfarb and Shanno) method. Peak power accumulation of determined points in selected area is sensitive to small changes of fluorescence spectral line, so the measurement of small changes of component content is sensitive. At the same time, compared with the single point measurement, measurement sensitivity is improved by the decrease influence of random error due to the selection of points. Three-dimensional fluorescence spectra and fluorescence contour spectra of single mineral oil and the mixture are measured by taking kerosene, diesel and gasoline as research objects, with a single mineral oil regarded whole, not considered each mineral oil components. Six characteristic peaks are selected for characteristic peak power integration to determine components content of mineral oil mixture of gasoline, kerosene and diesel by optimal algorithm. Compared with single point measurement of peak method and mean method, measurement sensitivity is improved about 50 times. The implementation of high precision measurement of mixture components content of gasoline, kerosene and diesel provides a practical algorithm for components content direct determination of spectra overlapping mixture without chemical separation.
FluorWPS: A Monte Carlo ray-tracing model to compute sun-induced chlorophyll fluorescence of three-dimensional canopy

USDA-ARS?s Scientific Manuscript database

A model to simulate radiative transfer (RT) of sun-induced chlorophyll fluorescence (SIF) of three-dimensional (3-D) canopy, FluorWPS, was proposed and evaluated. The inclusion of fluorescence excitation was implemented with the ‘weight reduction’ and ‘photon spread’ concepts based on Monte Carlo ra...
Simultaneous Visualization of Hydrogen Peroxide and Water Concentrations Using Photofragmentation Laser-Induced Fluorescence.

PubMed

Larsson, Kajsa; Aldén, Marcus; Bood, Joakim

2017-09-01

A concept based on photofragmentation laser-induced fluorescence (PFLIF) is for the first time demonstrated for simultaneous detection of hydrogen peroxide (H 2 O 2 ) and water (H 2 O) vapor in various mixtures containing the two constituents in a bath of argon gas. A photolysis laser pulse at 248 nm dissociates H 2 O 2 into OH fragments, whereupon a probe pulse, delayed 100 ns and tuned to an absorption line in the A 2 Σ + (v = 1) ← X 2 Π(v = 0) band of OH near 282 nm, induces fluorescence. The total OH fluorescence reflects the H 2 O 2 concentration, while its spectral shape is utilized to determine the H 2 O concentration via a model predicting the ratio between the fluorescence intensities of the A 2 Σ + (v = 1) → X 2 Π(v = 1) and the A 2 Σ + (v = 0) → X 2 Π(v = 0) bands. The H 2 O detection scheme requires that the bath gas has a collisional cross-section with OH(A) that is significantly lower than that of H 2 O, which is the case for argon. Spectrally dispersed OH fluorescence spectra were recorded for five different H 2 O 2 /H 2 O/Ar mixtures; the H 2 O 2 concentration in the range of 30-500 ppm and the H 2 O concentration in the range of 0-3%. Fluorescence intensity ratios predicted by the model for these mixtures agree very well with corresponding experimental data, which thus validates the model. The concept was also demonstrated for two-dimensional imaging, using two intensified charge-coupled device (CCD) cameras for signal detection. Water content was here sensed through the different temporal characteristics of the two fluorescence bands by triggering the two cameras so that one captures the total OH fluorescence while the other one captures only the early part, which mainly stems from A 2 Σ + (v = 1) → X 2 Π(v = 1) fluorescence. Hence, the H 2 O 2 concentration is reflected by the image of the camera recording the total OH fluorescence, whereas H 2 O concentration is extracted from the ratio between the two camera images. Quantification of the concentrations was carried out based on calibration measurements performed in known mixtures of H 2 O 2 (30-500 ppm) and H 2 O (0-3%) in bulk argon. The detection limits for single-shot imaging are estimated to be 20 ppm for H 2 O 2 and 0.05% for H 2 O. The authors believe that the concept provides a valuable asset in, for example, pharmaceutical or aseptic food packaging applications, where H 2 O 2 /H 2 O vapor is routinely used for sterilization.
Holographic fluorescence mapping using space-division matching method

NASA Astrophysics Data System (ADS)

Abe, Ryosuke; Hayasaki, Yoshio

2017-10-01

Three-dimensional mapping of fluorescence light sources was performed by using self-interference digital holography. The positions of the sources were quantitatively determined by using Gaussian fitting of the axial and lateral intensity distributions obtained from diffraction calculations through position calibration from the observation space to the sample space. A space-division matching method was developed to perform the mapping of many fluorescence light sources, in this experiment, 500 nm fluorescent nanoparticles fixed in gelatin. A fluorescence digital holographic microscope having a 60 × objective lens with a numerical aperture of 1.25 detected 13 fluorescence light sources in a measurable region with a radius of ∼ 20 μm and a height of ∼ 5 μm. It was found that the measurable region had a conical shape resulting from the overlap between two beams.
Two zinc(II) coordination complexes based on an asymmetric multidentate ligand: syntheses, structures, selective fluorescence sensing of iron(III) ions and thermal analyses.

PubMed

Liu, Yaru; Liu, Lan; Zhang, Xiao; Liang, Guorui; Gong, Xuebing

2018-01-01

The rational selection of ligands is vitally important in the construction of coordination complexes. Two novel Zn II complexes, namely bis(acetato-κO)bis[1-(1H-benzotriazol-1-ylmethyl)-2-propyl-1H-imidazole-κN 3 ]zinc(II) monohydrate, [Zn(C 13 H 15 N 5 ) 2 (C 2 H 3 O 2 ) 2 ]·H 2 O, (1), and bis(azido-κN 1 )bis[1-(1H-benzotriazol-1-ylmethyl)-2-propyl-1H-imidazole-κN 3 ]zinc(II), [Zn(C 13 H 15 N 5 ) 2 (N 3 ) 2 ], (2), constructed from the asymmetric multidentate imidazole ligand, have been synthesized under mild conditions and characterized by elemental analyses, IR spectroscopy and single-crystal X-ray diffraction analysis. Both complexes exhibit a three-dimensional supramolecular network directed by different intermolecular interactions between discrete mononuclear units. The complexes were also investigated by fluorescence and thermal analyses. The experimental results show that (1) is a promising fluorescence sensor for detecting Fe 3+ ions and (2) is effective as an accelerator of the thermal decomposition of ammonium perchlorate.
Three-Dimensional, Live-Cell Imaging of Chromatin Dynamics in Plant Nuclei Using Chromatin Tagging Systems.

PubMed

Hirakawa, Takeshi; Matsunaga, Sachihiro

2016-01-01

In plants, chromatin dynamics spatiotemporally change in response to various environmental stimuli. However, little is known about chromatin dynamics in the nuclei of plants. Here, we introduce a three-dimensional, live-cell imaging method that can monitor chromatin dynamics in nuclei via a chromatin tagging system that can visualize specific genomic loci in living plant cells. The chromatin tagging system is based on a bacterial operator/repressor system in which the repressor is fused to fluorescent proteins. A recent refinement of promoters for the system solved the problem of gene silencing and abnormal pairing frequencies between operators. Using this system, we can detect the spatiotemporal dynamics of two homologous loci as two fluorescent signals within a nucleus and monitor the distance between homologous loci. These live-cell imaging methods will provide new insights into genome organization, development processes, and subnuclear responses to environmental stimuli in plants.
Rich stochastic dynamics of co-doped Er:Yb fluorescence upconversion nanoparticles in the presence of thermal, non-conservative, harmonic and optical forces

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

PubMed

Seitzman, J M; Kychakoff, G; Hanson, R K

1985-09-01

A single-pulse, laser-induced-fluorescence diagnostic for the measurement of two-dimensional temperature fields in combustion flows is described. The method uses sheet illumination from a tunable laser to excite planar laserinduced fluorescence in a stable tracer molecule, seeded at constant mole fraction into the flow field. The temporal resolution of this technique is determined by the laser pulse length. Experimental results are presented for a rodstabilized, premixed methane-air flame, using the Q(1) (22) line of the nitric oxide A(2) Sigma(+) (v = 0) ? X(2)II((1/2))(v = 0) transition (lambda approximately 225.6 nm).
Dynamic characterization of hydrophobic and hydrophilic solutes in oleic-acid enhanced transdermal delivery using two-photon fluorescence microscopy

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

Tseng, Te-Yu; Yang, Chiu-Sheng; Chen, Yang-Fang

In this letter, we propose an efficient methodology of investigating dynamic properties of sulforhodamine B and rhodamine B hexyl ester molecules transporting across ex-vivo human stratum corneum with and without oleic acid enhancement. Three-dimensional, time-lapse fluorescence images of the stratum corneum can be obtained using two-photon fluorescence microscopy. Furthermore, temporal quantifications of transport enhancements in diffusion parameters can be achieved with the use of Fick's second law. Dynamic characterization of solutes transporting across the stratum corneum is an effective method for understanding transient phenomena in transdermal delivery of probe molecules, leading to improved delivery strategies of molecular species for therapeuticmore » purposes.« less
Direct reconstruction in CT-analogous pharmacokinetic diffuse fluorescence tomography: two-dimensional simulative and experimental validations

NASA Astrophysics Data System (ADS)

Wang, Xin; Zhang, Yanqi; Zhang, Limin; Li, Jiao; Zhou, Zhongxing; Zhao, Huijuan; Gao, Feng

2016-04-01

We present a generalized strategy for direct reconstruction in pharmacokinetic diffuse fluorescence tomography (DFT) with CT-analogous scanning mode, which can accomplish one-step reconstruction of the indocyanine-green pharmacokinetic-rate images within in vivo small animals by incorporating the compartmental kinetic model into an adaptive extended Kalman filtering scheme and using an instantaneous sampling dataset. This scheme, compared with the established indirect and direct methods, eliminates the interim error of the DFT inversion and relaxes the expensive requirement of the instrument for obtaining highly time-resolved date-sets of complete 360 deg projections. The scheme is validated by two-dimensional simulations for the two-compartment model and pilot phantom experiments for the one-compartment model, suggesting that the proposed method can estimate the compartmental concentrations and the pharmacokinetic-rates simultaneously with a fair quantitative and localization accuracy, and is well suitable for cost-effective and dense-sampling instrumentation based on the highly-sensitive photon counting technique.

Application of Tryptophan Fluorescence Bandwidth-Maximum Plot in Analysis of Monoclonal Antibody Structure.

PubMed

Huang, Cheng-Yen; Hsieh, Ming-Ching; Zhou, Qinwei

2017-04-01

Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.
Dual-modal three-dimensional imaging of single cells with isometric high resolution using an optical projection tomography microscope

NASA Astrophysics Data System (ADS)

Miao, Qin; Rahn, J. Richard; Tourovskaia, Anna; Meyer, Michael G.; Neumann, Thomas; Nelson, Alan C.; Seibel, Eric J.

2009-11-01

The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 μm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.
A detection method of vegetable oils in edible blended oil based on three-dimensional fluorescence spectroscopy technique.

PubMed

Xu, Jing; Liu, Xiao-Fei; Wang, Yu-Tian

2016-12-01

Edible blended vegetable oils are made from two or more refined oils. Blended oils can provide a wider range of essential fatty acids than single vegetable oils, which helps support good nutrition. Nutritional components in blended oils are related to the type and content of vegetable oils used, and a new, more accurate, method is proposed to identify and quantify the vegetable oils present using cluster analysis and a Quasi-Monte Carlo integral. Three-dimensional fluorescence spectra were obtained at 250-400nm (excitation) and 260-750nm (emission). Mixtures of sunflower, soybean and peanut oils were used as typical examples to validate the effectiveness of the method. Copyright © 2016 Elsevier Ltd. All rights reserved.
Single-photon and two-photon excited fluorescence behavior of a novel fluorene-based compound

NASA Astrophysics Data System (ADS)

Ma, Wenbo; Wu, Yiquan; Gu, Donghong; Gan, Fuxi

2005-09-01

A D-π-D type compound, 2,7-bis(4-methoxystyryl)-9,9-bis(2-ethylhexyl)-9H-fluorene (abbreviated as MO-Flu-MO), where electron-donor D is methoxy group andπis fluorene unit, has been synthesized. The molecular structures of the compound were characterized by elemental analyses, EI-MS and FT-IR spectra. UV-Vis spectra in the region 230--1000 nm and single-photon excited fluorescence in tetrahydrofuran (THF) of the compound were measured. It is found that the new compound exhibits strong two-photon excited fluorescence in the region 380--500 nm and moderate two-photon absorption (TPA) value in the femtoseconds regime (TPA cross-section as high as 55×10-50 cm4 s photon-1 with 13fs laser pulses). The results demonstrate that the compound is a promising candidate for two-photon three-dimensional (3D) optical data storage.
Cascaded two-photon nonlinearity in a one-dimensional waveguide with multiple two-level emitters

PubMed Central

Roy, Dibyendu

2013-01-01

We propose and theoretically investigate a model to realize cascaded optical nonlinearity with few atoms and photons in one-dimension (1D). The optical nonlinearity in our system is mediated by resonant interactions of photons with two-level emitters, such as atoms or quantum dots in a 1D photonic waveguide. Multi-photon transmission in the waveguide is nonreciprocal when the emitters have different transition energies. Our theory provides a clear physical understanding of the origin of nonreciprocity in the presence of cascaded nonlinearity. We show how various two-photon nonlinear effects including spatial attraction and repulsion between photons, background fluorescence can be tuned by changing the number of emitters and the coupling between emitters (controlled by the separation). PMID:23948782
Polarized two-photon photoselection in EGFP: Theory and experiment

NASA Astrophysics Data System (ADS)

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

2018-04-01

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

PubMed

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

2018-04-07

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

NASA Astrophysics Data System (ADS)

Panchadhyayee, Pradipta; Dutta, Bibhas Kumar; Das, Nityananda; Mahapatra, Prasanta Kumar

2018-02-01

A scheme is proposed to realize three-dimensional (3D) atom localization in a driven two-level atomic system via resonance fluorescence. The field arrangement for the atom localization involves the application of three mutually orthogonal standing-wave fields and an additional traveling-wave coupling field. We have shown the efficacy of such field arrangement in tuning the spatially modulated resonance in all directions. Under different parametric conditions, the 3D localization patterns originate with various shapes such as sphere, sheets, disk, bowling pin, snake flute, flower vase. High-precision localization is achieved when the radiation field detuning equals twice the combined Rabi frequencies of the standing-wave fields. Application of a traveling-wave field of suitable amplitude at optimum radiation field detuning under symmetric standing-wave configuration leads to 100% detection probability even in sub-wavelength domain. Asymmetric field configuration is also taken into consideration to exhibit atom localization with appreciable precision compared to that of the symmetric case. The momentum distribution of the localized atoms is found to follow the Heisenberg uncertainty principle under the validity of Raman-Nath approximation. The proposed field configuration is suitable for application in the study of atom localization in an optical lattice arrangement.
Temperature-dependent conformational variation of chromophoric dissolved organic matter and its consequent interaction with phenanthrene.

PubMed

Chen, Wei; Liu, Xiao-Yang; Yu, Han-Qing

2017-03-01

Temperature variation caused by climate change, seasonal variation and geographic locations affects the physicochemical compositions of chromophoric dissolved organic matter (CDOM), resulting in difference in the fates of CDOM-related environmental pollutants. Exploration into the thermal induced structural transition of CDOM can help to better understand their environmental impacts, but information on this aspect is still lacking. Through integrating fluorescence excitation-emission matrix coupled parallel factor analysis with synchronous fluorescence two-dimensional correlation spectroscopy, this study provides an in-depth insight into the temperature-dependent conformational transitions of CDOM and their impact on its hydrophobic interaction with persistent organic pollutants (with phenanthrene as an example) in water. The fluorescence components in CDOM change linearly to water temperature with different extents and different temperature regions. The thermal induced transition priority in CDOM is protein-like component → fulvic-like component → humic-like component. Furthermore, the impact of thermal-induced conformational transition of CDOM on its hydrophobic interaction with phenanthrene is observed and explored. The fluorescence-based analytic results reveal that the conjugation degree of the aromatic groups in the fulvic- and humic-like substances, and the unfolding of the secondary structure in the protein-like substances with aromatic structure, contribute to the conformation variation. This integrated approach jointly enhances the characterization of temperature-dependent conformational variation of CDOM, and provides a promising way to elucidate the environmental behaviours of CDOM. Copyright © 2017 Elsevier Ltd. All rights reserved.
Barriers to front propagation in laminar, three-dimensional fluid flows

NASA Astrophysics Data System (ADS)

Doan, Minh; Simons, J. J.; Lilienthal, Katherine; Solomon, Tom; Mitchell, Kevin A.

2018-03-01

We present experiments on one-way barriers that block reaction fronts in a fully three-dimensional (3D) fluid flow. Fluorescent Belousov-Zhabotinsky reaction fronts are imaged with laser-scanning in a laminar, overlapping vortex flow. The barriers are analyzed with a 3D extension to burning invariant manifold (BIM) theory that was previously applied to two-dimensional advection-reaction-diffusion processes. We discover tube and sheet barriers that guide the front evolution. The experimentally determined barriers are explained by BIMs calculated from a model of the flow.
Diffusion Tensor Analysis by Two-Dimensional Pair Correlation of Fluorescence Fluctuations in Cells.

PubMed

Di Rienzo, Carmine; Cardarelli, Francesco; Di Luca, Mariagrazia; Beltram, Fabio; Gratton, Enrico

2016-08-23

In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF yields an accurate description of the preferential diffusive routes. Furthermore, we combine this analysis with the image-derived mean-square displacement approach and gain information on the average nanoscopic molecular displacements in different directions. Through these quantities, we build a fluorescence-fluctuation-based diffusion tensor that contains information on speed and directionality of the local dynamical processes. Contrary to classical fluorescence correlation spectroscopy and related methods, this combined approach can distinguish between isotropic and anisotropic local diffusion. We argue that the measurement of this iMSD tensor will contribute to advance our understanding of the role played by the intracellular environment in the regulation of molecular diffusion at the nanoscale. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner

PubMed Central

Liu, Jonathan T. C.; Mandella, Michael J.; Ra, Hyejun; Wong, Larry K.; Solgaard, Olav; Kino, Gordon S.; Piyawattanametha, Wibool; Contag, Christopher H.; Wang, Thomas D.

2007-01-01

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5–7 μm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 × 100 μm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy. PMID:17215937
Nonuniform update for sparse target recovery in fluorescence molecular tomography accelerated by ordered subsets.

PubMed

Zhu, Dianwen; Li, Changqing

2014-12-01

Fluorescence molecular tomography (FMT) is a promising imaging modality and has been actively studied in the past two decades since it can locate the specific tumor position three-dimensionally in small animals. However, it remains a challenging task to obtain fast, robust and accurate reconstruction of fluorescent probe distribution in small animals due to the large computational burden, the noisy measurement and the ill-posed nature of the inverse problem. In this paper we propose a nonuniform preconditioning method in combination with L (1) regularization and ordered subsets technique (NUMOS) to take care of the different updating needs at different pixels, to enhance sparsity and suppress noise, and to further boost convergence of approximate solutions for fluorescence molecular tomography. Using both simulated data and phantom experiment, we found that the proposed nonuniform updating method outperforms its popular uniform counterpart by obtaining a more localized, less noisy, more accurate image. The computational cost was greatly reduced as well. The ordered subset (OS) technique provided additional 5 times and 3 times speed enhancements for simulation and phantom experiments, respectively, without degrading image qualities. When compared with the popular L (1) algorithms such as iterative soft-thresholding algorithm (ISTA) and Fast iterative soft-thresholding algorithm (FISTA) algorithms, NUMOS also outperforms them by obtaining a better image in much shorter period of time.
A method for three-dimensional quantitative observation of the microstructure of biological samples

NASA Astrophysics Data System (ADS)

Wang, Pengfei; Chen, Dieyan; Ma, Wanyun; Wu, Hongxin; Ji, Liang; Sun, Jialin; Lv, Danyu; Zhang, Lu; Li, Ying; Tian, Ning; Zheng, Jinggao; Zhao, Fengying

2009-07-01

Contemporary biology has developed into the era of cell biology and molecular biology, and people try to study the mechanism of all kinds of biological phenomena at the microcosmic level now. Accurate description of the microstructure of biological samples is exigent need from many biomedical experiments. This paper introduces a method for 3-dimensional quantitative observation on the microstructure of vital biological samples based on two photon laser scanning microscopy (TPLSM). TPLSM is a novel kind of fluorescence microscopy, which has excellence in its low optical damage, high resolution, deep penetration depth and suitability for 3-dimensional (3D) imaging. Fluorescent stained samples were observed by TPLSM, and afterward the original shapes of them were obtained through 3D image reconstruction. The spatial distribution of all objects in samples as well as their volumes could be derived by image segmentation and mathematic calculation. Thus the 3-dimensionally and quantitatively depicted microstructure of the samples was finally derived. We applied this method to quantitative analysis of the spatial distribution of chromosomes in meiotic mouse oocytes at metaphase, and wonderful results came out last.
Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection

PubMed Central

Dickerson, Jane A.; Ramsay, Lauren M.; Dada, Oluwatosin O.; Cermak, Nathan

2011-01-01

Capillary isoelectric focusing and capillary zone electrophoresis are coupled with laser-induced fluorescence detection to create an ultrasensitive two-dimensional separation method for proteins. In this method, two capillaries are joined through a buffer filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second dimension separation. A fraction was transferred to the second dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125. PMID:20603830
Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

PubMed

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

2017-06-01

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.
Insights into the Structural Changes Occurring upon Photoconversion in the Orange Carotenoid Protein from Broadband Two-Dimensional Electronic Spectroscopy

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

De Re, Eleonora; Schlau-Cohen, Gabriela S.; Leverenz, Ryan L.

Carotenoids play an essential role in photoprotection, interacting with other pigments to safely dissipate excess absorbed energy as heat. In cyanobacteria, the short time scale photoprotective mechanisms involve the photoactive orange carotenoid protein (OCP), which binds a single carbonyl carotenoid. Blue-green light induces the photoswitching of OCP from its ground state form (OCPO) to a metastable photoproduct (OCPR). OCPR can bind to the phycobilisome antenna and induce fluorescence quenching. The photoswitching is accompanied by structural and functional changes at the level of the protein and of the bound carotenoid. In this study, we use broadband two-dimensional electronic spectroscopy to lookmore » at the differences in excited state dynamics of the carotenoid in the two forms of OCP. Our results provide insight into the origin of the pronounced vibrational lineshape and oscillatory dynamics observed in linear absorption and 2D electronic spectroscopy of OCPO and the large inhomogeneous broadening in OCPR, with consequences for the chemical function of the two forms.« less
Three Dimensional Imaging of Cold Atoms in a Magneto Optical Trap with a Light Field Microscope

DTIC Science & Technology

2017-09-14

dimensional (3D) volume of the atoms is reconstructed using a modeled point spread function (PSF), taking into consideration the low magnification (1.25...axis fluorescence image. Optical axis separation between two atom clouds is measured to a 100µm accuracy in a 3mm deep volume , with a 16µm in-focus...79 vi Page 4.5 Phase Term Effects on the 3D Volume
Human thyroid specimen imaging by fluorescent x-ray computed tomography with synchrotron radiation

NASA Astrophysics Data System (ADS)

Takeda, Tohoru; Yu, Quanwen; Yashiro, Toru; Yuasa, Tetsuya; Hasegawa, Yasuo; Itai, Yuji; Akatsuka, Takao

1999-09-01

Fluorescent x-ray computed tomography (FXCT) is being developed to detect non-radioactive contrast materials in living specimens. The FXCT system consists of a silicon (111) channel cut monochromator, an x-ray slit and a collimator for fluorescent x ray detection, a scanning table for the target organ and an x-ray detector for fluorescent x-ray and transmission x-ray. To reduce Compton scattering overlapped on the fluorescent K(alpha) line, incident monochromatic x-ray was set at 37 keV. The FXCT clearly imaged a human thyroid gland and iodine content was estimated quantitatively. In a case of hyperthyroidism, the two-dimensional distribution of iodine content was not uniform, and thyroid cancer had a small amount of iodine. FXCT can be used to detect iodine within thyroid gland quantitatively and to delineate its distribution.
Assembly and property research on seven 0D–3D complexes derived from imidazole dicarboxylate and 1,2-bi(pyridin-4-yl)ethene

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

Mu, Bao; Li, Qian; Lv, Lei

2015-03-15

The hydrothermal reaction of transition metals, 1H-imidazole-4,5-dicarboxylic acid (H{sub 3}ImDC) and 1,2-bi(pyridin-4-yl)ethene (bpe) affords a series of new complexes, namely, [Mn(HImDC)(bpe)(H{sub 2}O)] (1), [M(H{sub 2}ImDC){sub 2}(H{sub 2}O){sub 2}]·(bpe) (M=Fe(2), Co(3), Zn(4), Cd(6)), [Zn{sub 3}(ImDC){sub 2}(bpe)(H{sub 2}O)]·3H{sub 2}O (5) and [Cd(H{sub 2}ImDC)(bpe)] (7), which are characterized by elemental analyses, IR, TG, XRPD and single crystal X-ray diffraction. Complex 1 exhibits a one dimensional (1D) zigzag chain with two types of irregular rings, and the 1D chains are linked to form a three dimensional (3D) supramolecular framework by the hydrogen bonding interactions (O–H∙∙∙O and O–H∙∙∙N). Complexes 2–4 and 6 are isomorphous, andmore » they display the mononuclear structures. In these complexes, the O–H∙∙∙O and O–H∙∙∙N hydrogen bonds play an important role in sustaining the whole 3D supramolecular frameworks. Complex 5 shows a (3,3)-connected 3D framework with (10{sup 3}) topology, and the lattice water molecules as guest molecules exist in the 3D framework. Complex 7 is a wave-like two dimensional (2D) structure, in which the adjacent 1D chains point at the opposite directions. Moreover, the fluorescent properties of complexes 1–7 and the magnetic property of 1 have been investigated. The water vapor adsorption for complex 5 has been researched at 298 K. - Graphical abstract: Seven new complexes based on different structural characteristics have been hydrothermally synthesized by the mixed ligands. The fluorescent properties, the magnetic property and the water vapor adsorption have been investigated. - Highlights: • The semi-rigid ligand with C=C bonds and imidazole dicarboxylates with some advantages have been used. • A series of new complexes with different structural characteristics have been discussed in detail. • The fluorescent properties, the magnetic property and the water vapor adsorption have been investigated.« less

Distance dependence of the energy transfer rate from a single semiconductor nanostructure to graphene.

PubMed

Federspiel, François; Froehlicher, Guillaume; Nasilowski, Michel; Pedetti, Silvia; Mahmood, Ather; Doudin, Bernard; Park, Serin; Lee, Jeong-O; Halley, David; Dubertret, Benoît; Gilliot, Pierre; Berciaud, Stéphane

2015-02-11

The near-field Coulomb interaction between a nanoemitter and a graphene monolayer results in strong Förster-type resonant energy transfer and subsequent fluorescence quenching. Here, we investigate the distance dependence of the energy transfer rate from individual, (i) zero-dimensional CdSe/CdS nanocrystals and (ii) two-dimensional CdSe/CdS/ZnS nanoplatelets to a graphene monolayer. For increasing distances d, the energy transfer rate from individual nanocrystals to graphene decays as 1/d(4). In contrast, the distance dependence of the energy transfer rate from a two-dimensional nanoplatelet to graphene deviates from a simple power law but is well described by a theoretical model, which considers a thermal distribution of free excitons in a two-dimensional quantum well. Our results show that accurate distance measurements can be performed at the single particle level using graphene-based molecular rulers and that energy transfer allows probing dimensionality effects at the nanoscale.
Study on the interaction between cinnamic acid and lysozyme

NASA Astrophysics Data System (ADS)

Zhang, Hong-Mei; Chen, Jian; Zhou, Qiu-Hua; Shi, Yue-Qin; Wang, Yan-Qing

2011-02-01

The interaction between lysozyme and cinnamic acid was investigated systematically by ultraviolet-vis absorbance, circular dichroism, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques at pH 7.40. The binding constants, quenching mechanism, and the number of binding sites were determined by the quenching of lysozyme fluorescence in presence of cinnamic acid. The results showed that the fluorescence quenching of lysozyme by cinnamic acid was a result of the formation of cinnamic acid-lysozyme complex. The hydrophobic and electrostatic interactions played major roles in stabilizing the complex; the distance r between donor and acceptor was obtained to be 2.07 nm according to Förster's theory; the effect of cinnamic acid on the conformation of lysozyme was analyzed using synchronous fluorescence, circular dichroism and three-dimensional fluorescence spectra.
Electrothermally Driven Fluorescence Switching by Liquid Crystal Elastomers Based On Dimensional Photonic Crystals.

PubMed

Lin, Changxu; Jiang, Yin; Tao, Cheng-An; Yin, Xianpeng; Lan, Yue; Wang, Chen; Wang, Shiqiang; Liu, Xiangyang; Li, Guangtao

2017-04-05

In this article, the fabrication of an active organic-inorganic one-dimensional photonic crystal structure to offer electrothermal fluorescence switching is described. The film is obtained by spin-coating of liquid crystal elastomers (LCEs) and TiO 2 nanoparticles alternatively. By utilizing the property of LCEs that can change their size and shape reversibly under external thermal stimulations, the λ max of the photonic band gap of these films is tuned by voltage through electrothermal conversion. The shifted photonic band gap further changes the matching degree between the photonic band gap of the film and the emission spectrum of organic dye mounting on the film. With rhodamine B as an example, the enhancement factor of its fluorescence emission is controlled by varying the matching degree. Thus, the fluorescence intensity is actively switched by voltage applied on the system, in a fast, adjustable, and reversible manner. The control chain of using the electrothermal stimulus to adjust fluorescence intensity via controlling the photonic band gap is proved by a scanning electron microscope (SEM) and UV-vis reflectance. This mechanism also corresponded to the results from the finite-difference time-domain (FDTD) simulation. The comprehensive usage of photonic crystals and liquid crystal elastomers opened a new possibility for active optical devices.
Quantitative 3-dimensional imaging of auxin and cytokinin levels in transgenic soybean and medicago truncatula roots via two-photon induced fluorescence imaging

NASA Astrophysics Data System (ADS)

Fisher, Jon; Gaillard, Paul; Nurmalasari, Ni Putu Dewi; Fellbaum, Carl; Subramaniam, Sen; Smith, Steve

2018-02-01

Industrial nitrogen fertilizers account for nearly 50% of the fossil fuel costs in modern agriculture and contribute to soil and water pollution. Therefore, significant interest exists in understanding and characterizing the efficiency of nitrogen fixation, and the biochemical signaling pathways which orchestrate the plant-microbial symbiosis through which plants fix nitrogen. Legume plant species exhibit a particularly efficient nitrogen uptake mechanism, using root nodules which house nitrogen-fixing rhizobial bacteria. While nodule development has been widely studied, there remain significant gaps in understanding the regulatory hormones' role in plant development. In this work, we produce 3-dimensional maps of auxin (AX) and cytokinin (CK) hormone concentrations within model plant root tips and nodules with respect to root architecture and cell type. Soybean and Medicago plants were transfected with a two-color fluorescent vector with AXsensitive green fluorescent protein (GFP) and CK-sensitive TdTomato (TdT). 3D images of soybean root nodules were captured using two-photon induced fluorescence microscopy. The resulting images were computationally analyzed using the localization code first developed by Weeks and later adapted by Kilfoil, and analyzed in the context of the root architecture. Statistical analysis of the resulting 3D hormone level maps reproduce-well the known roles of AX and CK in developing plant roots, and are the first quantitative description of these regulatory hormones tied to specific plant architecture. The analytical methods used, and the spatial distribution of these key regulatory hormones in plant roots, nodule primordia and root nodules, and their statistical interpretation are presented.
Fluorescence Intermittency and Nanodot Evolution in Graphene Oxide

NASA Astrophysics Data System (ADS)

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

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

PubMed

Weger, Lukas; Weidmann, Monika; Ali, Wael; Hildebrandt, Marcus; Gutmann, Jochen Stefan; Hoffmann-Jacobsen, Kerstin

2018-05-22

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) is applied to study the self-diffusion of polyethylene glycol solutions in the presence of weakly attractive interfaces. Glass coverslips modified with aminopropyl- and propyl-terminated silanes are used to study the influence of solid surfaces on polymer diffusion. A model of three phases of polymer diffusion allows to describe the experimental fluorescence autocorrelation functions. Besides the two-dimensional diffusion of adsorbed polymer on the substrate and three-dimensional free diffusion in bulk solution, a third diffusion time scale is observed with intermediate diffusion times. This retarded three-dimensional diffusion in solution is assigned to long range effects of solid surfaces on diffusional dynamics of polymers. The respective diffusion constants show Rouse scaling (D~N -1 ) indicating a screening of hydrodynamic interactions by the presence of the surface. Hence, the presented TIR-FCS method proves to be a valuable tool to investigate the effect of surfaces on polymer diffusion beyond the first adsorbed polymer layer on the 100 nm length scale.
Measuring the diffusion coefficient of ganglioside on cell membrane by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Dong, Shiqing; You, Minghai; Chen, Jianling; Zhou, Jie; Xie, Shusen; Yang, Hongqin

2017-06-01

The fluidity of proteins and lipids on cell membrane plays an important role in cell’s physiological functions. Fluorescence correlation spectroscopy (FCS) is an effective technique to detect the rapid dynamic behaviors of proteins and/or lipids in living cells. In this study, we used the rhodamine6G solution to optimize the FCS system. And, cholera toxin B subunit (CT-B) was used to label ganglioside on living Hela cell membranes. The diffusion time and coefficients of ganglioside can be obtained through fitting the autocorrelation curve based on the model of two-dimensional cell membrane. The results showed that the diffusion coefficients of ganglioside distributed within a wide range. It revealed the lateral diffusion of lipids on cell membrane was inhomogeneous, which was due to different microstructures of cytoplasmic membrane. The study provides a helpful method for further studying the dynamic characteristics of proteins and lipids molecules on living cell membrane.
[Three-dimensional Fluorescence Spectral Characteristics of Different Molecular Weight Fractionations of Dissolved Organic Matter in the Water-level Fluctuation Zones of Three Gorges Reservoir Areas].

PubMed

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

2016-03-15

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

NASA Astrophysics Data System (ADS)

Antonello, Jacopo; Burke, Daniel; Booth, Martin J.

2017-12-01

Like all methods of super-resolution microscopy, stimulated emission depletion (STED) microscopy can suffer from the effects of aberrations. The most important aspect of a STED microscope is that the depletion focus maintains a minimum, ideally zero, intensity point that is surrounded by a region of higher intensity. It follows that aberrations that cause a non-zero value of this minimum intensity are the most detrimental, as they inhibit fluorescence emission even at the centre of the depletion focus. We present analysis that elucidates the nature of these effects in terms of the different polarisation components at the focus for two-dimensional and three-dimensional STED resolution enhancement. It is found that only certain low-order aberration modes can affect the minimum intensity at the Gaussian focus. This has important consequences for the design of adaptive optics aberration correction systems.
A Method of Visualizing Three-Dimensional Distribution of Yeast in Bread Dough

NASA Astrophysics Data System (ADS)

Maeda, Tatsurou; Do, Gab-Soo; Sugiyama, Junichi; Oguchi, Kosei; Shiraga, Seizaburou; Ueda, Mitsuyoshi; Takeya, Koji; Endo, Shigeru

A novel technique was developed to monitor the change in three-dimensional (3D) distribution of yeast in frozen bread dough samples in accordance with the progress of mixing process. Application of a surface engineering technology allowed the identification of yeast in bread dough by bonding EGFP (Enhanced Green Fluorescent Protein) to the surface of yeast cells. The fluorescent yeast (a biomarker) was recognized as bright spots at the wavelength of 520 nm. A Micro-Slicer Image Processing System (MSIPS) with a fluorescence microscope was utilized to acquire cross-sectional images of frozen dough samples sliced at intervals of 1 μm. A set of successive two-dimensional images was reconstructed to analyze 3D distribution of yeast. Samples were taken from each of four normal mixing stages (i.e., pick up, clean up, development, and final stages) and also from over mixing stage. In the pick up stage yeast distribution was uneven with local areas of dense yeast. As the mixing progressed from clean up to final stages, the yeast became more evenly distributed throughout the dough sample. However, the uniformity in yeast distribution was lost in the over mixing stage possibly due to the breakdown of gluten structure within the dough sample.
Visualization and quantification of three-dimensional distribution of yeast in bread dough.

PubMed

Maeda, Tatsuro; DO, Gab-Soo; Sugiyama, Junichi; Araki, Tetsuya; Tsuta, Mizuki; Shiraga, Seizaburo; Ueda, Mitsuyoshi; Yamada, Masaharu; Takeya, Koji; Sagara, Yasuyuki

2009-07-01

A three-dimensional (3-D) bio-imaging technique was developed for visualizing and quantifying the 3-D distribution of yeast in frozen bread dough samples in accordance with the progress of the mixing process of the samples, applying cell-surface engineering to the surfaces of the yeast cells. The fluorescent yeast was recognized as bright spots at the wavelength of 520 nm. Frozen dough samples were sliced at intervals of 1 microm by an micro-slicer image processing system (MSIPS) equipped with a fluorescence microscope for acquiring cross-sectional images of the samples. A set of successive two-dimensional images was reconstructed to analyze the 3-D distribution of the yeast. The average shortest distance between centroids of enhanced green fluorescent protein (EGFP) yeasts was 10.7 microm at the pick-up stage, 9.7 microm at the clean-up stage, 9.0 microm at the final stage, and 10.2 microm at the over-mixing stage. The results indicated that the distribution of the yeast cells was the most uniform in the dough of white bread at the final stage, while the heterogeneous distribution at the over-mixing stage was possibly due to the destruction of the gluten network structure within the samples.
Fluorescence recovery after photobleaching measured by confocal microscopy as a tool for the analysis of vesicular lipid transport and plasma membrane mobility

NASA Astrophysics Data System (ADS)

Schmitz, Gerd; Goetz, Alexandra; Orso, Evelyn; Rothe, Gregor

1998-04-01

The vesicular transport of lipids from the endoplasmic reticulum via the Golgi apparatus affects the composition of the plasma membrane. The purpose of our study was to develop an in vitro test system for characterization of vesicular lipid transport kinetics by using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Fibroblasts from two patients homozygous for the hypercatabolic HDL deficiency syndrome Tangier disease and 4 control subjects were pulsed with the C6-NBD-ceramide for 30 minutes. Chase incubation at room temperature resulted in the metabolic accumulation of fluorescent C6-NBD-sphingolyelin and C6-NBD-glycosylceramides in the medial- and trans-Golgi region. Cells were analyzed with an inverted Leica TCS microscope. Calibration was performed through the analysis of diffusion of 50 nm microparticles embedded in media of different viscosity. An acousto optical tunable filter (AOTF) was used for the selective bleaching of the medial- and trans- Golgi region followed by analysis of the fluorescence recovery for 4 minutes. Post-bleach fluorescence recovery through the trans-Golgi-oriented transport of NBD-sphingomyelin was calculated from 2-dimensional scans. Tangier fibroblasts displayed a retarded recovery of fluorescence in the trans- Golgi region. This suggests that the vesicular transport of sphingomyelin and cholesterol is disturbed in Tangier disease confirming data from our laboratory generated with radiometabolites on whole cells. Our data suggest that FRAP analysis allows a sensitive kinetic and spatially resolved analysis of disturbances of vesicular lipid transport.
Construction of an efficient two-photon fluorescent probe for imaging nitroreductase in live cells and tissues

NASA Astrophysics Data System (ADS)

Zhou, Liyi; Gong, Liang; Hu, Shunqin

2018-06-01

Compared with traditional confocal microscopy, two-photon fluorescence microscopy (TPFM), which excites a two-photon (TP) fluorophore by near-infrared light, provides improved three-dimensional image resolution with increased tissue-image depth (>500 μm) and an extended observation time. Therefore, the development of novel functional TP fluorophores has attracted great attention in recent years. Herein, a novel TP fluorophore CM-NH2, which have the donor-π-acceptor (D-π-A)-structure, was designed and synthesized. We further used this dye developed a new type of TP fluorescent probe CM-NO2 for detecting nitroreductase (NTR). Upon incubated with NTR for 15 min, CM-NO2 displayed a 90-fold fluorescence enhancement at 505 nm and the maximal TP action cross-section value after reaction was detected and calculated to be 200 GM at 760 nm. The probe exhibited excellent properties such as high sensitivity, high selectivity, low cytotoxicity, and high photostability. Moreover, the probe was utilized to image the tumor hypoxia in live HeLa cells. Finally, using the CM-NO2 to image NTR in tissues was demonstrated.
Monte Carlo based method for fluorescence tomographic imaging with lifetime multiplexing using time gates

PubMed Central

Chen, Jin; Venugopal, Vivek; Intes, Xavier

2011-01-01

Time-resolved fluorescence optical tomography allows 3-dimensional localization of multiple fluorophores based on lifetime contrast while providing a unique data set for improved resolution. However, to employ the full fluorescence time measurements, a light propagation model that accurately simulates weakly diffused and multiple scattered photons is required. In this article, we derive a computationally efficient Monte Carlo based method to compute time-gated fluorescence Jacobians for the simultaneous imaging of two fluorophores with lifetime contrast. The Monte Carlo based formulation is validated on a synthetic murine model simulating the uptake in the kidneys of two distinct fluorophores with lifetime contrast. Experimentally, the method is validated using capillaries filled with 2.5nmol of ICG and IRDye™800CW respectively embedded in a diffuse media mimicking the average optical properties of mice. Combining multiple time gates in one inverse problem allows the simultaneous reconstruction of multiple fluorophores with increased resolution and minimal crosstalk using the proposed formulation. PMID:21483610
Mechanism of laser induced fluorescence signal generation in InCl3-ethanol mixture flames

NASA Astrophysics Data System (ADS)

Fang, Bolang; Hu, Zhiyun; Zhang, Zhenrong; Li, Guohua; Shao, Jun; Feng, Guobin

2017-05-01

Nonlinear regime Two-line Atomic Fluorescence (NTLAF) is a promising technique for two-dimensional thermometry. A key challenge is seeding of indium atoms into flame. This work aims at investigating the mechanism of Indium LIF signal generation in a fuel-rich InCl3-ethanol premixed flame. Several types of images including natural emission of the flame itself, natural emission of CH, natural emission of OH, natural emission at 410 nm/451 nm of indium atom, and laser induced fluorescence at 410 nm/451 nm were obtained. The indium atom was generated in the flame front, and could survive in the post-flame zone for a while which is benefit for making NTLAF measurements. Further detail mechanism of fluorescence signals generation in InCl3-ethanol solution burning was investigated. The conclusion which probable to be drew is that to gain high NTLAF signals, the size of liquid droplets should be well controlled, neither to be too large nor to be gasified.
Simultaneous acquisition of trajectory and fluorescence lifetime of moving single particles

NASA Astrophysics Data System (ADS)

Wu, Qianqian; Qi, Jing; Lin, Danying; Yan, Wei; Hu, Rui; Peng, Xiao; Qu, Junle

2017-02-01

Fluorescence lifetime imaging (FLIM) has been a powerful tool in life science because it can reveal the interactions of an excited fluorescent molecule and its environment. The combination with two-photon excitation (TPE) and timecorrelated single photon counting (TCSPC) provides it the ability of optical sectioning, high time resolution and detection efficiency. In previous work, we have introduced a two-dimensional acousto-optic deflector (AOD) into TCSPC-based FLIM to achieve fast and flexible FLIM. In this work, we combined the AOD-FLIM system with a single particle tracking (SPT) setup and algorithm and developed an SPT-FLIM system. Using the system, we acquired the trajectory and fluorescence lifetime of a moving particle simultaneously and reconstructed a life-time-marked pseudocolored trajectory, which might reflect dynamic interaction between the moving particle and its local environment along its motion trail. The results indicated the potential of the technique for studying the interaction between specific moving biological macromolecules and the ambient micro-environment in live cells.
Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex.

PubMed

Berggren, K; Chernokalskaya, E; Steinberg, T H; Kemper, C; Lopez, M F; Diwu, Z; Haugland, R P; Patton, W F

2000-07-01

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.
Detector Having A Transmission Grating Beam Splitter For Multi-Wavelength Sample Analysis.

DOEpatents

Liu, Changsheng; Li, Qingbo

2000-09-12

A detector for DNA sample identification is provided with a transmission grating beam splitter (TGBS). The TGBS split fluoresced light from a tagged DNA sample into 0th order and a 1st order components, both of which are detected on a two-dimensional detector array of a CCD camera. The 0th and 1st order components are detected along a column of pixels in the detector array, and are spaced apart from one another. The DNA samples are tagged with four fluorescent dyes, one dye specific for each nucleotide, and all four dyes responding in slightly different manner to the same monochromatic excitation signal. The TGBS splits fluoresced incoming light into 0th and 1st order components, which are then spread out among a number of pixels in the detector array. The 1st component of this light is received by pixels whose position relative to the 0th order component depends on the frequency of fluorescence. Thus, the position at which signal energy is detected on the array is indicative of the particular dye, and therefore, the corresponding nucleotide tagged by that dye. Monitoring signal energy at the 0th order pixel and selected 1st order pixels, provides a set of data from which one may then identify the particular nucleotide.
Detector Having A Transmission Grating Beam Splitter For Multi-Wavelength.

DOEpatents

Liu, Changsheng; Li, Qingbo (State College, PA

1999-12-07

A detector for DNA sample identification is provided with a transmission grating beam splitter (TGBS). The TGBS split fluoresced light from a tagged DNA sample into 0th order and a 1st order components, both of which are detected on a two-dimensional detector array of a CCD camera. The 0th and 1st order components are detected along a column of pixels in the detector array, and are spaced apart from one another. The DNA samples are tagged with four fluorescent dyes, one dye specific for each nucleotide, and all four dyes responding in slightly different manner to the same monochromatic excitation signal. The TGBS splits fluoresced incoming light into 0th and 1st order components, which are then spread out among a number of pixels in the detector array. The 1st component of this light is received by pixels whose position relative to the 0th order component depends on the frequency of fluorescence. Thus, the position at which signal energy is detected on the array is indicative of the particular dye, and therefore, the corresponding nucleotide tagged by that dye. Monitoring signal energy at the 0th order pixel and selected 1st order pixels, provides a set of data from which one may then identify the particular nucleotide.
Concentration Measurements in a Cold Flow Model Annular Combustor Using Laser Induced Fluorescence

NASA Technical Reports Server (NTRS)

Morgan, Douglas C.

1996-01-01

A nonintrusive concentration measurement method is developed for determining the concentration distribution in a complex flow field. The measurement method consists of marking a liquid flow with a water soluble fluorescent dye. The dye is excited by a two dimensional sheet of laser light. The fluorescent intensity is shown to be proportional to the relative concentration level. The fluorescent field is recorded on a video cassette recorder through a video camera. The recorded images are analyzed with image processing hardware and software to obtain intensity levels. Mean and root mean square (rms) values are calculated from these intensity levels. The method is tested on a single round turbulent jet because previous concentration measurements have been made on this configuration by other investigators. The previous results were used to comparison to qualify the current method. These comparisons showed that this method provides satisfactory results. 'Me concentration measurement system was used to measure the concentrations in the complex flow field of a model gas turbine annular combustor. The model annular combustor consists of opposing primary jets and an annular jet which discharges perpendicular to the primary jets. The mixing between the different jet flows can be visualized from the calculated mean and rms profiles. Concentration field visualization images obtained from the processing provide further qualitative information about the flow field.

Photoluminescence enhancement of monolayer tungsten disulfide in complicated plasmonic microstructures

NASA Astrophysics Data System (ADS)

Zhou, Yi; Hu, Xiaoyong; Gao, Wei; Song, Hanfa; Chu, Saisai; Yang, Hong; Gong, Qihuang

2018-06-01

Two-dimensional van der Waals materials are interesting for fundamental physics exploration and device applications because of their attractive physical properties. Here, we report a strategy to realize photoluminescence (PL) enhancement of two-dimensional transition-metal dichalcogenides (TMDCs) in the visible range using a plasmonic microstructure with patterned gold nanoantennas and a metal-insulator-semiconductor-insulator-metal structure. The PL intensity was enhanced by a factor of two under Y-polarization due to the increased radiative decay rate by the surface plasmon radiation channel in the gold nanoantennas and the decreased nonradiative decay rate by suppressing exciton quenching in the SiO2 isolation layer. The fluorescence lifetime of monolayer tungsten disulfide in this structure was shorter than that of a sample without patterned gold nanoantennas. Tailoring the light-matter interactions between two-dimensional TMDCs and plasmonic nanostructures may provide highly efficient optoelectronic devices such as TMDC-based light emitters.
Three-dimensional imaging of sulfides in silicate rocks at submicron resolution with multiphoton microscopy.

PubMed

Bénard, Antoine; Palle, Sabine; Doucet, Luc Serge; Ionov, Dmitri A

2011-12-01

We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (∼120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.
Volumetric Analysis of 3-D-Cultured Colonies in Wet Alginate Spots Using 384-Pillar Plate.

PubMed

Lee, Dong Woo; Choi, Yea-Jun; Lee, Sang-Yun; Kim, Myoung-Hee; Doh, Il; Ryu, Gyu Ha; Choi, Soo-Mi

2018-06-01

The volumetric analysis of three-dimensional (3-D)-cultured colonies in alginate spots has been proposed to increase drug efficacy. In a previously developed pillar/well chip platform, colonies within spots are usually stained and dried for analysis of cell viability using two-dimensional (2-D) fluorescent images. Since the number of viable cells in colonies is directly related to colony volume, we proposed the 3-D analysis of colonies for high-accuracy cell viability calculation. The spots were immersed in buffer, and the 3-D volume of each colony was calculated from the 2-D stacking fluorescent images of the spot with different focal positions. In the experiments with human gastric carcinoma cells and anticancer drugs, we compared cell viability values calculated using the 2-D area and 3-D volume of colonies in the wet and dried alginate spots, respectively. The IC 50 value calculated using the 3-D volume of the colonies (9.5 μM) was less than that calculated in the 2-D area analysis (121.5 μM). We observed that the colony showed a more sensitive drug response regarding volume calculated from the 3-D image reconstructed using several confocal images than regarding colony area calculated in the 2-D analysis.
Novel remodeling of the mouse heart mitochondrial proteome in response to acute insulin stimulation

PubMed Central

Pedersen, Brian A; Yazdi, Puya G; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Wang, Ping H

2015-01-01

Mitochondrial dysfunction contributes to the pathophysiology of diabetic cardiomyopathy. The aim of this study was to investigate the acute changes in the mitochondrial proteome in response to insulin stimulation. Cardiac mitochondria from C57BL/6 mice after insulin stimulation were analyzed using two-dimensional fluorescence difference gel electrophoresis. MALDI-TOF MS/MS was utilized to identify differences. Two enzymes involved in metabolism and four structural proteins were identified. Succinyl-CoA ligase [ADP forming] subunit beta was identified as one of the differentially regulated proteins. Upon insulin stimulation, a relatively more acidic isoform of this protein was increased by 53% and its functional activity was decreased by ∼32%. This proteomic remodeling in response to insulin stimulation may play an important role in the normal and diabetic heart. PMID:26610654
Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

PubMed Central

Beaumont, Kimberley A.; Anfosso, Andrea; Ahmed, Farzana

2015-01-01

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions. PMID:26779761
In-situ observation of NO and NO2 in streamer corona discharge by two-dimensional laser-induced fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Aramaki, M.; Kurakane, H.; Sasaki, K.

2003-10-01

It is known that most of nitrogenous oxides which are released from factories, cars, and so on are mainly composed NO and NO_2. Hence, it is especially important to examine dissociation processes of NO and NO2 in the research of the NOx removal using high-pressure non-equilibrium plasmas. In-situ observations of NO and NO2 in the plasma reactor are effective to investigate the reaction kinetics of NO_x. In the present work, we measured temporal variations of the density distributions of NO and NO2 in streamer corona discharges between needle and planar electrodes by two-dimensional laser induced fluorescence spectroscopy. The two-dimensional images of the density distributions of NO and NO2 were taken using an ICCD camera. The discharge atmosphere was air including NO2 (1500 ˜2000 ppm) at a pressure of 200 Torr. The NO2 density decreased locally in the region below the needle electrode at 10 ms after the discharge. At the same time, we observed the appearance of NO in the region corresponding to the decay of NO_2. The significant production of NO was not observed when the discharge atmosphere was composed of N2 and NO_2. Therefore, from these experimental results, it is suggested that NO is produced by a reaction NO2 + O arrow NO + O_2.
Investigation of the optical response of photonic crystal nanocavities in ferroelectric oxide thin film

NASA Astrophysics Data System (ADS)

Lin, Pao Tai; Russin, William A.; Joshi-Imre, Alexandra; Ocola, Leonidas E.; Wessels, B. W.

2015-10-01

The optical properties of BaTiO3 two dimensional photonic crystal (PhC) nanocavities were investigated. Two types of nanocavities consisting of dopants and vacancies with PhC periodicities ranging from 200 to 550 nm were evaluated. The images from laser scanning confocal microscopy show the optical scattering of the PhC cavities is highly wavelength dependent. An optical intensity reversal is observed when the wavelength of probe light shifts by 29 nm. Meanwhile, intensity contrast between the nanocavity and its adjacent PhCs is enhanced as the PhC periodicity becomes shorter than the probe wavelength. To determine the photonic band structures fluorescence from dye covered PhCs were imaged and analyzed. A strong enhancement of fluorescence is observed for the PhC with a period of 200 nm. Upon comparison to the 2D finite difference time domain calculations, the enhancement is attributed to strong light localization within the PhC nanocavity. As a result, the in-plane lightwave propagation is prohibited that results in an increase in the vertical light scattering.
Synthesis, structures and fluorescent properties of two novel lanthanide [Ln = Ce(III), Pr(III)] coordination polymers based on 1,3-benzenedicarboxylate and 2-(4-methoxyphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline ligands

NASA Astrophysics Data System (ADS)

Wang, Lei; Ni, Liang; yao, Jia

2012-09-01

Two structurally diverse coordination polymers [Ce2(m-BDC)2(m-HBDC)2(MOPIP)2·3/2H2O]n (1) and [Pr2(m-BDC)3(MOPIP)2·H2O]n(2) have been synthesized by hydrothermal reaction of lanthanide chloride with mixed ligands benzene-1,3-dicarboxylic acid and 2-(4-methoxyphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (MOPIP). The crystal structures of the complexes are zipper-like chains of octacoordinate Ln3+ ions, in which Ln3+ ions are bridged in different coordination modes by m-BDC2+ and decorated by MOPIP ligands. These chains are further assembled into three-dimensional supramolecular framework by π⋯π stacking and hydrogen bonding interactions. The fluorescent property and thermal stability were also investigated. Additionally, Natural bond orbital (NBO) analysis of complex 2 shows a weak covalent interaction between the coordinated atoms and Pr3+ ions.
Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases.

PubMed

Gráczer, Eva; Varga, Andrea; Melnik, Bogdan; Semisotnov, Gennady; Závodszky, Péter; Vas, Mária

2009-02-10

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trp's (located in separate domains) were systematically replaced with Phe's. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.
Three-Dimensional Reconstruction of Three-Way FRET Microscopy Improves Imaging of Multiple Protein-Protein Interactions.

PubMed

Scott, Brandon L; Hoppe, Adam D

2016-01-01

Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data.
Neural networks for dimensionality reduction of fluorescence spectra and prediction of drinking water disinfection by-products.

PubMed

Peleato, Nicolas M; Legge, Raymond L; Andrews, Robert C

2018-06-01

The use of fluorescence data coupled with neural networks for improved predictability of drinking water disinfection by-products (DBPs) was investigated. Novel application of autoencoders to process high-dimensional fluorescence data was related to common dimensionality reduction techniques of parallel factors analysis (PARAFAC) and principal component analysis (PCA). The proposed method was assessed based on component interpretability as well as for prediction of organic matter reactivity to formation of DBPs. Optimal prediction accuracies on a validation dataset were observed with an autoencoder-neural network approach or by utilizing the full spectrum without pre-processing. Latent representation by an autoencoder appeared to mitigate overfitting when compared to other methods. Although DBP prediction error was minimized by other pre-processing techniques, PARAFAC yielded interpretable components which resemble fluorescence expected from individual organic fluorophores. Through analysis of the network weights, fluorescence regions associated with DBP formation can be identified, representing a potential method to distinguish reactivity between fluorophore groupings. However, distinct results due to the applied dimensionality reduction approaches were observed, dictating a need for considering the role of data pre-processing in the interpretability of the results. In comparison to common organic measures currently used for DBP formation prediction, fluorescence was shown to improve prediction accuracies, with improvements to DBP prediction best realized when appropriate pre-processing and regression techniques were applied. The results of this study show promise for the potential application of neural networks to best utilize fluorescence EEM data for prediction of organic matter reactivity. Copyright © 2018 Elsevier Ltd. All rights reserved.
Investigating line- versus point-laser excitation for three-dimensional fluorescence imaging and tomography employing a trimodal imaging system

NASA Astrophysics Data System (ADS)

Cao, Liji; Peter, Jörg

2013-06-01

The adoption of axially oriented line illumination patterns for fluorescence excitation in small animals for fluorescence surface imaging (FSI) and fluorescence optical tomography (FOT) is being investigated. A trimodal single-photon-emission-computed-tomography/computed-tomography/optical-tomography (SPECT-CT-OT) small animal imaging system is being modified for employment of point- and line-laser excitation sources. These sources can be arbitrarily positioned around the imaged object. The line source is set to illuminate the object along its entire axial direction. Comparative evaluation of point and line illumination patterns for FSI and FOT is provided involving phantom as well as mouse data. Given the trimodal setup, CT data are used to guide the optical approaches by providing boundary information. Furthermore, FOT results are also being compared to SPECT. Results show that line-laser illumination yields a larger axial field of view (FOV) in FSI mode, hence faster data acquisition, and practically acceptable FOT reconstruction throughout the whole animal. Also, superimposed SPECT and FOT data provide additional information on similarities as well as differences in the distribution and uptake of both probe types. Fused CT data enhance further the anatomical localization of the tracer distribution in vivo. The feasibility of line-laser excitation for three-dimensional fluorescence imaging and tomography is demonstrated for initiating further research, however, not with the intention to replace one by the other.
Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides.

PubMed

Struwe, Weston B; Rudd, Pauline M

2012-08-01

In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λ(ex)=355 nm and λ(em)=440 nm. We concluded that the optimal wavelengths for 3-AQ were λ(ex)=355 nm and λ(em)=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λ(ex/em) values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent properties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.
Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations

NASA Astrophysics Data System (ADS)

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.
Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations.

PubMed

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.
Dual-color multiple-particle tracking at 50-nm localization and over 100-µm range in 3D with temporal focusing two-photon microscopy

PubMed Central

Ding, Yu; Li, Chunqiang

2016-01-01

Nanoscale particle tracking in three dimensions is crucial to directly observe dynamics of molecules and nanoparticles in living cells. Here we present a three-dimensional particle tracking method based on temporally focused two-photon excitation. Multiple particles are imaged at 30 frames/s in volume up to 180 × 180 × 100 µm3. The spatial localization precision can reach 50 nm. We demonstrate its capability of tracking fast swimming microbes at speed of ~200 µm/s. Two-photon dual-color tracking is achieved by simultaneously exciting two kinds of fluorescent beads at 800 nm to demonstrate its potential in molecular interaction studies. Our method provides a simple wide-field fluorescence imaging approach for deep multiple-particle tracking. PMID:27867724
3D mouse shape reconstruction based on phase-shifting algorithm for fluorescence molecular tomography imaging system.

PubMed

Zhao, Yue; Zhu, Dianwen; Baikejiang, Reheman; Li, Changqing

2015-11-10

This work introduces a fast, low-cost, robust method based on fringe pattern and phase shifting to obtain three-dimensional (3D) mouse surface geometry for fluorescence molecular tomography (FMT) imaging. We used two pico projector/webcam pairs to project and capture fringe patterns from different views. We first calibrated the pico projectors and the webcams to obtain their system parameters. Each pico projector/webcam pair had its own coordinate system. We used a cylindrical calibration bar to calculate the transformation matrix between these two coordinate systems. After that, the pico projectors projected nine fringe patterns with a phase-shifting step of 2π/9 onto the surface of a mouse-shaped phantom. The deformed fringe patterns were captured by the corresponding webcam respectively, and then were used to construct two phase maps, which were further converted to two 3D surfaces composed of scattered points. The two 3D point clouds were further merged into one with the transformation matrix. The surface extraction process took less than 30 seconds. Finally, we applied the Digiwarp method to warp a standard Digimouse into the measured surface. The proposed method can reconstruct the surface of a mouse-sized object with an accuracy of 0.5 mm, which we believe is sufficient to obtain a finite element mesh for FMT imaging. We performed an FMT experiment using a mouse-shaped phantom with one embedded fluorescence capillary target. With the warped finite element mesh, we successfully reconstructed the target, which validated our surface extraction approach.
Advancements of two dimensional correlation spectroscopy in protein researches

NASA Astrophysics Data System (ADS)

Tao, Yanchun; Wu, Yuqing; Zhang, Liping

2018-05-01

The developments of two-dimensional correlation spectroscopy (2DCOS) applications in protein studies are discussed, especially for the past two decades. The powerful utilities of 2DCOS combined with various analytical techniques in protein studies are summarized. The emphasis is on the vibration spectroscopic techniques including IR, NIR, Raman and optical activity (ROA), as well as vibration circular dichroism (VCD) and fluorescence spectroscopy. In addition, some new developments, such as hetero-spectral 2DCOS, moving-window correlation, and model based correlation, are also reviewed for their utility in the investigation of the secondary structure, denaturation, folding and unfolding changes of protein. Finally, the new possibility and challenges of 2DCOS in protein research are highlighted as well.
Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection

PubMed Central

Hospach, Ingeborg; Joseph, Yvonne; Mai, Michaela Kathrin; Krasteva, Nadejda; Nelles, Gabriele

2014-01-01

Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established. PMID:27600349
Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH.

PubMed

Palmes-Saloma, C; Saloma, C

2000-07-01

Long-depth imaging of specific gene expression in the midgestation whole-mount mouse embryo (WME) is demonstrated with single-photon excitation (1PE) confocal fluorescence microscopy and fluorescence in situ hybridization. Expression domains of Pax-6 mRNA transcripts were labeled with an in situ hybridization probe that is a RNA sequence complementary to the cloned gene fragment and were rendered visible using two fluorochrome-conjugated antibodies that fluoresce at peak wavelengths of lambda(F) = 0.525 microm and lambda(F) = 0. 580 microm, respectively. Distributions of Pax-6 mRNA domains as deep as 1000 microm in the day 9.5 WME were imaged with a long-working-distance (13.6 mm) objective lens (magnification 5x). The scattering problem posed by the optically thick WME sample is alleviated by careful control of the detector pinhole size and the application of simple but fast postdetection image enhancement techniques, such as space and wavelength averaging to produce high-quality fluorescence images. A three-dimensional reconstruction that clearly shows the Pax-6 mRNA expression domains in the forebrain, diencephalon, optic cup, and spinal cord of the day 9.5 WME is obtained. The advantages of 1PE confocal fluorescence imaging over two-photon excitation fluorescence imaging are discussed for the case of long-depth imaging in highly scattering media. Imaging in midgestation WMEs at optical depths of more than 350 microm has not yet been realized with two-photon fluorescence excitation. Copyright 2000 Academic Press.

Emergence of power-law scalings in shock-driven mixing transition

NASA Astrophysics Data System (ADS)

Vorobieff, Peter; Wayne, Patrick; Olmstead, Dell; Simons, Dylan; Truman, C. Randall; Kumar, Sanjay

2016-11-01

We present an experimental study of transition to turbulence due to shock-driven instability evolving on an initially cylindrical, diffuse density interface between air and a mixture of sulfur hexafluoride (SF6) and acetone. The plane of the shock is at an initial angle θ with the axis of the heavy-gas cylinder. We present the cases of planar normal (θ = 0) and oblique (θ =20°) shock interaction with the initial conditions. Flow is visualized in two perpendicular planes with planar laser-induced fluorescence (PLIF) triggered in acetone with a pulsed ultraviolet laser. Statistics of the flow are characterized in terms of the second-order structure function of the PLIF intensity. As instabilities in the flow evolve, the structure functions begin to develop power-law scalings, at late times manifesting over a range of scales spanning more than two orders of magnitude. We discuss the effects of the initial conditions on the emergence of these scalings, comparing the fully three-dimensional case (oblique shock interaction) with the quasi-two-dimensional case (planar normal shock interaction). We also discuss the flow anisotropy apparent in statistical differences in data from the two visualization planes. This work is funded by NNSA Grant DE-NA0002913.
Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

NASA Astrophysics Data System (ADS)

Ploetz, Evelyn; Lerner, Eitan; Husada, Florence; Roelfs, Martin; Chung, Sangyoon; Hohlbein, Johannes; Weiss, Shimon; Cordes, Thorben

2016-09-01

Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.
Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

PubMed Central

Ploetz, Evelyn; Lerner, Eitan; Husada, Florence; Roelfs, Martin; Chung, SangYoon; Hohlbein, Johannes; Weiss, Shimon; Cordes, Thorben

2016-01-01

Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble. PMID:27641327
Ratiometric, single-dye, pH-sensitive inhibited laser-induced fluorescence for the characterization of mixing and mass transfer

NASA Astrophysics Data System (ADS)

Lacassagne, Tom; Simoëns, Serge; El Hajem, Mahmoud; Champagne, Jean-Yves

2018-01-01

Inhibited planar laser-induced fluorescence (I-PLIF) techniques are widely used for heat and mass transfer studies in fluid mechanics. They allow the visualization of instantaneous two-dimensional field of a passive or reactive scalar, providing that this scalar acts as an inhibitor to the fluorescence of a specific molecule, and that this molecule is homogeneously mixed in the fluid at a known concentration. Local scalar values are deduced from fluorescence recordings thanks to preliminary calibration procedure. When confronted with non-optically thin systems, however, the knowledge of the excitation intensity distribution in the region of interest is also required, and this information is most of the time hard to obtain. To overcome that problem, two-color ratiometric PLIF techniques ( {I}^ {r}-PLIF) have been developed. In these methods, the ratio of two different fluorescence wavelengths triggered by the same excitation is used as an indicator of the scalar value. Such techniques have been used for temperature measurements in several studies but never, to the author's knowledge, for pH tracking and acid-base mixing, despite the frequent use of the one-color version in mass transfer studies. In the present work, a ratiometric pH-sensitive-inhibited PLIF technique ( {I}_ {pH}^ {r}-PLIF) using fluorescein sodium as a single dye and applicable to complex geometries and flows is developed. Theoretical considerations show that the ratio of the two-color fluorescence intensities should only depend on the dye's spectral quantum yield, itself pH-dependent. A detailed spectrofluorimetric study of fluorescein reveals that this ratio strictly increases with the pH for two well-chosen spectral bands (fluorescence colors). A similar trend is found when using sCmos cameras equipped with optical filters to record fluorescence signals. The method is then experimented on a test flow, a turbulent acidic jet injected in an initially pH-neutral volume of fluid. The results obtained using the ratiometric version are consistent with single-color technique measurements, but excitation intensity heterogeneity is more efficiently accounted for, with a much smaller time needed for data treatment and without requiring the knowledge of laser paths across the fluid. This new technique is also able to reduce the impact of some unwanted experimental features such as time-varying excitation intensity or reflections at interfaces. It can be of great interest for further applications to multiphase mass transfer studies.
Spectral reconstruction for shifted-excitation Raman difference spectroscopy (SERDS).

PubMed

Guo, Shuxia; Chernavskaia, Olga; Popp, Jürgen; Bocklitz, Thomas

2018-08-15

Fluorescence emission is one of the major obstacles to apply Raman spectroscopy in biological investigations. It is usually several orders more intense than Raman scattering and hampers further analysis. In cases where the fluorescence emission is too intense to be efficiently removed via routine mathematical baseline correction algorithms, an alternative approach is needed. One alternative approach is shifted-excitation Raman difference spectroscopy (SERDS), where two Raman spectra are recorded with two slightly different excitation wavelengths. Ideally, the fluorescence emission at the two excitations does not change while the Raman spectrum shifts according to the excitation wavelength. Hence the fluorescence is removed in the difference of the two recorded Raman spectra. For better interpretability a spectral reconstruction procedure is necessary to recover the fluorescence-free Raman spectrum. This is challenging due to the intensity variations between the two recorded Raman spectra caused by unavoidable experimental changes as well as the presence of noise. Existent approaches suffer from drawbacks like spectral resolution loss, fluorescence residual, and artefacts. In this contribution, we proposed a reconstruction method based on non-negative least squares (NNLS), where the intensity variations between the two measurements are utilized in the reconstruction model. The method achieved fluorescence-free reconstruction on three real-world SERDS datasets without significant information loss. Thereafter, we quantified the performance of the reconstruction based on artificial datasets from four aspects: reconstructed spectral resolution, precision of reconstruction, signal-to-noise-ratio (SNR), and fluorescence residual. The artificial datasets were constructed with varied Raman to fluorescence intensity ratio (RFIR), SNR, full-width at half-maximum (FWHM), excitation wavelength shift, and fluorescence variation between the two spectra. It was demonstrated that the NNLS approach provides a faithful reconstruction without significantly changing the spectral resolution. Meanwhile, the reconstruction is almost robust to fluorescence variations between the two spectra. Last but not the least the SNR was improved after reconstruction for extremely noisy SERDS datasets. Copyright © 2018 Elsevier B.V. All rights reserved.
Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

NASA Astrophysics Data System (ADS)

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

2016-03-01

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.
Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope.

PubMed

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

2016-03-01

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.
Single-Shot, Volumetrically Illuminated, Three-Dimensional, Tomographic Laser-Induced-Fluorescence Imaging in a Gaseous Free Jet

DTIC Science & Technology

2016-04-28

Single- shot , volumetrically illuminated, three- dimensional, tomographic laser-induced- fluorescence imaging in a gaseous free jet Benjamin R. Halls...us.af.mil Abstract: Single- shot , tomographic imaging of the three-dimensional concentration field is demonstrated in a turbulent gaseous free jet in co-flow...2001). 6. K. M. Tacina and W. J. A. Dahm, “Effects of heat release on turbulent shear flows, Part 1. A general equivalence principle for non-buoyant
Three-dimensional reconstruction of rat knee joint using episcopic fluorescence image capture.

PubMed

Takaishi, R; Aoyama, T; Zhang, X; Higuchi, S; Yamada, S; Takakuwa, T

2014-10-01

Development of the knee joint was morphologically investigated, and the process of cavitation was analyzed by using episcopic fluorescence image capture (EFIC) to create spatial and temporal three-dimensional (3D) reconstructions. Knee joints of Wister rat embryos between embryonic day (E)14 and E20 were investigated. Samples were sectioned and visualized using an EFIC. Then, two-dimensional image stacks were reconstructed using OsiriX software, and 3D reconstructions were generated using Amira software. Cavitations of the knee joint were constructed from five divided portions. Cavity formation initiated at multiple sites at E17; among them, the femoropatellar cavity (FPC) was the first. Cavitations of the medial side preceded those of the lateral side. Each cavity connected at E20 when cavitations around the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) were completed. Cavity formation initiated from six portions. In each portion, development proceeded asymmetrically. These results concerning anatomical development of the knee joint using EFIC contribute to a better understanding of the structural feature of the knee joint. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
A series of Cadmium(II) complexes with 2-substituted terephthalate building block and N-Donor co-ligands: Structural diversity and fluorescence properties

NASA Astrophysics Data System (ADS)

Ren, Yixia; Zhou, Shanhong; Wang, Zhixiang; Zhang, Meili; Wang, Jijiang; Cao, Jia

2017-11-01

Four new Cd(II) complexes have been prepared based on 1,2,4-trimellitic acid (H3tma) and monosodium 2-sulfoterephthalate (2-NaH2stp), formulated as [Cd2(Htma)2 (dpp)2(H2O)] (1), [Cd3 (tma)2 (2,4-bipy)4(H2O)2] (2), [Cd (2-Hstp) (2,2'-bipy)2]·2H2O (3) and [Cd (2-Hstp) (2,4-bipy) (H2O)2] (4) (dpp = dipyrido [3,2-a:2‧,3'-c] phenazine, 2,4-bipy = 2,4-bipyridine, 2,2'-bipy = 2,2'- bipyridine) by hydrothermal method. X-ray diffraction structural analyses show all these complexes crystallized in triclinic crystal system of Pī space group, but their structures are diverse. Complex 1 exhibits an infinite one-dimensional chain featuring the left- and right-handed stranded chains interweaved each other. For 2, the two-dimensional network is constructed by one-dimensional ladder-like chain linked by Cd2 ions. In complex 3, the cadmium ion is surrounded with one 2-Hstp2- anion and two 2,2'-bipy molecules. Complex 4 is also a discrete structure based on a metallic dimer unit. In all these complexes, the N-donor co-ligands take the important roles in the assembly of three-dimensional supramolecular structures. The fluorescence properties of complexes 1-4 could be assigned to the π - π* transition of organic ligands.
Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning.

PubMed

Ozbay, Baris N; Futia, Gregory L; Ma, Ming; Bright, Victor M; Gopinath, Juliet T; Hughes, Ethan G; Restrepo, Diego; Gibson, Emily A

2018-05-25

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.
Super-resolution three-dimensional fluorescence and optical diffraction tomography of live cells using structured illumination generated by a digital micromirror device.

PubMed

Shin, Seungwoo; Kim, Doyeon; Kim, Kyoohyun; Park, YongKeun

2018-06-15

We present a multimodal approach for measuring the three-dimensional (3D) refractive index (RI) and fluorescence distributions of live cells by combining optical diffraction tomography (ODT) and 3D structured illumination microscopy (SIM). A digital micromirror device is utilized to generate structured illumination patterns for both ODT and SIM, which enables fast and stable measurements. To verify its feasibility and applicability, the proposed method is used to measure the 3D RI distribution and 3D fluorescence image of various samples, including a cluster of fluorescent beads, and the time-lapse 3D RI dynamics of fluorescent beads inside a HeLa cell, from which the trajectory of the beads in the HeLa cell is analyzed using spatiotemporal correlations.
Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue.

PubMed

Gaertner, Maria; Cimalla, Peter; Meissner, Sven; Kuebler, Wolfgang M; Koch, Edmund

2012-07-01

Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.
Intravital Confocal and Two-photon Imaging of Dual-color Cells and Extracellular Matrix Mimics

PubMed Central

Bal, Ufuk; Andresen, Volker; Baggett, Brenda; Utzinger, Urs

2013-01-01

To optimize imaging of cells in three dimensional culture we studied confocal backscattering, Second Harmonic Generation (SHG) and autofluorescence as source of contrast in extracellular matrix (ECM) mimics and evaluated the attenuation as well as bleaching of endogenous cellular fluorescence signals. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence while still providing good reflectance to detect voids in the embedding medium. We labeled breast cancer cells’ outline with DsRed2 and nucleus with eGFP. DsRed2 can be excited with confocal imaging at 568nm, and with two photon excitation (TPE) in the red and longer NIR. eGFP was excited at 488nm for confocal and in the NIR for TPE. While there is small difference in the bleaching rate for eGFP between confocal and TPE we observed significant difference for DsRed2 where bleaching is strongest during TPE in the red wavelengths and smallest during confocal imaging. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence becomes twice as strong compared to confocal imaging. PMID:23380006
Fast elemental screening of soil and sediment profiles using small-spot energy-dispersive X-ray fluorescence: application to mining sediments geochemistry.

PubMed

Gonzalez-Fernandez, Oscar; Queralt, Ignacio

2010-09-01

Elemental analysis of different sediment cores originating from the Cartagena-La Union mining district in Spain was carried out by means of a programmable small-spot energy-dispersive X-ray fluorescence (EDXRF) spectrometer to study the distribution of heavy metals along soil profiles. Cores were obtained from upstream sediments of a mining creek, from the lowland sedimentation plain, and from a mining landfill dump (tailings pile). A programmable two-dimensional (2D) stage and a focal spot resolution of 600 μm allow us to obtain complete core mapping. Geochemical results were verified using a more powerful wavelength-dispersion X-ray fluorescence (WDXRF) technique. The data obtained was processed in order to study the statistical correlations within the elemental compositions. The results obtained allow us to observe the differential in-depth distribution of heavy metals among the sampled zones. Dump site cores exhibit a homogeneous distribution of heavy metals, whereas the alluvial plain core shows accumulation of heavy metals in the upper part. This approach can be useful for the fast screening of heavy metals in depositional environments around mining sites.
An integrated single- and two-photon non-diffracting light-sheet microscope

NASA Astrophysics Data System (ADS)

Lau, Sze Cheung; Chiu, Hoi Chun; Zhao, Luwei; Zhao, Teng; Loy, M. M. T.; Du, Shengwang

2018-04-01

We describe a fluorescence optical microscope with both single-photon and two-photon non-diffracting light-sheet excitations for large volume imaging. With a special design to accommodate two different wavelength ranges (visible: 400-700 nm and near infrared: 800-1200 nm), we combine the line-Bessel sheet (LBS, for single-photon excitation) and the scanning Bessel beam (SBB, for two-photon excitation) light sheet together in a single microscope setup. For a transparent thin sample where the scattering can be ignored, the LBS single-photon excitation is the optimal imaging solution. When the light scattering becomes significant for a deep-cell or deep-tissue imaging, we use SBB light-sheet two-photon excitation with a longer wavelength. We achieved nearly identical lateral/axial resolution of about 350/270 nm for both imagings. This integrated light-sheet microscope may have a wide application for live-cell and live-tissue three-dimensional high-speed imaging.
Feedback mechanism for smart nozzles and nebulizers

DOEpatents

Montaser, Akbar [Potomac, MD; Jorabchi, Kaveh [Arlington, VA; Kahen, Kaveh [Kleinburg, CA

2009-01-27

Nozzles and nebulizers able to produce aerosol with optimum and reproducible quality based on feedback information obtained using laser imaging techniques. Two laser-based imaging techniques based on particle image velocimetry (PTV) and optical patternation map and contrast size and velocity distributions for indirect and direct pneumatic nebulizations in plasma spectrometry. Two pulses from thin laser sheet with known time difference illuminate droplets flow field. Charge coupled device (CCL)) captures scattering of laser light from droplets, providing two instantaneous particle images. Pointwise cross-correlation of corresponding images yields two-dimensional velocity map of aerosol velocity field. For droplet size distribution studies, solution is doped with fluorescent dye and both laser induced florescence (LIF) and Mie scattering images are captured simultaneously by two CCDs with the same field of view. Ratio of LIF/Mie images provides relative droplet size information, then scaled by point calibration method via phase Doppler particle analyzer.
Construction of an efficient two-photon fluorescent probe for imaging nitroreductase in live cells and tissues.

PubMed

Zhou, Liyi; Gong, Liang; Hu, Shunqin

2018-06-15

Compared with traditional confocal microscopy, two-photon fluorescence microscopy (TPFM), which excites a two-photon (TP) fluorophore by near-infrared light, provides improved three-dimensional image resolution with increased tissue-image depth (>500μm) and an extended observation time. Therefore, the development of novel functional TP fluorophores has attracted great attention in recent years. Herein, a novel TP fluorophore CM-NH 2 , which have the donor-π-acceptor (D-π-A)-structure, was designed and synthesized. We further used this dye developed a new type of TP fluorescent probe CM-NO 2 for detecting nitroreductase (NTR). Upon incubated with NTR for 15min, CM-NO 2 displayed a ~90-fold fluorescence enhancement at 505nm and the maximal TP action cross-section value after reaction was detected and calculated to be 200 GM at 760nm. The probe exhibited excellent properties such as high sensitivity, high selectivity, low cytotoxicity, and high photostability. Moreover, the probe was utilized to image the tumor hypoxia in live HeLa cells. Finally, using the CM-NO 2 to image NTR in tissues was demonstrated. Copyright © 2018 Elsevier B.V. All rights reserved.
Mixed ligand two dimensional Cd(ii)/Ni(ii) metal organic frameworks containing dicarboxylate and tripodal N-donor ligands: Cd(ii) MOF is an efficient luminescent sensor for detection of picric acid in aqueous media.

PubMed

Rachuri, Yadagiri; Parmar, Bhavesh; Bisht, Kamal Kumar; Suresh, Eringathodi

2016-05-04

Two dimensional metal organic frameworks (MOFs) [Cd(5-BrIP)(TIB)]n () and [Ni2(5-BrIP)2(TIB)2]n (), involving the aromatic polycarboxylate ligand 5-bromo isophthalic acid (H2BrIP), flexible tripodal ligand 1,3,5-tris(imidazol-1-ylmethyl)benzene (TIB) and Cd(ii)/Ni(ii) metal nodes have been synthesized by different methods. These compounds were characterized by various analytical methods, and variable temperature X-ray diffraction data showed thermal stability of both MOFs up to 350 °C. Phase purity as well as water stability of the MOFs were established by powder X-ray diffraction, and the structural diversity of the compounds were investigated by single-crystal X-ray diffraction. Both the MOFs are mixed ligand 2D nets, and the topology of the network can be described as a binodal 3,5-c connected net with 3,5L2 topology having the point symbol {4(2)·6(7)·8}{4(2)·6}. Sensing of picric acid [2,4,6-trinitrophenol, TNP] by luminescence quenching among a large range of nitroanalytes in aqueous phase by the Cd(ii) luminescent MOF (LMOF) were been investigated. Structural studies on 1 : 1 co-crystals () of TIB and TNP were carried out. The selective and sensitive fluorescence quenching response of towards electron-deficient TNP over other nitro analytes in aqueous phase was demonstrated by fluorescence quenching titration. Concomitant occurrence of electron transfer/energy transfer processes and electrostatic interaction favours the selective sensing of TNP. A Cd(ii) LMOF ()-coated paper strip that we developed demonstrated fast and selective response to TNP, by the complete quenching of the blue fluorescence upon excitation of the paper strip at 365 nm radiation in its presence.
Fluorometric Biosniffer Camera "Sniff-Cam" for Direct Imaging of Gaseous Ethanol in Breath and Transdermal Vapor.

PubMed

Arakawa, Takahiro; Sato, Toshiyuki; Iitani, Kenta; Toma, Koji; Mitsubayashi, Kohji

2017-04-18

Various volatile organic compounds can be found in human transpiration, breath and body odor. In this paper, a novel two-dimensional fluorometric imaging system, known as a "sniffer-cam" for ethanol vapor released from human breath and palm skin was constructed and validated. This imaging system measures ethanol vapor concentrations as intensities of fluorescence through an enzymatic reaction induced by alcohol dehydrogenase (ADH). The imaging system consisted of multiple ultraviolet light emitting diode (UV-LED) excitation sheet, an ADH enzyme immobilized mesh substrate and a high-sensitive CCD camera. This imaging system uses ADH for recognition of ethanol vapor. It measures ethanol vapor by measuring fluorescence of nicotinamide adenine dinucleotide (NADH), which is produced by an enzymatic reaction on the mesh. This NADH fluorometric imaging system achieved the two-dimensional real-time imaging of ethanol vapor distribution (0.5-200 ppm). The system showed rapid and accurate responses and a visible measurement, which could lead to an analysis of metabolism function at real time in the near future.

Quantitative imaging of fibrotic and morphological changes in liver of non-alcoholic steatohepatitis (NASH) model mice by second harmonic generation (SHG) and auto-fluorescence (AF) imaging using two-photon excitation microscopy (TPEM).

PubMed

Yamamoto, Shin; Oshima, Yusuke; Saitou, Takashi; Watanabe, Takao; Miyake, Teruki; Yoshida, Osamu; Tokumoto, Yoshio; Abe, Masanori; Matsuura, Bunzo; Hiasa, Yoichi; Imamura, Takeshi

2016-12-01

Non-alcoholic steatohepatitis (NASH) is a common liver disorder caused by fatty liver. Because NASH is associated with fibrotic and morphological changes in liver tissue, a direct imaging technique is required for accurate staging of liver tissue. For this purpose, in this study we took advantage of two label-free optical imaging techniques, second harmonic generation (SHG) and auto-fluorescence (AF), using two-photon excitation microscopy (TPEM). Three-dimensional ex vivo imaging of tissues from NASH model mice, followed by image processing, revealed that SHG and AF are sufficient to quantitatively characterize the hepatic capsule at an early stage and parenchymal morphologies associated with liver disease progression, respectively.
Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation.

PubMed

He, Huaizhen; Zhan, Yingzhuan; Zhang, Yanmin; Zhang, Jie; He, Langchong

2012-01-01

Two novel taspine diphenyl derivatives (Ta-dD) were designed and synthesized by introducing different coumarin fluorescent groups into the basic structure of Ta-dD. The main advantage of these two compounds is that they can be used as fluorescence probes and inhibitors simultaneously. In the present study, the fluorescent properties of the probes were measured and their inhibition of four breast cancer cell lines was tested. Different concentrations of the fluorescence probe were added to MCF-7 breast cancer cells for fluorescence imaging analysis under normal conditions. The results suggested that both of the new compounds have not only fluorescence but also the ability to inhibit effects on different breast cancer cell lines, which indicates their possible further use as dual functional fluorescence probes in tracer analysis. Copyright © 2011 John Wiley & Sons, Ltd.
Difference in oxidative stress tolerance between rice cultivars estimated with chlorophyll fluorescence analysis.

PubMed

Kasajima, Ichiro

2017-04-26

Oxidative stress is considered to be involved in growth retardation of plants when they are exposed to a variety of biotic and abiotic stresses. Despite its potential importance in improving crop production, comparative studies on oxidative stress tolerance between rice (Oryza sativa L.) cultivars are limited. This work describes the difference in term of oxidative stress tolerance between 72 rice cultivars. 72 rice cultivars grown under naturally lit greenhouse were used in this study. Excised leaf discs were subjected to a low concentration of methyl viologen (paraquat), a chemical reagent known to generate reactive oxygen species in chloroplast. Chlorophyll fluorescence analysis using a two-dimensional fluorescence meter, ion leakage analysis as well as the measurement of chlorophyll contents were used to evaluate the oxidative stress tolerance of leaf discs. Furthermore, fluorescence intensities were finely analyzed based on new fluorescence theories that we have optimized. Treatment of leaf discs with methyl viologen caused differential decrease of maximum quantum yield of photosystem II (Fv/Fm) between cultivars. Decrease of Fv/Fm was also closely correlated with increase of ion leakage and decrease of chlorophyll a/b ratio. Fv/Fm was factorized into photochemical and non-photochemical parameters to classify rice cultivars into sensitive and tolerant ones. Among the 72 compared rice cultivars, the traditional cultivar Co13 was identified as the most tolerant to oxidative stress. Koshihikari, a dominant modern Japonica cultivar in Japan as well as IR58, one of the modern Indica breeding lines exhibited a strong tolerance to oxidative stress. Close correlation between Fv/Fm and chlorophyll a/b ratio provides a simple method to estimate oxidative stress tolerance, without measurement of chlorophyll fluorescence with special equipment. The fact that modern cultivars, especially major cultivars possessed tolerance to oxidative stress suggests that oxidative stress tolerance is one of the agricultural traits prerequisite for improvement of modern rice cultivars. Data presented in this study would enable breeding of rice cultivars having strong tolerance to oxidative stress.
Unraveling the equine lymphocyte proteome: differential septin 7 expression associates with immune cells in equine recurrent uveitis.

PubMed

Degroote, Roxane L; Hauck, Stefanie M; Amann, Barbara; Hirmer, Sieglinde; Ueffing, Marius; Deeg, Cornelia A

2014-01-01

Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease.
The increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: A potential for drug delivery system

NASA Astrophysics Data System (ADS)

Liu, Bing-Mi; Zhang, Jun; Hao, Ai-Jun; Xu, Liang; Wang, Dan; Ji, Hui; Sun, Shi-Jie; Chen, Bo-Qi; Liu, Bin

2016-02-01

The impacts of rutin and baicalin on the interaction of curcumin (CU) with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopies under imitated physiological conditions. The results showed that the fluorescence quenching of HSA by CU was a simultaneous static and dynamic quenching process, irrespective of the presence or absence of flavonoids. The binding constants between CU and HSA in the absence and presence of rutin and baicalin were 2.268 × 105 M- 1, 3.062 × 105 M- 1, and 3.271 × 105 M- 1, indicating that the binding affinity was increased in the case of two flavonoids. Furthermore, the binding distance determined according to Förster's theory was decreased in the presence of flavonoids. Combined with the fact that flavonoids and CU have the same binding site (site I), it can be concluded that they may simultaneously bind in different regions in site I, and formed a ternary complex of flavonoid-HSA-CU. Meanwhile, the results of fluorescence quenching, CD and three-dimensional fluorescence spectra revealed that flavonoids further strengthened the microenvironmental and conformational changes of HSA induced by CU binding. Therefore, it is possible to develop a novel complex involving CU, flavonoid and HSA for CU delivery. The work may provide some valuable information in terms of improving the poor bioavailabiliy of CU.
Real-time monitoring of anticancer drug release with highly fluorescent star-conjugated copolymer as a drug carrier.

PubMed

Qiu, Feng; Wang, Dali; Zhu, Qi; Zhu, Lijuan; Tong, Gangsheng; Lu, Yunfeng; Yan, Deyue; Zhu, Xinyuan

2014-04-14

Chemotherapy is one of the major systemic treatments for cancer, in which the drug release kinetics is a key factor for drug delivery. In the present work, a versatile fluorescence-based real-time monitoring system for intracellular drug release has been developed. First, two kinds of star-conjugated copolymers with different connections (e.g., pH-responsive acylhydrazone and stable ether) between a hyperbranched conjugated polymer (HCP) core and many linear poly(ethylene glycol) (PEG) arms were synthesized. Owing to the amphiphilic three-dimensional architecture, the star-conjugated copolymers could self-assemble into multimicelle aggregates from unimolecular micelles with excellent emission performance in the aqueous medium. When doxorubicin (DOX) as a model drug was encapsulated into copolymer micelles, the emission of star-conjugated copolymer and DOX was quenched. In vitro biological studies revealed that fluorescent intensities of both star-conjugated copolymer and DOX were activated when the drug was released from copolymeric micelles, resulting in the enhanced cellular proliferation inhibition against cancer cells. Importantly, pH-responsive feature of the star-conjugated copolymer with acylhydrazone linkage exhibited accelerated DOX release at a mildly acidic environment, because of the fast breakage of acylhydrazone in endosome or lysosome of tumor cells. Such fluorescent star-conjugated copolymers may open up new perspectives to real-time study of drug release kinetics of polymeric drug delivery systems for cancer therapy.
Imaging hydrogen flames by two-photon, laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Miles, R.; Lempert, W.; Kumar, V.; Diskin, G.

1991-01-01

A nonintrusive multicomponent imaging system is developed which can image hydrogen, hot oxygen, and air simultaneously. An Ar-F excimer laser is injection-locked to cover the Q1 two-photon transition in molecular hydrogen which allows the observation of both hot oxygen and cold hydrogen. Rayleigh scattering from the water molecules occurs at the same frequency as the illuminating laser allowing analysis of the air density. Images of ignited and nonignited hydrogen jets are recorded with a high-sensitivity gated video camera. The images permit the analysis of turbulent hydrogen-core jet, the combustion zone, and the surrounding air, and two-dimensional spatial correlations can be made to study the turbulent structure and couplings between different regions of the flow field. The method is of interest to the study of practical combustion systems which employ hydrogen-air diffusion flames.
High resolution multiple excitation spot optical microscopy

NASA Astrophysics Data System (ADS)

Dilipkumar, Shilpa; Mondal, Partha Pratim

2011-06-01

We propose fundamental improvements in three-dimensional (3D) resolution of multiple excitation spot optical microscopy. The excitation point spread function (PSF) is generated by two interfering counter-propagating depth-of-focus beams along the optical axis. Detection PSF is obtained by coherently interfering the emitted fluorescent light (collected by both the objectives) at the detector. System PSF shows upto 14-fold reduction in focal volume as compared to confocal, and almost 2-fold improvement in lateral resolution. Proposed PSF has the ability to simultaneously excite multiple 3D-spots of sub-femtoliter volume. Potential applications are in fluorescence microscopy and nanobioimaging.
Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.

PubMed Central

Axelrod, D; Koppel, D E; Schlessinger, J; Elson, E; Webb, W W

1976-01-01

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G. PMID:786399
Particle dynamics during nanoparticle synthesis by laser ablation in a background gas

NASA Astrophysics Data System (ADS)

Nakata, Yoshiki; Muramoto, Junichi; Okada, Tatsuo; Maeda, Mitsuo

2002-02-01

Particle dynamics during Si nanoparticle synthesis in a laser-ablation plume in different background gases were investigated by laser-spectroscopic imaging techniques. Two-dimensional laser induced fluorescence and ultraviolet Rayleigh scattering techniques were used to visualize the spatial distribution of the Si atoms and nanoparticles grown, respectively. We have developed a visualization technique called re-decomposition laser-induced fluorescence to observe small nanoparticles (hereafter called clusters) which are difficult to observe by the conventional imaging techniques. In this article, the whole process of nanoparticle synthesis in different background gases of He, Ne, Ar, N2 and O2 was investigated by these techniques. In He, Ne, Ar and N2 background gases at 10 Torr, the clustering of the Si atoms started 200, 250, 300 and 800 μs after ablation, respectively. The growth rate of the clusters in He background gas was much larger than that in the other gases. The spatial distributions of the Si nanoparticles were mushroom like in He, N2 and O2, and column like in Ne and Ar. It is thought that the difference in distribution was caused by differences in the flow characteristics of the background gases, which would imply that the viscosity of the background gas is one of the main governing parameters.
Capillary sieving electrophoresis and micellar electrokinetic capillary chromatography produce highly correlated separation of tryptic digests

PubMed Central

Dickerson, Jane A.; Dovichi, Norman J.

2011-01-01

We perform two-dimensional capillary electrophoresis on fluorescently labeled proteins and peptides. Capillary sieving electrophoresis was performed in the first dimension and micellar electrokinetic capillary chromatography was performed in the second. A cellular homogenate was labeled with the fluorogenic reagent FQ and separated using the system. This homogenate generated a pair of ridges; the first had essentially constant migration time in the CSE dimension, while the second had essentially constant migration time in the MEKC dimension. In addition a few spots were scattered through the electropherogram. The same homogenate was digested using trypsin, and then labeled and subjected to the two dimensional separation. In this case, the two ridges observed from the original two-dimensional separation disappeared, and were replaced by a set of spots that fell along the diagonal. Those spots were identified using a local-maximum algorithm and each was fit using a two-dimensional Gaussian surface by an unsupervised nonlinear least squares regression algorithm. The migration times of the tryptic digest components were highly correlated (r = 0.862). When the slowest migrating components were eliminated from the analysis, the correlation coefficient improved to r = 0.956. PMID:20564272
Combined SRCT & FXCT - The next steps

NASA Astrophysics Data System (ADS)

Hall, C.; Acres, R. G.; Winnett, A.; Wang, F.

2016-03-01

One of the goals in developing synchrotron radiation x-ray computed tomography (SRCT) for biomedical specimens, is allowing particular tissues and cell types to be marked in the images. This is equivalent to the staining in histology, which enables researchers to visualise and measure tissue structure and biochemical processes within the specimen. Some progress in this direction for SRCT is being made, using a variety of contrast agents that alter the natural x-ray attenuation of the marked tissue [1]. However there are limits to the usefulness of these attenuation altering techniques. Often high concentrations of potentially disruptive chemicals are required with reduced compatibility for in-vivo studies. Another image highlighting technique which might prove more sensitive is x-ray fluorescence imaging. In this case usually endogenous elemental markers are visualised. We would like to develop a lower resolution, but wider field of view means of three-dimensional (3-D) fluorescence imaging compatible with SRCT. We have previously proposed a technique in which x-ray fluorescence CT (FXCT) and SRCT data can be collected simultaneously [2]. This work resulted in proof of concept modelling, and a simple experiment test system. We show data here which demonstrate a two-dimensional (2-D) reconstruction of an iodine fluorescence map from a phantom. Measurements were performed with a fixed beam modulating mask using the Imaging and Medical beam line (IMBL) at the Australian Synchrotron. Fluorescence data was obtained during a CT scan using a single point detector, while transmission data was simultaneously collected using an area detector. A maximum likelihood expectation maximisation (MLEM) iterative algorithm was used to reconstruct the fluorescence map. We report on technique development and now believe compressive sensing (CS) imaging techniques suit SRCT and may overcome the issues encountered so far in combining SRCT and FXCT.
Direct three-dimensional observation of the microstructure and chemistry of C{sub 3}S hydration

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

Hu, Qinang; Aboustait, Mohammed; Kim, Taehwan

Disagreements about the mechanisms of cement hydration remain despite the fact that portland cement has been studied extensively for over 100 years. One reason for this is that direct observation of the change in microstructure and chemistry are challenging for many experimental techniques. This paper presents results from synchrotron nano X-ray tomography and fluorescence imaging. The data show unprecedented direct observations of small collections of C{sub 3}S particles before and after different periods of hydration in 15 mmol/L lime solution. X-ray absorption contrast is used to make three dimensional maps of the changes of these materials with time. The chemicalmore » compositions of hydration products are then identified with X-ray fluorescence mapping and scanning electron microscopy. These experiments are used to provide insight into the rate and morphology of the microstructure formation.« less
Lead(II) coordination polymers based on rigid-flexible 3,5-bis-oxyacetate-benzoic acid: Structural transition driven by temperature control

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

Chen, Yong-Qiang, E-mail: chenjzxy@126.com; Tian, Yuan

2017-03-15

Three Pb(II) complexes ([Pb{sub 3}(BOABA){sub 2}(H{sub 2}O)]·H{sub 2}O){sub n} (1), ([Pb{sub 4}(BOABA){sub 2}(µ{sub 4}-O)(H{sub 2}O){sub 2}]·H{sub 2}O){sub n} (2), and [Pb{sub 3}(BOABA){sub 2}(H{sub 2}O)]{sub n} (3) (H{sub 3}BOABA=3,5-bis-oxyacetate-benzoic acid) were obtained under the same reaction systems with different temperatures. Complexes 1 and 2 are two dimensional (2D) networks based on Pb-BOABA chains and Pb{sub 4}(µ{sub 4}-O)(COO){sub 6} SBUs, respectively. Complex 3 presents an interesting three dimensional (3D) framework, was obtained by increasing the reaction temperature. Structural transition of the crystallization products is largely dependent on the reaction temperature. Moreover, the fluorescence properties of complexes 1–3 have been investigated. - Graphicalmore » abstract: Three Pb(II) coordination polymers were obtained under the same reaction systems with different temperatures. Both of complexes 1 and 2 are 2D network. 3 presents a 3D framework based on Pb–O–C rods SBUs. The 2D to 3D structures transition between three complexes was achieved successfully by temperature control. - Highlights: • Three Pb(II) complexes were obtained under the same reaction systems with different temperatures. • Structural transition of the crystallization products is largely dependent on the reaction temperature. • The luminescence properties studies reveal that three complexes exhibit yellow fluorescence emission behavior, which might be good candidates for obtaining photoluminescent materials.« less
Three-dimensional rendering of segmented object using matlab - biomed 2010.

PubMed

Anderson, Jeffrey R; Barrett, Steven F

2010-01-01

The three-dimensional rendering of microscopic objects is a difficult and challenging task that often requires specialized image processing techniques. Previous work has been described of a semi-automatic segmentation process of fluorescently stained neurons collected as a sequence of slice images with a confocal laser scanning microscope. Once properly segmented, each individual object can be rendered and studied as a three-dimensional virtual object. This paper describes the work associated with the design and development of Matlab files to create three-dimensional images from the segmented object data previously mentioned. Part of the motivation for this work is to integrate both the segmentation and rendering processes into one software application, providing a seamless transition from the segmentation tasks to the rendering and visualization tasks. Previously these tasks were accomplished on two different computer systems, windows and Linux. This transition basically limits the usefulness of the segmentation and rendering applications to those who have both computer systems readily available. The focus of this work is to create custom Matlab image processing algorithms for object rendering and visualization, and merge these capabilities to the Matlab files that were developed especially for the image segmentation task. The completed Matlab application will contain both the segmentation and rendering processes in a single graphical user interface, or GUI. This process for rendering three-dimensional images in Matlab requires that a sequence of two-dimensional binary images, representing a cross-sectional slice of the object, be reassembled in a 3D space, and covered with a surface. Additional segmented objects can be rendered in the same 3D space. The surface properties of each object can be varied by the user to aid in the study and analysis of the objects. This inter-active process becomes a powerful visual tool to study and understand microscopic objects.
Dynamic measurement of fluorescent proteins spectral distribution on virus infected cells

NASA Astrophysics Data System (ADS)

Lee, Ja-Yun; Wu, Ming-Xiu; Kao, Chia-Yun; Wu, Tzong-Yuan; Hsu, I.-Jen

2006-09-01

We constructed a dynamic spectroscopy system that can simultaneously measure the intensity and spectral distributions of samples with multi-fluorophores in a single scan. The system was used to monitor the fluorescence distribution of cells infected by the virus, which is constructed by a recombinant baculoviruses, vAcD-Rhir-E, containing the red and green fluorescent protein gene that can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. The system was composed of an excitation light source, a scanning system and a spectrometer. We also developed an algorithm and fitting process to analyze the pattern of fluorescence distribution of the dual fluorescence produced in the recombinant virus-infected cells. All the algorithm and calculation are automatically processed in a visualized scanning program and can monitor the specific region of sample by calculating its intensity distribution. The spectral measurement of each pixel was performed at millisecond range and the two dimensional distribution of full spectrum was recorded within several seconds. We have constructed a dynamic spectroscopy system to monitor the process of virus-infection of cells. The distributions of the dual fluorescence were simultaneously measured at micrometer resolution.
Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots.

PubMed

Kakizuka, Taishi; Ikezaki, Keigo; Kaneshiro, Junichi; Fujita, Hideaki; Watanabe, Tomonobu M; Ichimura, Taro

2016-07-01

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery.
Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots

PubMed Central

Kakizuka, Taishi; Ikezaki, Keigo; Kaneshiro, Junichi; Fujita, Hideaki; Watanabe, Tomonobu M.; Ichimura, Taro

2016-01-01

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery. PMID:27446684
Influence of cell printing on biological characters of chondrocytes

PubMed Central

Qu, Miao; Gao, Xiaoyan; Hou, Yikang; Shen, Congcong; Xu, Yourong; Zhu, Ming; Wang, Hengjian; Xu, Haisong; Chai, Gang; Zhang, Yan

2015-01-01

Objective: To establish a two-dimensional biological printing technique of chondrocytes and compare the difference of related biological characters between printed chondrocytes and unprinted cells so as to control the cell transfer process and keep cell viability after printing. Methods: Primary chondrocytes were obtained from human mature and fetal cartilage tissues and then were regularly sub-cultured to harvest cells at passage 2 (P2), which were adjusted to the single cell suspension at a density of 1×106/mL. The experiment was divided into 2 groups: experimental group P2 chondrocytes were transferred by rapid prototype biological printer (driving voltage value 50 V, interval in x-axis 300 μm, interval in y-axis 1500 μm). Afterwards Live/Dead viability Kit and flow cytometry were respectively adopted to detect cell viability; CCK-8 Kit was adopted to detect cell proliferation viability; immunocytochemistry, immunofluorescence and RT-PCR was employed to identify related markers of chondrocytes; control group steps were the same as the printing group except that cell suspension received no printing. Results: Fluorescence microscopy and flow cytometry analyses showed that there was no significant difference between experimental group and control group in terms of cell viability. After 7-day in vitro culture, control group exhibited higher O.D values than experimental group from 2nd day to 7th day but there was no distinct difference between these two groups (P>0.05). Inverted microscope observation demonstrated that the morphology of these two groups had no significant difference either. Similarly, Immunocytochemistry, immunofluorescence and RT-PCR assays also showed that there was no significant difference in the protein and gene expression of type II collagen and aggrecan between these two groups (P>0.05). Conclusion Cell printing has no distinctly negative effect on cell vitality, proliferation and phenotype of chondrocytes. Biological printing technique may provide a novel approach for realizing the oriented, quantificational and regular distribution of chondrocytes in a two-dimensional plane and lay the foundation for the construction of three-dimensional cell printing or even organ printing system. PMID:26770337
Use of light absorbers to alter optical interrogation with epi-illumination and transillumination in three-dimensional cardiac models

NASA Astrophysics Data System (ADS)

Ramshesh, Venkat K.; Knisley, Stephen B.

2006-03-01

Cardiac optical mapping currently provides 2-D maps of transmembrane voltage-sensitive fluorescence localized near the tissue surface. Methods for interrogation at different depths are required for studies of arrhythmias and the effects of defibrillation shocks in 3-D cardiac tissue. We model the effects of coloading with a dye that absorbs excitation or fluorescence light on the radius and depth of the interrogated region with specific illumination and collection techniques. Results indicate radii and depths of interrogation are larger for transillumination versus epi-illumination, an effect that is more pronounced for broad-field excitation versus laser scanner. Coloading with a fluorescence absorber lessens interrogated depth for epi-illumination and increases it for transillumination, which is confirmed with measurements using transillumination of heart tissue slices. Coloading with an absorber of excitation light consistently decreases the interrogated depths. Transillumination and coloading also decrease the intensities of collected fluorescence. Thus, localization can be modified with wavelength-specific absorbers at the expense of a reduction in fluorescence intensity.

Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

NASA Technical Reports Server (NTRS)

Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

2004-01-01

Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.
[Rapid identification of hogwash oil by using synchronous fluorescence spectroscopy].

PubMed

Sun, Yan-Hui; An, Hai-Yang; Jia, Xiao-Li; Wang, Juan

2012-10-01

To identify hogwash oil quickly, the characteristic delta lambda of hogwash oil was analyzed by three dimensional fluorescence spectroscopy with parallel factor analysis, and the model was built up by using synchronous fluorescence spectroscopy with support vector machines (SVM). The results showed that the characteristic delta lambda of hogwash oil was 60 nm. Collecting original spectrum of different samples under the condition of characteristic delta lambda 60 nm, the best model was established while 5 principal components were selected from original spectrum and the radial basis function (RBF) was used as the kernel function, and the optimal penalty factor C and kernel function g were 512 and 0.5 respectively obtained by the grid searching and 6-fold cross validation. The discrimination rate of the model was 100% for both training sets and prediction sets. Thus, it is quick and accurate to apply synchronous fluorescence spectroscopy to identification of hogwash oil.
The interaction of C.I. acid red 27 with human hemoglobin in solution.

PubMed

Wang, Yan-Qing; Zhang, Hong-Mei; Tang, Bo-Ping

2010-08-02

The nature of the interaction between human hemoglobin and C.I. acid red 27 was investigated systematically by ultraviolet-vis absorbance, circular dichroism, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques at pH 7.40. The quenching mechanism, binding constants, and the number of binding sites were determined by the quenching of human hemoglobin fluorescence in presence of C.I. acid red 27. The results showed that the nature of the quenching was of static type and the process of binding acid red 27 on human hemoglobin was a spontaneous molecular interaction procedure. The electrostatic and hydrophobic interactions played a major role in stabilizing the complex; The distance r between donor and acceptor was obtained to be 4.40 nm according to Förster's theory; The effect of acid red 27 on the conformation of human hemoglobin was analyzed using synchronous fluorescence, circular dichroism and three-dimensional fluorescence spectra. 2010 Elsevier B.V. All rights reserved.
Three-dimensional quick response code based on inkjet printing of upconversion fluorescent nanoparticles for drug anti-counterfeiting.

PubMed

You, Minli; Lin, Min; Wang, Shurui; Wang, Xuemin; Zhang, Ge; Hong, Yuan; Dong, Yuqing; Jin, Guorui; Xu, Feng

2016-05-21

Medicine counterfeiting is a serious issue worldwide, involving potentially devastating health repercussions. Advanced anti-counterfeit technology for drugs has therefore aroused intensive interest. However, existing anti-counterfeit technologies are associated with drawbacks such as the high cost, complex fabrication process, sophisticated operation and incapability in authenticating drug ingredients. In this contribution, we developed a smart phone recognition based upconversion fluorescent three-dimensional (3D) quick response (QR) code for tracking and anti-counterfeiting of drugs. We firstly formulated three colored inks incorporating upconversion nanoparticles with RGB (i.e., red, green and blue) emission colors. Using a modified inkjet printer, we printed a series of colors by precisely regulating the overlap of these three inks. Meanwhile, we developed a multilayer printing and splitting technology, which significantly increases the information storage capacity per unit area. As an example, we directly printed the upconversion fluorescent 3D QR code on the surface of drug capsules. The 3D QR code consisted of three different color layers with each layer encoded by information of different aspects of the drug. A smart phone APP was designed to decode the multicolor 3D QR code, providing the authenticity and related information of drugs. The developed technology possesses merits in terms of low cost, ease of operation, high throughput and high information capacity, thus holds great potential for drug anti-counterfeiting.
Insights into the selective binding and toxic mechanism of microcystin to catalase

NASA Astrophysics Data System (ADS)

Hu, Yuandong; Da, Liangjun

2014-03-01

Microcystin is a sort of cyclic nonribosomal peptides produced by cyanobacteria. It is cyanotoxin, which can be very toxic for plants and animals including humans. The present study evaluated the interaction of microcystin and catalase, under physiological conditions by means of fluorescence, three-dimensional (3D) fluorescence, circular dichroism (CD), Fourier Transform infrared (FT-IR) spectroscopy, and enzymatic reactionkinetic techniques. The fluorescence data showed that microcystin could bind to catalase to form a complex. The binding process was a spontaneous molecular interaction procedure, in which electrostatic interactions played a major role. Energy transfer and fluorescence studies proved the existence of a static binding process. Additionally, as shown by the three-dimensional fluorescence, CD and FT-IR results, microcystin could lead to conformational and microenvironmental changes of the protein, which may affect the physiological functions of catalase. The work provides important insights into the toxicity mechanism of microcystin in vivo.
Effect of membrane microheterogeneity and domain size on fluorescence resonance energy transfer.

PubMed

Towles, Kevin B; Brown, Angela C; Wrenn, Steven P; Dan, Nily

2007-07-15

Studies of multicomponent membranes suggest lateral inhomogeneity in the form of membrane domains, but the size of small (nanoscale) domains in situ cannot be determined with current techniques. In this article, we present a model that enables extraction of membrane domain size from time-resolved fluorescence resonance energy transfer (FRET) data. We expand upon a classic approach to the infinite phase separation limit and formulate a model that accounts for the presence of disklike domains of finite dimensions within a two-dimensional infinite planar bilayer. The model was tested against off-lattice Monte Carlo calculations of a model membrane in the liquid-disordered (l(d)) and liquid-ordered (l(o)) coexistence regime. Simulated domain size was varied from 5 to 50 nm, and two fluorophores, preferentially partitioning into opposite phases, were randomly mixed to obtain the simulated time-resolved FRET data. The Monte Carlo data show clear differences in the efficiency of energy transfer as a function of domain size. The model fit of the data yielded good agreement for the domain size, especially in cases where the domain diameter is <20 nm. Thus, data analysis using the proposed model enables measurement of nanoscale membrane domains using time-resolved FRET.
Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ

PubMed Central

Perkins, Guy A.; Sun, Mei G.; Frey, Terrence G.

2009-01-01

Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of “virtual” 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts. PMID:19348881
Quantification of cellular autofluorescence of human skin using multiphoton tomography and fluorescence lifetime imaging in two spectral detection channels

PubMed Central

Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Neil, Mark A. A.; König, Karsten; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

2011-01-01

We explore the diagnostic potential of imaging endogenous fluorophores using two photon microscopy and fluorescence lifetime imaging (FLIM) in human skin with two spectral detection channels. Freshly excised benign dysplastic nevi (DN) and malignant nodular Basal Cell Carcinomas (nBCCs) were excited at 760 nm. The resulting fluorescence signal was binned manually on a cell by cell basis. This improved the reliability of fitting using a double exponential decay model and allowed the fluorescence signatures from different cell populations within the tissue to be identified and studied. We also performed a direct comparison between different diagnostic groups. A statistically significant difference between the median mean fluorescence lifetime of 2.79 ns versus 2.52 ns (blue channel, 300-500 nm) and 2.08 ns versus 1.33 ns (green channel, 500-640 nm) was found between nBCCs and DN respectively, using the Mann-Whitney U test (p < 0.01). Further differences in the distribution of fluorescence lifetime parameters and inter-patient variability are also discussed. PMID:22162820
Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

PubMed

Diaspro, A; Corosu, M; Ramoino, P; Robello, M

1999-11-01

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode. Copyright 1999 Wiley-Liss, Inc.
Multiple emitters in a waveguide: Nonreciprocity and correlated photons at perfect elastic transmission

NASA Astrophysics Data System (ADS)

Fang, Yao-Lung L.; Baranger, Harold U.

2017-07-01

We investigate interference and correlation effects when several detuned emitters are placed along a one-dimensional photonic waveguide. Such a setup allows multiple interactions between the photons and the strongly coupled emitters, and underlies proposed devices for quantum information processing. We show, first, that a pair of detuned two-level systems (2LS) separated by a half wavelength mimic a driven Λ -type three-level system (3LS) in both the single- and two-photon sectors. There is an interference-induced transparency peak at which the fluorescence is quenched, leaving the transmitted photons completely uncorrelated. Slightly away from this separation, we find that the inelastic scattering (fluorescence) is large, leading to nonlinear effects such as nonreciprocity (rectification). We connect this nonreciprocity to inelastic scattering caused by driving a dark pole and so derive a condition for maximum rectification. Finally, by placing a true 3LS midway between the two 2LS, we show that elastic scattering produces only transmission, but inelastic scattering nevertheless occurs (the fluorescence is not quenched) causing substantial photon correlations.
Super-resolution of fluorescence-free plasmonic nanoparticles using enhanced dark-field illumination based on wavelength-modulation

DOE PAGES

Zhang, Peng; Lee, Seungah; Yu, Hyunung; ...

2015-06-15

Super-resolution imaging of fluorescence-free plasmonic nanoparticles (NPs) was achieved using enhanced dark-field (EDF) illumination based on wavelength-modulation. Indistinguishable adjacent EDF images of 103-nm gold nanoparticles (GNPs), 40-nm gold nanorods (GNRs), and 80-nm silver nanoparticles (SNPs) were modulated at their wavelengths of specific localized surface plasmon scattering. The coordinates (x, y) of each NP were resolved by fitting their point spread functions with a two-dimensional Gaussian. The measured localization precisions of GNPs, GNRs, and SNPs were 2.5 nm, 5.0 nm, and 2.9 nm, respectively. From the resolved coordinates of NPs and the corresponding localization precisions, super-resolution images were reconstructed. Depending onmore » the spontaneous polarization of GNR scattering, the orientation angle of GNRs in two-dimensions was resolved and provided more elaborate localization information. This novel fluorescence-free super-resolution method was applied to live HeLa cells to resolve NPs and provided remarkable subdiffraction limit images.« less
Two-dimensional analysis of human lymphocyte proteins. III. Preliminary report on a marker for the early detection and diagnosis of infectious mononucleosis

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

Willard, K.E.

1982-04-01

Two-dimensional gel electrophoretic patterns of human peripheral blood leukocytes from 12 patients with infectious mononucleosis were prepared by use of the ISO-DALT system. Before the two-dimensional separation, the leukocytes were purified by Ficoll-Paque gradient centrifugation and labeled overnight with (/sup 35/S) methionine. Quantitative increases in two proteins were detected in the patterns of infected leukocytes from the patients as compared with controls. Fluorescence-activated cell sorting of leukocytes from normal human peripheral blood before subsequent two-dimensional gel analysis revealed that the dramatic increase in one of these proteins (Inmono:2) could be due to shifts in the population ratios of lymphocytes, monocytes,more » and granulocytes. In contrast, the appearance in the infected leukocytes of a second protein, Inmono:1, could not be accounted for by cell-population shifts. Increased amounts of these two proteins have been found in every patient studied who had clinically detectable infectious mononucleosis. In addition, a patient who displayed symptoms of infectious mononucleosis but who did not have a positive result in the MONOSPOT test (Ortho) until three weeks after our analysis also demonstrated increased relative amounts of these proteins in his leukocyte pattern.« less
Retention in porous layer pillar array planar separation platforms

DOE PAGES

Lincoln, Danielle R.; Lavrik, Nickolay V.; Kravchenko, Ivan I.; ...

2016-08-11

Here, this work presents the retention capabilities and surface area enhancement of highly ordered, high-aspect-ratio, open-platform, two-dimensional (2D) pillar arrays when coated with a thin layer of porous silicon oxide (PSO). Photolithographically prepared pillar arrays were coated with 50–250 nm of PSO via plasma-enhanced chemical vapor deposition and then functionalized with either octadecyltrichlorosilane or n-butyldimethylchlorosilane. Theoretical calculations indicate that a 50 nm layer of PSO increases the surface area of a pillar nearly 120-fold. Retention capabilities were tested by observing capillary-action-driven development under various conditions, as well as by running one-dimensional separations on varying thicknesses of PSO. Increasing the thicknessmore » of PSO on an array clearly resulted in greater retention of the analyte(s) in question in both experiments. In culmination, a two-dimensional separation of fluorescently derivatized amines was performed to further demonstrate the capabilities of these fabricated platforms.« less
Retention in porous layer pillar array planar separation platforms

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

Lincoln, Danielle R.; Lavrik, Nickolay V.; Kravchenko, Ivan I.

Here, this work presents the retention capabilities and surface area enhancement of highly ordered, high-aspect-ratio, open-platform, two-dimensional (2D) pillar arrays when coated with a thin layer of porous silicon oxide (PSO). Photolithographically prepared pillar arrays were coated with 50–250 nm of PSO via plasma-enhanced chemical vapor deposition and then functionalized with either octadecyltrichlorosilane or n-butyldimethylchlorosilane. Theoretical calculations indicate that a 50 nm layer of PSO increases the surface area of a pillar nearly 120-fold. Retention capabilities were tested by observing capillary-action-driven development under various conditions, as well as by running one-dimensional separations on varying thicknesses of PSO. Increasing the thicknessmore » of PSO on an array clearly resulted in greater retention of the analyte(s) in question in both experiments. In culmination, a two-dimensional separation of fluorescently derivatized amines was performed to further demonstrate the capabilities of these fabricated platforms.« less
Traffic Lights in Trichodesmium. Regulation of Photosynthesis for Nitrogen Fixation Studied by Chlorophyll Fluorescence Kinetic Microscopy1

PubMed Central

Küpper, Hendrik; Ferimazova, Naila; Šetlík, Ivan; Berman-Frank, Ilana

2004-01-01

We investigated interactions between photosynthesis and nitrogen fixation in the non-heterocystous marine cyanobacterium Trichodesmium IMS101 at the single-cell level by two-dimensional (imaging) microscopic measurements of chlorophyll fluorescence kinetics. Nitrogen fixation was closely associated with the appearance of cells with high basic fluorescence yield (F0), termed bright cells. In cultures aerated with normal air, both nitrogen fixation and bright cells appeared in the middle of the light phase. In cultures aerated with 5% oxygen, both processes occurred at a low level throughout most of the day. Under 50% oxygen, nitrogen fixation commenced at the beginning of the light phase but declined soon afterwards. Rapid reversible switches between fluorescence levels were observed, which indicated that the elevated F0 of the bright cells originates from reversible uncoupling of the photosystem II (PSII) antenna from the PSII reaction center. Two physiologically distinct types of bright cells were observed. Type I had about double F0 compared to the normal F0 in the dark phase and a PSII activity, measured as variable fluorescence (Fv = Fm − F0), similar to normal non-diazotrophic cells. Correlation of type I cells with nitrogen fixation, oxygen concentration, and light suggests that this physiological state is connected to an up-regulation of the Mehler reaction, resulting in oxygen consumption despite functional PSII. Type II cells had more than three times the normal F0 and hardly any PSII activity measurable by variable fluorescence. They did not occur under low-oxygen concentrations, but appeared under high-oxygen levels outside the diazotrophic period, suggesting that this state represents a reaction to oxidative stress not necessarily connected to nitrogen fixation. In addition to the two high-fluorescence states, cells were observed to reversibly enter a low-fluorescence state. This occurred mainly after a cell went through its bright phase and may represent a fluorescence-quenching recovery phase. PMID:15299119
Correlations of trace elements in breast human tissues: Evaluation of spatial distribution using μ-XRF

NASA Astrophysics Data System (ADS)

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

2012-05-01

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

NASA Astrophysics Data System (ADS)

Yamamoto, Kazuya; Ishimaru, Ichirou; Fujii, Yoshiki; Yasokawa, Toshiki; Kuriyama, Shigeki; Masaki, Tsutomu; Takegawa, Kaoru; Tanaka, Naotaka

2007-01-01

This letter reports on a method for accurately determining the component distribution in a cell membrane over the entire cell surface. This method involves exciting a fluorescent-dyed cell membrane using evanescent light and scanning the entire cell surface by rotating the cell using a noncontact technique, namely, proximal two-beam optical tweezers. To position the cell membrane in the thin evanescent field, the authors designed an optical system capable of precisely positioning the focal position. Using this method, they were able to measure the surface distribution of glycoprotein labeled by lectin in a breast cancer cell membrane.
In vitro three-dimensional coculturing poly3-hydroxybutyrate-co-3-hydroxyhexanoate with mouse-induced pluripotent stem cells for myocardial patch application.

PubMed

Shijun, Xu; Junsheng, Mu; Jianqun, Zhang; Ping, Bo

2016-03-01

Identifying a suitable polymeric biomaterial for myocardial patch repair following myocardial infarction, cerebral infarction, and cartilage injury is essential. This study aimed to investigate the effect of the novel polymer material, poly3-hydroxybutyrate-co-3-hydroxyhexanoate, on the adhesion, proliferation, and differentiation of mouse-induced pluripotent stem cells in vitro. Mouse-induced pluripotent stem cells were isolated, expanded, and cultured on either two-dimensional or three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films (membranes were perforated to imitate three-dimensional space). Following attachment onto the films, mouse-induced pluripotent stem cell morphology was visualized using scanning electron microscopy. Cell vitality was detected using the Cell Counting Kit-8 assay and cell proliferation was observed using fluorescent 4',6-diamidino-2-phenylindole (DAPI) staining. Mouse-induced pluripotent stem cells were induced into cardiomyocytes by differentiation medium containing vitamin C. A control group in the absence of an inducer was included. Mouse-induced pluripotent stem cell survival and differentiation were observed using immunofluorescence and flow cytometry, respectively. Mouse-induced pluripotent stem cells growth, proliferation, and differentiation were observed on both two-dimensional and three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Vitamin C markedly improved the efficiency of mouse-induced pluripotent stem cells differentiation into cardiomyocytes on poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Three-dimensional culture was better at promoting mouse-induced pluripotent stem cell proliferation and differentiation compared with two-dimensional culture. © The Author(s) 2016.
In situ monitoring of tracer tests: how to distinguish tracer recovery from natural background

NASA Astrophysics Data System (ADS)

Bailly-Comte, V.; Durepaire, X.; Batiot-Guilhe, C.; Schnegg, P.-A.

2018-03-01

Hydrogeological tracer tests are primarily conducted with fluorescent tracers. Field fluorometers make it possible to monitor tracers at very low concentrations (<1 ppb) and at high frequency. However, changes in natural fluorescence at a site resulting from variations of dissolved and suspended inorganic and organic material may compromise the measurement of useful signals, thereby limiting the chances of identifying or quantifying the real tracer recovery. An elevated natural signal can mask small concentrations of the tracer while its variability can give the impression of a false recovery. This article shows how the use of a combination of several continuous measurements at different wavelengths allows a better extraction of the natural signal. Field multispectral fluorometers were installed at two Mediterranean karst outlets; both drain carbonate systems but have different environmental conditions. The fluorometers functioned over several hydrologic cycles, in periods affected or not by artificial tracers, making it possible to observe natural signal variations at these sites. The optical properties of this type of field fluorometer were used to calculate the spectral response of the different optics of the measuring probe. These responses, superimposed on three-dimensional excitation/emission matrices produced from laboratory fluorescence measurements, allowed an understanding of what the fluorometer sees under natural flow conditions. The result is an innovative method for correcting artificial tracer results. This type of correction makes it possible to fine-tune the effect of natural background variation on tracer recovery curves for a clear identification of the tracer presence and a more precise quantification of its recovery.
[Fluorescence characterization of dissolved organic matter in the East China Sea after diatom red tide dispersion].

PubMed

Zhuo, Peng-ji; Zhao, Wei-hong

2009-05-01

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

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

PubMed

Göröcs, Zoltán; Ling, Yuye; Yu, Meng Dai; Karahalios, Dimitri; Mogharabi, Kian; Lu, Kenny; Wei, Qingshan; Ozcan, Aydogan

2013-11-21

We demonstrate a new fluorescent imaging technique that can screen for fluorescent micro-objects over an ultra-wide field-of-view (FOV) of ~532 cm(2), i.e., 19 cm × 28 cm, reaching a space-bandwidth product of more than 2 billion. For achieving such a large FOV, we modified the hardware and software of a commercially available flatbed scanner, and added a custom-designed absorbing fluorescent filter, a two-dimensional array of external light sources for computer-controlled and high-angle fluorescent excitation. We also re-programmed the driver of the scanner to take full control of the scanner hardware and achieve the highest possible exposure time, gain and sensitivity for detection of fluorescent micro-objects through the gradient index self-focusing lens array that is positioned in front of the scanner sensor chip. For example, this large FOV of our imaging platform allows us to screen more than 2.2 mL of undiluted whole blood for detection of fluorescent micro-objects within <5 minutes. This high-throughput fluorescent imaging platform could be useful for rare cell research and cytometry applications by enabling rapid screening of large volumes of optically dense media. Our results constitute the first time that a flatbed scanner has been converted to a fluorescent imaging system, achieving a record large FOV.
A Water-Stable Metal-Organic Framework for Highly Sensitive and Selective Sensing of Fe3+ Ion.

PubMed

Hou, Bing-Lei; Tian, Dan; Liu, Jiang; Dong, Long-Zhang; Li, Shun-Li; Li, Dong-Sheng; Lan, Ya-Qian

2016-10-17

A new metal-organic framework [Zn 5 (hfipbb) 4 (trz) 2 (H 2 O) 2 ] (NNU-1) [H 2 hfipbb = 4,4'-(hexafluoroisopropylidene)bis(benzoic acid), Htrz = 1H-1,2,3-triazole] was assembled by hydrothermal synthesis. Single-crystal X-ray diffraction analysis reveals that NNU-1 displays a twofold interpenetrating three-dimensional (3D) framework with a {4 24 ·6 4 }-bcu topology. Interestingly, the 3D framework contains a two-dimensional (2D) layered structure that consists of alternating left- and right-handed double helical chains. On the basis of the hydrophobic -CF 3 groups from H 2 hfipbb ligand, NNU-1 possesses excellent stability in water. It is worth noting that NNU-1 not only shows a highly selective fluorescence quenching effect to Fe 3+ ion in aqueous solution but also resists the interference of other metals including Fe 2+ ion. Accordingly, NNU-1 probably functions as a potential promising fluorescence sensor for detecting Fe 3+ ion with high sensitivity and selectivity.
A volumetric three-dimensional digital light photoactivatable dye display

NASA Astrophysics Data System (ADS)

Patel, Shreya K.; Cao, Jian; Lippert, Alexander R.

2017-07-01

Volumetric three-dimensional displays offer spatially accurate representations of images with a 360° view, but have been difficult to implement due to complex fabrication requirements. Herein, a chemically enabled volumetric 3D digital light photoactivatable dye display (3D Light PAD) is reported. The operating principle relies on photoactivatable dyes that become reversibly fluorescent upon illumination with ultraviolet light. Proper tuning of kinetics and emission wavelengths enables the generation of a spatial pattern of fluorescent emission at the intersection of two structured light beams. A first-generation 3D Light PAD was fabricated using the photoactivatable dye N-phenyl spirolactam rhodamine B, a commercial picoprojector, an ultraviolet projector and a custom quartz imaging chamber. The system displays a minimum voxel size of 0.68 mm3, 200 μm resolution and good stability over repeated `on-off' cycles. A range of high-resolution 3D images and animations can be projected, setting the foundation for widely accessible volumetric 3D displays.
A volumetric three-dimensional digital light photoactivatable dye display

PubMed Central

Patel, Shreya K.; Cao, Jian; Lippert, Alexander R.

2017-01-01

Volumetric three-dimensional displays offer spatially accurate representations of images with a 360° view, but have been difficult to implement due to complex fabrication requirements. Herein, a chemically enabled volumetric 3D digital light photoactivatable dye display (3D Light PAD) is reported. The operating principle relies on photoactivatable dyes that become reversibly fluorescent upon illumination with ultraviolet light. Proper tuning of kinetics and emission wavelengths enables the generation of a spatial pattern of fluorescent emission at the intersection of two structured light beams. A first-generation 3D Light PAD was fabricated using the photoactivatable dye N-phenyl spirolactam rhodamine B, a commercial picoprojector, an ultraviolet projector and a custom quartz imaging chamber. The system displays a minimum voxel size of 0.68 mm3, 200 μm resolution and good stability over repeated ‘on-off’ cycles. A range of high-resolution 3D images and animations can be projected, setting the foundation for widely accessible volumetric 3D displays. PMID:28695887
Two-dimensional imaging of molecular hydrogen in H2-air diffusion flames using two-photon laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Lempert, W.; Kumar, V.; Glesk, I.; Miles, R.; Diskin, G.

1991-01-01

The use of a tunable ArF laser at 193.26 nm to record simultaneous single-laser-shot, planar images of molecular hydrogen and hot oxygen in a turbulent H2-air diffusion flame. Excitation spectra of fuel and oxidant-rich flame zones confirm a partial overlap of the two-photon H2 and single-photon O2 Schumann-Runge absorption bands. UV Rayleigh scattering images of flame structure and estimated detection limits for the H2 two-photon imaging are also presented.
Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator.

PubMed

Shao, Yonghong; Qin, Wan; Liu, Honghai; Qu, Junle; Peng, Xiang; Niu, Hanben; Gao, Bruce Z

2012-07-01

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 μm × 136 μm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.
Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots*

PubMed Central

Li, Lei; Nelson, Clark J.; Solheim, Cory; Whelan, James; Millar, A. Harvey

2012-01-01

The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a 15N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (KS) and degradation (KD) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify KS and KD for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. KS and KD correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive 15N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability. PMID:22215636
Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

PubMed Central

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-01-01

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. PMID:21641327
Exploring the affinity binding of alkylmaltoside surfactants to bovine serum albumin and their effect on the protein stability: A spectroscopic approach.

PubMed

Hierrezuelo, J M; Carnero Ruiz, C

2015-08-01

Steady-state and time-resolved fluorescence together with circular dichroism (CD) spectroscopic studies was performed to examine the interactions between bovine serum albumin (BSA) and two alkylmaltoside surfactants, i.e. n-decyl-β-D-maltoside (β-C10G2) and n-dodecyl-β-D-maltoside (β-C12G2), having identical structures but different tail lengths. Changes in the intrinsic fluorescence of BSA from static as well as dynamic measurements revealed a weak protein-surfactant interaction and gave the corresponding binding curves, suggesting that the binding mechanism of surfactants to protein is essentially cooperative in nature. The behavior of both surfactants is similar, so that the differences detected were attributed to the more hydrophobic nature of β-C12G2, which favors the adsorption of micelle-like aggregates onto the protein surface. These observations were substantially demonstrated by data derived from synchronous, three-dimensional and anisotropy fluorescence experiments. Changes in the secondary structure of the protein induced by the interaction with surfactants were analyzed by CD to determine the contents of α-helix and β-strand. It was noted that whereas the addition of β-C10G2 appears to stabilize the secondary structure of the protein, β-C12G2 causes a marginal denaturation of BSA for a protein:surfactant molar ratio as high as 1 to 100. Copyright © 2015 Elsevier B.V. All rights reserved.
Three-dimensional confocal morphometry – a new approach for studying dynamic changes in cell morphology in brain slices

PubMed Central

Chvátal, Alexandr; Anděrová, Miroslava; Kirchhoff, Frank

2007-01-01

Pathological states in the central nervous system lead to dramatic changes in the activity of neuroactive substances in the extracellular space, to changes in ionic homeostasis and often to cell swelling. To quantify changes in cell morphology over a certain period of time, we employed a new technique, three-dimensional confocal morphometry. In our experiments, performed on enhanced green fluorescent protein/glial fibrillary acidic protein astrocytes in brain slices in situ and thus preserving the extracellular microenvironment, confocal morphometry revealed that the application of hypotonic solution evoked two types of volume change. In one population of astrocytes, hypotonic stress evoked small cell volume changes followed by a regulatory volume decrease, while in the second population volume changes were significantly larger without subsequent volume regulation. Three-dimensional cell reconstruction revealed that even though the total astrocyte volume increased during hypotonic stress, the morphological changes in various cell compartments and processes were more complex than have been previously shown, including swelling, shrinking and structural rearrangement. Our data show that astrocytes in brain slices in situ during hypotonic stress display complex behaviour. One population of astrocytes is highly capable of cell volume regulation, while the second population is characterized by prominent cell swelling, accompanied by plastic changes in morphology. It is possible to speculate that these two astrocyte populations play different roles during physiological and pathological states. PMID:17488344
Influence of depositional environment in fossil teeth: a micro-XRF and XAFS study

NASA Astrophysics Data System (ADS)

Zougrou, I. M.; Katsikini, M.; Pinakidou, F.; Papadopoulou, L.; Tsoukala, E.; Paloura, E. C.

2014-04-01

The formation of metal-rich phases during the fossilization of vertebrate fossil teeth, recovered from various deposition environments in northern Greece, is studied by means of synchrotron radiation X-ray fluorescence (SR-XRF) as well as Fe and Mn K edge X-ray absorption fine structure (XAFS) spectroscopy. XRF line-scans from the samples' cross-sections revealed different contamination paths for Mn and Fe. The two-dimensional XRF maps illustrate the spatial distribution of P, Ca, Mn and Fe as well as the precipitation of Fe-rich phases in cementum, dentin and dentinal tubules. Goethite, lepidocrocite and ferrihydrite were detected in the samples' cross-section by means of Fe K edge EXAFS spectroscopy. Moreover the Fe and Mn K edge EXAFS revealed the presence of vivianite and birnessite (MnO2) on the external surface of two samples.
5-ALA induced fluorescent image analysis of actinic keratosis

NASA Astrophysics Data System (ADS)

Cho, Yong-Jin; Bae, Youngwoo; Choi, Eung-Ho; Jung, Byungjo

2010-02-01

In this study, we quantitatively analyzed 5-ALA induced fluorescent images of actinic keratosis using digital fluorescent color and hyperspectral imaging modalities. UV-A was utilized to induce fluorescent images and actinic keratosis (AK) lesions were demarcated from surrounding the normal region with different methods. Eight subjects with AK lesion were participated in this study. In the hyperspectral imaging modality, spectral analysis method was utilized for hyperspectral cube image and AK lesions were demarcated from the normal region. Before image acquisition, we designated biopsy position for histopathology of AK lesion and surrounding normal region. Erythema index (E.I.) values on both regions were calculated from the spectral cube data. Image analysis of subjects resulted in two different groups: the first group with the higher fluorescence signal and E.I. on AK lesion than the normal region; the second group with lower fluorescence signal and without big difference in E.I. between two regions. In fluorescent color image analysis of facial AK, E.I. images were calculated on both normal and AK lesions and compared with the results of hyperspectral imaging modality. The results might indicate that the different intensity of fluorescence and E.I. among the subjects with AK might be interpreted as different phases of morphological and metabolic changes of AK lesions.
Characterization of spectral responses of dissolved organic matter (DOM) for atrazine binding during the sorption process onto black soil.

PubMed

Wang, Yifan; Zhang, Xinyuan; Zhang, Xing; Meng, Qingjuan; Gao, Fengjie; Zhang, Ying

2017-08-01

This study was aim to investigate the interaction between soil-derived dissolved organic matter (DOM) and atrazine as a kind of pesticides during the sorption process onto black soil. According to the experimental data, the adsorption capacity of Soil + DOM, Soil and DOM were 41.80, 31.45 and 9.35 mg kg -1 , separately, which indicated that DOM significantly enhanced the adsorption efficiency of atrazine by soil. Data implied that the pseudo-second-order kinetic equation could well explain the adsorption process. The adsorption isotherms (R 2 > 0.99) had a satisfactory fit in both Langmuir and Freundlich models. Three-dimensional excitation-emission matrix (3D-EEM), synchronous fluorescence, two-dimensional correlation spectroscopy (2D-COS) and Fourier transform infrared spectroscopy (FT-IR) were selected to analyze the interaction between DOM and atrazine. 3D-EEM showed that humic acid-like substances were the main component of DOM. The fluorescence of DOM samples were gradually quenched with the increased of atrazine concentrations. Synchronous fluorescence spectra showed that static fluorescence quenching was the main quenching process. 2D-COS indicated that the order of the spectral changes were as following: 336 nm > 282 nm. Furthermore, the fluorescence quenching of humic-like fraction occurred earlier than that of protein-like fraction under atrazine surroundings. FT-IR spectra indicated that main compositions of soil DOM include proteins, polysaccharides and humic substances. The findings of this study are significant to reveal DOM played an important role in the environmental fate of pesticides during sorption process onto black soil and also provide more useful information for understanding the interaction between DOM and pesticides by using spectral responses. Copyright © 2017. Published by Elsevier Ltd.
Fluorescence Molecular Tomography: Principles and Potential for Pharmaceutical Research

PubMed Central

Stuker, Florian; Ripoll, Jorge; Rudin, Markus

2011-01-01

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue's optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development. PMID:24310495
Capillary Array Waveguide Amplified Fluorescence Detector for mHealth

PubMed Central

Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

2013-01-01

Mobile Health (mHealth) analytical technologies are potentially useful for carrying out modern medical diagnostics in resource-poor settings. Effective mHealth devices for underserved populations need to be simple, low cost, and portable. Although cell phone cameras have been used for biodetection, their sensitivity is a limiting factor because currently it is too low to be effective for many mHealth applications, which depend on detection of weak fluorescent signals. To improve the sensitivity of portable phones, a capillary tube array was developed to amplify fluorescence signals using their waveguide properties. An array configured with 36 capillary tubes was demonstrated to have a ~100X increase in sensitivity, lowering the limit of detection (LOD) of mobile phones from 1000 nM to 10 nM for fluorescein. To confirm that the amplification was due to waveguide behavior, we coated the external surfaces of the capillaries with silver. The silver coating interfered with the waveguide behavior and diminished the fluorescence signal, thereby proving that the waveguide behavior was the main mechanism for enhancing optical sensitivity. The optical configuration described here is novel in several ways. First, the use of capillaries waveguide properties to improve detection of weak florescence signal is new. Second we describe here a three dimensional illumination system, while conventional angular laser waveguide illumination is spot (or line), which is functionally one-dimensional illumination, can illuminate only a single capillary or a single column (when a line generator is used) of capillaries and thus inherently limits the multiplexing capability of detection. The planar illumination demonstrated in this work enables illumination of a two dimensional capillary array (e.g. x columns and y rows of capillaries). In addition, the waveguide light propagation via the capillary wall provides a third dimension for illumination along the axis of the capillaries. Such an array can potentially be used for sensitive analysis of multiple fluorescent detection assays simultaneously. The simple phone based capillary array approach presented in this paper is capable of amplifying weak fluorescent signals thereby improving the sensitivity of optical detectors based on mobile phones. This may allow sensitive biological assays to be measured with low sensitivity detectors and may make mHealth practical for many diagnostics applications, especially in resource-poor and global health settings. PMID:24039345
Hindered diffusion of high molecular weight compounds in brain extracellular microenvironment measured with integrative optical imaging.

PubMed

Nicholson, C; Tao, L

1993-12-01

This paper describes the theory of an integrative optical imaging system and its application to the analysis of the diffusion of 3-, 10-, 40-, and 70-kDa fluorescent dextran molecules in agarose gel and brain extracellular microenvironment. The method uses a precisely defined source of fluorescent molecules pressure ejected from a micropipette, and a detailed theory of the intensity contributions from out-of-focus molecules in a three-dimensional medium to a two-dimensional image. Dextrans tagged with either tetramethylrhodamine or Texas Red were ejected into 0.3% agarose gel or rat cortical slices maintained in a perfused chamber at 34 degrees C and imaged using a compound epifluorescent microscope with a 10 x water-immersion objective. About 20 images were taken at 2-10-s intervals, recorded with a cooled CCD camera, then transferred to a 486 PC for quantitative analysis. The diffusion coefficient in agarose gel, D, and the apparent diffusion coefficient, D*, in brain tissue were determined by fitting an integral expression relating the measured two-dimensional image intensity to the theoretical three-dimensional dextran concentration. The measurements in dilute agarose gel provided a reference value of D and validated the method. Values of the tortuosity, lambda = (D/D*)1/2, for the 3- and 10-kDa dextrans were 1.70 and 1.63, respectively, which were consistent with previous values derived from tetramethylammonium measurements in cortex. Tortuosities for the 40- and 70-kDa dextrans had significantly larger values of 2.16 and 2.25, respectively. This suggests that the extracellular space may have local constrictions that hinder the diffusion of molecules above a critical size that lies in the range of many neurotrophic compounds.
Time-resolved multicolor two-photon excitation fluorescence microscopy of cells and tissues

NASA Astrophysics Data System (ADS)

Zheng, Wei

2014-11-01

Multilabeling which maps the distribution of different targets is an indispensable technique in many biochemical and biophysical studies. Two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with conventional fluorescence labeling techniques such as genetically encoded fluorescent protein (FP) and fluorescent dyes staining could be a powerful tool for imaging living cells. However, the challenge is that the excitation and emission wavelength of these endogenous fluorophores and fluorescent labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores, fluorescent proteins and fluorescent dyes were excited in their optimal wavelengths simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and wavelength domains. Cellular organelles such as nucleus, mitochondria, microtubule and endoplasmic reticulum, were clearly revealed in the TPEF images. The simultaneous imaging of multiple fluorophores of cells will greatly aid the study of sub-cellular compartments and protein localization.
A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy.

PubMed

Verveer, P. J; Gemkow, M. J; Jovin, T. M

1999-01-01

We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide-field data. In general, restored confocal data are superior to restored wide-field data, but given sufficient higher signal level for the wide-field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide-field data is presented.
Three-Dimensional Dynamic Bone Histomorphometry

PubMed Central

Slyfield, C.R.; Tkachenko, E.V.; Wilson, D.L.; Hernandez, C.J.

2011-01-01

Dynamic bone histomorphometry is the standard method for measuring bone remodeling at the level of individual events. While dynamic bone histomorphometry is an invaluable tool for understanding osteoporosis and other metabolic bone diseases, the technique’s two-dimensional nature requires the use of stereology and prevents measures of individual remodeling event number and size. Here, we use a novel three-dimensional fluorescence imaging technique to achieve measures of individual resorption cavities and formation events. We perform this three-dimensional histomorphometry approach using a common model of postmenopausal osteoporosis, the ovariectomized rat. The three-dimensional images demonstrate the spatial relationship between resorption cavities and formation events consistent with the hemi-osteonal model of cancellous bone remodeling. Established ovariectomy was associated with significant increases in the number of resorption cavities per unit bone surface (2.38 ± 0.24 mm−2 SHAM v. 3.86 ± 0.35 mm−2 OVX, mean ± SD, p < 0.05) and total volume occupied by cavities per unit bone volume (0.38 ± 0.06% SHAM v. 1.12 ± 0.18% OVX, p < 0.001), but no difference in surface area per resorption cavity, maximum cavity depth, or cavity volume. Additionally, we find that established ovariectomy is associated with increased size of bone formation events due to merging of formation events (23,700 ± 6,890 μm2 SHAM v. 33,300 ± 7,950 μm2 OVX). No differences in mineral apposition rate (determined in 3D) were associated with established ovariectomy. That established estrogen depletion is associated with increased number of remodeling events with only subtle changes in remodeling event size suggests that circulating estrogens may have their primary effect on the origination of new basic multicellular units with relatively little effect on the progression and termination of active remodeling events. PMID:22028195

Quantitative and qualitative measure of intralaboratory two-dimensional protein gel reproducibility and the effects of sample preparation, sample load, and image analysis.

PubMed

Choe, Leila H; Lee, Kelvin H

2003-10-01

We investigate one approach to assess the quantitative variability in two-dimensional gel electrophoresis (2-DE) separations based on gel-to-gel variability, sample preparation variability, sample load differences, and the effect of automation on image analysis. We observe that 95% of spots present in three out of four replicate gels exhibit less than a 0.52 coefficient of variation (CV) in fluorescent stain intensity (% volume) for a single sample run on multiple gels. When four parallel sample preparations are performed, this value increases to 0.57. We do not observe any significant change in quantitative value for an increase or decrease in sample load of 30% when using appropriate image analysis variables. Increasing use of automation, while necessary in modern 2-DE experiments, does change the observed level of quantitative and qualitative variability among replicate gels. The number of spots that change qualitatively for a single sample run in parallel varies from a CV = 0.03 for fully manual analysis to CV = 0.20 for a fully automated analysis. We present a systematic method by which a single laboratory can measure gel-to-gel variability using only three gel runs.
Dynamic Imaging of Mouse Embryos and Cardiodynamics in Static Culture.

PubMed

Lopez, Andrew L; Larina, Irina V

2018-01-01

The heart is a dynamic organ that quickly undergoes morphological and mechanical changes through early embryonic development. Characterizing these early moments is important for our understanding of proper embryonic development and the treatment of heart disease. Traditionally, tomographic imaging modalities and fluorescence-based microscopy are excellent approaches to visualize structural features and gene expression patterns, respectively, and connect aberrant gene programs to pathological phenotypes. However, these approaches usually require static samples or fluorescent markers, which can limit how much information we can derive from the dynamic and mechanical changes that regulate heart development. Optical coherence tomography (OCT) is unique in this circumstance because it allows for the acquisition of three-dimensional structural and four-dimensional (3D + time) functional images of living mouse embryos without fixation or contrast reagents. In this chapter, we focus on how OCT can visualize heart morphology at different stages of development and provide cardiodynamic information to reveal mechanical properties of the developing heart.
UV reactor flow visualization and mixing quantification using three-dimensional laser-induced fluorescence.

PubMed

Gandhi, Varun; Roberts, Philip J W; Stoesser, Thorsten; Wright, Harold; Kim, Jae-Hong

2011-07-01

Three-dimensional laser-induced fluorescence (3DLIF) was applied to visualize and quantitatively analyze mixing in a lab-scale UV reactor consisting of one lamp sleeve placed perpendicular to flow. The recirculation zone and the von Karman vortex shedding that commonly occur in flows around bluff bodies were successfully visualized. Multiple flow paths were analyzed by injecting the dye at various heights with respect to the lamp sleeve. A major difference in these pathways was the amount of dye that traveled close to the sleeve, i.e., a zone of higher residence time and higher UV exposure. Paths away from the center height had higher velocities and hence minimal influence by the presence of sleeve. Approach length was also characterized in order to increase the probability of microbes entering the region around the UV lamp. The 3DLIF technique developed in this study is expected to provide new insight on UV dose delivery useful for the design and optimization of UV reactors. Copyright © 2011 Elsevier Ltd. All rights reserved.
Identification of nitrate sources in groundwater using a stable isotope and 3DEEM in a landfill in Northeast China.

PubMed

Ma, Zhifei; Yang, Yu; Lian, Xinying; Jiang, Yonghai; Xi, Beidou; Peng, Xing; Yan, Kun

2016-09-01

The groundwater was sampled in a typical landfill area of the Northeast China. Coupled stable isotope and three dimensional excitation-emission matrix (3DEEM) were applied to dentify diffused NO3(-) inputs in the groundwater in this area. The results indicated that combined with the feature of groundwater hydrochemistry and three-dimensional fluorescence technology can effectively identify the nitrate pollution sources. The nitrate was derived from manure and sewage by δ(15)N and δ(18)O-NO3(-) values of groundwater in the different periods. The excitation-emission matrix fluorescence spectroscopy was further evidence of groundwater DOM mainly which comes from the landfill. The protein-like was very significant at the sampling points near the landfill (SPNL), but only fulvic acid-like appeared at downstream of the landfill groundwater sampling points (DLGSP) in the study area. Partial denitrification processes helped to attenuate nitrate concentration in anaerobic environment. Copyright © 2016 Elsevier B.V. All rights reserved.
Fluorescent component and complexation mechanism of extracellular polymeric substances during dye wastewater biotreatment by anaerobic granular sludge

NASA Astrophysics Data System (ADS)

Li, Na; Wei, Dong; Sun, Qunqun; Han, Xiao; Du, Bin; Wei, Qin

2018-02-01

In this study, methylene blue (MB) wastewater was biotreated by anaerobic granular sludge (AnGS), and the fluorescent components of extracellular polymeric substances (EPS) and complexation mechanism were evaluated. Based on the experimental data, the sorption of MB by both live and inactivated AnGS followed the pseudo-second-order model, and the adsorption isotherm conformed well to the Langmuir model. It was shown that the difference in the sorption of live and inactivated AnGS was not significant, indicating that the sorption is mainly a physical-chemical process and metabolically mediated diffusion is negligible. The interaction between EPS and MB was proved by three-dimensional excitation-emission matrix (3D-EEM) and synchronous fluorescence spectra. 3D-EEM indicated that protein (PN)-like substances were the main peaks of EPS, and gradually quenched with increase of MB concentrations. According to synchronous fluorescence spectra, the main fluorescence quenching was caused by PN-like and humic-like fractions, and belonged to the static type of quenching. FTIR spectra demonstrated that hydroxyl and amino groups played a major role in MB sorption.
Graphene and graphene-like 2D materials for optical biosensing and bioimaging: a review

NASA Astrophysics Data System (ADS)

Zhu, Chengzhou; Du, Dan; Lin, Yuehe

2015-09-01

The increasing demands of bioassay and biomedical applications have significantly promoted the rational design and fabrication of a wide range of functional nanomaterials. Coupling these advanced nanomaterials with biomolecule recognition events leads to novel sensing and diagnostic platforms. Because of their unique structures and multifunctionalities, two-dimensional nanomaterials, such as graphene and graphene-like materials (e.g., graphitic carbon nitride, transition metal dichalcogenides, boron nitride, and transition metal oxides), have stimulated great interest in the field of optical biosensors and imaging because of their innovative mechanical, physicochemical and optical properties. Depending on the different applications, the graphene and graphene-like nanomaterials can be tailored to form either fluorescent emitters or efficient fluorescence quenchers, making them powerful platforms for fabricating a series of optical biosensors to sensitively detect various targets including ions, small biomolecules, DNA/RNA and proteins. This review highlights the recent progress in optical biosensors based on graphene and graphene-like 2D materials and their imaging applications. Finally, the opportunities and some critical challenges in this field are also addressed.
Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines.

PubMed

Hearst, Scoty M; Gilder, Andrew S; Negi, Sandeep S; Davis, Misty D; George, Eric M; Whittom, Angela A; Toyota, Cory G; Husedzinovic, Alma; Gruss, Oliver J; Hebert, Michael D

2009-06-01

Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells.
Three-dimensional behavior of ice crystals and biological cells during freezing of cell suspensions.

PubMed

Ishiguro, H; Koike, K

1998-09-11

Behavior of ice crystals and human red blood cells during extracellular-freezing was investigated in three-dimensions using a confocal laser scanning microscope(CLSM), which noninvasively produces tomograms of biological materials. Physiological saline and physiological saline with 2.4 M glycerol were used for suspension. Various cooling rates for directional solidification were used for distinctive morphology of the ice crystals. Addition of acridine orange as a fluorescent dye into the cell suspension enabled ice crystal, cells and unfrozen solution to be distinguished by different colors. The results indicate that the microscopic structure is three-dimensional for flat, cellular, and dendritic solid-liquid interfaces and that a CLSM is very effective in studying three-dimensional structure during the freezing of cell suspensions.
Applications of Laser Scattering Probes to Turbulent Diffusion Flames

DTIC Science & Technology

1983-11-01

APPLICATIONS OF LASER SCATTERING PROBES TO TURBULENT DIFFUSION FLAMES u ^ j FINAL REPORT Contract N00014-80-C-0882 Submitted to Office of...Include Security Classification) Applications of Laser Scattering Probes to Turbulent Diffusion Flames PROJECT NO. TASK NO. WORK UNIT NO. 12...for a co-flowing jet turbulent diffusion flame, and planar laser-induced fluorescence to provide two- dimensional instantaneous images of the flame
Spectral ’Fingerprinting’ of Phytoplankton Populations by Two-Dimensional Fluorescence and Fourier-Transform-Based Pattern Recognition.

DTIC Science & Technology

1985-07-08

comparison to a library of known spectra. A preliminary study (Warner et al., 1984) of the application of this method to the pattern recognition of...case, the spectra from two blue-green algae are shown. Figure 3A indicates phycocyanin as the major fluorophore and 3B indicates phycoerythrin. Except...445. Ho, C.H., G.D. Christian, and E.R. Davidson, 1978. Application of the method of rank annihilation to quantitative analyses of multicomponent
Correlative FRET: new method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture

NASA Astrophysics Data System (ADS)

Shinogle-Decker, Heather; Martinez-Rivera, Noraida; O'Brien, John; Powell, Richard D.; Joshi, Vishwas N.; Connell, Samuel; Rosa-Molinar, Eduardo

2018-02-01

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence; in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies.
Fluorescent components and spatial patterns of chromophoric dissolved organic matters in Lake Taihu, a large shallow eutrophic lake in China.

PubMed

Yao, Bo; Hu, Chunming; Liu, Qingquan

2016-11-01

Water samples at both surface and bottom layers were taken from 102 sites in Lake Taihu to study the fluorescent components and spatial patterns of chromophoric dissolved organic matters (CDOM). Three-dimensional excitation-emission matrix data obtained from the samples were analyzed by parallel factor approach in which four humic-like and two protein-like fluorescent components (named C1-C6) were identified. The results showed that fluorescence intensities were higher in the northern and western lake regions, and notable declines of fluorescence maxima (F max ) were observed from the northwest to the center and then to the southeast of the lake. Calculated biological index (BIX) values ranged from 0.88 to 1.44 and humification index (HIX) values from 0.64 to 3.37 for all the samples. The spatial variations of BIX and HIX values suggested stronger allochthonous CDOM characteristics in Zhushan Bay and the western area and autochthonous characteristics in the southern and eastern areas. Vertically, the average F max value of the surface samples was about 6 % less than that of the bottom samples, but noticeable variations existed among different sampling sites and components. These distribution characteristics of CDOM were mainly attributed to the spatial heterogeneity of sources and wind-induced transportation process. Interestingly, the C6 component (Ex max /Em max = 250/455 nm) seemed to be unique in samples from Zhushan Bay and probably resulted from the discharge of the Taige River. Therefore, it could be used as an indicator of point-source discharge and a tracer to study the fate of CDOM in the lake.
Three-Dimensional Microstructure of Biological Tissues during Freezing and Thawing

NASA Astrophysics Data System (ADS)

Ishiguro, Hiroshi; Horimizu, Takashi; Kataori, Akinobu; Kajigaya, Hiroshi

Three-dimensional behavior of ice crystals and cells during the freezing and thawing of biological tissues was investigated microscopically in real time by using a confocal laser scanning microscope(CLSM) and a fluorescent dye, acridine orange (AO). Fresh tender meat (2nd pectoral muscles) of chicken was stained with the AO in physiological saline to distinguish ice crystals and cells by their different colors, and then frozen and thawed under two different thermal protocols: a) slow-cooling and rapid-warming and b) rapid-cooling and rapid-warming. The CLSM noninvasively produced optical tomograms of the tissues to clarify the pattern of freezing, morphology of ice crystals in the tissues, and the interaction between ice crystals and cells. Also, the tissues were morphologically investigated by pathological means after the freezing and thawing. Typical freezing pattern during the slow-cooling was extracellular-freezing, and those during the rapid-cooling were extracellular-freezing and intracellular freezing with a lot of fine ice crystals in the cells. Cracks caused by the extracellular and intracellular ice crystals remained in the muscle tissues after the thawing. The results obtained by using the CLSM/dye method were consistent with pathologically morphological changes in the tissues through freezing and thawing.
2D-DIGE in Proteomics.

PubMed

Pasquali, Matias; Serchi, Tommaso; Planchon, Sebastien; Renaut, Jenny

2017-01-01

The two-dimensional difference gel electrophoresis method is a valuable approach for proteomics. The method, using cyanine fluorescent dyes, allows the co-migration of multiple protein samples in the same gel and their simultaneous detection, thus reducing experimental and analytical time. 2D-DIGE, compared to traditional post-staining 2D-PAGE protocols (e.g., colloidal Coomassie or silver nitrate), provides faster and more reliable gel matching, limiting the impact of gel to gel variation, and allows also a good dynamic range for quantitative comparisons. By the use of internal standards, it is possible to normalize for experimental variations in spot intensities and gel patterns. Here we describe the experimental steps we follow in our routine 2D-DIGE procedure that we then apply to multiple biological questions.
A current-assisted CMOS photonic sampler with two taps for fluorescence lifetime sensing

NASA Astrophysics Data System (ADS)

Ingelberts, H.; Kuijk, M.

2016-04-01

Imaging based on fluorescence lifetime is becoming increasingly important in medical and biological applications. State-of- the-art fluorescence lifetime microscopes either use bulky and expensive gated image intensifiers coupled to a CCD or single-photon detectors in a slow scanning setup. Numerous attempts are being made to create compact, cost-effective all- CMOS imagers for fluorescence lifetime sensing. Single-photon avalanche diode (SPAD) imagers can have very good timing resolution and noise characteristics but have low detection efficiency. Another approach is to use CMOS imagers based on demodulation detectors. These imagers can be either very fast or very efficient but it remains a challenge to combine both characteristics. Recently we developed the current-assisted photonic sampler (CAPS) to tackle these problems and in this work, we present a new CAPS with two detection taps that can sample a fluorescence decay in two time windows. In the case of mono-exponential decays, two windows provide enough information to resolve the lifetime. We built an electro-optical setup to characterize the detector and use it for fluorescence lifetime measurements. It consists of a supercontinuum pulsed laser source, an optical system to focus light into the detector and picosecond timing electronics. We describe the structure and operation of the two-tap CAPS and provide basic characterization of the speed performance at multiple wavelengths in the visible and near-infrared spectrum. We also record fluorescence decays of different visible and NIR fluorescent dyes and provide different methods to resolve the fluorescence lifetime.
The feasibility study on 3-dimensional fluorescent x-ray computed tomography using the pinhole effect for biomedical applications.

PubMed

Sunaguchi, Naoki; Yuasa, Tetsuya; Hyodo, Kazuyuki; Zeniya, Tsutomu

2013-01-01

We propose a 3-dimensional fluorescent x-ray computed tomography (CT) pinhole collimator, aimed at providing molecular imaging with quantifiable measures and sub-millimeter spatial resolution. In this study, we demonstrate the feasibility of this concept and investigate imaging properties such as spatial resolution, contrast resolution and quantifiable measures, by imaging physical phantoms using a preliminary imaging system developed with monochromatic synchrotron x rays constructed at the BLNE-7A experimental line at KEK, Japan.
Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

PubMed

Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

2013-12-01

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Single-molecule imaging at high fluorophore concentrations by local activation of dye

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

Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.

Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use ofmore » short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.« less
Single-molecule imaging at high fluorophore concentrations by local activation of dye

DOE PAGES

Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.; ...

2015-02-17

Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use ofmore » short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.« less
Imaging and Radiography with Nuclear Resonance Fluorescence and Effective-Z (EZ-3D) Determination; SNM Detection Using Prompt Neutrons from Photon Induced Fission

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

Bertozzi, William; Hasty, Richard; Klimenko, Alexei

Four new technologies have been developed for use in non-intrusive inspection systems to detect nuclear materials, explosives and contraband. Nuclear Resonance Fluorescence (NRF) provides a three dimensional image of the isotopic content of a container. NRF determines the isotopic composition of a region and specifies the isotopic structure of the neighboring regions, thus providing the detailed isotopic composition of any threat. In transmission mode, NRF provides a two dimensional projection of the isotopic content of a container, much as standard X-ray radiography provides for density. The effective-Z method (EZ-3D) uses electromagnetic scattering processes to yield a three-dimensional map of themore » effective-Z and the density in a container. The EZ-3D method allows for a rapid discrimination based on effective Z and mass of materials such as those with high Z, as well as specifying regions of interest for other contraband. The energy spectrum of prompt neutrons from photon induced fission (PNPF) provides a unique identification of the presence of actinides and SNM. These four new technologies can be used independently or together to automatically determine the presence of hazardous materials or contraband. They can also be combined with other technologies to provide added specificity.« less

Preparation strategy and illumination of three-dimensional cell cultures in light sheet-based fluorescence microscopy

NASA Astrophysics Data System (ADS)

Bruns, Thomas; Schickinger, Sarah; Wittig, Rainer; Schneckenburger, Herbert

2012-10-01

A device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions, is described. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. Layers of the sample, of about 10 μm or less in diameter, are, thus, illuminated selectively and imaged by high resolution fluorescence microscopy. SPIM is operated at low light exposure even if a larger number of layers is imaged and is easily combined with laser scanning microscopy. Chinese hamster ovary cells expressing a membrane-associated green fluorescent protein are used for preliminary tests, and the uptake of the fluorescent marker, acridine orange via a microfluidic system, is visualized to demonstrate its potential in cancer research such as for the detection of cellular responses to anticancer drugs.
Microsecond protein dynamics observed at the single-molecule level

NASA Astrophysics Data System (ADS)

Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

2015-07-01

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

PubMed Central

Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

2015-01-01

How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767
Improving Cardiac Action Potential Measurements: 2D and 3D Cell Culture.

PubMed

Daily, Neil J; Yin, Yue; Kemanli, Pinar; Ip, Brian; Wakatsuki, Tetsuro

2015-11-01

Progress in the development of assays for measuring cardiac action potential is crucial for the discovery of drugs for treating cardiac disease and assessing cardiotoxicity. Recently, high-throughput methods for assessing action potential using induced pluripotent stem cell (iPSC) derived cardiomyocytes in both two-dimensional monolayer cultures and three-dimensional tissues have been developed. We describe an improved method for assessing cardiac action potential using an ultra-fast cost-effective plate reader with commercially available dyes. Our methods improve dramatically the detection of the fluorescence signal from these dyes and make way for the development of more high-throughput methods for cardiac drug discovery and cardiotoxicity.
3D time-lapse analysis of Rab11/FIP5 complex: spatiotemporal dynamics during apical lumen formation.

PubMed

Mangan, Anthony; Prekeris, Rytis

2015-01-01

Fluorescent imaging of fixed cells grown in two-dimensional (2D) cultures is one of the most widely used techniques for observing protein localization and distribution within cells. Although this technique can also be applied to polarized epithelial cells that form three-dimensional (3D) cysts when grown in a Matrigel matrix suspension, there are still significant limitations in imaging cells fixed at a particular point in time. Here, we describe the use of 3D time-lapse imaging of live cells to observe the dynamics of apical membrane initiation site (AMIS) formation and lumen expansion in polarized epithelial cells.
An automated real-time microscopy system for analysis of fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Bernardini, André; Wotzlaw, Christoph; Lipinski, Hans-Gerd; Fandrey, Joachim

2010-05-01

Molecular imaging based on Fluorescence Resonance Energy Transfer (FRET) is widely used in cellular physiology both for protein-protein interaction analysis and detecting conformational changes of single proteins, e.g. during activation of signaling cascades. However, getting reliable results from FRET measurements is still hampered by methodological problems such as spectral bleed through, chromatic aberration, focal plane shifts and false positive FRET. Particularly false positive FRET signals caused by random interaction of the fluorescent dyes can easily lead to misinterpretation of the data. This work introduces a Nipkow Disc based FRET microscopy system, that is easy to operate without expert knowledge of FRET. The system automatically accounts for all relevant sources of errors and provides various result presentations of two, three and four dimensional FRET data. Two examples are given to demonstrate the scope of application. An interaction analysis of the two subunits of the hypoxia-inducible transcription factor 1 demonstrates the use of the system as a tool for protein-protein interaction analysis. As an example for time lapse observations, the conformational change of the fluorophore labeled heat shock protein 33 in the presence of oxidant stress is shown.
Evidence of excited state localization and static disorder in LH2 investigated by 2D-polarization single-molecule imaging at room temperature.

PubMed

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

2013-12-07

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

PubMed

Haass-Koffler, Carolina L; Naeemuddin, Mohammad; Bartlett, Selena E

2012-08-31

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology even in complex tissue sections. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.
Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

PubMed

Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

2014-09-01

This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Fluorescence lifetime correlation spectroscopy for precise concentration detection in vivo by background subtraction

NASA Astrophysics Data System (ADS)

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

2009-07-01

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

NASA Astrophysics Data System (ADS)

Han, Chao; Yao, Lei; Xu, Di; Xie, Xianchuan; Zhang, Chaosheng

2016-05-01

A new dual-lumophore optical sensor combined with a robust RGB referencing method was developed for two-dimensional (2D) pH imaging in alkaline sediments and water. The pH sensor film consisted of a proton-permeable polymer (PVC) in which two dyes with different pH sensitivities and emission colors: (1) chloro phenyl imino propenyl aniline (CPIPA) and (2) the coumarin dye Macrolex® fluorescence yellow 10 GN (MFY-10 GN) were entrapped. Calibration experiments revealed the typical sigmoid function and temperature dependencies. This sensor featured high sensitivity and fast response over the alkaline working ranges from pH 7.5 to pH 10.5. Cross-sensitivity towards ionic strength (IS) was found to be negligible for freshwater when IS <0.1 M. The sensor had a spatial resolution of approximately 22 μm and aresponse time of <120 s when going from pH 7.0 to 9.0. The feasibility of the sensor was demonstrated using the pH microelectrode. An example of pH image obtained in the natrual freshwater sediment and water associated with the photosynthesis of Vallisneria spiral species was also presented, suggesting that the sensor held great promise for the field applications.
High-resolution Imaging of pH in Alkaline Sediments and Water Based on a New Rapid Response Fluorescent Planar Optode

PubMed Central

Han, Chao; Yao, Lei; Xu, Di; Xie, Xianchuan; Zhang, Chaosheng

2016-01-01

A new dual-lumophore optical sensor combined with a robust RGB referencing method was developed for two-dimensional (2D) pH imaging in alkaline sediments and water. The pH sensor film consisted of a proton-permeable polymer (PVC) in which two dyes with different pH sensitivities and emission colors: (1) chloro phenyl imino propenyl aniline (CPIPA) and (2) the coumarin dye Macrolex® fluorescence yellow 10 GN (MFY-10 GN) were entrapped. Calibration experiments revealed the typical sigmoid function and temperature dependencies. This sensor featured high sensitivity and fast response over the alkaline working ranges from pH 7.5 to pH 10.5. Cross-sensitivity towards ionic strength (IS) was found to be negligible for freshwater when IS <0.1 M. The sensor had a spatial resolution of approximately 22 μm and aresponse time of <120 s when going from pH 7.0 to 9.0. The feasibility of the sensor was demonstrated using the pH microelectrode. An example of pH image obtained in the natrual freshwater sediment and water associated with the photosynthesis of Vallisneria spiral species was also presented, suggesting that the sensor held great promise for the field applications. PMID:27199163
Luminescent silver(I) tert-butylethynide compounds with nicotinic/isonicotinic acid as ligands

NASA Astrophysics Data System (ADS)

Xie, Yi-Ming; Fan, Yue-Yue; Lin, Fu-Lin; Hu, Ting; Liu, Jia; Lu, Can-Zhong

2017-12-01

Solvothermal reaction of tBuC≡CAg, AgBF4 and nicotinic/isonicotinic acid in acetonitrile afforded two new silver(I) tert-butylethynide double salts, namely [(tBuC≡CAg)(AgL1)3] (HL1 = nicotinic acid) (1) and [(tBuC≡CAg)(AgL1)2] (HL2 = isonicotinic acid) (2). These compounds have been characterized by elemental analysis, infrared spectra, single-crystal X-ray analysis, X-ray powder diffraction, thermogravimetric analysis, UV-visible absorption spectra, and luminescent measurement. 1 exhibits a two-dimensional coordination network, and 2 features a three-dimensional coordination architecture. Luminescence measurements indicate that 1 shows a fluorescent emission band centered at 568 nm, and 2 exhibits an intense emission maximum at 550 nm and a shoulder peak at 436 nm.
Honeycomb-shaped coordination polymers based on the self-assembly of long flexible ligands and alkaline-earth ions

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

Lian, Chen; Liu, Liu; Guo, Xu

2016-01-15

Two novel coordination polymers, namely, [Ca(NCP){sub 2}]{sub ∞} (I) and [Sr(NCP){sub 2}]{sub ∞} (II) were synthesized under hydrothermal conditions based on 2-(4-carboxyphenyl)imidazo(4,5-f)-(1,10)phenanthroline (HNCP) and characterized by elemental analysis, infrared spectrometry, X-ray powder diffraction and single crystal X-ray diffraction. Findings indicate that I and II are isomorphous and isostructural, containing the unit of M(NCP{sup −}){sub 4} (M=Ca(II) and Sr(II)), based on which to assemble into three-dimensional (3D) porous 4-fold interpenetration honeycomb-shaped neutral coordination polymers (CPs). Between the adjacent lamellar structures in I and II, there exist π–π interactions between the pyridine rings belonging to phenanthroline of NCP{sup −} which stabilize themore » frameworks. Both I and II display stronger fluorescence emissions as well as high thermal stability. - Graphical abstract: One-dimensional nanotubular channels with the cross dimension of 37.1959(20)×23.6141(11)Å{sup 2} in the three-dimensional honeycomb-shaped coordination network of II are observed. The topological analysis of II indicates that there exists a typical diamond framework possessing large adamantanoid cages, which containing four cyclohexane-shaped patterns in chair conformations. - Highlights: • Two isomorphous and isostructural coordination polymers based on flexible ligand and two alkaline-earth metal salts have been synthesized and characterized. • Structural analysis indicates that I and II are assembled into 3D porous honeycomb-shaped metal-organic frameworks. • Both I and II display stronger fluorescence emissions and higher thermal stability.« less
Molecular engineering of two-photon fluorescent probes for bioimaging applications

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

PubMed

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-06-08

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Dual-Mode SERS-Fluorescence Immunoassay Using Graphene Quantum Dot Labeling on One-Dimensional Aligned Magnetoplasmonic Nanoparticles.

PubMed

Zou, Fengming; Zhou, Hongjian; Tan, Tran Van; Kim, Jeonghyo; Koh, Kwangnak; Lee, Jaebeom

2015-06-10

A novel dual-mode immunoassay based on surface-enhanced Raman scattering (SERS) and fluorescence was designed using graphene quantum dot (GQD) labels to detect a tuberculosis (TB) antigen, CFP-10, via a newly developed sensing platform of linearly aligned magnetoplasmonic (MagPlas) nanoparticles (NPs). The GQDs were excellent bilabeling materials for simultaneous Raman scattering and photoluminescence (PL). The one-dimensional (1D) alignment of MagPlas NPs simplified the immunoassay process and enabled fast, enhanced signal transduction. With a sandwich-type immunoassay using dual-mode nanoprobes, both SERS signals and fluorescence images were recognized in a highly sensitive and selective manner with a detection limit of 0.0511 pg mL(-1).
Synchronous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment based on a zwitterionic copper (II) metal-organic framework.

PubMed

Qiu, Gui-Hua; Weng, Zi-Hua; Hu, Pei-Pei; Duan, Wen-Jun; Xie, Bao-Ping; Sun, Bin; Tang, Xiao-Yan; Chen, Jin-Xiang

2018-04-01

From a three-dimensional (3D) metal-organic framework (MOF) of {[Cu(Cmdcp)(phen)(H 2 O)] 2 ·9H 2 O} n (1, H 3 CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide, phen = phenanthroline), a sensitive and selective fluorescence sensor has been developed for the simultaneous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded microRNA-like (miRNA-like) fragment. The results from molecular dynamics simulation confirmed that MOF 1 absorbs carboxyfluorescein (FAM)-tagged and 5(6)-carboxyrhodamine, triethylammonium salt (ROX)-tagged probe ss-DNA (probe DNA, P-DNA) by π … π stacking and hydrogen bonding, as well as additional electrostatic interactions to form a sensing platform of P-DNAs@1 with quenched FAM and ROX fluorescence. In the presence of targeted ebolavirus conserved RNA sequences or ebolavirus-encoded miRNA-like fragment, the fluorophore-labeled P-DNA hybridizes with the analyte to give a P-DNA@RNA duplex and released from MOF 1, triggering a fluorescence recovery. Simultaneous detection of two target RNAs has also been realized by single and synchronous fluorescence analysis. The formed sensing platform shows high sensitivity for ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment with detection limits at the picomolar level and high selectivity without cross-reaction between the two probes. MOF 1 thus shows the potential as an effective fluorescent sensing platform for the synchronous detection of two ebolavirus-related sequences, and offer improved diagnostic accuracy of Ebola virus disease. Copyright © 2017 Elsevier B.V. All rights reserved.
[The Influence of Runoff Pollution to DOM Features in an Urban Wastewater Treatment Plant].

PubMed

He, Li; Ji, Fang-ying; Lai, Ming-sheng; Xu, Xuan; Zhou, Wei-wei; Mao, Bo-lin; Yang, Ming-jia

2015-03-01

Combined with wastewater treatment process, the sewage in sunny and rainy day was collected from a wastewater treatment plant in Chongqing. Three-dimensional fluorescence spectra was used to investigate the characteristic fluorescence of dissolved organic matter (DOM). DOM dissolved organic carbon (DOC), chemical oxygen demand (COD), fluorescence index (ƒ450/500) and fluorescence intensity ratio γ (A, C) of fulvic acid in ultraviolet and visible region were used to analyze the impact of rain runoff pollution on sewage DOM. According to the experimental data, the DOM fluorescence fingerprints of this wastewater treatment plant were quite different from typical municipal sewage, and the main component was tryptophan with low excitation wavelength (Peak S), then the tryptophan with long wavelength excitation (Peak T) followed. A2/O process had an approximative degradation of the protein-like both in sunny day and rainy day, but had a better degradation of fulvic-like, DOC and COD in rainy day than that in sunny day. Morever, the fluorescence peaks got red-shifted after the biological treatment. The differences of DOM fluorescence fingerprint between sunny and rainy day were significant, the fluorescence center of UV fulvic (Peak A) in rainy day getting blue-shifted obviously, shifting from 240 - 248/390 - 440 to 240 - 250/370 - 400 nm. Although the DOM types in sunny and rainy day were the same, the source of fulvic got more complex by runoff and the component ratio of DOM also changed. Compared with the sunny day, the proportion of Peak S in DOM dereased by 10%, and the proportion of Peak A increased by 7% in rainy day.
Femtosecond/picosecond time-resolved fluorescence study of hydrophilic polymer fine particles.

PubMed

Nanjo, Daisuke; Hosoi, Haruko; Fujino, Tatsuya; Tahara, Tahei; Korenaga, Takashi

2007-03-22

Femtosecond/picosecond time-resolved fluorescence study of hydrophilic polymer fine particles (polyacrylamide, PAAm) was reported. Ultrafast fluorescence dynamics of polymer/water solution was monitored using a fluorescent probe molecule (C153). In the femtosecond time-resolved fluorescence measurement at 480 nm, slowly decay components having lifetimes of tau(1) approximately 53 ps and tau(2) approximately 5 ns were observed in addition to rapid fluorescence decay. Picosecond time-resolved fluorescence spectra of C153/PAAm/H2O solution were also measured. In the time-resolved fluorescence spectra of C153/PAAm/H2O, a peak shift from 490 to 515 nm was measured, which can be assigned to the solvation dynamics of polymer fine particles. The fluorescence peak shift was related to the solvation response function and two time constants were determined (tau(3) approximately 50 ps and tau(4) approximately 467 ps). Therefore, the tau(1) component observed in the femtosecond time-resolved fluorescence measurement was assigned to the solvation dynamics that was observed only in the presence of polymer fine particles. Rotational diffusion measurements were also carried out on the basis of the picosecond time-resolved fluorescence spectra. In the C153/PAAm/H2O solution, anisotropy decay having two different time constants was also derived (tau(6) approximately 76 ps and tau(7) approximately 676 ps), indicating the presence of two different microscopic molecular environments around the polymer surface. Using the Stokes-Einstein-Debye (SED) equation, microscopic viscosity around the polymer surface was evaluated. For the area that gave a rotational diffusion time of tau(6) approximately 76 ps, the calculated viscosity is approximately 1.1 cP and for tau(7) approximately 676 ps, it is approximately 10 cP. The calculated viscosity values clearly revealed that there are two different molecular environments around the polyacrylamide fine particles.

Effect of vesicle size on the prodan fluorescence in diheptadecanoylphosphatidylcholine bilayer membrane under atmospheric and high pressures.

PubMed

Goto, Masaki; Sawaguchi, Hiroshi; Tamai, Nobutake; Matsuki, Hitoshi; Kaneshina, Shoji

2010-08-17

The bilayer phase behavior of diheptadecanoylphosphatidylcholine (C17PC) with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under atmospheric and high pressures. The difference in phase transitions and thermodynamic quantities of the transition was hardly observed between LMV and GMV used here. On the contrary, the Prodan fluorescence in the bilayer membranes changed depending on the size of vesicles as well as on the phase states. From the second derivative of fluorescence spectra, the three-dimensional image plots in which we can see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under atmospheric pressure. The following characteristic behavior was found: (1) the Prodan molecules in GMV can be distributed to not only adjacent glycerol backbone region, but also near bulk-water region in the lamellar gel or ripple gel phase; (2) the blue valleys of GMV became deeper than those of LMV because of the greater surface density of the Prodan molecules per unit area of GMV than LMV; (3) the liquid crystalline phase of the bilayer excludes the Prodan molecules to a more hydrophilic region at the membrane surface with an increase in vesicle size; (4) the accurate information as to the phase transitions is gradually lost with increasing vesicle size. Under the high-pressure condition, the difference in Prodan fluorescence between LMV and GMV was essentially the same as the difference under atmospheric pressure except for the existence of the pressure-induced interdigitated gel phase. Further, we found that Prodan fluorescence spectra in the interdigitated gel phase were especially affected by the size of vesicles. This study revealed that the Prodan molecules can move around the headgroup region by responding not only to the phase state but also to the vesicle size, and they become a useful membrane probe, detecting important membrane properties such as the packing stress.
Impact of Cultivation Conditions, Ethylene Treatment, and Postharvest Storage on Selected Quality and Bioactivity Parameters of Kiwifruit "Hayward" Evaluated by Analytical and Chemometric Methods.

PubMed

Park, Yong Seo; Polovka, Martin; Ham, Kyung-Sik Ham; Park, Yang-Kyun; Vearasilp, Suchada; Namieśnik, Jacek; Toledo, Fernando; Arancibia-Avila, Patricia; Gorinstein, Shela

2016-09-01

Organic, semiorganic, and conventional "Hayward" kiwifruits, treated with ethylene for 24 h and stored during 10 days, were assessed by UV spectrometry, fluorometry, and chemometrical analysis for changes in selected characteristics of quality (firmness, dry matter and soluble solid contents, pH, and acidity) and bioactivity (concentration of polyphenols via Folin-Ciocalteu and p-hydroxybenzoic acid assays). All of the monitored qualitative parameters and characteristics related to bioactivity were affected either by cultivation practices or by ethylene treatment and storage. Results obtained, supported by statistical evaluation (Friedman two-way ANOVA) and chemometric analysis, clearly proved that the most significant impact on the majority of the evaluated parameters of quality and bioactivity of "Hayward" kiwifruit had the ethylene treatment followed by the cultivation practices and the postharvest storage. Total concentration of polyphenols expressed via p-hydroxybenzoic acid assay exhibited the most significant sensitivity to all three evaluated parameters, reaching a 16.5% increase for fresh organic compared to a conventional control sample. As a result of postharvest storage coupled with ethylene treatment, the difference increased to 26.3%. Three-dimensional fluorescence showed differences in the position of the main peaks and their fluorescence intensity for conventional, semiorganic, and organic kiwifruits in comparison with ethylene nontreated samples.
A Model-Based Approach for Microvasculature Structure Distortion Correction in Two-Photon Fluorescence Microscopy Images

PubMed Central

Dao, Lam; Glancy, Brian; Lucotte, Bertrand; Chang, Lin-Ching; Balaban, Robert S; Hsu, Li-Yueh

2015-01-01

SUMMARY This paper investigates a post-processing approach to correct spatial distortion in two-photon fluorescence microscopy images for vascular network reconstruction. It is aimed at in vivo imaging of large field-of-view, deep-tissue studies of vascular structures. Based on simple geometric modeling of the object-of-interest, a distortion function is directly estimated from the image volume by deconvolution analysis. Such distortion function is then applied to sub volumes of the image stack to adaptively adjust for spatially varying distortion and reduce the image blurring through blind deconvolution. The proposed technique was first evaluated in phantom imaging of fluorescent microspheres that are comparable in size to the underlying capillary vascular structures. The effectiveness of restoring three-dimensional spherical geometry of the microspheres using the estimated distortion function was compared with empirically measured point-spread function. Next, the proposed approach was applied to in vivo vascular imaging of mouse skeletal muscle to reduce the image distortion of the capillary structures. We show that the proposed method effectively improve the image quality and reduce spatially varying distortion that occurs in large field-of-view deep-tissue vascular dataset. The proposed method will help in qualitative interpretation and quantitative analysis of vascular structures from fluorescence microscopy images. PMID:26224257
Protochlorophyll complexes with similar steady-state fluorescence characteristics can differ in fluorescence lifetimes. A model study in Triton X-100.

PubMed

Myśliwa-Kurdziel, Beata; Solymosi, Katalin; Kruk, Jerzy; Böddi, Béla; Strzałka, Kazimierz

2007-03-01

The steady-state and time-resolved fluorescence characteristics of protochlorophyll (Pchl) dissolved in neat Triton X-100 and in Triton X-100 micelles were investigated, and the fluorescence lifetimes of different Pchl spectral forms were studied. Varying the concentration of Pchl or diluting the micellar solutions either with a buffer or with a micellar solution, 631-634, 645-655, 680-692 and above 700 nm emitting Pchl complexes were prepared, the ratios of which varied from one another. The fluorescence decay of the 631-634 nm emitting (monomeric) form had a mono-exponential character with a 5.4-ns fluorescence lifetime. The long-wavelength Pchl complexes (aggregates) had two fluorescence lifetime values within a range of 1.4-3.9 ns and 0.15-0.84 ns, which showed high variability in different environments. Depending on the conditions, either mono- or double-exponential fluorescence decay was found for a fluorescence band at 680-685 nm. These data show that despite their very similar steady-state fluorescence properties, Pchl complexes can differ in fluorescence lifetimes, which may reflect different molecular structures, intrinsic geometries or different molecular interactions. This underlines the importance of complex spectroscopic analysis for a precise description of native and artificial chlorophyllous pigment forms.
Towards metabolic mapping of the human retina.

PubMed

Schweitzer, D; Schenke, S; Hammer, M; Schweitzer, F; Jentsch, S; Birckner, E; Becker, W; Bergmann, A

2007-05-01

Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40 degrees fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 +/- 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510-560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 approximately 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus. Copyright 2007 Wiley-Liss, Inc.
One-Dimensional Spontaneous Raman Measurements Made in a Gas Turbine Combustor

NASA Technical Reports Server (NTRS)

DeGroot, Wilhelmus A.; Hicks, Yolanda R.; Locke, Randy J.; Anderson, Robert C.

2001-01-01

The NASA Glenn Research Center and the aerospace industry are designing and testing low-emission combustor concepts to build the next generation of cleaner, more fuel efficient aircraft powerplants. These combustors will operate at much higher inlet temperatures and at pressures that are up to 3 to 5 times greater than combustors in the current fleet. From a test and analysis viewpoint, there is an increasing need for measurements from these combustors that are nonintrusive, simultaneous, multipoint, and more quantitative. Glenn researchers have developed several unique test facilities (refs. 1 and 2) that allow, for the first time, optical interrogation of combustor flow fields, including subcomponent performance, at pressures ranging from 1 to 60 bar (1 to 60 atm). Experiments conducted at Glenn are the first application of a visible laser-pumped, one-dimensional, spontaneous Raman-scattering technique to analyze the flow in a high-pressure, advanced-concept fuel injector at pressures thus far reaching 12 bar (12 atm). This technique offers a complementary method to the existing two- and three-dimensional imaging methods used, such as planar laser-induced fluorescence. Raman measurements benefit from the fact that the signal from each species is a linear function of its density, and the relative densities of all major species can be acquired simultaneously with good precision. The Raman method has the added potential to calibrate multidimensional measurements by providing an independent measurement of species number-densities at known points within the planar laser-induced fluorescence images. The visible Raman method is similar to an ultraviolet-Raman technique first tried in the same test facility (ref. 3). However, the visible method did not suffer from the ultraviolet technique's fuel-born polycyclic aromatic hydrocarbon fluorescence interferences.
Quantitative characterization of 3D deformations of cell interactions with soft biomaterials

NASA Astrophysics Data System (ADS)

Franck, Christian

In recent years, the importance of mechanical forces in directing cellular function has been recognized as a significant factor in biological and physiological processes. In fact, these physical forces are now viewed equally as important as biochemical stimuli in controlling cellular response. Not only do these cellular forces, or cell tractions, play an important role in cell migration, they are also significant to many other physiological and pathological processes, both at the tissue and organ level, including wound healing, inflammation, angiogenesis, and embryogenesis. A complete quantification of cell tractions during cell-material interactions can lead to a deeper understanding of the fundamental role these forces play in cell biology. Thus, understanding the function and role of a cell from a mechanical framework can have important implications towards the development of new implant materials and drug treatments. Previous research has contributed significant descriptions of cell-tissue interactions by quantifying cell tractions in two-dimensional environments; however, most physiological processes are three-dimensional in nature. Recent studies have shown morphological differences in cells cultured on two-dimensional substrates versus three-dimensional matrices, and that the intrinsic extracellular matrix interactions and migration behavior are different in three dimensions versus two dimensions. Hence, measurement techniques are needed to investigate cellular behavior in all three dimensions. This thesis presents a full-field imaging technique capable of quantitatively measuring cell traction forces in all three spatial dimensions, and hence addresses the need of a three-dimensional quantitative imaging technique to gain insight into the fundamental role of physical forces in biological processes. The technique combines laser scanning confocal microscopy (LSCM) with digital volume correlation (DVC) to track the motion of fluorescent particles during cell-induced or externally applied deformations. This method is validated by comparing experimentally measured non-uniform deformation fields near hard and soft spherical inclusions under uniaxial compression with the corresponding analytical solution. Utilization of a newly developed computationally efficient stretch-correlation and deconvolution algorithm is shown to improve the overall measurement accuracy, in particular under large deformations. Using this technique, the full three-dimensional substrate displacement fields are experimentally determined during the migration of individual fibroblast cells on polyacrylamide gels. This is the first study to show the highly three-dimensional structure of cell-induced displacement and traction fields. These new findings suggest a three-dimensional push-pull cell motility, which differs from the traditional theories based on two-dimensional data. These results provide new insight into the dynamic cell-matrix force exchange or mechanotransduction of migrating cells, and will aid in the development of new three-dimensional cell motility and adhesion models. As this study reveals, the mechanical interactions of cells and their extracellular matrix appear to be highly three-dimensional. It also shows that the LSCM-DVC technique is well suited for investigating the mechanics of cell-matrix interactions while providing a platform to access detailed information of the intricate biomechanical coupling for many cellular responses. Thus, this method has the capability to provide direct quantitative experimental data showing how cells interact with their surroundings in three dimensions and might stimulate new avenues of scientific thought in understanding the fundamental role physical forces play in regulating cell behavior.
Multi-spectroscopic investigation on the complexation of tetracycline with dissolved organic matter derived from algae and macrophyte.

PubMed

Bai, Leilei; Zhao, Zhen; Wang, Chunliu; Wang, Changhui; Liu, Xin; Jiang, Helong

2017-11-01

Interactions of antibiotics with algae-derived dissolved organic matter (ADOM) and macrophyte-derived dissolved organic matter (MDOM) are of vital importance to the transport and ecotoxicity of antibiotics in eutrophic freshwater lakes. Multi-spectroscopic techniques were used to investigate the complexation of tetracycline (TTC) with ADOM and MDOM collected from Lake Taihu (China). The 3 fluorescent components, tyrosine-, tryptophan-, and humic-like component, were identified by excitation emission matrix spectra with parallel factor analysis. Their fluorescence was quenched at different degree by TTC titration through static quenching. The complexation of TTC induced conformational changes in DOM fractions. Synchronous fluorescence spectra combined with two dimensional correlation spectroscopy further suggested that the formation of TTC-DOM complexes occurred on the sequential order of tryptophan-like→tyrosine-like→humic-like component. The effective quenching constants of tryptophan- and tyrosine-like component were similar, higher than those of humic-like component. The strong binding ability and abundant content of protein-like substances indicated their prominent role in the TTC-DOM complexation. Fourier transform infrared spectroscopy further revealed that the heterogeneous functional groups, including amide I and II, aromatics, and aliphatics, were responsible for the complexation. These results highlight the significant impact of the overgrowth of algae and macrophyte on the environmental behavior of antibiotics in waters. Copyright © 2017 Elsevier Ltd. All rights reserved.
Two-dimensional fingerprinting approach for comparison of complex substances analysed by HPLC-UV and fluorescence detection.

PubMed

Ni, Yongnian; Liu, Ying; Kokot, Serge

2011-02-07

This work is concerned with the research and development of methodology for analysis of complex mixtures such as pharmaceutical or food samples, which contain many analytes. Variously treated samples (swill washed, fried and scorched) of the Rhizoma atractylodis macrocephalae (RAM) traditional Chinese medicine (TCM) as well as the common substitute, Rhizoma atractylodis (RA) TCM were chosen as examples for analysis. A combined data matrix of chromatographic 2-D HPLC-DAD-FLD (two-dimensional high performance liquid chromatography with diode array and fluorescence detectors) fingerprint profiles was constructed with the use of the HPLC-DAD and HPLC-FLD individual data matrices; the purpose was to collect maximum information and to interpret this complex data with the use of various chemometrics methods e.g. the rank-ordering multi-criteria decision making (MCDM) PROMETHEE and GAIA, K-nearest neighbours (KNN), partial least squares (PLS), back propagation-artificial neural networks (BP-ANN) methods. The chemometrics analysis demonstrated that the combined 2-D HPLC-DAD-FLD data matrix does indeed provide more information and facilitates better performing classification/prediction models for the analysis of such complex samples as the RAM and RA ones noted above. It is suggested that this fingerprint approach is suitable for analysis of other complex, multi-analyte substances.
Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA

NASA Astrophysics Data System (ADS)

Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e.

2016-03-01

Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.
Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

PubMed

Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

2016-03-05

Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. Copyright © 2015 Elsevier B.V. All rights reserved.
Intracellular Localization of Arabidopsis Sulfurtransferases1

PubMed Central

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D.; Papenbrock, Jutta

2004-01-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism. PMID:15181206
Intracellular localization of Arabidopsis sulfurtransferases.

PubMed

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D; Papenbrock, Jutta

2004-06-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.
Multivariate functional response regression, with application to fluorescence spectroscopy in a cervical pre-cancer study.

PubMed

Zhu, Hongxiao; Morris, Jeffrey S; Wei, Fengrong; Cox, Dennis D

2017-07-01

Many scientific studies measure different types of high-dimensional signals or images from the same subject, producing multivariate functional data. These functional measurements carry different types of information about the scientific process, and a joint analysis that integrates information across them may provide new insights into the underlying mechanism for the phenomenon under study. Motivated by fluorescence spectroscopy data in a cervical pre-cancer study, a multivariate functional response regression model is proposed, which treats multivariate functional observations as responses and a common set of covariates as predictors. This novel modeling framework simultaneously accounts for correlations between functional variables and potential multi-level structures in data that are induced by experimental design. The model is fitted by performing a two-stage linear transformation-a basis expansion to each functional variable followed by principal component analysis for the concatenated basis coefficients. This transformation effectively reduces the intra-and inter-function correlations and facilitates fast and convenient calculation. A fully Bayesian approach is adopted to sample the model parameters in the transformed space, and posterior inference is performed after inverse-transforming the regression coefficients back to the original data domain. The proposed approach produces functional tests that flag local regions on the functional effects, while controlling the overall experiment-wise error rate or false discovery rate. It also enables functional discriminant analysis through posterior predictive calculation. Analysis of the fluorescence spectroscopy data reveals local regions with differential expressions across the pre-cancer and normal samples. These regions may serve as biomarkers for prognosis and disease assessment.
A fluorescence model of the murine lung for optical detection of pathogenic bacteria

NASA Astrophysics Data System (ADS)

Durkee, Madeleine S.; Cirillo, Jeffrey D.; Maitland, Kristen C.

2017-07-01

We present a computer model of intravital excitation and external fluorescence detection in the murine lungs validated with a three-dimensional lung tissue phantom. The model is applied to optical detection of pulmonary tuberculosis infection.
Apoferritin fibers: a new template for 1D fluorescent hybrid nanostructures

NASA Astrophysics Data System (ADS)

Jurado, Rocío; Castello, Fabio; Bondia, Patricia; Casado, Santiago; Flors, Cristina; Cuesta, Rafael; Domínguez-Vera, José M.; Orte, Angel; Gálvez, Natividad

2016-05-01

Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials.Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials. Electronic supplementary information (ESI) available: TEM images of ferritin protein fiber formation, and apoferritin after 18 days of heat treatment; FLIM-PIE technique details; fluorescence emission spectra of apoferritin and β-lactoglobulin fibers functionalized with different QDs. See DOI: 10.1039/c6nr01044j
Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

NASA Astrophysics Data System (ADS)

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.
Trends in fluorescence imaging and related techniques to unravel biological information.

PubMed

Haustein, Elke; Schwille, Petra

2007-09-01

Optical microscopy is among the most powerful tools that the physical sciences have ever provided biology. It is indispensable for basic lab work, as well as for cutting edge research, as the visual monitoring of life processes still belongs to the most compelling evidences for a multitude of biomedical applications. Along with the rapid development of new probes and methods for the analysis of laser induced fluorescence, optical microscopy over past years experienced a vast increase of both new techniques and novel combinations of established methods to study biological processes with unprecedented spatial and temporal precision. On the one hand, major technical advances have significantly improved spatial resolution. On the other hand, life scientists are moving toward three- and even four-dimensional cell biology and biophysics involving time as a crucial coordinate to quantitatively understand living specimen. Monitoring the whole cell or tissue in real time, rather than producing snap-shot-like two-dimensional projections, will enable more physiological and, thus, more clinically relevant experiments, whereas an increase in temporal resolution facilitates monitoring fast nonperiodic processes as well as the quantitative analysis of characteristic dynamics.
Trends in fluorescence imaging and related techniques to unravel biological information

PubMed Central

Haustein, Elke; Schwille, Petra

2007-01-01

Optical microscopy is among the most powerful tools that the physical sciences have ever provided biology. It is indispensable for basic lab work, as well as for cutting edge research, as the visual monitoring of life processes still belongs to the most compelling evidences for a multitude of biomedical applications. Along with the rapid development of new probes and methods for the analysis of laser induced fluorescence, optical microscopy over past years experienced a vast increase of both new techniques and novel combinations of established methods to study biological processes with unprecedented spatial and temporal precision. On the one hand, major technical advances have significantly improved spatial resolution. On the other hand, life scientists are moving toward three- and even four-dimensional cell biology and biophysics involving time as a crucial coordinate to quantitatively understand living specimen. Monitoring the whole cell or tissue in real time, rather than producing snap-shot-like two-dimensional projections, will enable more physiological and, thus, more clinically relevant experiments, whereas an increase in temporal resolution facilitates monitoring fast nonperiodic processes as well as the quantitative analysis of characteristic dynamics. PMID:19404444
Phase retrieval and 3D imaging in gold nanoparticles based fluorescence microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Ilovitsh, Tali; Ilovitsh, Asaf; Weiss, Aryeh M.; Meir, Rinat; Zalevsky, Zeev

2017-02-01

Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume, and is therefore time consuming, and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold nanoparticles tagged sample using only two images. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is then further enhanced also for tracking of single fluorescent particles within a three dimensional (3D) cellular environment based on image processing algorithms that can significantly increases localization accuracy of the 3D point spread function in respect to regular Gaussian fitting. All proposed concepts are validated both on simulated data as well as experimentally.

In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function

PubMed Central

Lew, Matthew D.; Thompson, Michael A.; Badieirostami, Majid; Moerner, W. E.

2010-01-01

The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional super-resolution imaging. We characterize the localization precision of a unique method for 3D superresolution imaging featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the standard PSF throughout a 2 μm depth of field. Comparisons between the DH-PSF and other methods for 3D super-resolution are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in biological systems by tracking the movement of quantum dots in glycerol and in live cells. PMID:20563317
Succinate dehydrogenase activity and soma size of motoneurons innervating different portions of the rat tibialis anterior

NASA Technical Reports Server (NTRS)

Ishihara, A.; Roy, R. R.; Edgerton, V. R.

1995-01-01

The spatial distribution, soma size and oxidative enzyme activity of gamma and alpha motoneurons innervating muscle fibres in the deep (away from the surface of the muscle) and superficial (close to the surface of the muscle) portions of the tibialis anterior in normal rats were determined. The deep portion had a higher percentage of high oxidative fibres than the superficial portion of the muscle. Motoneurons were labelled by retrograde neuronal transport of fluorescent tracers: Fast Blue and Nuclear Yellow were injected into the deep portion and Nuclear Yellow into the superficial portion of the muscle. Therefore, motoneurons innervating the deep portion were identified by both a blue fluorescent cytoplasm and a golden-yellow fluorescent nucleus, while motoneurons innervating the superficial portion were identified by only a golden-yellow fluorescent nucleus. After staining for succinate dehydrogenase activity on the same section used for the identification of the motoneurons, soma size and succinate dehydrogenase activity of the motoneurons were measured. The gamma and alpha motoneurons innervating both the deep and superficial portions were located primarily at L4 and were intermingled within the same region of the dorsolateral portion of the ventral horn in the spinal cord. Mean soma size was similar for either gamma or alpha motoneurons in the two portions of the muscle. The alpha motoneurons innervating the superficial portion had a lower mean succinate dehydrogenase activity than those innervating the deep portion of the muscle. An inverse relationship between soma size and succinate dehydrogenase activity of alpha, but not gamma, motoneurons innervating both the deep and superficial portions was observed. Based on three-dimensional reconstructions within the spinal cord, there were no apparent differences in the spatial distribution of the motoneurons, either gamma or alpha, associated with the deep and superficial compartments of the muscle. The data provide evidence for an interdependence in the oxidative capacity between a motoneuron and its target muscle fibres in two subpopulations of motoneurons from the same motor pool, i.e. the same muscle.
Two-Photon Microscopy Analysis of Gold Nanoparticle Uptake in 3D Cell Spheroids.

PubMed

Rane, Tushar D; Armani, Andrea M

2016-01-01

Nanomaterials can be synthesized from a wide range of material systems in numerous morphologies, creating an extremely diverse portfolio. As result of this tunability, these materials are emerging as a new class of nanotherapeutics and imaging agents. One particularly interesting nanomaterial is the gold nanoparticle. Due to its inherent biocompatibility and tunable photothermal behavior, it has made a rapid transition from the lab setting to in vivo testing. In most nanotherapeutic applications, the efficacy of the agent is directly related to the target of interest. However, the optimization of the AuNP size and shape for efficacy in vitro, prior to testing in in vivo models of a disease, has been largely limited to two dimensional monolayers of cells. Two dimensional cell cultures are unable to reproduce conditions experienced by AuNP in the body. In this article, we systematically investigate the effect of different properties of AuNP on the penetration depth into 3D cell spheroids using two-photon microscopy. The 3D spheroids are formed from the HCT116 cell line, a colorectal carcinoma cell line. In addition to studying different sizes and shapes of AuNPs, we also study the effect of an oligo surface chemistry. There is a significant difference between AuNP uptake profiles in the 2D monolayers of cells as compared to the 3D cell spheroids. Additionally, the range of sizes and shapes studied here also exhibit marked differences in uptake penetration depth and efficacy. Finally, our results demonstrate that two-photon microscopy enables quantitative AuNP localization and concentration data to be obtained at the single spheroid level without fluorescent labeling of the AuNP, thus, providing a viable technique for large scale screening of AuNP properties in 3D cell spheroids as compared to tedious and time consuming techniques like electron microscopy.
Reaction-time-resolved measurements of laser-induced fluorescence in a shock tube with a single laser pulse

NASA Astrophysics Data System (ADS)

Zabeti, S.; Fikri, M.; Schulz, C.

2017-11-01

Shock tubes allow for the study of ultra-fast gas-phase reactions on the microsecond time scale. Because the repetition rate of the experiments is low, it is crucial to gain as much information as possible from each individual measurement. While reaction-time-resolved species concentration and temperature measurements with fast absorption methods are established, conventional laser-induced fluorescence (LIF) measurements with pulsed lasers provide data only at a single reaction time. Therefore, fluorescence methods have rarely been used in shock-tube diagnostics. In this paper, a novel experimental concept is presented that allows reaction-time-resolved LIF measurements with one single laser pulse using a test section that is equipped with several optical ports. After the passage of the shock wave, the reactive mixture is excited along the center of the tube with a 266-nm laser beam directed through a window in the end wall of the shock tube. The emitted LIF signal is collected through elongated sidewall windows and focused onto the entrance slit of an imaging spectrometer coupled to an intensified CCD camera. The one-dimensional spatial resolution of the measurement translates into a reaction-time-resolved measurement while the species information can be gained from the spectral axis of the detected two-dimensional image. Anisole pyrolysis was selected as the benchmark reaction to demonstrate the new apparatus.
Reflecting microscope system with a 0.99 numerical aperture designed for three-dimensional fluorescence imaging of individual molecules at cryogenic temperatures

PubMed Central

Inagawa, H.; Toratani, Y.; Motohashi, K.; Nakamura, I.; Matsushita, M.; Fujiyoshi, S.

2015-01-01

We have developed a cryogenic fluorescence microscope system, the core of which is a reflecting objective that consists of spherical and aspherical mirrors. The use of an aspherical mirror allows the reflecting objective to have a numerical aperture (NA) of up to 0.99, which is close to the maximum possible NA of 1.03 in superfluid helium. The performance of the system at a temperature of 1.7 K was tested by recording a three-dimensional fluorescence image of individual quantum dots using excitation wavelengths (λex) of 532 nm and 635 nm. At 1.7 K, the microscope worked with achromatic and nearly diffraction-limited performance. The 1/e2 radius (Γ) of the point spread function of the reflecting objective in the lateral (xy) direction was 0.212 ± 0.008 μm at λex = 532 nm and was less than 1.2 times the simulated value for a perfectly polished objective. The radius Γ in the axial (z) direction was 0.91 ± 0.04 μm at λex = 532 nm and was less than 1.4 times the simulated value of Γ. The chromatic aberrations between the two wavelengths were one order of magnitude smaller than Γ in each direction. PMID:26239746
Three-dimensional quick response code based on inkjet printing of upconversion fluorescent nanoparticles for drug anti-counterfeiting

NASA Astrophysics Data System (ADS)

You, Minli; Lin, Min; Wang, Shurui; Wang, Xuemin; Zhang, Ge; Hong, Yuan; Dong, Yuqing; Jin, Guorui; Xu, Feng

2016-05-01

Medicine counterfeiting is a serious issue worldwide, involving potentially devastating health repercussions. Advanced anti-counterfeit technology for drugs has therefore aroused intensive interest. However, existing anti-counterfeit technologies are associated with drawbacks such as the high cost, complex fabrication process, sophisticated operation and incapability in authenticating drug ingredients. In this contribution, we developed a smart phone recognition based upconversion fluorescent three-dimensional (3D) quick response (QR) code for tracking and anti-counterfeiting of drugs. We firstly formulated three colored inks incorporating upconversion nanoparticles with RGB (i.e., red, green and blue) emission colors. Using a modified inkjet printer, we printed a series of colors by precisely regulating the overlap of these three inks. Meanwhile, we developed a multilayer printing and splitting technology, which significantly increases the information storage capacity per unit area. As an example, we directly printed the upconversion fluorescent 3D QR code on the surface of drug capsules. The 3D QR code consisted of three different color layers with each layer encoded by information of different aspects of the drug. A smart phone APP was designed to decode the multicolor 3D QR code, providing the authenticity and related information of drugs. The developed technology possesses merits in terms of low cost, ease of operation, high throughput and high information capacity, thus holds great potential for drug anti-counterfeiting.Medicine counterfeiting is a serious issue worldwide, involving potentially devastating health repercussions. Advanced anti-counterfeit technology for drugs has therefore aroused intensive interest. However, existing anti-counterfeit technologies are associated with drawbacks such as the high cost, complex fabrication process, sophisticated operation and incapability in authenticating drug ingredients. In this contribution, we developed a smart phone recognition based upconversion fluorescent three-dimensional (3D) quick response (QR) code for tracking and anti-counterfeiting of drugs. We firstly formulated three colored inks incorporating upconversion nanoparticles with RGB (i.e., red, green and blue) emission colors. Using a modified inkjet printer, we printed a series of colors by precisely regulating the overlap of these three inks. Meanwhile, we developed a multilayer printing and splitting technology, which significantly increases the information storage capacity per unit area. As an example, we directly printed the upconversion fluorescent 3D QR code on the surface of drug capsules. The 3D QR code consisted of three different color layers with each layer encoded by information of different aspects of the drug. A smart phone APP was designed to decode the multicolor 3D QR code, providing the authenticity and related information of drugs. The developed technology possesses merits in terms of low cost, ease of operation, high throughput and high information capacity, thus holds great potential for drug anti-counterfeiting. Electronic supplementary information (ESI) available: Calculating details of UCNP content per 3D QR code and decoding process of the 3D QR code. See DOI: 10.1039/c6nr01353h
Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

NASA Technical Reports Server (NTRS)

Shrestha, Suresh; Salins, Lyndon L E.; Mark Ensor, C.; Daunert, Sylvia

2002-01-01

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 517-526, 2002.
As-prepared MoS2 quantum dot as a facile fluorescent probe for long-term tracing of live cells

NASA Astrophysics Data System (ADS)

Zhou, Kai; Zhang, Yue; Xia, Zhining; Wei, Weili

2016-07-01

Recently, the newly emerged two-dimensional nanomaterials, layered transition metal dichalcogenide (e.g. MoS2) nanosheets, have drawn tremendous attentions due to their extraordinary electronic and optical properties, and MoS2 quantum dots (MoS2 QDs) with lateral sizes less than 10 nm have been found to be highly luminescent. In the present study, a facile approach for large-scale preparation of MoS2 QDs by Na intercalation reaction without using any toxic organic reagents is proposed. MoS2 QDs were carefully characterized by various techniques including transmission electron microscopy, atomic force microscopy, dynamic light scattering, spectroscopy, in vitro cytotoxicology, and capillary electrophoresis. The as-prepared MoS2 QDs were strongly fluorescent, highly photo-stable, low in cytotoxicity, and readily reactive to thiols. These inherent properties of MoS2 QDs make them excellent fluorescent probes for long-term live cell tracing. The results of live cells imaging indicated that MoS2 QD stained cells remained highly fluorescent after long-term culture, and could be easily traced from other co-cultured cell lines.
Non-fluorescent nanoscopic monitoring of a single trapped nanoparticle via nonlinear point sources.

PubMed

Yoon, Seung Ju; Lee, Jungmin; Han, Sangyoon; Kim, Chang-Kyu; Ahn, Chi Won; Kim, Myung-Ki; Lee, Yong-Hee

2018-06-07

Detection of single nanoparticles or molecules has often relied on fluorescent schemes. However, fluorescence detection approaches limit the range of investigable nanoparticles or molecules. Here, we propose and demonstrate a non-fluorescent nanoscopic trapping and monitoring platform that can trap a single sub-5-nm particle and monitor it with a pair of floating nonlinear point sources. The resonant photon funnelling into an extremely small volume of ~5 × 5 × 7 nm 3 through the three-dimensionally tapered 5-nm-gap plasmonic nanoantenna enables the trapping of a 4-nm CdSe/ZnS quantum dot with low intensity of a 1560-nm continuous-wave laser, and the pumping of 1560-nm femtosecond laser pulses creates strong background-free second-harmonic point illumination sources at the two vertices of the nanoantenna. Under the stable trapping conditions, intermittent but intense nonlinear optical spikes are observed on top of the second-harmonic signal plateau, which is identified as the 3.0-Hz Kramers hopping of the quantum dot trapped in the 5-nm gap.
Hyperspectral Image Analysis for Skin Tumor Detection

NASA Astrophysics Data System (ADS)

Kong, Seong G.; Park, Lae-Jeong

This chapter presents hyperspectral imaging of fluorescence for nonin-vasive detection of tumorous tissue on mouse skin. Hyperspectral imaging sensors collect two-dimensional (2D) image data of an object in a number of narrow, adjacent spectral bands. This high-resolution measurement of spectral information reveals a continuous emission spectrum for each image pixel useful for skin tumor detection. The hyperspectral image data used in this study are fluorescence intensities of a mouse sample consisting of 21 spectral bands in the visible spectrum of wavelengths ranging from 440 to 640 nm. Fluorescence signals are measured using a laser excitation source with the center wavelength of 337 nm. An acousto-optic tunable filter is used to capture individual spectral band images at a 10-nm resolution. All spectral band images are spatially registered with the reference band image at 490 nm to obtain exact pixel correspondences by compensating the offsets caused during the image capture procedure. The support vector machines with polynomial kernel functions provide decision boundaries with a maximum separation margin to classify malignant tumor and normal tissue from the observed fluorescence spectral signatures for skin tumor detection.
Couplings between hierarchical conformational dynamics from multi-time correlation functions and two-dimensional lifetime spectra: Application to adenylate kinase

NASA Astrophysics Data System (ADS)

Ono, Junichi; Takada, Shoji; Saito, Shinji

2015-06-01

An analytical method based on a three-time correlation function and the corresponding two-dimensional (2D) lifetime spectrum is developed to elucidate the time-dependent couplings between the multi-timescale (i.e., hierarchical) conformational dynamics in heterogeneous systems such as proteins. In analogy with 2D NMR, IR, electronic, and fluorescence spectroscopies, the waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra can provide a quantitative description of the dynamical correlations between the conformational motions with different lifetimes. The present method is applied to intrinsic conformational changes of substrate-free adenylate kinase (AKE) using long-time coarse-grained molecular dynamics simulations. It is found that the hierarchical conformational dynamics arise from the intra-domain structural transitions among conformational substates of AKE by analyzing the one-time correlation functions and one-dimensional lifetime spectra for the donor-acceptor distances corresponding to single-molecule Förster resonance energy transfer experiments with the use of the principal component analysis. In addition, the complicated waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra for the donor-acceptor distances is attributed to the fact that the time evolution of the couplings between the conformational dynamics depends upon both the spatial and temporal characters of the system. The present method is expected to shed light on the biological relationship among the structure, dynamics, and function.
Couplings between hierarchical conformational dynamics from multi-time correlation functions and two-dimensional lifetime spectra: Application to adenylate kinase

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

Ono, Junichi; Takada, Shoji; Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502

2015-06-07

An analytical method based on a three-time correlation function and the corresponding two-dimensional (2D) lifetime spectrum is developed to elucidate the time-dependent couplings between the multi-timescale (i.e., hierarchical) conformational dynamics in heterogeneous systems such as proteins. In analogy with 2D NMR, IR, electronic, and fluorescence spectroscopies, the waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra can provide a quantitative description of the dynamical correlations between the conformational motions with different lifetimes. The present method is applied to intrinsic conformational changes of substrate-free adenylate kinase (AKE) using long-time coarse-grained molecular dynamics simulations. It is found that the hierarchicalmore » conformational dynamics arise from the intra-domain structural transitions among conformational substates of AKE by analyzing the one-time correlation functions and one-dimensional lifetime spectra for the donor-acceptor distances corresponding to single-molecule Förster resonance energy transfer experiments with the use of the principal component analysis. In addition, the complicated waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra for the donor-acceptor distances is attributed to the fact that the time evolution of the couplings between the conformational dynamics depends upon both the spatial and temporal characters of the system. The present method is expected to shed light on the biological relationship among the structure, dynamics, and function.« less
Theoretical Analysis of Novel Quasi-3D Microscopy of Cell Deformation

PubMed Central

Qiu, Jun; Baik, Andrew D.; Lu, X. Lucas; Hillman, Elizabeth M. C.; Zhuang, Zhuo; Guo, X. Edward

2012-01-01

A novel quasi-three-dimensional (quasi-3D) microscopy technique has been developed to enable visualization of a cell under dynamic loading in two orthogonal planes simultaneously. The three-dimensional (3D) dynamics of the mechanical behavior of a cell under fluid flow can be examined at a high temporal resolution. In this study, a numerical model of a fluorescently dyed cell was created in 3D space, and the cell was subjected to uniaxial deformation or unidirectional fluid shear flow via finite element analysis (FEA). Therefore, the intracellular deformation in the simulated cells was exactly prescribed. Two-dimensional fluorescent images simulating the quasi-3D technique were created from the cell and its deformed states in 3D space using a point-spread function (PSF) and a convolution operation. These simulated original and deformed images were processed by a digital image correlation technique to calculate quasi-3D-based intracellular strains. The calculated strains were compared to the prescribed strains, thus providing a theoretical basis for the measurement of the accuracy of quasi-3D and wide-field microscopy-based intracellular strain measurements against the true 3D strains. The signal-to-noise ratio (SNR) of the simulated quasi-3D images was also modulated using additive Gaussian noise, and a minimum SNR of 12 was needed to recover the prescribed strains using digital image correlation. Our computational study demonstrated that quasi-3D strain measurements closely recovered the true 3D strains in uniform and fluid flow cellular strain states to within 5% strain error. PMID:22707985
FRET Imaging in Three-dimensional Hydrogels

PubMed Central

Taboas, Juan M.

2016-01-01

Imaging of Förster resonance energy transfer (FRET) is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination. PMID:27500354
Perovskite nanocrystals: across-dimensional attachment, film-scale assembly on a flexible substrate and their fluorescence properties

NASA Astrophysics Data System (ADS)

Huang, Wenyi; Liu, Jiajia; Bai, Bing; Huang, Liu; Xu, Meng; Liu, Jia; Rong, Hongpan; Zhang, Jiatao

2018-03-01

Perovskite nanocrystals (NCs), which are a good fluorescence candidate with excellent photoelectric properties, have opened new avenues in the fabrication of highly efficient solar cells, light-emitting diodes (LEDs), and other optoelectronic devices. Further advances will rely on the multitude of compositional, structural variants that enable the formation of lower-dimensionality layered and three-dimensional (3D) perovskites with architectural innovations. In this work, the perovskite film was fabricated on a flexible substrate using simple dip-coating technology and 3D assemblies of perovskite NCs were obtained through an attachment process. Original perovskite NCs had a rectangular or square morphology with high particle uniformity and the narrow and symmetric fluorescence emission peak was adjustable at 515-527 nm. The controllable self-assembly of the micron size cuboid-like 3D assembly had an apparent enhancement on peak (111) in the x-ray diffraction (XRD) pattern. Surface ligands not only play a role in the attachment process but also keep the independence of each NC in 3D assemblies. Such assembly of the perovskite film maintained the original perovskite NCs fluorescence emission peak and narrow full width at the half-maximum (FWHM), which is of great importance for the investigation of future devices.
Biosensing with Förster Resonance Energy Transfer Coupling between Fluorophores and Nanocarbon Allotropes

PubMed Central

Ding, Shaowei; Cargill, Allison A.; Das, Suprem R.; Medintz, Igor L.; Claussen, Jonathan C.

2015-01-01

Nanocarbon allotropes (NCAs), including zero-dimensional carbon dots (CDs), one-dimensional carbon nanotubes (CNTs) and two-dimensional graphene, exhibit exceptional material properties, such as unique electrical/thermal conductivity, biocompatibility and high quenching efficiency, that make them well suited for both electrical/electrochemical and optical sensors/biosensors alike. In particular, these material properties have been exploited to significantly enhance the transduction of biorecognition events in fluorescence-based biosensing involving Förster resonant energy transfer (FRET). This review analyzes current advances in sensors and biosensors that utilize graphene, CNTs or CDs as the platform in optical sensors and biosensors. Widely utilized synthesis/fabrication techniques, intrinsic material properties and current research examples of such nanocarbon, FRET-based sensors/biosensors are illustrated. The future outlook and challenges for the research field are also detailed. PMID:26110411
A detailed study of gold-nanoparticle loaded cells using X-ray based techniques for cell-tracking applications with single-cell sensitivity

NASA Astrophysics Data System (ADS)

Astolfo, Alberto; Arfelli, Fulvia; Schültke, Elisabeth; James, Simon; Mancini, Lucia; Menk, Ralf-Hendrik

2013-03-01

In the present study complementary high-resolution imaging techniques on different length scales are applied to elucidate a cellular loading protocol of gold nanoparticles and subsequently its impact on long term and high-resolution cell-tracking utilizing X-ray technology. Although demonstrated for malignant cell lines the results can be applied to non-malignant cell lines as well. In particular the accumulation of the gold marker per cell has been assessed quantitatively by virtue of electron microscopy, two-dimensional X-ray fluorescence imaging techniques and X-ray CT with micrometric and sub-micrometric resolution. Moreover, utilizing these techniques the three dimensional distribution of the incorporated nanoparticles, which are sequestered in lysosomes as a permanent marker, could be determined. The latter allowed elucidation of the gold partition during mitosis and the cell size, which subsequently enabled us to define the optimal instrument settings of a compact microCT system to visualize gold loaded cells. The results obtained demonstrate the feasibility of cell-tracking using X-ray CT with compact sources.
Nonlinear microscopy of collagen fibers

NASA Astrophysics Data System (ADS)

Strupler, M.; Pena, A.-M.; Hernest, M.; Tharaux, P.-L.; Fabre, A.; Marchal-Somme, J.; Crestani, B.; Débarre, D.; Martin, J.-L.; Beaurepaire, E.; Schanne-Klein, M.-C.

2007-02-01

We used intrinsic Second Harmonic Generation (SHG) by fibrillar collagen to visualize the three-dimensional architecture of collagen fibrosis at the micrometer scale using laser scanning nonlinear microscopy. We showed that SHG signals are highly specific to fibrillar collagen and provide a sensitive probe of the micrometer-scale structural organization of collagen in tissues. Moreover, recording simultaneously other nonlinear optical signals in a multimodal setup, we visualized the tissue morphology using Two-Photon Excited Fluorescence (2PEF) signals from endogenous chromophores such as NADH or elastin. We then compared different methods to determine accurate indexes of collagen fibrosis using nonlinear microscopy, given that most collagen fibrils are smaller than the microscope resolution and that second harmonic generation is a coherent process. In order to define a robust method to process our three-dimensional images, we either calculated the fraction of the images occupied by a significant SHG signal, or averaged SHG signal intensities. We showed that these scores provide an estimation of the extension of renal and pulmonary fibrosis in murine models, and that they clearly sort out the fibrotic mice.
Three-Dimensional Simulation of Ultrasound-Induced Microalgal Cell Disruption.

PubMed

Wang, M; Yuan, W; Hale, Andy

2016-03-01

The three-dimensional distribution (x, y, and z) of ultrasound-induced microalgal cell disruption in a sonochemical reactor was predicted by solving the Helmholtz equation using a three-dimensional acoustic module in the COMSOL Multiphysics software. The simulated local ultrasound pressure at any given location (x, y, and z) was found to correlate with cell disruption of a freshwater alga, Scenedesmus dimorphus, represented by the change of algal cell particle/debris concentration, chlorophyll-a fluorescence density (CAFD), and Nile red stained lipid fluorescence density (LFD), which was also validated by the model reaction of potassium iodide oxidation (the Weissler reaction). Furthermore, the effect of ultrasound power intensity and processing duration on algal cell disruption was examined to address the limitation of the model.
Chemical structure-nonlinear optical property relationships for a series of two-photon absorbing fluorene molecules

NASA Astrophysics Data System (ADS)

Hales, Joel Mccajah

This dissertation reports on the investigation of two-photon absorption (2PA) in a series of fluorenyl molecules. Several current and emerging technologies exploit this optical nonlinearity including two-photon fluorescence imaging, three-dimensional microfabrication, site-specific photodynamic cancer therapy and biological caging studies. The two key features of this nonlinearity which make it an ideal candidate for the above applications are its quadratic dependence on the incident irradiance and the improved penetration into absorbing media that it affords. As a consequence of the burgeoning field which exploits 2PA, it is a goal to find materials that exhibit strong two-photon absorbing capabilities. Organic materials are promising candidates for 2PA applications because their material properties can be tailored through molecular engineering thereby facilitating optimization of their nonlinear optical properties. Fluorene derivatives are particularly interesting since they possess high photochemical stability for organic molecules and are generally strongly fluorescent. By systematically altering the structural properties in a series of fluorenyl molecules, we have determined how these changes affect their two-photon absorbing capabilities. This was accomplished through characterization of both the strength and location of their 2PA spectra. In order to ensure the validity of these results, three separate nonlinear characterization techniques were employed: two-photon fluorescence spectroscopy, white-light continuum pump-probe spectroscopy, and the Z-scan technique. In addition, full linear spectroscopic characterization was performed on these molecules along with supplementary quantum chemical calculations to obtain certain molecular properties that might impact the nonlinearity. Different designs in chemical architecture allowed investigation of the effects of symmetry, solvism, donor-acceptor strengths, conjugation length, and multi-branched geometries on the two-photon absorbing properties of these molecules. In addition, the means to enhance 2PA via intermediate state resonances was investigated. To provide plausible explanations for the experimentally observed trends, a conceptually simple three level model was employed. The subsequent correlations found between chemical structure and the linear and nonlinear optical properties of these molecules provided definitive conclusions on how to properly optimize their two-photon absorbing capabilities. The resulting large nonlinearities found in these molecules have already shown promise in a variety of the aforementioned applications.

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

NASA Astrophysics Data System (ADS)

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

2011-04-01

We present protoporphyrin IX (PpIX) fluorescence measurements acquired from patients presenting with superficial basal cell carcinoma during photodynamic therapy (PDT) treatment, facilitating in vivo photobleaching to be monitored. Monte Carlo (MC) simulations, taking into account photobleaching, are performed on a three-dimensional cube grid, which represents the treatment geometry. Consequently, it is possible to determine the spatial and temporal changes to the origin of collected fluorescence and generated singlet oxygen. From our clinical results, an in vivo photobleaching dose constant, β of 5-aminolaevulinic acid-induced PpIX fluorescence is found to be 14 +/- 1 J/cm2. Results from our MC simulations suggest that an increase from our typical administered treatment light dose of 75-150 J/cm2 could increase the effective PDT treatment initially achieved at a depth of 2.7-3.3 mm in the tumor, respectively. Moreover, this increase reduces the surface PpIX fluorescence from 0.00012 to 0.000003 of the maximum value recorded before treatment. The recommendation of administrating a larger light dose, which advocates an increase in the treatment time after surface PpIX fluorescence has diminished, remains valid for different sets of optical properties and therefore should have a beneficial outcome on the total treatment effect.
A threefold interpenetrated two-dimensional zinc(II) supramolecular architecture based on 3-nitrobenzoic acid and 4,4'-bipyridine.

PubMed

Tang, Long; Wang, Ji-Jiang; Fu, Feng; Wang, Sheng-Wen; Liu, Qi-Rui

2016-02-01

With regard to crystal engineering, building block or modular assembly methodologies have shown great success in the design and construction of metal-organic coordination polymers. The critical factor for the construction of coordination polymers is the rational choice of the organic building blocks and the metal centre. The reaction of Zn(OAc)2·2H2O (OAc is acetate) with 3-nitrobenzoic acid (HNBA) and 4,4'-bipyridine (4,4'-bipy) under hydrothermal conditions produced a two-dimensional zinc(II) supramolecular architecture, catena-poly[[bis(3-nitrobenzoato-κ(2)O,O')zinc(II)]-μ-4,4'-bipyridine-κ(2)N:N'], [Zn(C7H4NO4)2(C10H8N2)]n or [Zn(NBA)2(4,4'-bipy)]n, which was characterized by elemental analysis, IR spectroscopy, thermogravimetric analysis and single-crystal X-ray diffraction analysis. The Zn(II) ions are connected by the 4,4'-bipy ligands to form a one-dimensional zigzag chain and the chains are decorated with anionic NBA ligands which interact further through aromatic π-π stacking interactions, expanding the structure into a threefold interpenetrated two-dimensional supramolecular architecture. The solid-state fluorescence analysis indicates a slight blue shift compared with pure 4,4'-bipyridine and HNBA.
Fluorescence characterization of fractionated dissolved organic matter in the five tributaries of Poyang Lake, China.

PubMed

Yan, Caixia; Liu, Huihui; Sheng, Yanru; Huang, Xian; Nie, Minghua; Huang, Qi; Baalousha, Mohammed

2018-10-01

Characterization of natural colloids is the key to understand pollutant fate and transport in the environment. The present study investigates the relationship between size and fluorescence properties of colloidal organic matter (COM) from five tributaries of Poyang Lake. Colloids were size-fractionated using cross-flow ultrafiltration and their fluorescence properties were measured by three-dimensional excitation-emission matrix fluorescence spectroscopy (3D-EEM). Parallel factor analysis (PARAFAC) and/or Self-organizing map (SOM) were applied to assess fluorescence properties as proxy indicators for the different size of colloids. PARAFAC analysis identified four fluorescence components including three humic-like components (C1-C3) and a protein-like component (C4). These four fluorescence components, and in particular the protein-like component, are primarily present in <1 kDa phase. For the colloidal fractions (1-10 kDa, 10-100 kDa, and 100 kDa-0.7 μm), the majority of fluorophores are associated with the smallest size fraction. SOM analysis demonstrated that relatively high fluorescence intensity and aromaticity occur primarily in <1 kDa phase, followed by 1-10 kDa colloids. Coupling PARAFAC and SOM facilitate the visualization and interpretation of the relationship between colloidal size and fluorescence properties with fewer input variables, shorter running time, higher reliability, and nondestructive results. Fluorescence indices analysis reveals that the smallest colloidal fraction (1-10 kDa) was dominated by higher humified and less autochthonous COM. Copyright © 2018 Elsevier B.V. All rights reserved.
Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo

NASA Astrophysics Data System (ADS)

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

2012-07-01

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

PubMed

Martín-Pacheco, Ana; Del Río Castillo, Antonio Esaú; Martín, Cristina; Herrero, María Antonia; Merino, Sonia; García Fierro, José Luis; Díez-Barra, Enrique; Vázquez, Ester

2018-05-30

Fluorescence based on quantum confinement is a property restricted to the nanoscopic range. The incorporation of nanoparticles in a three-dimensional polymeric network could afford macroscopic scaffolds that show nanoscopic properties. Moreover, if these scaffolds are based on strong bonds, the stability of the resulting materials can be preserved, thus enhancing their final applications. We report for the first time the preparation of a graphene quantum dot (GQD) composite based on a cationic covalent network. This new material has unusual features: (i) the final composite remains stable after several swelling-deswelling cycles, thus demonstrating strong interactions between GQDs and the polymeric material, and therefore it could be used as a portable system. (ii) Fluorescence emission in the composite and in solution is quasi-independent to the excitation wavelength. (iii) However, and in contrast to the behavior observed in GQD solutions, the fluorescence of the composite remains unaltered over a wide pH range and in the presence of different ions commonly found in tap water. (iv) Fluorescence quenching is only observed as a consequence of molecules that bear aromatic systems, and this could be applied to the preparation of in situ water sensors.
Particle visualization in high-power impulse magnetron sputtering. I. 2D density mapping

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

Britun, Nikolay, E-mail: nikolay.britun@umons.ac.be; Palmucci, Maria; Konstantinidis, Stephanos

2015-04-28

Time-resolved characterization of an Ar-Ti high-power impulse magnetron sputtering discharge has been performed. This paper deals with two-dimensional density mapping in the discharge volume obtained by laser-induced fluorescence imaging. The time-resolved density evolution of Ti neutrals, singly ionized Ti atoms (Ti{sup +}), and Ar metastable atoms (Ar{sup met}) in the area above the sputtered cathode is mapped for the first time in this type of discharges. The energetic characteristics of the discharge species are additionally studied by Doppler-shift laser-induced fluorescence imaging. The questions related to the propagation of both the neutral and ionized discharge particles, as well as to theirmore » spatial density distributions, are discussed.« less
Sensitivity and accuracy of hybrid fluorescence-mediated tomography in deep tissue regions.

PubMed

Rosenhain, Stefanie; Al Rawashdeh, Wa'el; Kiessling, Fabian; Gremse, Felix

2017-09-01

Fluorescence-mediated tomography (FMT) enables noninvasive assessment of the three-dimensional distribution of near-infrared fluorescence in mice. The combination with micro-computed tomography (µCT) provides anatomical data, enabling improved fluorescence reconstruction and image analysis. The aim of our study was to assess sensitivity and accuracy of µCT-FMT under realistic in vivo conditions in deeply-seated regions. Accordingly, we acquired fluorescence reflectance images (FRI) and µCT-FMT scans of mice which were prepared with rectal insertions with different amounts of fluorescent dye. Default and high-sensitivity scans were acquired and background signal was analyzed for three FMT channels (670 nm, 745 nm, and 790 nm). Analysis was performed for the original and an improved FMT reconstruction using the µCT data. While FRI and the original FMT reconstruction could detect 100 pmol, the improved FMT reconstruction could detect 10 pmol and significantly improved signal localization. By using a finer sampling grid and increasing the exposure time, the sensitivity could be further improved to detect 0.5 pmol. Background signal was highest in the 670 nm channel and most prominent in the gastro-intestinal tract and in organs with high relative amounts of blood. In conclusion, we show that µCT-FMT allows sensitive and accurate assessment of fluorescence in deep tissue regions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

2014-09-01

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.
Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

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

Lin, Jian; Zheng, Wei; Wang, Zi

2014-09-08

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.
Comparison of femtosecond- and nanosecond-two-photon-absorption laser-induced fluorescence (TALIF) of atomic oxygen in atmospheric-pressure plasmas

NASA Astrophysics Data System (ADS)

Schmidt, Jacob B.; Sands, Brian; Scofield, James; Gord, James R.; Roy, Sukesh

2017-05-01

Absolute number densities of atomic species produced by nanosecond (ns)-duration, repetitively pulsed electric discharges are measured by two-photon-absorption laser-induced fluorescence (TALIF). Unique to this work is the development of femtosecond-laser-based TALIF (fs-TALIF) that offers a number of advantages over more conventional nanosecond (ns)-pulse-duration laser techniques, such as higher-fidelity quenching rate measurements over a wide pressure range, significantly reduced photolytic interference (including photo-dissociation and photo-ionization), ability to collect two-dimensional images of atomic-species number densities with high spatial resolution aided by higher signal level, and efficient and accurate measurements of atomic-species number densities due to the higher repetition rates of the laser. For full quantification of these advantages, atomic-oxygen TALIF signals are collected from an atmospheric-pressure plasma jet employing both ns- and fs-duration laser-excitation pulses and the results are compared and contrasted.
In vivo three-dimensional optical coherence tomography and multiphoton microscopy in a mouse model of ovarian neoplasia

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.; Marion, Samuel L.; Rice, Photini Faith; Bentley, David L.; Besselsen, David; Utzinger, Urs; Hoyer, Patricia B.; Barton, Jennifer K.

2013-03-01

Our goal is to use optical coherence tomography (OCT) and multiphoton microscopy (MPM) to detect early tumor development in a mouse model of ovarian neoplasia. We hope to use information regarding early tumor development to create a diagnostic test for high-risk patients. In this study we collect in vivo images using OCT, second harmonic generation and two-photon excited fluorescence from non-vinylcyclohexene diepoxide (VCD)-dosed and VCD-dosed mice. VCD causes follicular apoptosis (simulating menopause) and leads to tumor development. Using OCT and MPM we visualized the ovarian microstructure and were able to see differences between non-VCD-dosed and VCD-dosed animals. This leads us to believe that OCT and MPM may be useful for detecting changes due to early tumor development.
Proton beam spatial distribution and Bragg peak imaging by photoluminescence of color centers in lithium fluoride crystals at the TOP-IMPLART linear accelerator

NASA Astrophysics Data System (ADS)

Piccinini, M.; Ronsivalle, C.; Ampollini, A.; Bazzano, G.; Picardi, L.; Nenzi, P.; Trinca, E.; Vadrucci, M.; Bonfigli, F.; Nichelatti, E.; Vincenti, M. A.; Montereali, R. M.

2017-11-01

Solid-state radiation detectors based on the photoluminescence of stable point defects in lithium fluoride crystals have been used for advanced diagnostics during the commissioning of the segment up to 27 MeV of the TOP-IMPLART proton linear accelerator for proton therapy applications, under development at ENEA C.R. Frascati, Italy. The LiF detectors high intrinsic spatial resolution and wide dynamic range allow obtaining two-dimensional images of the beam transverse intensity distribution and also identifying the Bragg peak position with micrometric precision by using a conventional optical fluorescence microscope. Results of the proton beam characterization, among which, the estimation of beam energy components and dynamics, are reported and discussed for different operating conditions of the accelerator.
Improved Underwater Excitation-Emission Matrix Fluorometer

NASA Technical Reports Server (NTRS)

Moore, Casey; daCunha, John; Rhoades, Bruce; Twardowski, Michael

2007-01-01

A compact, high-resolution, two-dimensional excitation-emission matrix fluorometer (EEMF) has been designed and built specifically for use in identifying and measuring the concentrations of organic compounds, including polluting hydrocarbons, in natural underwater settings. Heretofore, most EEMFs have been designed and built for installation in laboratories, where they are used to analyze the contents of samples collected in the field and brought to the laboratories. Because the present EEMF can be operated in the field, it is better suited to measurement of spatially and temporally varying concentrations of substances of interest. In excitation-emission matrix (EEM) fluorometry, fluorescence is excited by irradiating a sample at one or more wavelengths, and the fluorescent emission from the sample is measured at multiple wavelengths. When excitation is provided at only one wavelength, the technique is termed one-dimensional (1D) EEM fluorometry because the resulting matrix of fluorescence emission data (the EEM) contains only one row or column. When excitation is provided at multiple wavelengths, the technique is termed two-dimensional (2D) EEM fluorometry because the resulting EEM contains multiple rows and columns. EEM fluorometry - especially the 2D variety - is well established as a means of simultaneously detecting numerous dissolved and particulate compounds in water. Each compound or pool of compounds has a unique spectral fluorescence signature, and each EEM is rich in information content, in that it can contain multiple fluorescence signatures. By use of deconvolution and/or other mixture-analyses techniques, it is often possible to isolate the spectral signature of compounds of interest, even when their fluorescence spectra overlap. What distinguishes the present 2D EEMF over prior laboratory-type 2D EEMFs are several improvements in packaging (including a sealed housing) and other aspects of design that render it suitable for use in natural underwater settings. In addition, the design of the present 2D EEMF incorporates improvements over the one prior commercial underwater 2D EEMF, developed in 1994 by the same company that developed the present one. Notable advanced features of the present EEMF include the following: 1) High sensitivity and spectral resolution are achieved by use of an off-the-shelf grating spectrometer equipped with a sensor in the form of a commercial astronomical- grade 256 532-pixel charge-coupled-device (CCD) array. 2) All of the power supply, timing, control, and readout circuits for the illumination source and the CCD, ancillary environmental monitoring sensors, and circuitry for controlling a shutter or filter motor are custom-designed and mounted compactly on three circuit boards below a fourth circuit board that holds the CCD (see figure). 3) The compactness of the grating spectrometer, CCD, and circuit assembly makes it possible to fit the entire instrument into a compact package that is intended to be maneuverable underwater by one person. 4) In mass production, the cost of the complete instrument would be relatively low - estimated at approximately $30,000 at 2005 prices.
Comparison of the longissimus muscle proteome between obese and lean pigs at 180 days.

PubMed

Li, Anning; Mo, Delin; Zhao, Xiao; Jiang, Wei; Cong, Peiqing; He, Zuyong; Xiao, Shuqi; Liu, Xiaohong; Chen, Yaosheng

2013-02-01

Production of high-quality meat is important to satisfy the consumer and make the pig industry competitive. Obese and lean breeds of pig show clear differences in adipogenic capacity and meat quality, but the underlying molecular mechanism remains unclear. We have compared protein expression of the longissimus muscle between Lantang (LT, obese) and Landrace (LR, lean) pigs at the age of 180 days using two-dimensional fluorescence difference gel electrophoresis. Of the 1,400 protein spots detected per gel, 18 were differentially expressed between the two breeds. Using peptide mass fingerprint and tandem mass spectrometry, 17 protein spots were identified, corresponding to ten different proteins that could be divided into four groups: metabolism-related, structure-related, stress-related, and other (unclassified). Among the metabolism-related proteins, COX5A and ATP5B, which participate in oxidative phosphorylation, were highly expressed in LT, whereas ENO3, which is involved in glycolysis, was highly expressed in LR. These results may contribute valuable information to our understanding of the molecular mechanism responsible for differences between obese and lean pigs, such as growth rate and meat quality.
4Pi-confocal microscopy of live cells

NASA Astrophysics Data System (ADS)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.
Two novel zinc(II) coordination polymers constructed from in situ amidation ligands

NASA Astrophysics Data System (ADS)

Yu, Xiao-Yang; Fu, Yao; Fu, Jian-Tao; Xu, Jia-Ning; Luo, Ya-Nan; Yang, Yan-Yan; Qu, Xiao-Shu; Zhang, Jing; Lu, Shu-Lai

2018-04-01

Two novel coordination compounds, [Zn(Hbimh) (H2O)]·H2O (1) and [Zn(Hbimh)]·(4,4ʹ-bpy)0.5 (2) (H3bimh = benzimidazole-5,6-hydrazide, 4,4ʹ-bpy = 4,4ʹ-bipyridine), have been prepared from the hydrothermal in situ amidation cyclization reactions of H3bimdc (H3bimdc = benzimidazole-5,6-dicarboxylic acid) and hydrazine hydrate (N2H4·H2O). Compound 1 exhibits a one-dimensional (1D) hexagon channel structure. Compound 2 is a three-dimensional (3D) framework structure, with 4,4ʹ-bpy fill the channels. We also obtained the ligand H3bimh. The compounds were characterized by IR, PXRD, TGA and elemental analysis. The fluorescence properties in the solid state at room temperature were also investigated.
Use of Complementary Approaches to Imaging Biomolecules and Endogenous and Exogenous Trace Elements and Nanoparticles in Biological Samples

NASA Astrophysics Data System (ADS)

Brown, Koshonna Dinettia

X-ray Fluorescence Microscopy (XFM) is a useful technique for study of biological samples. XFM was used to map and quantify endogenous biological elements as well as exogenous materials in biological samples, such as the distribution of titanium dioxide (TiO2) nanoparticles. TiO 2 nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic particles for cancer detection and treatment, drug delivery, and induction of DNA breaks. Delivery of such nanoparticles can be targeted to specific cells and subcellular structures. In this work, we develop two novel approaches to stain TiO2 nanoparticles for optical microscopy and to confirm that staining by XFM. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called CLICK chemistry, for labeling of azide conjugated TiO2 nanoparticles with "clickable" dyes such as alkyne Alexa Fluor dyes with a high fluorescent yield. To confirm that the optical fluorescence signals of nanoparticles stained in situ match the distribution of the Ti element, we used high resolution synchrotron X-Ray Fluorescence Microscopy (XFM) using the Bionanoprobe instrument at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific X-ray fluorescence showed excellent overlap with the location of Alexa Fluor optical fluorescence detected by confocal microscopy. In this work XFM was also used to investigate native elemental differences between two different types of head and neck cancer, one associated with human papilloma virus infection, the other virus free. Future work may see a cross between these themes, for example, exploration of TiO2 nanoparticles as anticancer treatment for these two different types of head and neck cancer.
Ns-scaled time-gated fluorescence lifetime imaging for forensic document examination

NASA Astrophysics Data System (ADS)

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

NASA Astrophysics Data System (ADS)

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

2014-10-01

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

PubMed

Lidke, Diane S; Lidke, Keith A

2012-06-01

A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

Facile synthesis of a two-photon fluorescent probe based on pyrimidine 2-isothiocyanate and its application in bioimaging.

PubMed

Yang, Jie; Hu, Wei; Li, Huirong; Hou, Hanna; Tu, Yi; Liu, Bo

2018-04-18

Two-photon microscopy imaging has been widely applied in biological imaging, but the development of two-photon absorption probes is obviously lagging behind in the development of imaging technology. In this paper, a two-photon fluorescent probe (1) based on pyrimidine 2-isothiocyanate has been designed and synthesized through a simple method for two-photon biological imaging. Probe 1 was able to couple effectively with the amino groups on biomolecules. To verify the reactivity of the isothiocyanate group on probe 1 and the amine groups on the biomolecules, d-glucosamine was chosen as a model biomolecule to conjugate with probe 1. The result showed that probe 1 could effectively conjugate with d-glucosamine to synthesize probe 2, and the yield of probe 2 was 83%. After conjugating with d-glucosamine, linear absorption spectra, single-photon fluorescence spectra, and two-photon fluorescence spectra of probes 1 and 2 did not present significant changes. Probes 1 and 2 exhibited high fluorescence quantum yields (0.71-0.79) in toluene and chloroform. They also exhibited different photo-physical properties in solvents with different polarities. The two-photon absorption cross-section of probe 1 was 953 GM in toluene. In addition, probe 1 could be effectively conjugated with transferrin, and the conjugated probe (Tf-1) could be transported into Hep G2 cells through a receptor-mediated process for biological imaging. These results demonstrate that such probes are expected to have great potential applications in two-photon fluorescence bioimaging.
Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells

PubMed Central

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-01-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. Three dimensional-Magnetic Twisting Cytometry (3D-MTC) is a technique for applying local mechanical stresses on living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real time acquisition of a living cell’s mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC – microscopy platform takes around 20 days to construct and the experimental procedures require ~4 days when carried out by a life sciences graduate student. PMID:28686583
Using two-dimensional correlation size exclusion chromatography (2D-CoSEC) to explore the size-dependent heterogeneity of humic substances for copper binding.

PubMed

Lee, Yun-Kyung; Hur, Jin

2017-08-01

Knowledge of the heterogeneous distribution of humic substances (HS) reactivities along a continuum of molecular weight (MW) is crucial for the systems where the HS MW is subject to change. In this study, two dimensional correlation spectroscopy combined with size exclusion chromatography (2D-CoSEC) was first utilized to obtain a continuous and heterogeneous presence of copper binding characteristics within bulk HS with respect to MW. HS solutions with varying copper concentrations were directly injected into a size exclusion chromatography (SEC) system with Tris-HCl buffer as a mobile phase. Several validation tests confirmed neither structural disruption of HS nor competition effect of the mobile phase used. Similar to batch systems, fluorescence quenching was observed in the chromatograms over a wide range of HS MW. 2D-CoSEC maps of a soil-derived HS (Elliot soil humic acid) showed the greater fluorescence quenching degrees with respect to the apparent MW on the order of 12500 Da > 10600 Da > 7000 Da > 15800 Da. The binding constants calculated based on modified Stern-Volmer equation were consistent with the 2D-CoSEC results. More heterogeneity of copper binding affinities within bulk HS was found for the soil-derived HS versus an aquatic HS. The traditional fluorescence quenching titration method using ultrafiltered HS size fractions failed to delineate detailed distribution of the copper binding characteristics, exhibiting a much shorter range of the binding constants than those obtained from the 2D-CoSEC. Our proposed technique demonstrated a great potential to describe metal binding characteristics of HS at high MW resolution, providing a clear picture of the size-dependent metal-HS interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.
Two-dimensional correlation spectroscopic analysis on the interaction between humic acids and TiO2 nanoparticles.

PubMed

Chen, Wei; Qian, Chen; Liu, Xiao-Yang; Yu, Han-Qing

2014-10-07

The elucidation of the interaction between TiO2 nanoparticles (NPs) and natural organic matter (NOM) can help one to better understand the fates, features, and environmental impacts of NPs. In this work, two-dimensional (2D) Fourier transformation infrared (FTIR) correlation spectroscopy (CoS) assisted by the fluorescence excitation-emission matrix (EEM) method is used to explore the interaction mechanism of humic acid (HA) with TiO2 NPs at a molecular level. The results show that the C═O bonds (carboxylate, amide, quinone, or ketone) and C-O bonds (phenol, aliphatic C-OH, and polysaccharide) of HA play important roles in their interaction with TiO2 NPs. The adsorption process of HA onto the surface of TiO2 NPs is different from the bonding process of the two species in solution. The forms of the relevant groups of HA and their consequent reaction with TiO2 NPs are affected to a great extent by the solution pH and the surface charge of NPs. The 2D-FTIR-CoS method is found to be able to construct a comprehensive picture about the NOM-TiO2 NPs interaction process. This 2D-FTIR-CoS approach might also be used to probe other complicated interaction processes in natural and engineered environments.
Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS–PAGE coupled with nanoLC–ESI–MS/MS bottom-up proteomics

PubMed Central

Clement, Cristina C.; Aphkhazava, David; Nieves, Edward; Callaway, Myrasol; Olszewski, Waldemar; Rotzschke, Olaf; Santambrogio, Laura

2013-01-01

In this study a proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC Orbitrap–ESI–MS/MS), and two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC–ESI–MS/MS). The 253 significantly identified proteins (p<0.05), obtained from the tandem mass spectrometry data, were further analyzed with pathway analysis (IPA) to define the functional signature of prenodal lymph and matched plasma. The 1DEF coupled with nanoLC–MS–MS revealed that the common proteome between the two biological fluids (144 out of 253 proteins) was dominated by complement activation and blood coagulation components, transporters and protease inhibitors. The enriched proteome of human lymph (72 proteins) consisted of products derived from the extracellular matrix, apoptosis and cellular catabolism. In contrast, the enriched proteome of human plasma (37 proteins) consisted of soluble molecules of the coagulation system and cell–cell signaling factors. The functional networks associated with both common and source-distinctive proteomes highlight the principal biological activity of these immunologically relevant body fluids. PMID:23202415
Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system.

PubMed

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.
Morphological spot counting from stacked images for automated analysis of gene copy numbers by fluorescence in situ hybridization.

PubMed

Grigoryan, Artyom M; Dougherty, Edward R; Kononen, Juha; Bubendorf, Lukas; Hostetter, Galen; Kallioniemi, Olli

2002-01-01

Fluorescence in situ hybridization (FISH) is a molecular diagnostic technique in which a fluorescent labeled probe hybridizes to a target nucleotide sequence of deoxyribose nucleic acid. Upon excitation, each chromosome containing the target sequence produces a fluorescent signal (spot). Because fluorescent spot counting is tedious and often subjective, automated digital algorithms to count spots are desirable. New technology provides a stack of images on multiple focal planes throughout a tissue sample. Multiple-focal-plane imaging helps overcome the biases and imprecision inherent in single-focal-plane methods. This paper proposes an algorithm for global spot counting in stacked three-dimensional slice FISH images without the necessity of nuclei segmentation. It is designed to work in complex backgrounds, when there are agglomerated nuclei, and in the presence of illumination gradients. It is based on the morphological top-hat transform, which locates intensity spikes on irregular backgrounds. After finding signals in the slice images, the algorithm groups these together to form three-dimensional spots. Filters are employed to separate legitimate spots from fluorescent noise. The algorithm is set in a comprehensive toolbox that provides visualization and analytic facilities. It includes simulation software that allows examination of algorithm performance for various image and algorithm parameter settings, including signal size, signal density, and the number of slices.
Single shot, three-dimensional fluorescence microscopy with a spatially rotating point spread function

PubMed Central

Wang, Zhaojun; Cai, Yanan; Liang, Yansheng; Zhou, Xing; Yan, Shaohui; Dan, Dan; Bianco, Piero R.; Lei, Ming; Yao, Baoli

2017-01-01

A wide-field fluorescence microscope with a double-helix point spread function (PSF) is constructed to obtain the specimen’s three-dimensional distribution with a single snapshot. Spiral-phase-based computer-generated holograms (CGHs) are adopted to make the depth-of-field of the microscope adjustable. The impact of system aberrations on the double-helix PSF at high numerical aperture is analyzed to reveal the necessity of the aberration correction. A modified cepstrum-based reconstruction scheme is promoted in accordance with properties of the new double-helix PSF. The extended depth-of-field images and the corresponding depth maps for both a simulated sample and a tilted section slice of bovine pulmonary artery endothelial (BPAE) cells are recovered, respectively, verifying that the depth-of-field is properly extended and the depth of the specimen can be estimated at a precision of 23.4nm. This three-dimensional fluorescence microscope with a framerate-rank time resolution is suitable for studying the fast developing process of thin and sparsely distributed micron-scale cells in extended depth-of-field. PMID:29296483
Characterization of dissolved organic matter in a coral reef ecosystem subjected to anthropogenic pressures (La Réunion Island, Indian Ocean) using multi-dimensional fluorescence spectroscopy.

PubMed

Tedetti, Marc; Cuet, Pascale; Guigue, Catherine; Goutx, Madeleine

2011-05-01

La Saline fringing reef is the most important coral reef complex of La Réunion Island (southwestern Indian Ocean; 21°07'S, 55°32'E). This ecosystem is subjected to anthropogenic pressures through river inputs and submarine groundwater discharge (SGD). The goal of this study was to characterize the pool of fluorescent dissolved organic matter (FDOM) in different water bodies of La Saline fringing reef ecosystem using excitation-emission matrix (EEM) spectrofluorometry. From EEMs, we identified the different fluorophores by the peak picking technique and determined two fluorescence indices issued from the literature: the humification index (HIX) and the biological index (BIX). The main known fluorophores were present within the sample set: humic-like A, humic-like C, marine humic-like M, tryptophan-like T1 and T2, and tyrosine-like B1 and B2. In some samples, unknown fluorophores ("U") were also detected. The surface oceanic waters located beyond the reef front displayed a typical oligotrophic marine signature, with a dominance of autochthonous/biological material (presence of peaks: T1>B1>A>T2>M>C; HIX: 0.9±0.4; BIX: 2.3±1.1). In the reef waters, the autochthonous/biological fingerprint also dominated even though the content in humic substances was higher (same relative distribution of peaks; HIX: 1.6±0.6; BIX: 1.0±0.1). Sedimentary and volcanic SGD showed very different patterns with a strong terrestrial source for the former (A>T1>C>B1 and A>C>B1; HIX: 9.8±2.0; BIX: 0.8±0.0) and a weak terrestrial source for the latter (A>B1>U3>B2>C and A>U4>C; HIX: 2.4±0.3; BIX: 0.9±0.0). In the Hermitage River, both humic substances and protein-like material were abundant (T1>A>U5>B1>C>B2; HIX: 2.3; BIX: 1.4). We provide evidences for the presence of anthropogenic DOM in some of these water bodies. Some oceanic samples (presence of peaks U1 and U2) were likely contaminated by oil-derived PAHs from ships navigating around the reef front, whereas the Hermitage River was highly impacted by sewage effluents, numerous in this coastal area of La Réunion Island. We conclude that multi-dimensional fluorescence spectroscopy (EEM) coupled to the determination of HIX and BIX is a good tool for assessing the origin and distribution of DOM in the coral reef ecosystems submitted to anthropogenic impacts. Copyright © 2011 Elsevier B.V. All rights reserved.
Optical properties of chromophoric dissolved organic matter (CDOM) in surface and pore waters adjacent to an oil well in a southern California salt marsh.

PubMed

Bowen, Jennifer C; Clark, Catherine D; Keller, Jason K; De Bruyn, Warren J

2017-01-15

Chromophoric dissolved organic matter (CDOM) optical properties were measured in surface and pore waters as a function of depth and distance from an oil well in a southern California salt marsh. Higher fluorescence and absorbances in pore vs. surface waters suggest soil pore water is a reservoir of CDOM in the marsh. Protein-like fluorophores in pore waters at distinct depths corresponded to variations in sulfate depletion and Fe(II) concentrations from anaerobic microbial activity. These variations were supported by fluorescence indexes and are consistent with differences in optical molecular weight and aromaticity indicators. Fluorescence indices were consistent with autochthonous material of aquatic origin in surface waters, with more terrestrial, humified allochthonous material in deeper pore waters. CDOM optical properties were consistent with significantly enhanced microbial activity in regions closest to the oil well, along with a three-dimensional excitation/emission matrix fluorescence spectrum peak attributable to oil, suggesting anaerobic microbial degradation of oil. Copyright © 2016 Elsevier Ltd. All rights reserved.
A 21 000-year record of fluorescent organic matter markers in the WAIS Divide ice core

NASA Astrophysics Data System (ADS)

D'Andrilli, Juliana; Foreman, Christine M.; Sigl, Michael; Priscu, John C.; McConnell, Joseph R.

2017-05-01

Englacial ice contains a significant reservoir of organic material (OM), preserving a chronological record of materials from Earth's past. Here, we investigate if OM composition surveys in ice core research can provide paleoecological information on the dynamic nature of our Earth through time. Temporal trends in OM composition from the early Holocene extending back to the Last Glacial Maximum (LGM) of the West Antarctic Ice Sheet Divide (WD) ice core were measured by fluorescence spectroscopy. Multivariate parallel factor (PARAFAC) analysis is widely used to isolate the chemical components that best describe the observed variation across three-dimensional fluorescence spectroscopy (excitation-emission matrices; EEMs) assays. Fluorescent OM markers identified by PARAFAC modeling of the EEMs from the LGM (27.0-18.0 kyr BP; before present 1950) through the last deglaciation (LD; 18.0-11.5 kyr BP), to the mid-Holocene (11.5-6.0 kyr BP) provided evidence of different types of fluorescent OM composition and origin in the WD ice core over 21.0 kyr. Low excitation-emission wavelength fluorescent PARAFAC component one (C1), associated with chemical species similar to simple lignin phenols was the greatest contributor throughout the ice core, suggesting a strong signature of terrestrial OM in all climate periods. The component two (C2) OM marker, encompassed distinct variability in the ice core describing chemical species similar to tannin- and phenylalanine-like material. Component three (C3), associated with humic-like terrestrial material further resistant to biodegradation, was only characteristic of the Holocene, suggesting that more complex organic polymers such as lignins or tannins may be an ecological marker of warmer climates. We suggest that fluorescent OM markers observed during the LGM were the result of greater continental dust loading of lignin precursor (monolignol) material in a drier climate, with lower marine influences when sea ice extent was higher and continents had more expansive tundra cover. As the climate warmed, the record of OM markers in the WD ice core changed, reflecting shifts in carbon productivity as a result of global ecosystem response.
Dynamic Assessment of the Endothelialization of Tissue-Engineered Blood Vessels Using an Optical Coherence Tomography Catheter-Based Fluorescence Imaging System.

PubMed

Gurjarpadhye, Abhijit Achyut; DeWitt, Matthew R; Xu, Yong; Wang, Ge; Rylander, Marissa Nichole; Rylander, Christopher G

2015-07-01

Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization. C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system. No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15 ± 4% to 89 ± 6% over 5 days. In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation.
Two-dimensional concentration and temperature measurements in extended flames of industrial burners using PLIF

NASA Astrophysics Data System (ADS)

Mueller, Dirk; Triebel, Wolfgang; Bochmann, Arne; Schmidl, Gabriele; Eckardt, Daniel; Burkert, Alfons; Roeper, Juergen; Schwerin, Malte

2003-11-01

Concentration profiles of OH, O2 and NO as well as temperature fields in diffusion flames of a length of approx. 300 mm and 40 mm in diameter used for gas-phase synthesis of fused silica have been determined by Planar Laser Induced Fluorescence (PLIF). The measurements have been carried out using a tunable spectrally narrowed KrF laser, whose wavelengths could be switched pulse-to-pulse. The laser beam was shaped as a light sheet into the flame at a fixed position. The flame area under investigation was monitored by moving the burner mounted on a stepper motor. By adapted synchronization the laser induced fluorescence was continuously recorded over the height of the flame perpendicular to the laser light sheet with an intensified CCD camera (10 fps, 8 bit dynamic range, 768 x 576 pixels). By image processing the spatial offset between images was corrected and superposed images were averaged and analyzed. This method allows to investigate the flame by recording 2D-fluorescence images including an automatic correction of intensity inhomogeneities of the laser light sheet. Based on the excited radical or molecule the fluorescence images were used to determine concentration and temperature distributions to build up a 2D-map of the flame. The PLIF experiment was calibrated with precise determination of the temperature at one coordinate of the flame by Spontaneous Vibrational Raman Scattering (VRS) of N2. As a result temperatures up to 3200 K could be determined with an accuracy better than 3% and a spatial resolution better than 1 mm. Temperature variations in the flame at different gas flows of fuel and oxidizer could be monitored sensitively. Also, the influence of different carrier gases like N2, Ar and He on the temperature distribution was investigated. Fluctuations in gas flow caused by turbulence could be monitored as well.
Computational cell quantification in the human brain tissues based on hard x-ray phase-contrast tomograms

NASA Astrophysics Data System (ADS)

Hieber, Simone E.; Bikis, Christos; Khimchenko, Anna; Schulz, Georg; Deyhle, Hans; Thalmann, Peter; Chicherova, Natalia; Rack, Alexander; Zdora, Marie-Christine; Zanette, Irene; Schweighauser, Gabriel; Hench, Jürgen; Müller, Bert

2016-10-01

Cell visualization and counting plays a crucial role in biological and medical research including the study of neurodegenerative diseases. The neuronal cell loss is typically determined to measure the extent of the disease. Its characterization is challenging because the cell density and size already differs by more than three orders of magnitude in a healthy cerebellum. Cell visualization is commonly performed by histology and fluorescence microscopy. These techniques are limited to resolve complex microstructures in the third dimension. Phase- contrast tomography has been proven to provide sufficient contrast in the three-dimensional imaging of soft tissue down to the cell level and, therefore, offers the basis for the three-dimensional segmentation. Within this context, a human cerebellum sample was embedded in paraffin and measured in local phase-contrast mode at the beamline ID19 (ESRF, Grenoble, France) and the Diamond Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK). After the application of Frangi-based filtering the data showed sufficient contrast to automatically identify the Purkinje cells and to quantify their density to 177 cells per mm3 within the volume of interest. Moreover, brain layers were segmented in a region of interest based on edge detection. Subsequently performed histological analysis validated the presence of the cells, which required a mapping from the two- dimensional histological slices to the three-dimensional tomogram. The methodology can also be applied to further tissue types and shows potential for the computational tissue analysis in health and disease.
Two-Dimensional Fluidization of Nanomaterials via Biomimetic Membranes towards Assisted Self Assembly

NASA Astrophysics Data System (ADS)

Kelly, Kathleen

Materials that take advantage of the exceptional properties of nano-meter sized aggregates of atoms are poised to play an important role in future technologies. Prime examples for such nano-materials that have an extremely large surface to volume ratio and thus are physically determined by surface related effects are quantum dots (qdots) and carbon nanotubes (CNTs). The production of such manmade nano-objects has by now become routine and even commercialized. However, the controlled assembly of individual nano-sized building blocks into larger structures of higher geometric and functional complexity has proven to be much more challenging. Yet, this is exactly what is required for many applications that have transformative potential for new technologies. If the tedious procedure to sequentially position individual nano-objects is to be forgone, the assembly of such objects into larger structures needs to be implicitly encoded and many ways to bestow such self-assembly abilities onto nano objects are being developed. Yet, as overall size and complexity of such self-assembled structures increases, kinetic and geometric frustration begin to prevent the system to achieve the desired configuration. In nature, this problem is solved by relying on guided or forced variants of the self-assembly approach. To translate such concepts into the realm of man-made nano-technology, ways to dynamically manipulate nano-materials need to be devised. Thus, in the first part of this work, I provide a proof of concept that supported lipid bilayers (SLBs) that exhibit free lateral diffusion of their constituents can be utilized as a two-dimensional platform for active nano-material manipulation. We used streptavidin coated quantum dots (Q-dots) as a model nano-building-block. Q-dots are 0-dimensional nanomaterials engineered to be fluorescent based solely on their diameter making visualization convenient. Biotinylated lipids were used to tether Q-dots to a SLB and we observed that the 2-dimensional fluidity of the bilayer was translated to the quantum dots as they freely diffused. The quantum dots were visualized using wide-field fluorescent microscopy and single particle tracking techniques were employed to analyze their dynamic behavior. Next, an electric field was applied to the system to induce electroosmotic flow (EOF) which creates a bulk flow of the buffer solution. The quantum dots were again tracked and ballistic motion was observed in the particle tracks due to the electroosmosis in the system. This proved that SLBs could be used as a two-dimensional fluid platform for nanomaterials and electroosmosis can be used to manipulate the motion of the Q-dots once they are tethered to the membrane. Next, we set out to employ the same technique to carbon nanotubes (CNTs), which are known for their highly versatile mechanical and electrical properties. However, carbon nanotubes are extremely hydrophobic and tend to aggregate in aqueous solutions which negatively impacts the viability of tethering the CNTs to the bilayer, fluorescently staining and then imaging them. First, we had to solubilize the CNTs such that they were monodisperse and characterize the CNT-detergent solutions. We were able to create monodisperse solutions of CNTs such that the detergent levels were low enough that the integrity of the bilayer was intact. We were also able to fluorescently label the CNTs in order to visualize them, and tether them to a SLB using a peptide sequence. Future directions of this project would include employing EOF to mobilize the CNTs and use a more sophisticated single particle tracking software to track individual CNTs and analyze their motion.
Towards High Density 3-D Memory in Diamond

NASA Astrophysics Data System (ADS)

Henshaw, Jacob; Dhomkar, Siddharth; Meriles, Carlos; Jayakumar, Harishankar

The nitrogen-vacancy (NV) center in diamond is presently the focus of widespread attention for applications ranging from quantum information processing to nanoscale metrology. Of great utility is the ability to optically initialize the NV charge state, which has an immediate impact on the center's light emission properties. Here, we use two-color microscopy in NV-rich, type-1b diamond to demonstrate fluorescence-encoded long-term storage of classical information. As a proof of principle, we write, reset, and rewrite various patterns with 2-D binary bit density comparable to present DVD-ROM technology. The strong fluorescence signal originating from the diffraction-limited bit volume allows us to transition from binary to multi-valued encoding, which translates into a significant storage capacity boost. Finally, we show that our technique preserves information written on different planes of the diamond crystal and thus serves as a platform for three-dimensional storage. Substantial enhancement in the bit density could be achieved with the aid of super resolution microscopy techniques already employed to discriminate between NVs with sub-diffraction, nanometer accuracy, a regime where the storage capacity could exceed 1017 bytes/cm3 We acknowledge support from the National Science Foundation through Grant NSF-1314205.
Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines

PubMed Central

Hearst, Scoty M.; Gilder, Andrew S.; Negi, Sandeep S.; Davis, Misty D.; George, Eric M.; Whittom, Angela A.; Toyota, Cory G.; Husedzinovic, Alma; Gruss, Oliver J.; Hebert, Michael D.

2009-01-01

Summary Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells. PMID:19435804
Speckle correlation resolution enhancement of wide-field fluorescence imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yilmaz, Hasan

2016-03-01

Structured illumination enables high-resolution fluorescence imaging of nanostructures [1]. We demonstrate a new high-resolution fluorescence imaging method that uses a scattering layer with a high-index substrate as a solid immersion lens [2]. Random scattering of coherent light enables a speckle pattern with a very fine structure that illuminates the fluorescent nanospheres on the back surface of the high-index substrate. The speckle pattern is raster-scanned over the fluorescent nanospheres using a speckle correlation effect known as the optical memory effect. A series of standard-resolution fluorescence images per each speckle pattern displacement are recorded by an electron-multiplying CCD camera using a commercial microscope objective. We have developed a new phase-retrieval algorithm to reconstruct a high-resolution, wide-field image from several standard-resolution wide-field images. We have introduced phase information of Fourier components of standard-resolution images as a new constraint in our algorithm which discards ambiguities therefore ensures convergence to a unique solution. We demonstrate two-dimensional fluorescence images of a collection of nanospheres with a deconvolved Abbe resolution of 116 nm and a field of view of 10 µm × 10 µm. Our method is robust against optical aberrations and stage drifts, therefore excellent for imaging nanostructures under ambient conditions. [1] M. G. L. Gustafsson, J. Microsc. 198, 82-87 (2000). [2] H. Yilmaz, E. G. van Putten, J. Bertolotti, A. Lagendijk, W. L. Vos, and A. P. Mosk, Optica 2, 424-429 (2015).
Time reversal optical tomography locates fluorescent targets in a turbid medium

NASA Astrophysics Data System (ADS)

Wu, Binlin; Cai, W.; Gayen, S. K.

2013-03-01

A fluorescence optical tomography approach that extends time reversal optical tomography (TROT) to locate fluorescent targets embedded in a turbid medium is introduced. It uses a multi-source illumination and multi-detector signal acquisition scheme, along with TR matrix formalism, and multiple signal classification (MUSIC) to construct pseudo-image of the targets. The samples consisted of a single or two small tubes filled with water solution of Indocyanine Green (ICG) dye as targets embedded in a 250 mm × 250 mm × 60 mm rectangular cell filled with Intralipid-20% suspension as the scattering medium. The ICG concentration was 1μM, and the Intralipid-20% concentration was adjusted to provide ~ 1-mm transport length for both excitation wavelength of 790 nm and fluorescence wavelength around 825 nm. The data matrix was constructed using the diffusely transmitted fluorescence signals for all scan positions, and the TR matrix was constructed by multiplying data matrix with its transpose. A pseudo spectrum was calculated using the signal subspace of the TR matrix. Tomographic images were generated using the pseudo spectrum. The peaks in the pseudo images provided locations of the target(s) with sub-millimeter accuracy. Concurrent transmission TROT measurements corroborated fluorescence-TROT findings. The results demonstrate that TROT is a fast approach that can be used to obtain accurate three-dimensional position information of fluorescence targets embedded deep inside a highly scattering medium, such as, a contrast-enhanced tumor in a human breast.
Resonant fluorescence for multilevel systems in intense nonmonochromatic fields: possibilities for applications in laser medicine

NASA Astrophysics Data System (ADS)

Karagodova, Tamara Y.

1999-03-01

The theory of resonant fluorescence of multilevel system in two monochromatic intense laser fields has been applied for investigating the temporal decay of magnetic sublevels of an atom. As for two-level system the triplet of resonant fluorescence is observed, for real atom being the multilevel system the multiplet of resonant fluorescence can be observed. The excitation spectra, defining the intensities of lines in the multiplet of resonant fluorescence, and shifts of components of spectra are shown. Typical temporal dependence of fluorescence intensity for magnetic sublevels of an atom having different relaxation constants is shown. The computer simulation of resonant fluorescence for simple systems can help to understand the regularities in temporal decay curves of atherosclerotic plaque, malignant tumor compared to normal surrounding tissue.

Halide/pseudohalide complexes of cadmium(II) with benzimidazole: Synthesis, crystal structures and fluorescence properties

NASA Astrophysics Data System (ADS)

Zhao, Hai-Yan; Yang, Fu-Li; Li, Na; Wang, Xiao-Jing

2017-11-01

Two new dinuclear Cd(II) complexes, [CdL1Cl2]2·H2O (1) and [CdL1(N3)2]2·CH3OH (2) and one dicyanamide bridged one-dimensional polynuclear network [CdL1(μ1,5-dca)dca]n (3) of the potentially tridentate NNN-donor Schiff base 2-((1H-benzimidazol-2-yl-ethylimino)-methyl)pyridine (L1) and another dinucler Cd(II) complex [CdL2Cl(dca)]2 (4) of a similar NNN-donor Schiff base ligand 2-((1H-benzimidazol-2-yl-propylimino)-methyl)pyridine (L2), have been synthesized and characterized by elemental analyses, IR and single crystal X-ray crystallography. The ligands L1 and L2 are [1 + 1] condensation products of pyridine-2-carbaldehyde with 2-aminoethyl-1H-benzimidazole and 2-aminopropyl-1H-benzimidazole, respectively. In the complexes 1 and 4 the two Cd(II) centers are held together by the bridged chloride ligands, while in 2 the two Cd(II) centers are bridged by μ1,1-azide ions. Complex 3 has a one-dimensional infinite chain structure in which Cd(II) ions are bridged by single dicyanamide groups in end-to-end fashion. All the metal centers have a distorted octahedral geometry and H-bonding or π⋯π interactions are operative to bind the complex units in the solid state. Furthermore, these complexes have been investigated by thermogravimetric analyses and fluorescence spectra.
DNA Hairpins Containing the Cytidine Analog Pyrrolo-dC: Structural, Thermodynamic, and Spectroscopic Studies

PubMed Central

Zhang, Xu; Wadkins, Randy M.

2009-01-01

Structures formed by single-strand DNA have become increasingly interesting because of their roles in a number of biological processes, particularly transcription and its regulation. Of particular importance is the fact that antitumor drugs such as Actinomycin D can selectively bind DNA hairpins over fully paired, double-strand DNA. A new fluorescent base analog, pyrrolo-deoxycytidine (PdC), can now be routinely incorporated into single-strand DNA. The fluorescence of PdC is particularly useful for studying the formation of single-strand DNA in regions of double-strand DNA. The fluorescence is quenched when PdC is paired with a complementary guanine residue, and thus is greatly enhanced upon formation of single-strand DNA. Hence, any process that results in melting or opening of DNA strands produces an increase in the fluorescence intensity of this base analog. In this study we measured the structural effects of incorporating PdC into DNA hairpins, and the effect of this incorporation on the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. Two hairpin DNAs were used: one with PdC in the stem (basepaired) and one with PdC in the loop (unpaired). The thermal stability, 7-aminoactinomycin D binding, and three-dimensional structures of PdC incorporated into these DNA hairpins were all quite similar as compared to the hairpins containing an unmodified dC residue. Fluorescence lifetime measurements indicate that two lifetimes are present in PdC, and that the increase in fluorescence of the unpaired PdC residue compared to the basepaired PdC is due to an increase in the contribution of the longer lifetime to the average fluorescence lifetime. Our data indicate that PdC can be used effectively to differentiate paired and unpaired bases in DNA hairpin secondary structures, and should be similarly applicable for related structures such as cruciforms and quadruplexes. Further, our data indicate that PdC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D. PMID:19254547
DNA hairpins containing the cytidine analog pyrrolo-dC: structural, thermodynamic, and spectroscopic studies.

PubMed

Zhang, Xu; Wadkins, Randy M

2009-03-04

Structures formed by single-strand DNA have become increasingly interesting because of their roles in a number of biological processes, particularly transcription and its regulation. Of particular importance is the fact that antitumor drugs such as Actinomycin D can selectively bind DNA hairpins over fully paired, double-strand DNA. A new fluorescent base analog, pyrrolo-deoxycytidine (PdC), can now be routinely incorporated into single-strand DNA. The fluorescence of PdC is particularly useful for studying the formation of single-strand DNA in regions of double-strand DNA. The fluorescence is quenched when PdC is paired with a complementary guanine residue, and thus is greatly enhanced upon formation of single-strand DNA. Hence, any process that results in melting or opening of DNA strands produces an increase in the fluorescence intensity of this base analog. In this study we measured the structural effects of incorporating PdC into DNA hairpins, and the effect of this incorporation on the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. Two hairpin DNAs were used: one with PdC in the stem (basepaired) and one with PdC in the loop (unpaired). The thermal stability, 7-aminoactinomycin D binding, and three-dimensional structures of PdC incorporated into these DNA hairpins were all quite similar as compared to the hairpins containing an unmodified dC residue. Fluorescence lifetime measurements indicate that two lifetimes are present in PdC, and that the increase in fluorescence of the unpaired PdC residue compared to the basepaired PdC is due to an increase in the contribution of the longer lifetime to the average fluorescence lifetime. Our data indicate that PdC can be used effectively to differentiate paired and unpaired bases in DNA hairpin secondary structures, and should be similarly applicable for related structures such as cruciforms and quadruplexes. Further, our data indicate that PdC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D.
A two-dimensional layered Cd(II) coordination polymer with a three-dimensional supramolecular architecture incorporating mixed multidentate N- and O-donor ligands.

PubMed

Huang, Qiu-Ying; Su, Ming-Yang; Meng, Xiang-Ru

2015-06-01

The combination of N-heterocyclic and multicarboxylate ligands is a good choice for the construction of metal-organic frameworks. In the title coordination polymer, poly[bis{μ2-1-[(1H-benzimidazol-2-yl)methyl]-1H-tetrazole-κ(2)N(3):N(4)}(μ4-butanedioato-κ(4)O(1):O(1'):O(4):O(4'))(μ2-butanedioato-κ(2)O(1):O(4))dicadmium], [Cd(C4H4O4)(C9H8N6)]n, each Cd(II) ion exhibits an irregular octahedral CdO4N2 coordination geometry and is coordinated by four O atoms from three carboxylate groups of three succinate (butanedioate) ligands and two N atoms from two 1-[(1H-benzimidazol-2-yl)methyl]-1H-tetrazole (bimt) ligands. Cd(II) ions are connected by two kinds of crystallographically independent succinate ligands to generate a two-dimensional layered structure with bimt ligands located on each side of the layer. Adjacent layers are further connected by hydrogen bonding, leading to a three-dimensional supramolecular architecture in the solid state. Thermogravimetric analysis of the title polymer shows that it is stable up to 529 K and then loses weight from 529 to 918 K, corresponding to the decomposition of the bimt ligands and succinate groups. The polymer exhibits a strong fluorescence emission in the solid state at room temperature.
Fluorescence multispectral imaging-based diagnostic system for atherosclerosis.

PubMed

Ho, Cassandra Su Lyn; Horiuchi, Toshikatsu; Taniguchi, Hiroaki; Umetsu, Araya; Hagisawa, Kohsuke; Iwaya, Keiichi; Nakai, Kanji; Azmi, Amalina; Zulaziz, Natasha; Azhim, Azran; Shinomiya, Nariyoshi; Morimoto, Yuji

2016-08-20

Composition of atherosclerotic arterial walls is rich in lipids such as cholesterol, unlike normal arterial walls. In this study, we aimed to utilize this difference to diagnose atherosclerosis via multispectral fluorescence imaging, which allows for identification of fluorescence originating from the substance in the arterial wall. The inner surface of extracted arteries (rabbit abdominal aorta, human coronary artery) was illuminated by 405 nm excitation light and multispectral fluorescence images were obtained. Pathological examination of human coronary artery samples were carried out and thickness of arteries were calculated by measuring combined media and intima thickness. The fluorescence spectra in atherosclerotic sites were different from those in normal sites. Multiple regions of interest (ROI) were selected within each sample and a ratio between two fluorescence intensity differences (where each intensity difference is calculated between an identifier wavelength and a base wavelength) from each ROI was determined, allowing for discrimination of atherosclerotic sites. Fluorescence intensity and thickness of artery were found to be significantly correlated. These results indicate that multispectral fluorescence imaging provides qualitative and quantitative evaluations of atherosclerosis and is therefore a viable method of diagnosing the disease.
Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens.

PubMed

Sohn, Miryeong; Himmelsbach, David S; Barton, Franklin E; Fedorka-Cray, Paula J

2009-11-01

This study deals with the rapid detection and differentiation of Escherichia coli, Salmonella, and Campylobacter, which are the most commonly identified commensal and pathogenic bacteria in foods, using fluorescence spectroscopy and multivariate analysis. Each bacterial sample cultured under controlled conditions was diluted in physiologic saline for analysis. Fluorescence spectra were collected over a range of 200-700 nm with 0.5 nm intervals on the PerkinElmer Fluorescence Spectrometer. The synchronous scan technique was employed to find the optimum excitation (lambda(ex)) and emission (lambda(em)) wavelengths for individual bacteria with the wavelength interval (Deltalambda) being varied from 10 to 200 nm. The synchronous spectra and two-dimensional plots showed two maximum lambda(ex) values at 225 nm and 280 nm and one maximum lambda(em) at 335-345 nm (lambda(em) = lambda(ex) + Deltalambda), which correspond to the lambda(ex) = 225 nm, Deltalambda = 110-120 nm, and lambda(ex) = 280 nm, Deltalambda = 60-65 nm. For all three bacterial genera, the same synchronous scan results were obtained. The emission spectra from the three bacteria groups were very similar, creating difficulty in classification. However, the application of principal component analysis (PCA) to the fluorescence spectra resulted in successful classification of the bacteria by their genus as well as determining their concentration. The detection limit was approximately 10(3)-10(4) cells/mL for each bacterial sample. These results demonstrated that fluorescence spectroscopy, when coupled with PCA processing, has the potential to detect and to classify bacterial pathogens in liquids. The methodology is rapid (>10 min), inexpensive, and requires minimal sample preparation compared to standard analytical methods for bacterial detection.
Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids.

PubMed

Schmitz, Alexander; Fischer, Sabine C; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H K

2017-03-03

Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid's size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 10 5 to 1 × 10 6 cells/mm 3 . Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture.
Two-photon microscopy of fungal keratitis-affected rabbit cornea ex vivo using moxifloxacin as a labeling agent.

PubMed

Lee, Jun Ho; Le, Viet-Hoan; Lee, Seunghun; Park, Jin Hyoung; Lee, Jin Ah; Tchah, Hungwon; Kim, Sungjee; Kim, Myoung Joon; Kim, Ki Hean

2018-05-19

Two-photon microscopy (TPM) is a three dimensional (3D) microscopic technique based on nonlinear two-photon fluorescence, which has been tested as an alternative to reflectance confocal microscopy (RCM) for detecting fungal keratitis via optical imaging. Although TPM provided images with better contrast than RCM for fungal keratitis, its imaging speed was relatively low because of weak intrinsic signal. Moxifloxacin, a Food and Drug Administration (FDA)-approved antibiotic, was recently used as a cell-labeling agent for TPM. In this study, moxifloxacin was used to label fungal cells for TPM imaging of fungal keratitis models. Fungal cell suspensions and ex vivo fungal keratitis-affected rabbit corneas were prepared using two types of fungal pathogens, Aspergillus fumigatus and Candida albicans, and TPM imaging was performed both with and without moxifloxacin treatment. Fungal cells with enhanced fluorescence were clearly visible by TPM of moxifloxacin-treated fungal cell suspensions. TPM of moxifloxacin-treated fungal keratitis rabbit corneas revealed both the infecting fungal cells and corneal cells similar to those observed in TPM without moxifloxacin treatment, albeit with approximately 10-times enhanced fluorescence. Fungal cells were distinguished from corneal cells on the basis of their distinct morphologies. Thus, TPM with moxifloxacin labeling might be useful for the detection of fungal keratitis at the improved imaging speed. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.
Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography

NASA Astrophysics Data System (ADS)

Favicchio, Rosy; Psycharakis, Stylianos; Schönig, Kai; Bartsch, Dusan; Mamalaki, Clio; Papamatheakis, Joseph; Ripoll, Jorge; Zacharakis, Giannis

2016-02-01

Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture.
Using fluorescence spectroscopy coupled with chemometric analysis to investigate the origin, composition, and dynamics of dissolved organic matter in leachate-polluted groundwater.

PubMed

He, Xiao-Song; Xi, Bei-Dou; Gao, Ru-Tai; Wang, Lei; Ma, Yan; Cui, Dong-Yu; Tan, Wen-Bing

2015-06-01

Groundwater was collected in 2011 and 2012, and fluorescence spectroscopy coupled with chemometric analysis was employed to investigate the composition, origin, and dynamics of dissolved organic matter (DOM) in the groundwater. The results showed that the groundwater DOM comprised protein-, fulvic-, and humic-like substances, and the protein-like component originated predominantly from microbial production. The groundwater pollution by landfill leachate enhanced microbial activity and thereby increased microbial by-product-like material such as protein-like component in the groundwater. Excitation-emission matrix fluorescence spectra combined with parallel factor analysis showed that the protein-like matter content increased from 2011 to 2012 in the groundwater, whereas the fulvic- and humic-like matter concentration exhibited no significant changes. In addition, synchronous-scan fluorescence spectra coupled with two-dimensional correlation analysis showed that the change of the fulvic- and humic-like matter was faster than that of the protein-like substances, as the groundwater flowed from upstream to downstream in 2011, but slower than that of the protein-like substance in 2012 due to the enhancement of microbial activity. Fluorescence spectroscopy combined with chemometric analysis can investigate groundwater pollution characteristics and monitor DOM dynamics in groundwater.
Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.

PubMed Central

Iino, R; Koyama, I; Kusumi, A

2001-01-01

Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions). PMID:11371443
Spectral characterization of biological aerosol particles using two-wavelength excited laser-induced fluorescence and elastic scattering measurements.

PubMed

Sivaprakasam, Vasanthi; Lin, Horn-Bond; Huston, Alan L; Eversole, Jay D

2011-03-28

A two-wavelength laser-induced fluorescence (LIF) instrument has been developed and used to characterize individual biological aerosol particles, including biological warfare (BW) agent surrogates. Fluorescence in discrete spectral bands from widely different species, and also from similar species under different growth conditions were measured and compared. The two-wavelength excitation approach was found to increase discrimination among several biological materials, and especially with respect to diesel exhaust particles, a common interferent for LIF BW detection systems. The spectral characteristics of a variety of biological materials and ambient air components have been studied as a function of aerosol particle size and incident fluence.
New solutions for standardization, monitoring and quality management of fluorescence-based imaging systems (Conference Presentation)

NASA Astrophysics Data System (ADS)

Royon, Arnaud; Papon, Gautier

2016-03-01

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

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

Garcia-Lechuga, M.; Laser Processing Group, Instituto de Óptica “Daza de Valdés,” CSIC, 28006-Madrid; Fuentes, L. M.

2014-10-07

We report a detailed characterization of the spatial resolution provided by two-photon absorption spectroscopy suited for plasma diagnosis via the 1S-2S transition of atomic hydrogen for optogalvanic detection and laser induced fluorescence (LIF). A precise knowledge of the spatial resolution is crucial for a correct interpretation of measurements, if the plasma parameters to be analysed undergo strong spatial variations. The present study is based on a novel approach which provides a reliable and realistic determination of the spatial resolution. Measured irradiance distribution of laser beam waists in the overlap volume, provided by a high resolution UV camera, are employed tomore » resolve coupled rate equations accounting for two-photon excitation, fluorescence decay and ionization. The resulting three-dimensional yield distributions reveal in detail the spatial resolution for optogalvanic and LIF detection and related saturation due to depletion. Two-photon absorption profiles broader than the Fourier transform-limited laser bandwidth are also incorporated in the calculations. The approach allows an accurate analysis of the spatial resolution present in recent and future measurements.« less
Three-dimensional photogrammetric measurement of magnetic field lines in the WEGA stellarator

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

Drewelow, Peter; Braeuer, Torsten; Otte, Matthias

2009-12-15

The magnetic confinement of plasmas in fusion experiments can significantly degrade due to perturbations of the magnetic field. A precise analysis of the magnetic field in a stellarator-type experiment utilizes electrons as test particles following the magnetic field line. The usual fluorescent detector for this electron beam limits the provided information to two-dimensional cut views at certain toroidal positions. However, the technique described in this article allows measuring the three-dimensional structure of the magnetic field by means of close-range photogrammetry. After testing and optimizing the main diagnostic components, measurements of the magnetic field lines were accomplished with a spatial resolutionmore » of 5 mm. The results agree with numeric calculations, qualifying this technique as an additional tool to investigate magnetic field configurations in a stellarator. For a possible future application, ways are indicated on how to reduce experimental error sources.« less
Novel method for the measurement of liquid film thickness during fuel spray impingement on surfaces.

PubMed

Henkel, S; Beyrau, F; Hardalupas, Y; Taylor, A M K P

2016-02-08

This paper describes the development and application of a novel optical technique for the measurement of liquid film thickness formed on surfaces during the impingement of automotive fuel sprays. The technique makes use of the change of the light scattering characteristics of a metal surface with known roughness, when liquid is deposited. Important advantages of the technique over previously established methods are the ability to measure the time-dependent spatial distribution of the liquid film without a need to add a fluorescent tracer to the liquid, while the measurement principle is not influenced by changes of the pressure and temperature of the liquid or the surrounding gas phase. Also, there is no need for non-fluorescing surrogate fuels. However, an in situ calibration of the dependence of signal intensity on liquid film thickness is required. The developed method can be applied to measure the time-dependent and two-dimensional distribution of the liquid fuel film thickness on the piston or the liner of gasoline direct injection (GDI) engines. The applicability of this technique was evaluated with impinging sprays of several linear alkanes and alcohols with different thermo-physical properties. The surface temperature of the impingement plate was controlled to simulate the range of piston surface temperatures inside a GDI engine. Two sets of liquid film thickness measurements were obtained. During the first set, the surface temperature of the plate was kept constant, while the spray of different fuels interacted with the surface. In the second set, the plate temperature was adjusted to match the boiling temperature of each fuel. In this way, the influence of the surface temperature on the liquid film created by the spray of different fuels and their evaporation characteristics could be demonstrated.
Methods to Assess Mitochondrial Morphology in Mammalian Cells Mounting Autophagic or Mitophagic Responses.

PubMed

Marchi, S; Bonora, M; Patergnani, S; Giorgi, C; Pinton, P

2017-01-01

It is widely acknowledged that mitochondria are highly active structures that rapidly respond to cellular and environmental perturbations by changing their shape, number, and distribution. Mitochondrial remodeling is a key component of diverse biological processes, ranging from cell cycle progression to autophagy. In this chapter, we describe different methodologies for the morphological study of the mitochondrial network. Instructions are given for the preparation of samples for fluorescent microscopy, based on genetically encoded strategies or the employment of synthetic fluorescent dyes. We also propose detailed protocols to analyze mitochondrial morphometric parameters from both three-dimensional and bidimensional datasets. Finally, we describe a protocol for the visualization and quantification of mitochondrial structures through electron microscopy. © 2017 Elsevier Inc. All rights reserved.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindberg, David J.; Wranne, Moa S.; Gilbert Gatty, Mélina

Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concentration matched samples preparedmore » under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation. - Highlights: • ThT emission is more intense with Aβ(1-40) fibrils than with Aβ(1-42) fibrils. • Aβ(1-42) fibrils induce longer ThT fluorescence lifetimes and higher quantum yield. • ThT emission intensity in Aβ fibril samples reports on fibril morphology. • The ThT fluorescence lifetime is a characteristic feature of each Aβ fibril type.« less
Temperature dependence of Ti:Sapphire fluorescence spectra for the design of cryogenic cooled Ti:Sapphire CPA laser.

PubMed

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

2017-03-20

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

NASA Astrophysics Data System (ADS)

Marrocco, Michele

2007-11-01

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

Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

PubMed Central

Mergny, J L; Boutorine, A S; Garestier, T; Belloc, F; Rougée, M; Bulychev, N V; Koshkin, A A; Bourson, J; Lebedev, A V; Valeur, B

1994-01-01

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes. PMID:8152922
Accurate quantification of fluorescent targets within turbid media based on a decoupled fluorescence Monte Carlo model.

PubMed

Deng, Yong; Luo, Zhaoyang; Jiang, Xu; Xie, Wenhao; Luo, Qingming

2015-07-01

We propose a method based on a decoupled fluorescence Monte Carlo model for constructing fluorescence Jacobians to enable accurate quantification of fluorescence targets within turbid media. The effectiveness of the proposed method is validated using two cylindrical phantoms enclosing fluorescent targets within homogeneous and heterogeneous background media. The results demonstrate that our method can recover relative concentrations of the fluorescent targets with higher accuracy than the perturbation fluorescence Monte Carlo method. This suggests that our method is suitable for quantitative fluorescence diffuse optical tomography, especially for in vivo imaging of fluorophore targets for diagnosis of different diseases and abnormalities.
Is There Excitation Energy Transfer between Different Layers of Stacked Photosystem-II-Containing Thylakoid Membranes?

PubMed

Farooq, Shazia; Chmeliov, Jevgenij; Trinkunas, Gediminas; Valkunas, Leonas; van Amerongen, Herbert

2016-04-07

We have compared picosecond fluorescence decay kinetics for stacked and unstacked photosystem II membranes in order to evaluate the efficiency of excitation energy transfer between the neighboring layers. The measured kinetics were analyzed in terms of a recently developed fluctuating antenna model that provides information about the dimensionality of the studied system. Independently of the stacking state, all preparations exhibited virtually the same value of the apparent dimensionality, d = 1.6. Thus, we conclude that membrane stacking does not affect the efficiency of the delivery of excitation energy toward the reaction centers but ensures a more compact organization of the thylakoid membranes within the chloroplast and separation of photosystems I and II.
A naphthalimide fluorophore with efficient intramolecular PET and ICT processes: application in molecular logic.

PubMed

Wang, Haixia; Wu, Haixia; Xue, Lin; Shi, Yan; Li, Xiyou

2011-08-07

A novel 4-amino-1,8-naphthalimide (NDI) with two different metal cation receptors connected at 4-amino or imide nitrogen positions respectively was designed and prepared. Significant internal charge transfer (ICT) as well as photoinduced electron transfer (PET) from the receptors to NDI is revealed by the shifted UV-vis absorption spectra and significant fluorescence quenching. Both Zn(2+) and Cu(2+) can coordinate selectively with the two cation receptors in this molecule with different affinities. The coordination of Zn(2+) with the receptor at imide nitrogen hindered the PET process and accordingly restored the quenched fluorescence of NDI. But the coordination of Zn(2+) at 4-amino position blocked the ICT process and caused significant blue-shift on the absorption peak with the fluorescence intensity unaffected. Similarly, coordination of Cu(2+) with the receptor at imide nitrogen can block the PET process, but can not restore the quenched fluorescence of compound 3 due to the paramagnetic properties of Cu(2+), which quench the fluorescence significantly instead. With Cu(2+) and Zn(2+) as two chemical inputs and absorption or fluorescence as output, several logic gate operations, such as OR, NOR and INHIBIT, can be achieved.
Fluorescence spectroscopy and molecular weight distribution of extracellular polymers from full-scale activated sludge biomass.

PubMed

Esparza-Soto, M; Westerhoff, P K

2001-01-01

Two fractions of extracellular polymer substances (EPSs), soluble and readily extractable (RE), were characterised in terms of their molecular weight distributions (MWD) and 3-D excitation-emission-matrix (EEM) fluorescence spectroscopy signatures. The EPS fractions were different: the soluble EPSs were composed mainly of high molecular weight compounds, while the RE EPSs were composed of small molecular weight compounds. Contrary to previous thought, EPS may not be considered only as macromolecular because most organic matter present in both fractions had low molecular weight. Three different fluorophore peaks were identified in the EEM fluorescence spectra. Two peaks were attributed to protein-like fluorophores, and the third to a humic-like fluorophore. Fluorescence signatures were different from other previously published signatures for marine and riverine environments. EEM spectroscopy proved to be a suitable method that may be used to characterise and trace organic matter of bacterial origin in wastewater treatment operations.
Investigation of Real-Time Two-Dimensional Visualization of Fuel Spray Liquid/Vapor Distribution via Exciplex Fluorescence.

DTIC Science & Technology

1987-08-30

Unlimited 4 PERFORMIG ORGANIZATION REPORT NUMBER(S) S. MONITORING ORGANIZATION REPORT NUMBER(S) R87-9. 875-F l__/_____- 4- 6. NAME OF PERFORMING... ORGANIZATION 6b OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION United Technologies (If applcable) Department of the Army Research Center U.S. Army Research...Triangle Park, NC 27709-2211 .Ba NAME OF FUNDING JSPONSORING 8b OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER ORGANIZATION (If
An experimental study of a quasi-two dimensional rising foam

NASA Astrophysics Data System (ADS)

Bennani, Nora; Fujiwara, Akiko; Takagi, Shu; Matsumoto, Yoichiro

2006-11-01

Motivated by the use of the flotation process to clean a non-homogeneous liquid, we here report on an experimental study of quasi-two dimensional flowing foam. Conditions are free-drainage which is driven by gravity and capillarity. The coarsening process, which is due to the aging of the foam, is also occurring, changing the general shape of this polydispersed foam cells. Tea seed saponin was used as surfactant, and Rhodamine-B fluorescent particles were tracked using the Particle Tracking Velocimetry technique. Experiments were performed in an acrylic tank filled with tap water (height H= 1m, width W= 0.15 m and Depth D= 8mm). The air was injected from its bottom part with a fixed flow rate, and went through a porous plate (size of the pores was 10μm), and created 3mm diameter non-spherical bubbles. The void fraction, in the liquid phase, was estimated to be around 1%. Fluorescent particles were beforehand added in the liquid phase in order to trace wastewater particle motion. The generated foam gas cells sizes were in the range of 0.5 to 5 cm, depending on the surfactant concentration and the coarsening process. The behaviours of these particle tracers and of the liquid, with these herein foaming conditions, are here presented and are compared to available data and theories.
Permeabilization assay for antimicrobial peptides based on pore-spanning lipid membranes on nanoporous alumina.

PubMed

Neubacher, Henrik; Mey, Ingo; Carnarius, Christian; Lazzara, Thomas D; Steinem, Claudia

2014-04-29

Screening tools to study antimicrobial peptides (AMPs) with the aim to optimize therapeutic delivery vectors require automated and parallelized sampling based on chip technology. Here, we present the development of a chip-based assay that allows for the investigation of the action of AMPs on planar lipid membranes in a time-resolved manner by fluorescence readout. Anodic aluminum oxide (AAO) composed of cylindrical pores with a diameter of 70 nm and a thickness of up to 10 μm was used as a support to generate pore-spanning lipid bilayers from giant unilamellar vesicle spreading, which resulted in large continuous membrane patches sealing the pores. Because AAO is optically transparent, fluid single lipid bilayers and the underlying pore cavities can be readily observed by three-dimensional confocal laser scanning microscopy (CLSM). To assay the membrane permeabilizing activity of the AMPs, the translocation of the water-soluble dyes into the AAO cavities and the fluorescence of the sulforhodamine 101 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanol-l-amine triethylammonium salt (Texas Red DHPE)-labeled lipid membrane were observed by CLSM in a time-resolved manner as a function of the AMP concentration. The effect of two different AMPs, magainin-2 and melittin, was investigated, showing that the concentrations required for membrane permeabilization and the kinetics of the dye entrance differ significantly. Our results are discussed in light of the proposed permeabilization models of the two AMPs. The presented data demonstrate the potential of this setup for the development of an on-chip screening platform for AMPs.
Evaluation of natural organic matter changes from Lake Hohloh by three-dimensional excitation-emission matrix fluorescence spectroscopy during TiO(2)/UV process.

PubMed

Valencia, Sergio; Marín, Juan M; Restrepo, Gloria; Frimmel, Fritz H

2014-03-15

This study shows the changes of natural organic matter (NOM) from Lake Hohloh, (Black Forest, Germany) during heterogeneous photocatalysis with TiO2 (TiO2/UV). The effect of pH on the adsorption of NOM onto TiO2 in the dark and TiO2/UV degradation of NOM was followed using three-dimensional excitation-emission matrix (EEM) fluorescence. At pH values between 4 and 9, the NOM was adsorbed onto TiO2 in the dark with a greater decrease in the fluorescence intensity and in the spectral shapes, especially under acidic pH conditions. However, at pH = 10 there was not adsorption on NOM which led to a negligible changes the fluorescence intensity. A significant high linear correlation was observed between the DOC adsorption onto TiO2 and the maximum fluorescence intensity. Additionally, the NOM adsorption onto TiO2 and its TiO2/UV degradation shifted the fluorescence maxima toward shorter wavelengths in the EEM contour plots, with a decrease in aromaticity. These changes were accompanied by a substantial decrease in the organically bound halogens adsorbable on activated carbon (AOXFP) and the trihalomethane formation potential (THMFP). Thus, the decrease in maximum fluorescence intensity can be used as an indicator of AOXFP and TTHMFP removal efficiency. Therefore, fluorescence spectroscopy is a robust analytical technique for evaluate TiO2/UV removal of NOM. Copyright © 2013 Elsevier Ltd. All rights reserved.
Resonant Mode-hopping Micromixing

PubMed Central

Jang, Ling-Sheng; Chao, Shih-Hui; Holl, Mark R.; Meldrum, Deirdre R.

2009-01-01

A common micromixer design strategy is to generate interleaved flow topologies to enhance diffusion. However, problems with these designs include complicated structures and dead volumes within the flow fields. We present an active micromixer using a resonating piezoceramic/silicon composite diaphragm to generate acoustic streaming flow topologies. Circulation patterns are observed experimentally and correlate to the resonant mode shapes of the diaphragm. The dead volumes in the flow field are eliminated by rapidly switching from one discrete resonant mode to another (i.e., resonant mode-hop). Mixer performance is characterized by mixing buffer with a fluorescence tracer containing fluorescein. Movies of the mixing process are analyzed by converting fluorescent images to two-dimensional fluorescein concentration distributions. The results demonstrate that mode-hopping operation rapidly homogenized chamber contents, circumventing diffusion-isolated zones. PMID:19551159
Effect of Annealing on Exciton Diffusion in a High Performance Small Molecule Organic Photovoltaic Material.

PubMed

Long, Yun; Hedley, Gordon J; Ruseckas, Arvydas; Chowdhury, Mithun; Roland, Thomas; Serrano, Luis A; Cooke, Graeme; Samuel, Ifor D W

2017-05-03

Singlet exciton diffusion was studied in the efficient organic photovoltaic electron donor material DTS(FBTTh 2 ) 2 . Three complementary time-resolved fluorescence measurements were performed: quenching in planar heterojunctions with an electron acceptor, exciton-exciton annihilation, and fluorescence depolarization. The average exciton diffusivity increases upon annealing from 1.6 × 10 -3 to 3.6 × 10 -3 cm 2 s -1 , resulting in an enhancement of the mean two-dimensional exciton diffusion length (L D = (4Dτ) 1/2 ) from 15 to 27 nm. About 30% of the excitons get trapped very quickly in as-cast films. The high exciton diffusion coefficient of the material leads to it being able to harvest excitons efficiently from large donor domains in bulk heterojunctions.
Effect of Annealing on Exciton Diffusion in a High Performance Small Molecule Organic Photovoltaic Material

PubMed Central

2017-01-01

Singlet exciton diffusion was studied in the efficient organic photovoltaic electron donor material DTS(FBTTh2)2. Three complementary time-resolved fluorescence measurements were performed: quenching in planar heterojunctions with an electron acceptor, exciton–exciton annihilation, and fluorescence depolarization. The average exciton diffusivity increases upon annealing from 1.6 × 10–3 to 3.6 × 10–3 cm2 s–1, resulting in an enhancement of the mean two-dimensional exciton diffusion length (LD = (4Dτ)1/2) from 15 to 27 nm. About 30% of the excitons get trapped very quickly in as-cast films. The high exciton diffusion coefficient of the material leads to it being able to harvest excitons efficiently from large donor domains in bulk heterojunctions. PMID:28358189
A monoclonal IgM directed against immunodominant catalase B of cell wall of Aspergillus fumigatus exerts anti-A. fumigatus activities.

PubMed

Chaturvedi, Ashok K; Kumar, Rohitashw; Kumar, Awanit; Shukla, Praveen K

2009-11-01

Aspergillus fumigatus, a ubiquitous fungus, has been reported to cause human diseases like allergic pulmonary aspergillosis, aspergilloma and invasive infection. Limited spectrum and emergence of resistance has become a serious problem with available antifungals. Therefore, an alternative approach is required for successful treatment of mycoses. In the present study, immunogenic protein profile of A. fumigatus cell wall was generated using two-dimensional-gel electrophoresis and three hybridomas producing monoclonal antibodies (MAbs; IgM) were selected after fusion experiments. Of these three MAbs, MAb-7 exhibited potent in vitro inhibitory activity, which was confirmed by MTT assay, fluorescence-activated cell sorter analysis and immuno-fluorescence studies, and the protein was identified as catalase B using MALDI-TOF-MS.
Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds.

PubMed

Kim, Min Jeong; Shin, Yong Cheol; Lee, Jong Ho; Jun, Seung Won; Kim, Chang-Seok; Lee, Yunki; Park, Jong-Chul; Lee, Soo-Hong; Park, Ki Dong; Han, Dong-Wook

2016-01-01

Hydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM. The physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope. Our findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.
Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

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

Yinfa, Ma.

Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will bemore » described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.« less
Spectral and time-resolved studies on ocular structures

NASA Astrophysics Data System (ADS)

Schweitzer, D.; Jentsch, S.; Schenke, S.; Hammer, M.; Biskup, C.; Gaillard, E.

2007-07-01

Measurements of endogeous fluorophores open the possibility for evaluation of metabolic state at the eye. For interpretation of 2-dimensional measurements of time-resolved auto fluorescence in 2 separate spectral ranges at the human eye, comparing measurements were performed on porcine eyes. Determining excitation and emission spectra, attention was drawn of proof of coenzymes NADH and FAD in isolated anatomical structures cornea, aqueous humor, lens, vitreous, neuronal retina, retinal pigment epithelium (RPE), choroid, and sclera. All these structures exhibit auto fluorescence, highest in lens. Excitation at 350 nm results in local fluorescence maxima at 460 nm, corresponding to NADH, in all structures. This short-wave excitation allows metabolic studies only at the anterior eye, because of the limited transmission of the ocular media. During excitation at 446 nm the existence of FAD is expressed by local fluorescence maxima at 530 nm. The composition fluorescence spectra allow no discrimination between single ocular structures. Approximating the dynamic fluorescence by a double exponential function, the shortest lifetimes were detected in RPE and neuronal retina. The histograms of mean lifetime t M cover each other on lens with cornea and also on sclera with choroid. Despite the lifetimes are close between RPE and neuronal retina, the relative contributions Q I are wide different. The gradient of trend lines in cluster diagrams of amplitudes α II vs. α I allows a discrimination of ocular structures.
3D in-vivo imaging of GFP-expressing T-cells in mice with non-contact fluorescence molecular tomography (Invited Paper)

NASA Astrophysics Data System (ADS)

Garofalakis, Anikitos; Meyer, Heiko; Zacharakis, Giannis; Economou, Eleftherios N.; Mamalaki, Clio; Papamatheakis, Joseph; Ntziachristos, Vasilis; Ripoll, Jorge

2005-06-01

Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores in-vivo. Due to this fact, there has been great interest in developing imaging systems offering quantitative information on the location and concentration of chromophores and fluorescent probes. In this study we present a novel imaging system that enables three dimensional (3D) imaging of fluorescent signals in bodies of arbitrary shapes in a non-contact geometry, in combination with a 3D surface reconstruction algorithm, which is appropriate for in-vivo small animal imaging of fluorescent probes. The system consists of a rotating sample holder and a lens coupled Charge Coupled Device (CCD) camera in combination with a fiber coupled laser scanning device. An Argon ion laser is used as the source and different filters are used for the detection of various fluorophores or fluorescing proteins. With this new setup a large measurements dataset can be achieved while the use of inversion models give a high capacity for quantitative 3D reconstruction of fluorochrome distributions as well as high spatial resolution. The system has already been tested in the observation of the distribution of Green Fluorescent Protein (GFP) expressing T-lymphocytes in order to study the function of the immune system in a murine model, which can then be related to the function of the human immune system.
Enhancement of fluorescence using nanoplasmonic and photonic structures

NASA Astrophysics Data System (ADS)

Arya, Akash; Tagore, Amit K.; Dantham, Venkata R.

2018-05-01

Two different nanoplasmonic structures have been synthesized using wet-chemistry method and demonstrated the enhancement of fluorescence of dye molecules. The difficulties associated with the plasmonic enhancement of fluorescence are discussed and to overcome these, an efficient approach has been proposed to enhance the fluorescence of a few molecules with the help of a high quality factor photonic microstructure (whispering gallery mode microresonator). The fabrication details of microresonators and experimental arrangement for enhancing the fluorescence are reported here.
Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

PubMed

Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

2015-11-17

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.
Application of a temperature-dependent fluorescent dye (Rhodamine B) to the measurement of radiofrequency radiation-induced temperature changes in biological samples.

PubMed

Chen, Yuen Y; Wood, Andrew W

2009-10-01

We have applied a non-contact method for studying the temperature changes produced by radiofrequency (RF) radiation specifically to small biological samples. A temperature-dependent fluorescent dye, Rhodamine B, as imaged by laser scanning confocal microscopy (LSCM) was used to do this. The results were calibrated against real-time temperature measurements from fiber optic probes, with a calibration factor of 3.4% intensity change degrees C(-1) and a reproducibility of +/-6%. This non-contact method provided two-dimensional and three-dimensional images of temperature change and distributions in biological samples, at a spatial resolution of a few micrometers and with an estimated absolute precision of around 1.5 degrees C, with a differential precision of 0.4 degree C. Temperature rise within tissue was found to be non-uniform. Estimates of specific absorption rate (SAR) from absorbed power measurements were greater than those estimated from rate of temperature rise, measured at 1 min intervals, probably because this interval is too long to permit accurate estimation of initial temperature rise following start of RF exposure. Future experiments will aim to explore this.

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

Kim, M; Finlay, J; Zhu, T

Purpose: Photosensitizer concentration during photodynamic therapy (PDT) is an important parameter for accurate dosimetry. Fluorescence signal can be used as a measure of photosensitizer concentration. Two methods of data acquisition were compared to an ex vivo study both for in vivo and phantom models. Methods: Fluorescence signal of commonly used photosensitizer benzoporphyrin derivative monoacid ring A (BPD) was obtained in phantoms and mouse tumors using an excitation light of 405 nm. Interstitial fluorescence signal was obtained using a side-cut fiber inserted into the tumor tissue of interest. Using a previously developed multi-fiber probe, tumor surface fluorescence measurements were also collected.more » Signals were calibrated according to optical phantoms with known sensitizer fluorescence. Optical properties for each sample were determined and the influence of different absorption and scattering properties on the fluorescence signals was investigated. Using single value decomposition of the spectra, the sensitizer concentration was determined using the two different measurement geometries. An ex vivo analysis was also performed for tumor samples to determine the sensitizer concentration. Results: The two fluorescence signals obtained from the surface multi-fiber probe and the interstitial measurements were compared and were corresponding for both phantoms and mouse models. The values obtained were comparable to the ex vivo measurements as well. Despite the difference in geometry, the surface probe measurements can still be used as a metric for determining the presence of sensitizer in small volume tumors. Conclusion: The multi-fiber contact probe can be used as a tool to measure fluorescence at the surface of the treatment area for PDT and predict sensitizer concentration throughout the tumor. This is advantageous in that the measurement does not damage any tissue. Future work will include investigating the dependence of these results on intratumor sensitizer distribution.« less
Differential high-speed digital micromirror device based fluorescence speckle confocal microscopy.

PubMed

Jiang, Shihong; Walker, John

2010-01-20

We report a differential fluorescence speckle confocal microscope that acquires an image in a fraction of a second by exploiting the very high frame rate of modern digital micromirror devices (DMDs). The DMD projects a sequence of predefined binary speckle patterns to the sample and modulates the intensity of the returning fluorescent light simultaneously. The fluorescent light reflecting from the DMD's "on" and "off" pixels is modulated by correlated speckle and anticorrelated speckle, respectively, to form two images on two CCD cameras in parallel. The sum of the two images recovers a widefield image, but their difference gives a near-confocal image in real time. Experimental results for both low and high numerical apertures are shown.
Ultrathin Two-Dimensional Covalent Organic Framework Nanosheets: Preparation and Application in Highly Sensitive and Selective DNA Detection.

PubMed

Peng, Yongwu; Huang, Ying; Zhu, Yihan; Chen, Bo; Wang, Liying; Lai, Zhuangchai; Zhang, Zhicheng; Zhao, Meiting; Tan, Chaoliang; Yang, Nailiang; Shao, Fangwei; Han, Yu; Zhang, Hua

2017-06-28

The ability to prepare ultrathin two-dimensional (2D) covalent organic framework (COF) nanosheets (NSs) in high yield is of great importance for the further exploration of their unique properties and potential applications. Herein, by elaborately designing and choosing two flexible molecules with C 3v molecular symmetry as building units, a novel imine-linked COF, namely, TPA-COF, with a hexagonal layered structure and sheet-like morphology, is synthesized. Since the flexible building units are integrated into the COF skeletons, the interlayer stacking becomes weak, resulting in the easy exfoliation of TPA-COF into ultrathin 2D NSs. Impressively, for the first time, the detailed structural information, i.e., the pore channels and individual building units in the NSs, is clearly visualized by using the recently developed low-dose imaging technique of transmission electron microscopy (TEM). As a proof-of-concept application, the obtained ultrathin COF NSs are used as a novel fluorescence sensing platform for the highly sensitive and selective detection of DNA.
Study of interactions of an anticancer drug neratinib with bovine serum albumin: Spectroscopic and molecular docking approach

NASA Astrophysics Data System (ADS)

Wani, Tanveer A.; Bakheit, Ahmed H.; Abounassif, M. A.; Zargar, Seema

2018-03-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.
Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach.

PubMed

Wani, Tanveer A; Bakheit, Ahmed H; Abounassif, M A; Zargar, Seema

2018-01-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.
Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach

PubMed Central

Wani, Tanveer A.; Bakheit, Ahmed H.; Abounassif, M. A.; Zargar, Seema

2018-01-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes. PMID:29564326
Probing the behavior of bovine serum albumin upon binding to atenolol: insights from spectroscopic and molecular docking approaches.

PubMed

Jiang, Tuo-Ying; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi; Shi, Jie-Hua

2018-04-01

Molecular interaction of atenolol, a selective β 1 receptor antagonist with the major carrier protein, bovine serum albumin (BSA), was investigated under imitated physiological conditions (pH 7.4) by means of fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and molecular modeling studies. The steady-state fluorescence spectra manifested that static type, due to formation of the atenolol-BSA complex, was the dominant mechanism for fluorescence quenching. The characteristic information about the binding interaction of atenolol with BSA in terms of binding constant (K b ) were determined by the UV-vis absorption titration, and were found to be in the order of 10 3 M -1 at different temperatures, indicating the existence of a weak binding in this system. Thermodynamic analysis revealed that the binding process was primarily mediated by van der Waals force and hydrogen bonds due to the negative sign for enthalpy change (ΔH 0 ), entropy change (ΔS 0 ). The molecular docking results elucidated that atenolol preferred binding on the site II of BSA according to the findings observed in competitive binding experiments. Moreover, via alterations in synchronous fluorescence, three-dimensional fluorescence and FT-IR spectral properties, it was concluded that atenolol could arouse slight configurational and micro-environmental changes of BSA.
Characterization, phase solubility and molecular modeling of α-cyclodextrin/pyrimethamine inclusion complex

NASA Astrophysics Data System (ADS)

Araujo, Marcia Valeria Gaspar de; Macedo, Osmir F. L.; Nascimento, Cristiane da Cunha; Conegero, Leila Souza; Barreto, Ledjane Silva; Almeida, Luis Eduardo; Costa, Nivan Bezerra da; Gimenez, Iara F.

2009-02-01

An inclusion complex between the dihydrofolate reductase inhibitor pyrimethamine (PYR) and α-cyclodextrin (α-CD) was prepared and characterized. From the phase-solubility diagram, a linear increase of PYR solubility was verified as a function of α-CD concentration, suggesting the formation of a soluble complex. A 1:1 host-guest stoichiometry can be proposed according to the Job's plot, obtained from the difference of PYR fluorescence intensity in the presence and absence of α-CD. Differential scanning calorimetry (DSC) measurements provided additional evidences of complexation such as the absence of the endothermic peak assigned to the melting of the drug. The inclusion mode characterized by two-dimensional 1H NMR spectroscopy (ROESY) involves penetration of the p-chlorophenyl ring into the α-CD cavity, in agreement to the orientation optimized by molecular modeling methods.
Two dimensional PMMA nanofluidic device fabricated by hot embossing and oxygen plasma assisted thermal bonding methods

NASA Astrophysics Data System (ADS)

Yin, Zhifu; Sun, Lei; Zou, Helin; Cheng, E.

2015-05-01

A method for obtaining a low-cost and high-replication precision two-dimensional (2D) nanofluidic device with a polymethyl methacrylate (PMMA) sheet is proposed. To improve the replication precision of the 2D PMMA nanochannels during the hot embossing process, the deformation of the PMMA sheet was analyzed by a numerical simulation method. The constants of the generalized Maxwell model used in the numerical simulation were calculated by experimental compressive creep curves based on previously established fitting formula. With optimized process parameters, 176 nm-wide and 180 nm-deep nanochannels were successfully replicated into the PMMA sheet with a replication precision of 98.2%. To thermal bond the 2D PMMA nanochannels with high bonding strength and low dimensional loss, the parameters of the oxygen plasma treatment and thermal bonding process were optimized. In order to measure the dimensional loss of 2D nanochannels after thermal bonding, a dimension loss evaluating method based on the nanoindentation experiments was proposed. According to the dimension loss evaluating method, the total dimensional loss of 2D nanochannels was 6 nm and 21 nm in width and depth, respectively. The tensile bonding strength of the 2D PMMA nanofluidic device was 0.57 MPa. The fluorescence images demonstrate that there was no blocking or leakage over the entire microchannels and nanochannels.
Phosphorene quantum dots

NASA Astrophysics Data System (ADS)

Vishnoi, Pratap; Mazumder, Madhulika; Barua, Manaswee; Pati, Swapan K.; Rao, C. N. R.

2018-05-01

Phosphorene, a two-dimensional material, has been a subject of recent investigations. In the present study, we have prepared blue fluorescent phosphorene quantum dots (PQDs) by liquid phase exfoliation of black phosphorus in two non-polar solvents, toluene and mesitylene. The average particle sizes of PQDs decrease from 5.0 to 1.0 nm on increasing the sonicator power from 150 to 225 W. The photoluminescence spectrum of the PQDs is red-shifted in the 395-470 nm range on increasing the excitation-wavelength from 300 to 480 nm. Electron donor and acceptor molecules quench the photoluminescence, with the acceptors showing more marked effects.
Cardiovascular Imaging Using Two-Photon Microscopy

PubMed Central

Scherschel, John A.; Rubart, Michael

2008-01-01

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer. PMID:18986603
X-ray Fluorescence Holography: Principles, Apparatus, and Applications

NASA Astrophysics Data System (ADS)

Hayashi, Kouichi; Korecki, Pawel

2018-06-01

X-ray fluorescence holography (XFH) is an atomic structure determination technique that combines the capabilities of X-ray diffraction and X-ray fluorescence spectroscopy. It provides a unique means of gaining fully three-dimensional information about the local atomic structure and lattice site positions of selected elements inside compound samples. In this work, we discuss experimental and theoretical aspects that are essential for the efficient recording and analysis of X-ray fluorescence holograms and review the most recent advances in XFH. We describe experiments performed with brilliant synchrotron radiation as well as with tabletop setups that employ conventional X-ray tubes.
[Laser Induced Fluorescence Spectroscopic Analysis of Aromatics from One Ring to Four Rings].

PubMed

Zhang, Peng; Liu, Hai-feng; Yue, Zong-yu; Chen, Bei-ling; Yao, Ming-fa

2015-06-01

In order to distinguish small aromatics preferably, a Nd : YAG Laser was used to supply an excitation laser, which was adjusted to 0.085 J x cm(-2) at 266 nm. Benzene, toluene, naphthalene, phenanthrene, anthracene, pyrene and chrysene were used as the representative of different rings aromatics. The fluorescence emission spectra were researched for each aromatic hydrocarbon and mixtures by Laser induced fluorescence (LIF). Results showed that the rings number determined the fluorescence emission spectra, and the structure with same rings number did not affect the emission fluorescence spectrum ranges. This was due to the fact that the absorption efficiency difference at 266 nm resulted in that the fluorescence intensities of each aromatic hydrocarbon with same rings number were different and the fluorescence intensities difference were more apparently with aromatic ring number increasing. When the absorption efficiency was similar at 266 nm and the concentrations of each aromatic hydrocarbon were same, the fluorescence intensities were increased with aromatic ring number increasing. With aromatic ring number increasing, the fluorescence spectrum and emission peak wavelength were all red-shifted from ultraviolet to visible and the fluorescence spectrum range was also wider as the absorption efficiency was similar. The fluorescence emission spectra from one to four rings could be discriminated in the following wavelengths, 275 to 320 nm, 320 to 375 nm, 375 to 425 nm, 425 to 556 nm, respectively. It can be used for distinguish the type of the polycyclic aromatic hydrocarbons (PAHs) as it exists in single type. As PAHs are usually exist in a variety of different rings number at the same time, the results for each aromatic hydrocarbon may not apply to the aromatic hydrocarbon mixtures. For the aromatic hydrocarbon mixtures, results showed that the one- or two-ring PAHs in mixtures could not be detected by fluorescence as three- or four-ring PAHs existed in mixture. This was caused by radiation energy transfer mechanism, in which the ultraviolet light was lost in mixtures but the fluorescence intensities were increased with the one- or two-ring PAHs adding. When the mixture only contained three- and four-ring PAHs, the fluorescence emission spectrum showed the both characteristics of three- and four-ring PAHs fluorescence. When three- and four-ring PAHs existed in mixtures at the same time, the fluorescence emission spectra were related to each concentration, so the rings number could be discriminated to a certain extent.
Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

NASA Astrophysics Data System (ADS)

Deng, Fengyuan; Ding, Changqin; Martin, Jerald C.; Scarborough, Nicole M.; Song, Zhengtian; Eakins, Gregory S.; Simpson, Garth J.

2018-02-01

Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam's position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.
Optical Characterization of Paper Aging Based on Laser-Induced Fluorescence (LIF) Spectroscopy.

PubMed

Zhang, Hao; Wang, Shun; Chang, Keke; Sun, Haifeng; Guo, Qingqian; Ma, Liuzheng; Yang, Yatao; Zou, Caihong; Wang, Ling; Hu, Jiandong

2018-06-01

Paper aging and degradation are growing concerns for those who are responsible for the conservation of documents, archives, and libraries. In this study, the paper aging was investigated using laser-induced fluorescence spectroscopy (LIFS), where the fluorescence properties of 47 paper samples with different ages were explored. The paper exhibits fluorescence in the blue-green spectral region with two peaks at about 448 nm and 480 nm under the excitation of 405 nm laser. Both fluorescence peaks changed in absolute intensities and thus the ratio of peak intensities was also influenced with the increasing ages. By applying principal component analysis (PCA) and k-means clustering algorithm, all 47 paper samples were classified into nine groups based on the differences in paper age. Then the first-derivative fluorescence spectral curves were proposed to figure out the relationship between the spectral characteristic and the paper age, and two quantitative models were established based on the changes of first-derivative spectral peak at 443 nm, where one is an exponential fitting curve with an R-squared value of 0.99 and another is a linear fitting curve with an R-squared value of 0.88. The results demonstrated that the combination of fluorescence spectroscopy and PCA can be used for the classification of paper samples with different ages. Moreover, the first-derivative fluorescence spectral curves can be used to quantitatively evaluate the age-related changes of paper samples.
An introduction to MATLAB.

PubMed

Sobie, Eric A

2011-09-13

This two-part lecture introduces students to the scientific computing language MATLAB. Prior computer programming experience is not required. The lectures present basic concepts of computer programming logic that tend to cause difficulties for beginners in addition to concepts that relate specifically to the MATLAB language syntax. The lectures begin with a discussion of vectors, matrices, and arrays. Because many types of biological data, such as fluorescence images and DNA microarrays, are stored as two-dimensional objects, processing these data is a form of array manipulation, and MATLAB is especially adept at handling such array objects. The students are introduced to basic commands in MATLAB, as well as built-in functions that provide useful shortcuts. The second lecture focuses on the differences between MATLAB scripts and MATLAB functions and describes when one method of programming organization might be preferable to the other. The principles are illustrated through the analysis of experimental data, specifically measurements of intracellular calcium concentration in live cells obtained using confocal microscopy.
An Introduction to MATLAB

PubMed Central

Sobie, Eric A.

2014-01-01

This two-part lecture introduces students to the scientific computing language MATLAB. Prior computer programming experience is not required. The lectures present basic concepts of computer programming logic that tend to cause difficulties for beginners in addition to concepts that relate specifically to the MATLAB language syntax. The lectures begin with a discussion of vectors, matrices, and arrays. Because many types of biological data, such as fluorescence images and DNA microarrays, are stored as two-dimensional objects, processing these data is a form of array manipulation, and MATLAB is especially adept at handling such array objects. The students are introduced to basic commands in MATLAB, as well as built-in functions that provide useful shortcuts. The second lecture focuses on the differences between MATLAB scripts and MATLAB functions and describes when one method of programming organization might be preferable to the other. The principles are illustrated through the analysis of experimental data, specifically measurements of intracellular calcium concentration in live cells obtained using confocal microscopy. PMID:21934110
Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition.

PubMed

Mathew, B; Schmitz, A; Muñoz-Descalzo, S; Ansari, N; Pampaloni, F; Stelzer, E H K; Fischer, S C

2015-06-08

Due to the large amount of data produced by advanced microscopy, automated image analysis is crucial in modern biology. Most applications require reliable cell nuclei segmentation. However, in many biological specimens cell nuclei are densely packed and appear to touch one another in the images. Therefore, a major difficulty of three-dimensional cell nuclei segmentation is the decomposition of cell nuclei that apparently touch each other. Current methods are highly adapted to a certain biological specimen or a specific microscope. They do not ensure similarly accurate segmentation performance, i.e. their robustness for different datasets is not guaranteed. Hence, these methods require elaborate adjustments to each dataset. We present an advanced three-dimensional cell nuclei segmentation algorithm that is accurate and robust. Our approach combines local adaptive pre-processing with decomposition based on Lines-of-Sight (LoS) to separate apparently touching cell nuclei into approximately convex parts. We demonstrate the superior performance of our algorithm using data from different specimens recorded with different microscopes. The three-dimensional images were recorded with confocal and light sheet-based fluorescence microscopes. The specimens are an early mouse embryo and two different cellular spheroids. We compared the segmentation accuracy of our algorithm with ground truth data for the test images and results from state-of-the-art methods. The analysis shows that our method is accurate throughout all test datasets (mean F-measure: 91%) whereas the other methods each failed for at least one dataset (F-measure≤69%). Furthermore, nuclei volume measurements are improved for LoS decomposition. The state-of-the-art methods required laborious adjustments of parameter values to achieve these results. Our LoS algorithm did not require parameter value adjustments. The accurate performance was achieved with one fixed set of parameter values. We developed a novel and fully automated three-dimensional cell nuclei segmentation method incorporating LoS decomposition. LoS are easily accessible features that ensure correct splitting of apparently touching cell nuclei independent of their shape, size or intensity. Our method showed superior performance compared to state-of-the-art methods, performing accurately for a variety of test images. Hence, our LoS approach can be readily applied to quantitative evaluation in drug testing, developmental and cell biology.
[Development and evaluation of a novel three-dimensional adjustable confocal laser-induced fluorescence detector].

PubMed

Liu, Fan; Yu, Shuxin; Tang, Tao; Sun, Yuanshe; Zhang, Weibing; Li, Tong

2011-09-01

Laser-induced fluorescence detector (LIFD) is one of the most sensitive detectors in analytical chemical files. Confocal optical configuration is widely used in LIFDs. Two effective approaches used to achieve the best signal to noise ratio (S/N) are increasing the confocal precision and minimizing the background noise. A novel three-dimensional adjustable confocal LIFD was developed, using a new three-dimensional adjustable supporter of reflector and modularized optical system. A detection limit (S/N = 3) of 1 x 10(-12) mol/L and a linear dynamic range of 3 orders of magnitude were obtained using fluorescein isothiocyanate (FITC) standard as the test sample. The noise level and drift levels were 8.0 x 10(-3) mV and 1.4 x 10(-3) mV/h, respectively, which were almost 10 times lower than before. And the stability of the LIFD was evaluated by five replicate injections of 5 x 10(-9) mol/L FITC, and the relative standard deviations (RSDs) of peak height and peak area were 0.38% and 0.41%, respectively. Further more, three biogenic amines, which were derivatized by FITC, were separated by high performance liquid chromatography (HPLC) and then detected by the novel LIFD. And the detection limits (S/N = 3) ranged 0.01 to 0.02 nmol/L, which were better than other methods. Therefore, the LIFD is highly sensitive, as well as shows a real low noise level and good reproducibility.
Phosphorylation-Induced Conformational Changes of Photoactivated Rhodopsin Probed by Fluorescent Labeling at Cys140 and Cys316.

PubMed

Rodríguez, Sheerly; Silva, May-Li; Benaím, Gustavo; Bubis, José

2018-05-03

In order to monitor conformational changes following photoactivation and phosphorylation of bovine rhodopsin, the two reactive sulfhydryl groups at Cys 140 and Cys 316 were specifically labeled with the monobromobimane (mBBr) fluorophore. Although alterations in conformation after light exposure of rhodopsin were not detected by fluorescence excitation scans (300-450 nm) of the mBBr-labeled protein, the fluorescence signal was reduced ∼ 90% in samples containing photoactivated phosphorhodopsin. Predominant labeling at either Cys 140 or Cys 316 in light-activated and phosphorylated rhodopsin merely generated a decrease of ∼ 38% and 28%, respectively, in the fluorescence excitation intensity. Thus, neither mBBr-modified Cys 140 nor mBBr-modified Cys 316 were involved single-handedly in the remarkable fall seen on the signal following phosphorylation of the protein; rather, the incorporation of phosphate groups on the mBBr-labeled light-activated rhodopsin appeared to affect its fluorescence signal in a cooperative or synergistic manner. These findings demonstrated that the phosphorylation of specific hydroxyl groups at the carboxyl terminal tail of rhodopsin causes definite conformational changes in the three-dimensional fold of the protein. Apparently, amino acid residues that are buried in the interior of the inactive protein become accessible following bleaching and phosphorylation of rhodopsin, quenching in turn the fluorescence excitation signal of mBBr-modified rhodopsin. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

2D fluorescence spectroscopy for monitoring ion-exchange membrane based technologies - Reverse electrodialysis (RED).

PubMed

Pawlowski, Sylwin; Galinha, Claudia F; Crespo, João G; Velizarov, Svetlozar

2016-01-01

Reverse electrodialysis (RED) is one of the emerging, membrane-based technologies for harvesting salinity gradient energy. In RED process, fouling is an undesirable operation constraint since it leads to a decrease of the obtainable net power density due to increasing stack electric resistance and pressure drop. Therefore, early fouling detection is one of the main challenges for successful RED technology implementation. In the present study, two-dimensional (2D) fluorescence spectroscopy was used, for the first time, as a tool for fouling monitoring in RED. Fluorescence excitation-emission matrices (EEMs) of ion-exchange membrane surfaces and of natural aqueous streams were acquired during one month of a RED stack operation. Fouling evolvement on the ion-exchange membrane surfaces was successfully followed by 2D fluorescence spectroscopy and quantified using principal components analysis (PCA). Additionally, the efficiency of cleaning strategy was assessed by measuring the membrane fluorescence emission intensity before and after cleaning. The anion-exchange membrane (AEM) surface in contact with river water showed to be significantly affected due to fouling by humic compounds, which were found to cross through the membrane from the lower salinity (river water) to higher salinity (sea water) stream. The results obtained show that the combined approach of using 2D fluorescence spectroscopy and PCA has a high potential for studying fouling development and membrane cleaning efficiency in ion exchange membrane processes. Copyright © 2015 Elsevier Ltd. All rights reserved.
Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

PubMed

Cooper, Mark S; Szeto, Daniel P; Sommers-Herivel, Greg; Topczewski, Jacek; Solnica-Krezel, Lila; Kang, Hee-Chol; Johnson, Iain; Kimelman, David

2005-02-01

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Copyright 2004 Wiley-Liss, Inc.
Zinc Oxide Nanomaterials for Biomedical Fluorescence Detection

PubMed Central

Hahm, Jong-in

2014-01-01

One-dimensional zinc oxide nanomaterials have been recently developed into novel, extremely effective, optical signal-enhancing bioplatforms. Their usefulness has been demonstrated in various biomedical fluorescence assays. Fluorescence is extensively used in biology and medicine as a sensitive and noninvasive detection method for tracking and analyzing biological molecules. Achieving high sensitivity via improving signal-to-noise ratio is of paramount importance in fluorescence-based, trace-level detection. Recent advances in the development of optically superior one-dimensional materials have contributed to this important biomedical area of detection. This review article will discuss major research developments that have so far been made in this emerging and exciting topical field. The discussion will cover a broad range of subjects including synthesis of zinc oxide nanorods (ZnO NRs), various properties differentiating them as suitable optical biodetection platforms, their demonstrated applicability in DNA and protein detection, and the nanomaterial characteristics relevant for biomolecular fluorescence enhancement. This review will then summarize the current status of ZnO NR-based biodetection and further elaborate future utility of ZnO NR platforms for advanced biomedical assays, based on their proven advantages. Lastly, present challenges experienced in this topical area will be identified and focal subject areas for future research will be suggested as well. PMID:24730276
Novel insights into anoxic/aerobic(1)/aerobic(2) biological fluidized-bed system for coke wastewater treatment by fluorescence excitation-emission matrix spectra coupled with parallel factor analysis.

PubMed

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

2014-10-01

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

PubMed

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.
Interface of physics and biology: engineering virus-based nanoparticles for biophotonics.

PubMed

Wen, Amy M; Infusino, Melissa; De Luca, Antonio; Kernan, Daniel L; Czapar, Anna E; Strangi, Giuseppe; Steinmetz, Nicole F

2015-01-21

Virus-based nanoparticles (VNPs) have been used for a wide range of applications, spanning basic materials science and translational medicine. Their propensity to self-assemble into precise structures that offer a three-dimensional scaffold for functionalization has led to their use as optical contrast agents and related biophotonics applications. A number of fluorescently labeled platforms have been developed and their utility in optical imaging demonstrated, yet their optical properties have not been investigated in detail. In this study, two VNPs of varying architectures were compared side-by-side to determine the impact of dye density, dye localization, conjugation chemistry, and microenvironment on the optical properties of the probes. Dyes were attached to icosahedral cowpea mosaic virus (CPMV) and rod-shaped tobacco mosaic virus (TMV) through a range of chemistries to target particular side chains displayed at specific locations around the virus. The fluorescence intensity and lifetime of the particles were determined, first using photochemical experiments on the benchtop, and second in imaging experiments using tissue culture experiments. The virus-based optical probes were found to be extraordinarily robust under ultrashort, pulsed laser light conditions with a significant amount of excitation energy, maintaining structural and chemical stability. The most effective fluorescence output was achieved through dye placement at optimized densities coupled to the exterior surface avoiding conjugated ring systems. Lifetime measurements indicate that fluorescence output depends not only on spacing the fluorophores, but also on dimer stacking and configurational changes leading to radiationless relaxation-and these processes are related to the conjugation chemistry and nanoparticle shape. For biological applications, the particles were also examined in tissue culture, from which it was found that the optical properties differed from those found on the benchtop due to effects from cellular processes and uptake kinetics. Data indicate that fluorescent cargos are released in the endolysosomal compartment of the cell targeted by the virus-based optical probes. These studies provide insight into the optical properties and fates of fluorescent proteinaceous imaging probes. The cellular release of cargo has implications not only for virus-based optical probes, but also for drug delivery and release systems.
A one-dimensional zinc(II) coordination polymer with a three-dimensional supramolecular architecture incorporating 1-[(1H-benzimidazol-2-yl)methyl]-1H-tetrazole and adipate.

PubMed

Liu, Chun Li; Huang, Qiu Ying; Meng, Xiang Ru

2016-12-01

The synthesis of coordination polymers or metal-organic frameworks (MOFs) has attracted considerable interest owing to the interesting structures and potential applications of these compounds. It is still a challenge to predict the exact structures and compositions of the final products. A new one-dimensional coordination polymer, catena-poly[[[bis{1-[(1H-benzimidazol-2-yl)methyl]-1H-tetrazole-κN 3 }zinc(II)]-μ-hexane-1,6-dicarboxylato-κ 4 O 1 ,O 1' :O 6 ,O 6' ] monohydrate], {[Zn(C 6 H 8 O 4 )(C 9 H 8 N 6 ) 2 ]·H 2 O} n , has been synthesized by the reaction of Zn(Ac) 2 (Ac is acetate) with 1-[(1H-benzimidazol-2-yl)methyl]-1H-tetrazole (bimt) and adipic acid (H 2 adi) at room temperature. In the polymer, each Zn II ion exhibits an irregular octahedral ZnN 2 O 4 coordination geometry and is coordinated by two N atoms from two symmetry-related bimt ligands and four O atoms from two symmetry-related dianionic adipate ligands. Zn II ions are connected by adipate ligands into a one-dimensional chain which runs parallel to the c axis. The bimt ligands coordinate to the Zn II ions in a monodentate mode on both sides of the main chain. In the crystal, the one-dimensional chains are further connected through N-H...O hydrogen bonds, leading to a three-dimensional supramolecular architecture. In addition, the title polymer exhibits fluorescence, with emissions at 334 and 350 nm in the solid state at room temperature.
Fluorescence spectroscopy of trapped molecular ions

NASA Astrophysics Data System (ADS)

Wright, Kenneth Charles

This thesis describes the development of a unique instrument capable of detecting fluorescence emission from large gas phase molecular ions trapped in a three-dimensional quadrupole ion trap. The hypothesis that has formed the basis of this work is the belief that fluorescence spectroscopy can be combined with ion trap mass spectrometry to probe the structure of gas phase molecular ions. The ion trap provides a rarefied environment where fluorescence experiments can be conducted without interference from solvent molecules or impurities. Although fluorescence was not detected during preliminary experiments, two significant experimental challenges associated with detecting the gas phase fluorescence of ions were discovered. First, gas phase ions were vulnerable to photodissociation and low laser powers were necessary to avoid photodissociation. Since fluorescence emission is directly proportional to laser intensity, a lower laser power limits the fluorescence signal. Second, the fluorescence emission was not significantly Stokes shifted from the excitation. The lack of Stokes shift meant the small fluorescence signal must be detected in the presence of a large amount of background scatter generated by the excitation. Initially, this background was seven orders of magnitude higher than the analytical signal ultimately detected. A specially designed fiber optic probe was inserted between the electrodes of the ion trap to stop light scattered off the outside surfaces of the trap from reaching the detector. The inside surfaces of the ion trap were coated black to further reduce the amount of scattered light collected. These innovations helped reduced the background by six orders of magnitude and fluorescence emission from rhodamine-6G was detected. Pulse counting experiments were used to optimize fluorescence detection. The effects of trapping level, laser power, and irradiation time were investigated and optimized. The instrument developed in this work not only allows for the detection of fluorescent photons, but the sensitivity is high enough for the light to be dispersed and an emission spectrum recorded. The emission spectra of rhodamine-6G and 5-carboxyrhodamine-6G ions reported in this thesis represent the first spectra recorded from large molecular ions confined in a quadrupole ion trap. Finally, anti-Stokes fluorescence from rhodamine-6G was also detected.
Fast and Selective Two-Stage Ratiometric Fluorescent Probes for Imaging of Glutathione in Living Cells.

PubMed

Gong, Deyan; Han, Shi-Chong; Iqbal, Anam; Qian, Jing; Cao, Ting; Liu, Wei; Liu, Weisheng; Qin, Wenwu; Guo, Huichen

2017-12-19

Two fluorescent, m-nitrophenol-substituted difluoroboron dipyrromethene dyes have been designed by nucleophilic substitution reaction of 3,5-dichloro-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). Nonsymmetric and symmetric probes, that is. BODIPY 1 (with one nitrophenol group at the position 3) and BODIPY 2 (with two nitrophenol groups at the positions 3 and 5) were applied to ratiometric fluorescent glutathione detection. The detection is based on the two-step nucleophilic aromatic substitution of the nitrophenol groups of the probes by glutathione in buffer solution containing CTAB. In the first stage, probe 1 showed ratiometric fluorescent color change from green (λ em = 530 nm) to yellow (λ em = 561 nm) because of monosubstitution with glutathione (I 561nm /I 530nm ). Addition of excess glutathione caused the second stage of ratiometric fluorescent color change from yellow to reddish orange (λ em = 596 nm, I 596nm /I 561nm ) due to disubstitution with glutathione. Therefore, different concentration ranges of glutathione (from less to excess) could be rapidly detected by the two-stage ratiometric fluorescent probe 1 in 5 min. While, probe 2 shows single-stage ratiometric fluorescent detection to GSH (from green to reddish orange, I 596nm /I 535nm ). Probes 1 and 2 exhibit excellent properties with sensitive, specific colorimetric response and ratiometric fluorescent response to glutathione over other sulfur nucleophiles. Application to cellular ratiometric fluorescence imaging indicated that the probes were highly responsive to intracellular glutathione.
Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation.

PubMed

Muraki, Michiro

2016-01-01

Human Fas ligand extracellular domain has been investigated as an important target protein in the field of medical biotechnology. In a recent study, the author developed an effective method to produce biologically active human Fas ligand extracellular domain derivatives using site-specific chemical modifications. A human Fas ligand extracellular domain derivative containing a reactive cysteine residue within its N-terminal tag sequence, which locates not proximal to the binding interface between the ligand and the receptor in terms of the three-dimensional structure, was modified by Fluorescein-5-Maleimide without impairing the specific binding activity toward human Fas receptor extracellular domain. The purified protein sample free of low molecular-weight contaminants showed a characteristic fluorescence spectrum derived from the attached Fluorescein moieties, and formed a stable binding complex with human Fas receptor extracellular domain-human IgG1 Fc domain fusion protein in solution. The conjugation number of the fluorochrome was estimated to be 2.5 per a single human Fas ligand extracellular domain trimer from the ratio of the absorbance value at 280 nm to that at 495 nm. A functional fluorescent human Fas ligand extracellular domain derivative was prepared via a site-specific conjugation of fluorochrome, which was guided by the three-dimensional structure information on the ligand-receptor complex. Fluorescent derivatives created by this method may contribute to the development of an improved diagnosis system for the diseases related to Fas receptor.
Analysis of the binding interaction in uric acid - Human hemoglobin system by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Makarska-Bialokoz, Magdalena

2017-05-01

The binding interaction between human hemoglobin and uric acid has been studied for the first time, by UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence techniques. Characteristic effects observed for human hemoglobin intrinsic fluorescence during interaction with uric acid at neutral pH point at the formation of stacking non-covalent and non-fluorescent complexes. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as Förster resonance energy transfer parameters confirm the existence of static quenching. The results of synchronous fluorescence measurements indicate that the fluorescence quenching of human hemoglobin originates both from Trp and Tyr residues and that the addition of uric acid could significantly hinder the physiological functions of human hemoglobin.
Separation and identification of anthocyanin extracted from mulberry fruit and the pigment binding properties toward human serum albumin.

PubMed

Sheng, Feng; Wang, Yuning; Zhao, Xingchen; Tian, Na; Hu, Huali; Li, Pengxia

2014-07-16

Purple pigments were isolated from mulberry extracts using preparative high-speed countercurrent chromatography (HSCCC) and identified by ESI-MS/MS and high performance liquid chromatography (HPLC) techniques. The solvent system containing methyl tert-butyl ether, 1-butanol, acetonitrile, water, and trifluoroacetic acid (10:30:10:50:0.05; %, v/v) was developed in order to separate anthocyanins with different polarities. Cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-galactopyranoside) (also known as keracyanin) is the major component present in mulberry (41.3%). Other isolated pigments are cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) and petunidin 3-O-β-glucopyranoside. The binding characteristics of keracyanin with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopy. Spectroscopic analysis reveals that HSA fluorescence quenched by keracyanin follows a static mode. Binding of keracyanin to HSA mainly depends on van der Waals force or H-bonds with average binding distance of 2.82 nm. The results from synchronous fluorescence, three-dimensional fluorescence, and CD spectra show that adaptive structure rearrangement and decrease of α-helical structure occur in the presence of keracyanin.
Second Harmonic Generation Imaging Analysis of Collagen Arrangement in Human Cornea.

PubMed

Park, Choul Yong; Lee, Jimmy K; Chuck, Roy S

2015-08-01

To describe the horizontal arrangement of human corneal collagen bundles by using second harmonic generation (SHG) imaging. Human corneas were imaged with an inverted two photon excitation fluorescence microscope. The excitation laser (Ti:Sapphire) was tuned to 850 nm. Backscatter signals of SHG were collected through a 425/30-nm bandpass emission filter. Multiple, consecutive, and overlapping image stacks (z-stacks) were acquired to generate three dimensional data sets. ImageJ software was used to analyze the arrangement pattern (irregularity) of collagen bundles at each image plane. Collagen bundles in the corneal lamellae demonstrated a complex layout merging and splitting within a single lamellar plane. The patterns were significantly different in the superficial and limbal cornea when compared with deep and central regions. Collagen bundles were smaller in the superficial layer and larger in deep lamellae. By using SHG imaging, the horizontal arrangement of corneal collagen bundles was elucidated at different depths and focal regions of the human cornea.
Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

PubMed Central

Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

2016-01-01

We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944
Coregistered fluorescence-enhanced tumor resection of malignant glioma: relationships between δ-aminolevulinic acid–induced protoporphyrin IX fluorescence, magnetic resonance imaging enhancement, and neuropathological parameters

PubMed Central

Roberts, David W.; Valdés, Pablo A.; Harris, Brent T.; Fontaine, Kathryn M.; Hartov, Alexander; Fan, Xiaoyao; Ji, Songbai; Lollis, S. Scott; Pogue, Brian W.; Leblond, Frederic; Tosteson, Tor D.; Wilson, Brian C.; Paulsen, Keith D.

2010-01-01

Object The aim of this study was to investigate the relationships between intraoperative fluorescence, features on MR imaging, and neuropathological parameters in 11 cases of newly diagnosed glioblastoma multiforme (GBM) treated using protoporphyrin IX (PpIX) fluorescence-guided resection. Methods In 11 patients with a newly diagnosed GBM, δ-aminolevulinic acid (ALA) was administered to enhance endogenous synthesis of the fluorophore PpIX. The patients then underwent fluorescence-guided resection, coregistered with conventional neuronavigational image guidance. Biopsy specimens were collected at different times during surgery and assigned a fluorescence level of 0–3 (0, no fluorescence; 1, low fluorescence; 2, moderate fluorescence; or 3, high fluorescence). Contrast enhancement on MR imaging was quantified using two image metrics: 1) Gd-enhanced signal intensity (GdE) on T1-weighted subtraction MR image volumes, and 2) normalized contrast ratios (nCRs) in T1-weighted, postGd-injection MR image volumes for each biopsy specimen, using the biopsy-specific image-space coordinate transformation provided by the navigation system. Subsequently, each GdE and nCR value was grouped into one of two fluorescence categories, defined by its corresponding biopsy specimen fluorescence assessment as negative fluorescence (fluorescence level 0) or positive fluorescence (fluorescence level 1, 2, or 3). A single neuropathologist analyzed the H & E–stained tissue slides of each biopsy specimen and measured three neuropathological parameters: 1) histopathological score (0–IV); 2) tumor burden score (0–III); and 3) necrotic burden score (0–III). Results Mixed-model analyses with random effects for individuals show a highly statistically significant difference between fluorescing and nonfluorescing tissue in GdE (mean difference 8.33, p = 0.018) and nCRs (mean difference 5.15, p < 0.001). An analysis of association demonstrated a significant relationship between the levels of intraoperative fluorescence and histopathological score (χ2 = 58.8, p < 0.001), between fluorescence levels and tumor burden (χ2 = 42.7, p < 0.001), and between fluorescence levels and necrotic burden (χ2 = 30.9, p < 0.001). The corresponding Spearman rank correlation coefficients were 0.51 (p < 0.001) for fluorescence and histopathological score, and 0.49 (p < 0.001) for fluorescence and tumor burden, suggesting a strongly positive relationship for each of these variables. Conclusions These results demonstrate a significant relationship between contrast enhancement on preoperative MR imaging and observable intraoperative PpIX fluorescence. The finding that preoperative MR image signatures are predictive of intraoperative PpIX fluorescence is of practical importance for identifying candidates for the procedure. Furthermore, this study provides evidence that a strong relationship exists between tumor aggressiveness and the degree of tissue fluorescence that is observable intraoperatively, and that observable fluorescence has an excellent positive predictive value but a low negative predictive value. PMID:20380535
Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria).

PubMed

Wiedenmann, J; Elke, C; Spindler, K D; Funke, W

2000-12-19

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like beta-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of beta-can that could be formed in a multimerization process.
Cracks in the β-can: Fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria)

PubMed Central

Wiedenmann, Jörg; Elke, Carsten; Spindler, Klaus-Dieter; Funke, Werner

2000-01-01

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like β-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of β-can that could be formed in a multimerization process. PMID:11121018
Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

PubMed

Petersen, Timothy W; Ibrahim, Sherrif F; Diercks, Alan H; van den Engh, Ger

2004-08-01

Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.
Two-Dimensional Standing Wave Total Internal Reflection Fluorescence Microscopy: Superresolution Imaging of Single Molecular and Biological Specimens

PubMed Central

Chung, Euiheon; Kim, Daekeun; Cui, Yan; Kim, Yang-Hyo; So, Peter T. C.

2007-01-01

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or ∼100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy. PMID:17483188
Synthesis-identification integration: One-pot hydrothermal preparation of fluorescent nitrogen-doped carbon nanodots for differentiating nucleobases with the aid of multivariate chemometrics analysis.

PubMed

Zhuang, Qianfen; Cao, Wei; Ni, Yongnian; Wang, Yong

2018-08-01

Most of the conventional multidimensional differential sensors currently need at least two-step fabrication, namely synthesis of probe(s) and identification of multiple analytes by mixing of analytes with probe(s), and were conducted using multiple sensing elements or several devices. In the study, we chose five different nucleobases (adenine, cytosine, guanine, thymine, and uracil) as model analytes, and found that under hydrothermal conditions, sodium citrate could react directly with various nucleobases to yield different nitrogen-doped carbon nanodots (CDs). The CDs synthesized from different nucleobases exhibited different fluorescent properties, leading to their respective characteristic fluorescence spectra. Hence, we combined the fluorescence spectra of the CDs with advanced chemometrics like principle component analysis (PCA), hierarchical cluster analysis (HCA), K-nearest neighbor (KNN) and soft independent modeling of class analogy (SIMCA), to present a conceptually novel "synthesis-identification integration" strategy to construct a multidimensional differential sensor for nucleobase discrimination. Single-wavelength excitation fluorescence spectral data, single-wavelength emission fluorescence spectral data, and fluorescence Excitation-Emission Matrices (EEMs) of the CDs were respectively used as input data of the differential sensor. The results showed that the discrimination ability of the multidimensional differential sensor with EEM data set as input data was superior to those with single-wavelength excitation/emission fluorescence data set, suggesting that increasing the number of the data input could improve the discrimination power. Two supervised pattern recognition methods, namely KNN and SIMCA, correctly identified the five nucleobases with a classification accuracy of 100%. The proposed "synthesis-identification integration" strategy together with a multidimensional array of experimental data holds great promise in the construction of differential sensors. Copyright © 2018 Elsevier B.V. All rights reserved.

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