Super-resolution imaging based on the temperature-dependent electron-phonon collision frequency effect of metal thin films

NASA Astrophysics Data System (ADS)

Ding, Chenliang; Wei, Jingsong; Xiao, Mufei

2018-05-01

We herein propose a far-field super-resolution imaging with metal thin films based on the temperature-dependent electron-phonon collision frequency effect. In the proposed method, neither fluorescence labeling nor any special properties are required for the samples. The 100 nm lands and 200 nm grooves on the Blu-ray disk substrates were clearly resolved and imaged through a laser scanning microscope of wavelength 405 nm. The spot size was approximately 0.80 μm , and the imaging resolution of 1/8 of the laser spot size was experimentally obtained. This work can be applied to the far-field super-resolution imaging of samples with neither fluorescence labeling nor any special properties.

Garry Rumbles | NREL

Science.gov Websites

, colloidal quantum dots, and single-walled carbon nanotubes. Laser-based experiments (time-resolved fluorescence spectroscopy; time-resolved resonance Raman spectroscopy; laser-induced fluorescence spectroscopy ; time-resolved evanescent wave-induced fluorescence spectroscopy; picosecond coherent anti-Stokes Raman

Development of a New Time-Resolved Laser-Induced Fluorescence Technique

NASA Astrophysics Data System (ADS)

Durot, Christopher; Gallimore, Alec

2012-10-01

We are developing a time-resolved laser-induced fluorescence (LIF) technique to interrogate the ion velocity distribution function (VDF) of EP thruster plumes down to the microsecond time scale. Better measurements of dynamic plasma processes will lead to improvements in simulation and prediction of thruster operation and erosion. We present the development of the new technique and results of initial tests. Signal-to-noise ratio (SNR) is often a challenge for LIF studies, and it is only more challenging for time-resolved measurements since a lock-in amplifier cannot be used with a long time constant. The new system uses laser modulation on the order of MHz, which enables the use of electronic filtering and phase-sensitive detection to improve SNR while preserving time-resolved information. Statistical averaging over many cycles to further improve SNR is done in the frequency domain. This technique can have significant advantages, including (1) larger spatial maps enabled by shorter data acquisition time and (2) the ability to average data without creating a phase reference by modifying the thruster operating condition with a periodic cutoff in discharge current, which can modify the ion velocity distribution.

Energy resolved actinometry for simultaneous measurement of atomic oxygen densities and local mean electron energies in radio-frequency driven plasmas

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

Greb, Arthur, E-mail: ag941@york.ac.uk; Niemi, Kari; O'Connell, Deborah

2014-12-08

A diagnostic method for the simultaneous determination of atomic oxygen densities and mean electron energies is demonstrated for an atmospheric pressure radio-frequency plasma jet. The proposed method is based on phase resolved optical emission measurements of the direct and dissociative electron-impact excitation dynamics of three distinct emission lines, namely, Ar 750.4 nm, O 777.4 nm, and O 844.6 nm. The energy dependence of these lines serves as basis for analysis by taking into account two line ratios. In this frame, the method is highly adaptable with regard to pressure and gas composition. Results are benchmarked against independent numerical simulations and two-photon absorption laser-inducedmore » fluorescence experiments.« less

Detecting aromatic compounds on planetary surfaces using ultraviolet time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Eshelman, E.; Daly, M. G.; Slater, G.; Cloutis, E.

2018-02-01

Many aromatic organic molecules exhibit strong and characteristic fluorescence when excited with ultraviolet radiation. As laser excitation in the ultraviolet generates both fluorescence and resonantly enhanced Raman scattering of aromatic vibrational modes, combined Raman and fluorescence instruments have been proposed to search for organic compounds on Mars. In this work the time-resolved fluorescence of a suite of 24 compounds composed of 2-5 ringed alternant, non-alternant, and heterocyclic PAHs was measured. Fluorescence instrumentation with similar specifications to a putative flight instrument was capable of observing the fluorescence decay of these compounds with a sub-ns resolution. Incorporating time-resolved capabilities was also found to increase the ability to discriminate between individual PAHs. Incorporating time-resolved fluorescence capabilities into an ultraviolet gated Raman system intended for a rover or lander can increase the ability to detect and characterize PAHs on planetary surfaces.

Lagrangian 3D tracking of fluorescent microscopic objects in motion

NASA Astrophysics Data System (ADS)

Darnige, T.; Figueroa-Morales, N.; Bohec, P.; Lindner, A.; Clément, E.

2017-05-01

We describe the development of a tracking device, mounted on an epi-fluorescent inverted microscope, suited to obtain time resolved 3D Lagrangian tracks of fluorescent passive or active micro-objects in microfluidic devices. The system is based on real-time image processing, determining the displacement of a x, y mechanical stage to keep the chosen object at a fixed position in the observation frame. The z displacement is based on the refocusing of the fluorescent object determining the displacement of a piezo mover keeping the moving object in focus. Track coordinates of the object with respect to the microfluidic device as well as images of the object are obtained at a frequency of several tenths of Hertz. This device is particularly well adapted to obtain trajectories of motile micro-organisms in microfluidic devices with or without flow.

Lagrangian 3D tracking of fluorescent microscopic objects in motion.

PubMed

Darnige, T; Figueroa-Morales, N; Bohec, P; Lindner, A; Clément, E

2017-05-01

We describe the development of a tracking device, mounted on an epi-fluorescent inverted microscope, suited to obtain time resolved 3D Lagrangian tracks of fluorescent passive or active micro-objects in microfluidic devices. The system is based on real-time image processing, determining the displacement of a x, y mechanical stage to keep the chosen object at a fixed position in the observation frame. The z displacement is based on the refocusing of the fluorescent object determining the displacement of a piezo mover keeping the moving object in focus. Track coordinates of the object with respect to the microfluidic device as well as images of the object are obtained at a frequency of several tenths of Hertz. This device is particularly well adapted to obtain trajectories of motile micro-organisms in microfluidic devices with or without flow.

Underdetermined blind separation of three-way fluorescence spectra of PAHs in water

NASA Astrophysics Data System (ADS)

Yang, Ruifang; Zhao, Nanjing; Xiao, Xue; Zhu, Wei; Chen, Yunan; Yin, Gaofang; Liu, Jianguo; Liu, Wenqing

2018-06-01

In this work, underdetermined blind decomposition method is developed to recognize individual components from the three-way fluorescent spectra of their mixtures by using sparse component analysis (SCA). The mixing matrix is estimated from the mixtures using fuzzy data clustering algorithm together with the scatters corresponding to local energy maximum value in the time-frequency domain, and the spectra of object components are recovered by pseudo inverse technique. As an example, using this method three and four pure components spectra can be blindly extracted from two samples of their mixture, with similarities between resolved and reference spectra all above 0.80. This work opens a new and effective path to realize monitoring PAHs in water by three-way fluorescence spectroscopy technique.

Detection of experimental brain tumors using time-resolved laser-induced fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Thompson, Reid C.; Black, Keith L.; Kateb, Babak; Marcu, Laura

2002-05-01

Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) has the potential to provide a non- invasive characterization and detection of tumors. We utilized TR-LIFS to detect gliomas in-vivo in the rat C6 glioma model. Time-resolved emission spectra of both normal brain and tumor were analyzed to determine if unique fluorescence signatures could be used to distinguish the two. Fluorescence parameters derived from both spectral and time domain were used for tissue characterization. Our results show that in the rat C6 glioma model, TR-LIFS can be used to differentiate brain tumors from normal tissue (gray and white mater) based upon time- resolved fluorescence signatures seen in brain tumors.

Depth-resolved fluorescence of biological tissue

NASA Astrophysics Data System (ADS)

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

2005-06-01

The depth-resolved autofluorescence ofrabbit oral tissue, normal and dysplastic human ectocervical tissue within l20μm depth were investigated utilizing a confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of oral and ectocervical tissue, strong keratin fluorescence with the spectral characteristics similar to collagen was observed. The fluorescence signal from epithelial tissue between the keratinizing layer and stroma can be well resolved. Furthermore, NADH and FADfluorescence measured from the underlying non-keratinizing epithelial layer were strongly correlated to the tissue pathology. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

Novel tissue phantom for testing a dual-modality diagnostic system: time-resolved fluorescence spectroscopy and high frequency ultrasound

NASA Astrophysics Data System (ADS)

Sun, Yang; Liao, Kuo-Chih; Sun, Yinghua; Park, Jesung; Marcu, Laura

2008-02-01

A unique tissue phantom is reported here that mimics the optical and acoustical properties of biological tissue and enables testing and validation of a dual-modality clinical diagnostic system combining time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasound backscatter microscopy (UBM). The phantom consisted of contrast agents including silicon dioxide particles with a range of diameters from 0.5 to 10 μm acting as optical and acoustical scatterers, and FITC-conjugated dextran mimicking the endogenous fluorophore in tissue. The agents were encapsulated in a polymer bead attached to the end of an optical fiber with a 200 μm diameter using a UV-induced polymerization technique. A set of beads with fibers were then implanted into a gel-based matrix with controlled patterns including a design with lateral distribution and a design with successively changing depth. The configuration presented here allowed the validation of the hybrid fluorescence spectroscopic and ultrasonic system by detecting the lateral and depth distribution of the contrast agents, as well as for coregistration of the ultrasonic image with spectroscopic data. In addition, the depth of the beads in the gel matrix was changed to explore the effect of different concentration ratio of the mixture on the fluorescence signal emitted.

Measurements of population densities of metastable and resonant levels of argon using laser induced fluorescence

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

Nikolić, M.; Newton, J.; Sukenik, C. I.

2015-01-14

We present a new approach to measure population densities of Ar I metastable and resonant excited states in low temperature Ar plasmas at pressures higher than 1 Torr. This approach combines the time resolved laser induced fluorescence technique with the kinetic model of Ar. The kinetic model of Ar is based on calculating the population rates of metastable and resonant levels by including contributions from the processes that affect population densities of Ar I excited states. In particular, we included collisional quenching processes between atoms in the ground state and excited states, since we are investigating plasma at higher pressures. Wemore » also determined time resolved population densities of Ar I 2 p excited states by employing optical emission spectroscopy technique. Time resolved Ar I excited state populations are presented for the case of the post-discharge of the supersonic flowing microwave discharge at pressures of 1.7 and 2.3 Torr. The experimental set-up consists of a pulsed tunable dye laser operating in the near infrared region and a cylindrical resonance cavity operating in TE{sub 111} mode at 2.45 GHz. Results show that time resolved population densities of Ar I metastable and resonant states oscillate with twice the frequency of the discharge.« less

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.

Time-resolved fluorescence microscopy to study biologically related applications using sol-gel derived and cellular media

NASA Astrophysics Data System (ADS)

Toury, Marion; Chandler, Lin; Allison, Archie; Campbell, David; McLoskey, David; Holmes-Smith, A. Sheila; Hungerford, Graham

2011-03-01

Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA Scientific DeltaDiode and DynaMyc).

Underdetermined blind separation of three-way fluorescence spectra of PAHs in water.

PubMed

Yang, Ruifang; Zhao, Nanjing; Xiao, Xue; Zhu, Wei; Chen, Yunan; Yin, Gaofang; Liu, Jianguo; Liu, Wenqing

2018-06-15

In this work, underdetermined blind decomposition method is developed to recognize individual components from the three-way fluorescent spectra of their mixtures by using sparse component analysis (SCA). The mixing matrix is estimated from the mixtures using fuzzy data clustering algorithm together with the scatters corresponding to local energy maximum value in the time-frequency domain, and the spectra of object components are recovered by pseudo inverse technique. As an example, using this method three and four pure components spectra can be blindly extracted from two samples of their mixture, with similarities between resolved and reference spectra all above 0.80. This work opens a new and effective path to realize monitoring PAHs in water by three-way fluorescence spectroscopy technique. Copyright © 2018 Elsevier B.V. All rights reserved.

Emerging biomedical applications of time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

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

1994-07-01

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

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

PubMed

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

2014-07-09

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

Halide (Cl(super -)) Quenching of Quinine Sulfate Fluorescence: A Time-Resolved Fluorescence Experiment for Physical Chemistry

ERIC Educational Resources Information Center

Gutow, Jonathan H.

2005-01-01

The time-resolved fluorescence experiment investigating the halide quenching of fluorescence from quinine sulfate in water is described. The objectives of the experiment include reinforcing student understanding of the kinetics of competing pathways, making connections with microscopic theories of kinetics through comparison of experimental and…

Time-dependent photon migration imaging

NASA Astrophysics Data System (ADS)

Sevick, Eva M.; Wang, NaiGuang; Chance, Britton

1992-02-01

Recently, the application of both time- and frequency-resolved fluorescence techniques for the determination of photon migration characteristics in strongly scattering media has been used to characterize the optical properties in strongly scattering media. Specifically, Chance and coworkers have utilized measurement of photon migration characteristics to determine tissue hemoglobin absorbance and ultimately oxygenation status in homogeneous tissues. In this study, we present simulation results and experimental measurements for both techniques to show the capacity of time-dependent photon migration characteristics to image optically obscure absorbers located in strongly scattering media. The applications of time-dependent photon imaging in the biomedical community include imaging of light absorbing hematomas, tumors, hypoxic tissue volumes, and other tissue abnormalities. Herein, we show that the time-resolved parameter of mean photon path length, , and the frequency- resolved parameter of phase-shift, (theta) , can be used similarly to obtain three dimensional information of absorber position from two-dimensional measurements. Finally, we show that unlike imaging techniques that monitor the intensity of light without regard to the migration characteristics, the resolution of time-dependent photon migration measurements is enhanced by tissue scattering, further potentiating their use for biomedical imaging.

Depth-resolved fluorescence of human ectocervical tissue

NASA Astrophysics Data System (ADS)

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

2005-04-01

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

Interaction and dynamics of (alkylamide + electrolyte) deep eutectics: Dependence on alkyl chain-length, temperature, and anion identity

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

Guchhait, Biswajit; Das, Suman; Daschakraborty, Snehasis

Here we investigate the solute-medium interaction and solute-centered dynamics in (RCONH{sub 2} + LiX) deep eutectics (DEs) via carrying out time-resolved fluorescence measurements and all-atom molecular dynamics simulations at various temperatures. Alkylamides (RCONH{sub 2}) considered are acetamide (CH{sub 3}CONH{sub 2}), propionamide (CH{sub 3}CH{sub 2}CONH{sub 2}), and butyramide (CH{sub 3}CH{sub 2}CH{sub 2}CONH{sub 2}); the electrolytes (LiX) are lithium perchlorate (LiClO{sub 4}), lithium bromide (LiBr), and lithium nitrate (LiNO{sub 3}). Differential scanning calorimetric measurements reveal glass transition temperatures (T{sub g}) of these DEs are ∼195 K and show a very weak dependence on alkyl chain-length and electrolyte identity. Time-resolved and steady statemore » fluorescence measurements with these DEs have been carried out at six-to-nine different temperatures that are ∼100–150 K above their individual T{sub g}s. Four different solute probes providing a good spread of fluorescence lifetimes have been employed in steady state measurements, revealing strong excitation wavelength dependence of probe fluorescence emission peak frequencies. Extent of this dependence, which shows sensitivity to anion identity, has been found to increase with increase of amide chain-length and decrease of probe lifetime. Time-resolved measurements reveal strong fractional power dependence of average rates for solute solvation and rotation with fraction power being relatively smaller (stronger viscosity decoupling) for DEs containing longer amide and larger (weaker decoupling) for DEs containing perchlorate anion. Representative all-atom molecular dynamics simulations of (CH{sub 3}CONH{sub 2} + LiX) DEs at different temperatures reveal strongly stretched exponential relaxation of wavevector dependent acetamide self dynamic structure factor with time constants dependent both on ion identity and temperature, providing justification for explaining the fluorescence results in terms of temporal heterogeneity and amide clustering in these multi-component melts.« less

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

Microviscosity of supercooled water confined within aminopropyl-modified mesoporous silica as studied by time-resolved fluorescence spectroscopy.

PubMed

Yamaguchi, Akira; Namekawa, Manato; Itoh, Tetsuji; Teramae, Norio

2012-01-01

The fluorescence dynamics of rhodamine B (RhB) immobilized on the pore surface of aminopropyl (AP)-modified mesoporous silica (diameter of the silica framework, 3.1 nm) was examined at temperatures between 293 and 193 K to study the microviscosity of supercooled water confined inside the pores. The mesoporous silica specimen with a dense AP layer (2.1 molecules nm(-2)) was prepared, and RhB isothiocyanate was covalently bound to part of the surface AP groups. The fluorescence lifetime of the surface RhB increased with decreasing temperature from 293 to 223 K, indicating that freezing of the confined water did not occur in this temperature range. The microviscosity of the supercooled confined water was evaluated from an analysis of the lifetime data based on a frequency-dependent friction model.

Approximate description of Stokes shifts in ICT fluorescence emission

NASA Astrophysics Data System (ADS)

Saielli, Giacomo; Braun, David; Polimeno, Antonino; Nordio, Pier Luigi

1996-07-01

The time-resolved emission spectrum of a dual fluorescent prototype system like DMABN is associated with an intramolecular adiabatic charge-transfer reaction and the simultaneous relaxation of the polarization coordinate describing the dynamic behaviour of the polar solvent. The dynamic Stokes shift of the frequency maximum of the long-wavelength emission band related to the charge-transfer (CT) state towards the red region is interpreted as a consequence of a kinetic pathway which deviates from steepest descent to the CT state, the rate-determining step being the solvent relaxation. The present stochastic treatment is based on the assumption that internal and solvent coordinates could be described separately, neglecting coupling elements in the case of slow solvent relaxation.

Time-Resolved Fluorescent Immunochromatography of Aflatoxin B1 in Soybean Sauce: A Rapid and Sensitive Quantitative Analysis.

PubMed

Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Zhang, Wen

2016-07-14

Rapid and quantitative sensing of aflatoxin B1 with high sensitivity and specificity has drawn increased attention of studies investigating soybean sauce. A sensitive and rapid quantitative immunochromatographic sensing method was developed for the detection of aflatoxin B1 based on time-resolved fluorescence. It combines the advantages of time-resolved fluorescent sensing and immunochromatography. The dynamic range of a competitive and portable immunoassay was 0.3-10.0 µg·kg(-1), with a limit of detection (LOD) of 0.1 µg·kg(-1) and recoveries of 87.2%-114.3%, within 10 min. The results showed good correlation (R² > 0.99) between time-resolved fluorescent immunochromatographic strip test and high performance liquid chromatography (HPLC). Soybean sauce samples analyzed using time-resolved fluorescent immunochromatographic strip test revealed that 64.2% of samples contained aflatoxin B1 at levels ranging from 0.31 to 12.5 µg·kg(-1). The strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique in food safety analysis.

Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

NASA Astrophysics Data System (ADS)

Horilova, Julia; Cunderlikova, Beata; Marcek Chorvatova, Alzbeta

2015-05-01

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

Multi-channel lock-in amplifier assisted femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy with efficient rejection of superfluorescence background.

PubMed

Mao, Pengcheng; Wang, Zhuan; Dang, Wei; Weng, Yuxiang

2015-12-01

Superfluorescence appears as an intense background in femtosecond time-resolved fluorescence noncollinear optical parametric amplification spectroscopy, which severely interferes the reliable acquisition of the time-resolved fluorescence spectra especially for an optically dilute sample. Superfluorescence originates from the optical amplification of the vacuum quantum noise, which would be inevitably concomitant with the amplified fluorescence photons during the optical parametric amplification process. Here, we report the development of a femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectrometer assisted with a 32-channel lock-in amplifier for efficient rejection of the superfluorescence background. With this spectrometer, the superfluorescence background signal can be significantly reduced to 1/300-1/100 when the seeding fluorescence is modulated. An integrated 32-bundle optical fiber is used as a linear array light receiver connected to 32 photodiodes in one-to-one mode, and the photodiodes are further coupled to a home-built 32-channel synchronous digital lock-in amplifier. As an implementation, time-resolved fluorescence spectra for rhodamine 6G dye in ethanol solution at an optically dilute concentration of 10(-5)M excited at 510 nm with an excitation intensity of 70 nJ/pulse have been successfully recorded, and the detection limit at a pump intensity of 60 μJ/pulse was determined as about 13 photons/pulse. Concentration dependent redshift starting at 30 ps after the excitation in time-resolved fluorescence spectra of this dye has also been observed, which can be attributed to the formation of the excimer at a higher concentration, while the blueshift in the earlier time within 10 ps is attributed to the solvation process.

Multi-channel lock-in amplifier assisted femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy with efficient rejection of superfluorescence background

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

Mao, Pengcheng; Wang, Zhuan; Dang, Wei

Superfluorescence appears as an intense background in femtosecond time-resolved fluorescence noncollinear optical parametric amplification spectroscopy, which severely interferes the reliable acquisition of the time-resolved fluorescence spectra especially for an optically dilute sample. Superfluorescence originates from the optical amplification of the vacuum quantum noise, which would be inevitably concomitant with the amplified fluorescence photons during the optical parametric amplification process. Here, we report the development of a femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectrometer assisted with a 32-channel lock-in amplifier for efficient rejection of the superfluorescence background. With this spectrometer, the superfluorescence background signal can be significantly reduced to 1/300–1/100more » when the seeding fluorescence is modulated. An integrated 32-bundle optical fiber is used as a linear array light receiver connected to 32 photodiodes in one-to-one mode, and the photodiodes are further coupled to a home-built 32-channel synchronous digital lock-in amplifier. As an implementation, time-resolved fluorescence spectra for rhodamine 6G dye in ethanol solution at an optically dilute concentration of 10{sup −5}M excited at 510 nm with an excitation intensity of 70 nJ/pulse have been successfully recorded, and the detection limit at a pump intensity of 60 μJ/pulse was determined as about 13 photons/pulse. Concentration dependent redshift starting at 30 ps after the excitation in time-resolved fluorescence spectra of this dye has also been observed, which can be attributed to the formation of the excimer at a higher concentration, while the blueshift in the earlier time within 10 ps is attributed to the solvation process.« less

Multi-scale spectrally resolved quantitative fluorescence imaging system: towards neurosurgical guidance in glioma resection

NASA Astrophysics Data System (ADS)

Xie, Yijing; Thom, Maria; Miserocchi, Anna; McEvoy, Andrew W.; Desjardins, Adrien; Ourselin, Sebastien; Vercauteren, Tom

2017-02-01

In glioma resection surgery, the detection of tumour is often guided by using intraoperative fluorescence imaging notably with 5-ALA-PpIX, providing fluorescent contrast between normal brain tissue and the gliomas tissue to achieve improved tumour delineation and prolonged patient survival compared with the conventional white-light guided resection. However, the commercially available fluorescence imaging system relies on surgeon's eyes to visualise and distinguish the fluorescence signals, which unfortunately makes the resection subjective. In this study, we developed a novel multi-scale spectrally-resolved fluorescence imaging system and a computational model for quantification of PpIX concentration. The system consisted of a wide-field spectrally-resolved quantitative imaging device and a fluorescence endomicroscopic imaging system enabling optical biopsy. Ex vivo animal tissue experiments as well as human tumour sample studies demonstrated that the system was capable of specifically detecting the PpIX fluorescent signal and estimate the true concentration of PpIX in brain specimen.

Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array

PubMed Central

Luthman, Anna Siri; Dumitru, Sebastian; Quiros‐Gonzalez, Isabel; Joseph, James

2017-01-01

Abstract The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter‐based imaging systems. Hyperspectral imaging (HSI) facilitates the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for HSI is typically complex, bulky and expensive. We sought to overcome these limitations using a novel robust and low cost HSI camera based on a spectrally resolved detector array (SRDA). We integrated this HSI camera into a wide‐field reflectance‐based imaging system operating in the near‐infrared range to assess the suitability for in vivo imaging of exogenous fluorescent contrast agents. Using this fluorescence HSI (fHSI) system, we were able to accurately resolve the presence and concentration of at least 7 fluorescent dyes in solution. We also demonstrate high spectral unmixing precision, signal linearity with dye concentration and at depth in tissue mimicking phantoms, and delineate 4 fluorescent dyes in vivo. Our approach, including statistical background removal, could be directly generalised to broader spectral ranges, for example, to resolve tissue reflectance or autofluorescence and in future be tailored to video rate applications requiring snapshot HSI data acquisition. PMID:28485130

Effects of tissue optical properties on time-resolved fluorescence measurements from brain tumors: an experimental and computational study

NASA Astrophysics Data System (ADS)

Butte, Pramod V.; Vishwanath, Karthik; Pikul, Brian K.; Mycek, Mary-Ann; Marcu, Laura

2003-07-01

Time-Resolved Laser-Induced Fluorescence Spectroscopy (tr-LIFS) offers the potential for intra-operative diagnosis of primary brain tumors. However, both the intrinsic properties of endogenous fluorophores and the optical properties of brain tissue could affect the fluorescence measurements from brain. Scattering has been demonstrated to increase, for instance, detected lifetimes by 10-20% in media less scattering than the brain. The overall goal of this study is to investigate experimentally and computationally how optical properties of distinct types of brain tissue (normal porcine white and gray matter) affect the propagation of the excitation pulse and fluorescent transients and the detected fluorescence lifetime. A time-domain tr-LIFS apparatus (fast digitizer and gated detection) was employed to measure the propagation of ultra-short pulsed light through brain specimens (1-2.5-mm source-detector separation; 0.100-mm increment). A Monte Carlo model for semi-infinite turbid media was used to simulate time-resolved light propagation for arbitrary source-detector fiber geometries and optical fiber specifications; and to record spatially- and temporally resolved information. We determined a good correlation between experimental and computational results. Our findings provide means for quantification of time-resolved fluorescence spectra from healthy and diseased brain tissue.

Time-Resolved Measurements in Optoelectronic Microbioanalysis

NASA Technical Reports Server (NTRS)

Bearman, Gregory; Kossakovski, Dmitri

2003-01-01

A report presents discussion of time-resolved measurements in optoelectronic microbioanalysis. Proposed microbioanalytical laboratory-on-a-chip devices for detection of microbes and toxic chemicals would include optoelectronic sensors and associated electronic circuits that would look for fluorescence or phosphorescence signatures of multiple hazardous biomolecules in order to detect which ones were present in a given situation. The emphasis in the instant report is on gating an active-pixel sensor in the time domain, instead of filtering light in the wavelength domain, to prevent the sensor from responding to a laser pulse used to excite fluorescence or phosphorescence while enabling the sensor to respond to the decaying fluorescence or phosphorescence signal that follows the laser pulse. The active-pixel sensor would be turned on after the laser pulse and would be used to either integrate the fluorescence or phosphorescence signal over several lifetimes and many excitation pulses or else take time-resolved measurements of the fluorescence or phosphorescence. The report also discusses issues of multiplexing and of using time-resolved measurements of fluorophores with known different fluorescence lifetimes to distinguish among them.

Conformational explosion: Understanding the complexity of short chain para-dialkylbenzene potential energy surfaces

NASA Astrophysics Data System (ADS)

Mishra, Piyush; Hewett, Daniel M.; Zwier, Timothy S.

2018-05-01

The single-conformation ultraviolet and infrared spectroscopy of three short-chain para-dialkylbenzenes (para-diethylbenzene, para-dipropylbenzene, and para-dibutylbenzene) is reported for the jet-cooled, isolated molecules. The present study builds off previous work on single-chain n-alkylbenzenes, where an anharmonic local mode Hamiltonian method was developed to account for stretch-bend Fermi resonance in the alkyl CH stretch region [D. P. Tabor et al., J. Chem. Phys. 144, 224310 (2016)]. The jet-cooled molecules are interrogated using laser-induced fluorescence (LIF) excitation, fluorescence dip infrared spectroscopy, and dispersed fluorescence. The LIF spectra in the S1 ← S0 origin region show a dramatic increase in the number of resolved transitions with increasing length of the alkyl chains, reflecting an explosion in the number of unique low-energy conformations formed when two independent alkyl chains are present. Since the barriers to isomerization of the alkyl chain are similar in size, this results in an "egg carton" shaped potential energy surface. A combination of electronic frequency shift and alkyl CH stretch infrared spectra is used to generate a consistent set of conformational assignments. Using these experimental techniques in conjunction with computational methods, subsets of origin transitions in the LIF excitation spectrum can be classified into different conformational families. Two conformations are resolved in para-diethylbenzene, seven in para-dipropylbenzene, and about nineteen in para-dibutylbenzene. These chains are largely independent of each other as there are no new single-chain conformations induced by the presence of a second chain. A cursory LIF excitation scan of para-dioctylbenzene shows a broad congested spectrum at frequencies consistent with interactions of alkyl chains with the phenyl π cloud.

Conformational explosion: Understanding the complexity of short chain para-dialkylbenzene potential energy surfaces.

PubMed

Mishra, Piyush; Hewett, Daniel M; Zwier, Timothy S

2018-05-14

The single-conformation ultraviolet and infrared spectroscopy of three short-chain para-dialkylbenzenes (para-diethylbenzene, para-dipropylbenzene, and para-dibutylbenzene) is reported for the jet-cooled, isolated molecules. The present study builds off previous work on single-chain n-alkylbenzenes, where an anharmonic local mode Hamiltonian method was developed to account for stretch-bend Fermi resonance in the alkyl CH stretch region [D. P. Tabor et al., J. Chem. Phys. 144, 224310 (2016)]. The jet-cooled molecules are interrogated using laser-induced fluorescence (LIF) excitation, fluorescence dip infrared spectroscopy, and dispersed fluorescence. The LIF spectra in the S 1 ← S 0 origin region show a dramatic increase in the number of resolved transitions with increasing length of the alkyl chains, reflecting an explosion in the number of unique low-energy conformations formed when two independent alkyl chains are present. Since the barriers to isomerization of the alkyl chain are similar in size, this results in an "egg carton" shaped potential energy surface. A combination of electronic frequency shift and alkyl CH stretch infrared spectra is used to generate a consistent set of conformational assignments. Using these experimental techniques in conjunction with computational methods, subsets of origin transitions in the LIF excitation spectrum can be classified into different conformational families. Two conformations are resolved in para-diethylbenzene, seven in para-dipropylbenzene, and about nineteen in para-dibutylbenzene. These chains are largely independent of each other as there are no new single-chain conformations induced by the presence of a second chain. A cursory LIF excitation scan of para-dioctylbenzene shows a broad congested spectrum at frequencies consistent with interactions of alkyl chains with the phenyl π cloud.

Laser-induced dental caries and plaque diagnosis on patients by sensitive autofluorescence spectroscopy and time-gated video imaging: preliminary studies

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Schneckenburger, Herbert

1994-09-01

The laser-induced in vivo autofluorescence of human teeth was investigated by means of time- resolved/time-gated fluorescence techniques. The aim of these studies was non-contact caries and plaque detection. Carious lesions and dental plaque fluoresce in the red spectral region. This autofluorescence seems to be based on porphyrin-producing bacteria. We report on preliminary studies on patients using a novel method of autofluorescence imaging. A special device was constructed for time-gated video imaging. Nanosecond laser pulses for fluorescence excitation were provided by a frequency-doubled, Q-switched Nd:YAG laser. Autofluorescence was detected in an appropriate nanosecond time window using a video camera with a time-gated image intensifier (minimal time gate: 5 ns). Laser-induced autofluorescence based on porphyrin-producing bacteria seems to be an appropriate tool for detecting dental lesions and for creating `caries-images' and `dental plaque' images.

Quantitative, spectrally-resolved intraoperative fluorescence imaging

PubMed Central

Valdés, Pablo A.; Leblond, Frederic; Jacobs, Valerie L.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.

2012-01-01

Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field. PMID:23152935

Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics.

PubMed

Wei, Liping; Yan, Wenrong; Ho, Derek

2017-12-04

Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices.

Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics

PubMed Central

Yan, Wenrong; Ho, Derek

2017-01-01

Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices. PMID:29207568

Microlensed dual-fiber probe for depth-resolved fluorescence measurements

NASA Astrophysics Data System (ADS)

Choi, Hae Young; Ryu, Seon Young; Kim, Jae Young; Kim, Geon Hee; Park, Seong Jun; Lee, Byeong Ha; Chang, Ki Soo

2011-07-01

We propose and demonstrate a compact microlensed dual-fiber probe that has a good collection efficiency and a high depth-resolution ability for fluorescence measurements. The probe is formed with a conventional fusion splicer creating a common focusing lens on two fibers placed side by side. The collection efficiency of the fabricated probe was evaluated by measuring the fluorescence signal of a fresh ginkgo leaf. It was shown experimentally that the proposed probe could effectively collect the fluorescence signal with a six-fold increase compared to that of a general flat-tipped probe. The beam propagation method was used to design a probe with an optimized working distance and an improved resolving depth. It was found that the working distance depends mainly on the radius of curvature of the lens, whereas the resolving depth is determined by the core diameters of the illumination and collection fibers. The depth-resolved ability of probes with working distances of ~100 μm and 300 μm was validated by using a two-layer tissue phantom. The experimental results demonstrate that the microlensed dual-fiber probe has the potential to facilitate depth-resolved fluorescence detection of epithelial tissue.

High-performance time-resolved fluorescence by direct waveform recording.

PubMed

Muretta, Joseph M; Kyrychenko, Alexander; Ladokhin, Alexey S; Kast, David J; Gillispie, Gregory D; Thomas, David D

2010-10-01

We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1 μJ/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 10(5) times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats.

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

Fluorescence Lifetime Techniques in Medical Applications

PubMed Central

Marcu, Laura

2012-01-01

This article presents an overview of time-resolved (lifetime) fluorescence techniques used in biomedical diagnostics. In particular, we review the development of time-resolved fluorescence spectroscopy (TRFS) and fluorescence lifetime imaging (FLIM) instrumentation and associated methodologies which allows for in vivo characterization and diagnosis of biological tissues. Emphasis is placed on the translational research potential of these techniques and on evaluating whether intrinsic fluorescence signals provide useful contrast for the diagnosis of human diseases including cancer (gastrointestinal tract, lung, head and neck, and brain), skin and eye diseases, and atherosclerotic cardiovascular disease. PMID:22273730

Time-resolved fluorescence decay measurements for flowing particles

DOEpatents

Deka, C.; Steinkamp, J.A.

1999-06-01

Time-resolved fluorescence decay measurements are disclosed for flowing particles. An apparatus and method for the measurement and analysis of fluorescence for individual cells and particles in flow are described, wherein the rapid measurement capabilities of flow cytometry and the robust measurement and analysis procedures of time-domain fluorescence lifetime spectroscopy are combined. A pulse-modulated CW laser is employed for excitation of the particles. The characteristics and the repetition rate of the excitation pulses can be readily adjusted to accommodate for fluorescence decays having a wide range of lifetimes. 12 figs.

Time-resolved fluorescence decay measurements for flowing particles

DOEpatents

Deka, Chiranjit; Steinkamp, John A.

1999-01-01

Time-resolved fluorescence decay measurements for flowing particles. An apparatus and method for the measurement and analysis of fluorescence for individual cells and particles in flow are described, wherein the rapid measurement capabilities of flow cytometry and the robust measurement and analysis procedures of time-domain fluorescence lifetime spectroscopy are combined. A pulse-modulated cw laser is employed for excitation of the particles. The characteristics and the repetition rate of the excitation pulses can be readily adjusted to accommodate for fluorescence decays having a wide range of lifetimes.

Validation of a time-resolved fluorescence spectroscopy apparatus in a rabbit atherosclerosis model

NASA Astrophysics Data System (ADS)

Fang, Qiyin; Jo, Javier A.; Papaioannou, Thanassis; Dorafshar, Amir; Reil, Todd; Qiao, Jian-Hua; Fishbein, Michael C.; Freischlag, Julie A.; Marcu, Laura

2004-07-01

Time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) has been studied as a potential tool for in vivo diagnosis of atherosclerotic lesions. This study is to evaluate the potential of a compact fiber-optics based tr-LIFS instrument developed in our laboratory for in vivo analysis of atherosclerotic plaque composition. Time-resolved fluorescence spectroscopy studies were performed in vivo on fifteen New Zealand White rabbits (atherosclerotic: N=8, control: N=7). Time-resolved fluorescence spectra were acquired (range: 360-600 nm, increment: 5 nm, total acquisition time: 65 s) from normal aorta wall and lesions in the abdominal aorta. Data were analyzed in terms of fluorescence emission spectra and wavelength specific lifetimes. Following trichrome staining, tissue specimens were analyzed histopathologically in terms of intima/media thickness and biochemical composition (collagen, elastin, foam cells, and etc). Based on intimal thickness, the lesions were divided into thin and thick lesions. Each group was further separated into two categories: collagen rich lesions and foam cell rich lesions based on their biochemical composition. The obtained spectral and time domain fluorescence signatures were subsequently correlated to the histopathological findings. The results have shown that time-domain fluorescence spectral features can be used in vivo to separate atherosclerotic lesions from normal aorta wall as well discrimination within certain types of lesions.

Use of Time-Resolved Fluorescence to Monitor Bioactive Compounds in Plant Based Foodstuffs

PubMed Central

Lemos, M. Adília; Sárniková, Katarína; Bot, Francesca; Anese, Monica; Hungerford, Graham

2015-01-01

The study of compounds that exhibit antioxidant activity has recently received much interest in the food industry because of their potential health benefits. Most of these compounds are plant based, such as polyphenolics and carotenoids, and there is a need to monitor them from the field through processing and into the body. Ideally, a monitoring technique should be non-invasive with the potential for remote capabilities. The application of the phenomenon of fluorescence has proved to be well suited, as many plant associated compounds exhibit fluorescence. The photophysical behaviour of fluorescent molecules is also highly dependent on their microenvironment, making them suitable probes to monitor changes in pH, viscosity and polarity, for example. Time-resolved fluorescence techniques have recently come to the fore, as they offer the ability to obtain more information, coupled with the fact that the fluorescence lifetime is an absolute measure, while steady state just provides relative and average information. In this work, we will present illustrative time-resolved measurements, rather than a comprehensive review, to show the potential of time-resolved fluorescence applied to the study of bioactive substances. The aim is to help assess if any changes occur in their form, going from extraction via storage and cooking to the interaction with serum albumin, a principal blood transport protein. PMID:26132136

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 1. Mono-frequency phase and modulation analysis reveals a conformational change of a PSII pigment complex during the IP thermal phase.

PubMed

Moise, Nicolae; Moya, Ismaël

2004-06-28

The relationship between the fluorescence lifetime (tau) and yield (Phi) obtained in phase and modulation fluorometry at 54 MHz during the chlorophyll fluorescence induction in dark-adapted leaves under low actinic light has been investigated. Three typical phases have been identified: (i) linear during the OI photochemical rise, (ii) convex curvature during the subsequent IP thermal rise, and (iii) linear during the PS slow decay. A similar relationship has been obtained in the fluorescence induction for the fluorescence yield measured at 685 nm plotted versus the fluorescence yield measured at 735 nm. A spectrally resolved analysis shows that the curvature of the tau-Phi relationship is not due to chlorophyll fluorescence reabsorption effects. Several other hypotheses are discussed and we conclude that the curvature of the tau-Phi relationship is due to a variable and transitory nonphotochemical quenching. We tentatively propose that this quenching results from a conformational change of a pigment-protein complex of Photosystem II core antenna during the IP phase and could explain both spectral and temporal transitory changes of the fluorescence. A variable blue shift of the 685 nm peak of the fluorescence spectrum during the IP phase has been observed, supporting this hypothesis.

Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence.

PubMed

Li, Dong; Zheng, Wei; Qu, Jianan Y

2008-10-15

A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments.

PubMed

Neumann, M; Herten, D P; Dietrich, A; Wolfrum, J; Sauer, M

2000-02-25

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.

Time Resolved Raman and Fluorescence Spectrometer for Planetary Mineralogy

NASA Astrophysics Data System (ADS)

Blacksberg, Jordana; Rossman, George

2010-05-01

Raman spectroscopy is a prime candidate for the next generation of planetary instruments, as it addresses the primary goal of mineralogical analysis which is structure and composition. It does not require sample preparation and provides unique mineral fingerprints, even for mixed phase samples. However, large fluorescence return from many mineral samples under visible light excitation can seriously compromise the quality of the spectra or even render Raman spectra unattainable. Fluorescence interference is likely to be a problem on Mars and is evident in Raman spectra of Martian Meteorites[1]. Our approach uses time resolution for elimination of fluorescence from Raman spectra, allowing for traditional visible laser excitation (532 nm). Since Raman occurs instantaneously with the laser pulse and fluorescence lifetimes vary from nsec to msec depending on the mineral, it is possible to separate them out in time. Complementary information can also be obtained simultaneously using the time resolved fluorescence data. The Simultaneous Spectral Temporal Adaptive Raman Spectrometer (SSTARS) is a planetary instrument under development at the Jet Propulsion Laboratory, capable of time-resolved in situ Raman and fluorescence spectroscopy. A streak camera and pulsed miniature microchip laser provide psec scale time resolution. Our ability to observe the complete time evolution of Raman and fluorescence in minerals provides a foundation for design of pulsed Raman and fluorescence spectrometers in diverse planetary environments. We will discuss the SSTARS instrument design and performance capability. We will also present time-resolved pulsed Raman spectra collected from a relevant set of minerals selected using available data on Mars mineralogy[2]. Of particular interest are minerals resulting from aqueous alteration on Mars. For comparison, we will present Raman spectra obtained using a commercial continuous wave (CW) green (514 nm) Raman system. In many cases using a CW laser the strong mineral fluorescence saturates the detector and Raman spectra are unattainable. This problem is overcome by using time resolved Raman where fluorescence is eliminated. [1]Frosch et al., Anal. Chem. 2007, 79, 1101-1108 [2]Bell, J.,ed, The Martian Surface: Composition, Mineralogy, and physical Properties, Cambridge University Press, 2008

Time-resolvable fluorescent conjugates for the detection of pathogens in environmental samples containing autofluorescent material

NASA Astrophysics Data System (ADS)

Connally, Russell; Veal, Duncan; Piper, James A.

2003-07-01

Water is routinely monitored for environmental pathogens such a Cryptosporidium and Giardia using immunofluorescence microscopy (IFM). Autofluorescence can greatly diminish an operators capacity to resolve labeled pathogens from non-specific background. Naturally fluorescing components (autofluorophores) encountered in biological samples typically have fluorescent lifetimes (τ) of less than 100 nanoseconds and their emissions may be excluded through use of time-resolved fluorescence microscopy (TRFM). TRFM relies on the large differences in τ between autofluorescent molecules and long-lived lanthanide chelates. In TRFM, targets labeled with a time-resolvable fluorescent immunoconjugate are excited by an intense (UV) light pulse. A short delay is imposed to permit the decay of autofluorescence before capture of luminescence from the excited chelate using an image intensified CCD camera. In our experience, autofluorescence can be reduced to insignificant levels with a consequent 30-fold increase in target visibility using TRFM techniques. We report conjugation of a novel europium chelate to a monoclonal antibody specific for Giardia lamblia and use of the immunoconjugate for TRFM studies. Initial attempts to conjugate the same chelate to a monoclonal antibody directed against Cryptosporidium parvum led to poorly fluorescent constructs that were prone to denature and precipitate. We successfully conjugated BHHCT to anti-mouse polyvalent immunoglobulin and used this construct to overcome the difficulties in direct labeling of the anti-Cryptosporidium antibody. Both Giardia and Cryptosporidium were labeled using the anti-mouse protocol with a subsequent 20-fold and 6.6-fold suppression of autofluorescence respectively. A rapid protocol for conjugating and purifying the immunoconjugate was found and methods of quantifying the fluorescence to protein ratio determined. Performance of our TRFM was dependent on the quality and brightness of the immunoconjugate and optimization of the conjugation process is necessary to reap the full benefit of time-resolved techniques.

Highly sensitive detection of human papillomavirus type 16 DNA using time-resolved fluorescence microscopy and long lifetime probes

NASA Astrophysics Data System (ADS)

Wang, Xue F.; Periasamy, Ammasi; Wodnicki, Pawel; Siadat-Pajouh, M.; Herman, Brian

1995-04-01

We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using fluorescence in situ hybridization image is useful for the diagnosis of many other type of diseases, the system we have developed should find numerous applications for the diagnosis of disease states.

Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult's brain

NASA Astrophysics Data System (ADS)

Gerega, Anna; Milej, Daniel; Weigl, Wojciech; Botwicz, Marcin; Zolek, Norbert; Kacprzak, Michal; Wierzejski, Wojciech; Toczylowska, Beata; Mayzner-Zawadzka, Ewa; Maniewski, Roman; Liebert, Adam

2012-08-01

Optical technique based on diffuse reflectance measurement combined with indocyanine green (ICG) bolus tracking is extensively tested as a method for clinical assessment of brain perfusion in adults at the bedside. Methodology of multiwavelength and time-resolved detection of fluorescence light excited in the ICG is presented and advantages of measurements at multiple wavelengths are discussed. Measurements were carried out: 1. on a physical homogeneous phantom to study the concentration dependence of the fluorescence signal, 2. on the phantom to simulate the dynamic inflow of ICG at different depths, and 3. in vivo on surface of the human head. Pattern of inflow and washout of ICG in the head of healthy volunteers after intravenous injection of the dye was observed for the first time with time-resolved instrumentation at multiple emission wavelengths. The multiwavelength detection of fluorescence signal confirms that at longer emission wavelengths, probability of reabsorption of the fluorescence light by the dye itself is reduced. Considering different light penetration depths at different wavelengths, and the pronounced reabsorption at longer wavelengths, the time-resolved multiwavelength technique may be useful in signal decomposition, leading to evaluation of extra- and intracerebral components of the measured signals.

BHHST: An improved lanthanide chelate for time-resolved fluorescence applications

NASA Astrophysics Data System (ADS)

Connally, Russell; Jin, Dayong; Piper, James

2005-04-01

The detection of the waterborne pathogens Giardia lamblia and Cryptosporidium parvum in environmental water bodies requires concentration of large volumes of water due to the low dose required for infection. The highly concentrated (10,000-fold) water sample is often rich in strongly autofluorescent algae, organic debris and mineral particles that can obscure immunofluorescently labeled (oo)cysts during analysis. Time-resolved fluorescence techniques exploit the long fluorescence lifetimes of lanthanide chelates (ms) to differentiate target fluorescence from background autofluorescence (ns). Relatively simple instrumentation can be used to enhance the signal-to-noise ratio (S/N) of labelled target. Time-resolved fluorescence techniques exploit the large difference in lifetime by briefly exciting fluorescence from the sample using a pulsed excitation source. Capture of the resulting fluorescence emission is delayed until the more rapidly decaying autofluorescence has faded beyond detection, whereon the much stronger and slower fading emission from labelled target is collected. BHHCT is a tetradentate beta-diketone chelate that is activated to bind with protein (antibody) as the chlorosulfonate. The high activity of this residue makes conjugations difficult to control and can lead to the formation of unstable immunoconjugates. To overcome these limitations a 5-atom hydrophylic molecular tether was attached to BHHCT via the chlorosulfonate and the BHHCT derivative was then activated to bind to proteins as the succinimide. The new compound (BHHST) could be prepared in high purity and was far more stable than the chlorosulfonate on storage. A high activity immunocojugate was prepared against Cryptosporidium that yielded an 8-fold increase in SNR using a lab-built time-resolved fluorescence microscope.

Time-resolved resonance fluorescence spectroscopy for study of chemical reactions in laser-induced plasmas.

PubMed

Liu, Lei; Deng, Leimin; Fan, Lisha; Huang, Xi; Lu, Yao; Shen, Xiaokang; Jiang, Lan; Silvain, Jean-François; Lu, Yongfeng

2017-10-30

Identification of chemical intermediates and study of chemical reaction pathways and mechanisms in laser-induced plasmas are important for laser-ablated applications. Laser-induced breakdown spectroscopy (LIBS), as a promising spectroscopic technique, is efficient for elemental analyses but can only provide limited information about chemical products in laser-induced plasmas. In this work, time-resolved resonance fluorescence spectroscopy was studied as a promising tool for the study of chemical reactions in laser-induced plasmas. Resonance fluorescence excitation of diatomic aluminum monoxide (AlO) and triatomic dialuminum monoxide (Al 2 O) was used to identify these chemical intermediates. Time-resolved fluorescence spectra of AlO and Al 2 O were used to observe the temporal evolution in laser-induced Al plasmas and to study their formation in the Al-O 2 chemistry in air.

Time-resolved UV-excited microarray reader for fluorescence energy transfer (FRET) measurements

NASA Astrophysics Data System (ADS)

Orellana, Adelina; Hokkanen, Ari P.; Pastinen, Tomi; Takkinen, Kristina; Soderlund, Hans

2001-05-01

Analytical systems based on immunochemistry are largely used in medical diagnostics and in biotechnology. There is a significant pressure to develop the present assay formats to become easier to use, faster, and less reagent consuming. Further developments towards high density array--like multianalyte measurement systems would be valuable. To this aim we have studied the applicability of fluorescence resonance energy transfer and time-resolved fluorescence resonance energy transfer in immunoassays on microspots and in microwells. We have used engineered recombinant antibodies detecting the pentameric protein CRP as a model analyte system, and tested different assay formats. We describe also the construction of a time-resolved scanning epifluorometer with which we could measure the FRET interaction between the slow fluorescence decay from europium chelates and its energy transfer to the rapidly decaying fluorophore Cy5.

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.

[Assessment of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H].

PubMed

Cheng, Ying; Ren, Mingming; Niu, Yanyan; Qiao, Jianhua; Aneba, S; Chorvat, D; Chorvatova, A

2009-12-01

The primary function of cardiac mitochondria is the production of ATP to support heart contraction. Examination of the mitochondrial redox state is therefore crucially important to sensitively detect early signs of mitochondrial function in pathophysiological conditions, such as ischemia, diabetes and heart failure. We study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply. Here NAD(P)H is studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD(P) H fluorescence is recorded in cardiac cells following excitation with 375nm UV-light and detection by spectrally-resolved time-correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and fluorescence lifetimes. Modulation of NADH production and/or mitochondrial respiration is tested to study dynamic characteristics of NAD(P) H fluorescence decay. Our results show that at least a 3-exponential decay model, with 0.4-0.7ns, 1.2-1.9ns and 8.0-13. Ons lifetime pools is necessary to describe cardiomyocyte autofluorescence (AF) within 420-560nm spectral range. Increased mitochondrial NADH production by ketone bodies enhanced the fluorescence intensity, without significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol (DNP), an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF intensity, broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects are comparable to the study of NADH fluorescence decay in vitro. In the present contribution we demonstrated that spectrally-resolved fluorescence lifetime technique provides promising new tool for analysis of mitochondrial NAD(P) H fluorescence with good reproducibility in living cardiomyocytes. This approach will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level. In the future, this approach can prove helpful in the clinical diagnosis and treatment of mitochondrial disorder.

Capturing Structural Snapshots during Photochemical Reactions with Ultrafast Raman Spectroscopy: From Materials Transformation to Biosensor Responses.

PubMed

Fang, Chong; Tang, Longteng; Oscar, Breland G; Chen, Cheng

2018-06-21

Chemistry studies the composition, structure, properties, and transformation of matter. A mechanistic understanding of the pertinent processes is required to translate fundamental knowledge into practical applications. The current development of ultrafast Raman as a powerful time-resolved vibrational technique, particularly femtosecond stimulated Raman spectroscopy (FSRS), has shed light on the structure-energy-function relationships of various photosensitive systems. This Perspective reviews recent work incorporating optical innovations, including the broad-band up-converted multicolor array (BUMA) into a tunable FSRS setup, and demonstrates its resolving power to watch metal speciation and photolysis, leading to high-quality thin films, and fluorescence modulation of chimeric protein biosensors for calcium ion imaging. We discuss advantages of performing FSRS in the mixed time-frequency domain and present strategies to delineate mechanisms by tracking low-frequency modes and systematically modifying chemical structures with specific functional groups. These unique insights at the chemical-bond level have started to enable the rational design and precise control of functional molecular machines in optical, materials, energy, and life sciences.

A flash-lamp based device for fluorescence detection and identification of individual pollen grains.

PubMed

Kiselev, Denis; Bonacina, Luigi; Wolf, Jean-Pierre

2013-03-01

We present a novel optical aerosol particle detector based on Xe flash lamp excitation and spectrally resolved fluorescence acquisition. We demonstrate its performances on three natural pollens acquiring in real-time scattering intensity at two wavelengths, sub-microsecond time-resolved scattering traces of the particles' passage in the focus, and UV-excited fluorescence spectra. We show that the device gives access to a rather specific detection of the bioaerosol particles.

CMOS Time-Resolved, Contact, and Multispectral Fluorescence Imaging for DNA Molecular Diagnostics

PubMed Central

Guo, Nan; Cheung, Ka Wai; Wong, Hiu Tung; Ho, Derek

2014-01-01

Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA) detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS) technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art. PMID:25365460

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

NASA Astrophysics Data System (ADS)

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

2014-12-01

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

Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

USDA-ARS?s Scientific Manuscript database

Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved t...

Application of spectroscopy and super-resolution microscopy: Excited state

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

Bhattacharjee, Ujjal

Photophysics of inorganic materials and organic molecules in complex systems have been extensively studied with absorption and emission spectroscopy.1-4 Steady-state and time-resolved fluorescence studies are commonly carried out to characterize excited-state properties of fluorophores. Although steady-state fluorescence measurements are widely used for analytical applications, time-resolved fluorescence measurements provide more detailed information about excited-state properties and the environment in the vicinity of the fluorophore. Many photophysical processes, such as photoinduced electron transfer (PET), rotational reorientation, solvent relaxation, and energy transfer, occur on a nanosecond (10 -9 s) timescale, thus affecting the lifetime of the fluorophores. Moreover, time-resolved microscopy methods, such asmore » lifetimeimaging, combine the benefits of the microscopic measurement and information-rich, timeresolved data. Thus, time-resolved fluorescence spectroscopy combined with microscopy can be used to quantify these processes and to obtain a deeper understanding of the chemical surroundings of the fluorophore in a small area under investigation. This thesis discusses various photophysical and super-resolution microscopic studies of organic and inorganic materials, which have been outlined below.« less

Ion Dynamics of a BHT-600 Hall Thruster Measured with Time-Resolved Laser-Induced Fluorescence

DTIC Science & Technology

2016-01-14

LIF  Method V = 0 V V λ = λ0 λ > λ0 λ < λ0 Ion Velocity from  Doppler Shift: 0 5 10 15 20 25 30 0 0.1 0.2 0.3 0.4...0.5 0.6 0.7 0.8 0.9 1 Frequency (GHz) N o r m a l i z e d S i g n a l xneg28z6_AVG OG LIF OG Fit LIF Fit AFRL | BHT‐600 TRLIF | Jan 14, 2016 10...Distribution A: Approved for Public Release; Distribution Unlimited. PA# 16204 Time‐Resolved  LIF  Method Laser  Tapered Amp  D1  BS1  BS2  D2  O G C  

Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector.

PubMed

Rojalin, Tatu; Kurki, Lauri; Laaksonen, Timo; Viitala, Tapani; Kostamovaara, Juha; Gordon, Keith C; Galvis, Leonardo; Wachsmann-Hogiu, Sebastian; Strachan, Clare J; Yliperttula, Marjo

2016-01-01

In this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the pharmaceutical setting, process analytical technology (PAT), and the life sciences.

Reorientational Dynamics of Enzymes Adsorbed on Quartz: A Temperature-Dependent Time-Resolved TIRF Anisotropy Study

PubMed Central

Czeslik, C.; Royer, C.; Hazlett, T.; Mantulin, W.

2003-01-01

The preservation of enzyme activity and protein binding capacity upon protein adsorption at solid interfaces is important for biotechnological and medical applications. Because these properties are partly related to the protein flexibility and mobility, we have studied the internal dynamics and the whole-body reorientational rates of two enzymes, staphylococcal nuclease (SNase) and hen egg white lysozyme, over the temperature range of 20–80°C when the proteins are adsorbed at the silica/water interface and, for comparison, when they are dissolved in buffer. The data were obtained using a combination of two experimental techniques, total internal reflection fluorescence spectroscopy and time-resolved fluorescence anisotropy measurements in the frequency domain, with the protein Trp residues as intrinsic fluorescence probes. It has been found that the internal dynamics and the whole-body rotation of SNase and lysozyme are markedly reduced upon adsorption over large temperature ranges. At elevated temperatures, both protein molecules appear completely immobilized and the fractional amplitudes for the whole-body rotation, which are related to the order parameter for the local rotational freedom of the Trp residues, remain constant and do not approach zero. This behavior indicates that the angular range of the Trp reorientation within the adsorbed proteins is largely restricted even at high temperatures, in contrast to that of the dissolved proteins. The results of this study thus provide a deeper understanding of protein activity at solid surfaces. PMID:12668461

DNA sequencing using fluorescence background electroblotting membrane

DOEpatents

Caldwell, Karin D.; Chu, Tun-Jen; Pitt, William G.

1992-01-01

A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.

DNA sequencing using fluorescence background electroblotting membrane

DOEpatents

Caldwell, K.D.; Chu, T.J.; Pitt, W.G.

1992-05-12

A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through amino groups contained on the surface. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to the target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membranes may be reprobed numerous times. No Drawings

Saturation-resolved-fluorescence spectroscopy of Cr3+:mullite glass ceramic

NASA Astrophysics Data System (ADS)

Liu, Huimin; Knutson, Robert; Yen, W. M.

1990-01-01

We present a saturation-based technique designed to isolate and uncouple individual components of inhomogeneously broadened spectra that are simultaneously coupled to each other through spectral overlap and energy-transfer interactions. We have termed the technique saturation-resolved-fluorescence spectroscopy; we demonstrate its usefulness in deconvoluting the complex spectra of Cr3+:mullite glass ceramic.

Spatially Resolved Analysis of Amines Using a Fluorescence Molecular Probe: Molecular Analysis of IDPs

NASA Technical Reports Server (NTRS)

Clemett, S. J.; Messenger, S.; Thomas-Keprta, K. L.; Wentworth, S. J.; Robinson, G. A.; McKay, D. S.

2002-01-01

Some Interplanetary Dust Particles (IDPs) have large isotope anomalies in H and N. To address the nature of the carrier phase, we are developing a procedure to spatially resolve the distribution of organic species on IDP thin sections utilizing fluorescent molecular probes. Additional information is contained in the original extended abstract.

Molecular Imaging for Breast Cancer Using Magnetic Resonance-Guided Positron Emission Mammography and Excitation-Resolved Near-Infrared Fluorescence Imaging

NASA Astrophysics Data System (ADS)

Cho, Jaedu

The aim of this work is to develop novel breast-specific molecular imaging techniques for management of breast cancer. In this dissertation, we describe two novel molecular imaging approaches for breast cancer management. In Part I, we introduce our multimodal molecular imaging approach for breast cancer therapy monitoring using magnetic resonance imaging and positron emission mammography (MR/PEM). We have focused on the therapy monitoring technique for aggressive cancer molecular subtypes, which is challenging due to time constraint. Breast cancer therapy planning relies on a fast and accurate monitoring of functional and anatomical change. We demonstrate a proof-of-concept of sequential dual-modal magnetic resonance and positron emission mammography (MR/PEM) for the cancer therapy monitoring. We have developed dedicated breast coils with breast compression mechanism equipped with MR-compatible PEM detector heads. We have designed a fiducial marker that allows straightforward image registration of data obtained from MRI and PEM. We propose an optimal multimodal imaging procedure for MR/PEM. In Part II, we have focused on the development of a novel intraoperative near-infrared fluorescence imaging system (NIRF) for image-guided breast cancer surgery. Conventional spectrally-resolved NIRF systems are unable to resolve various NIR fluorescence dyes for the following reasons. First, the fluorescence spectra of viable NIR fluorescence dyes are heavily overlapping. Second, conventional emission-resolved NIRF suffers from a trade-off between the fluence rate and the spectral resolution. Third, the multiple scattering in tissue degrades not only the spatial information but also the spectral contents by the red-shift. We develop a wavelength-swept laser-based NIRF system that can resolve the excitation shift of various NIR fluorescence dyes without substantial loss of the fluence rate. A linear ratiometric model is employed to measure the relative shift of the excitation spectrum of a fluorescence dye.

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.

Time-resolved and steady-state fluorescence studies of excited-state proton-transfer reactions of proflavine

NASA Astrophysics Data System (ADS)

De Silvestri, S.; Laporta, P.

1984-01-01

Time-resolved and steady-state fluorescence studies of proflavine in aqueous solution are presented. The observation of a monoexponential fluorescence decay with a time constant decreasing with increasing pH and the presence of an anomalous red-shift in the fluorescence spectrum as a function of pH indicate the existence of a complex proton-transfer mechanism in the excited state. A reaction scheme is proposed and the corresponding proton-transfer rates are evaluated. An excited-state pK value of 12.85 is obtained for the equilibrium between the cationic form of proflavine and the same form dissociated at an amino group.

Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

NASA Astrophysics Data System (ADS)

Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

2003-07-01

Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

Time-resolved fluorescence of thioredoxin single-tryptophan mutants: modeling experimental results with minimum perturbation mapping

NASA Astrophysics Data System (ADS)

Silva, Norberto D., Jr.; Haydock, Christopher; Prendergast, Franklyn G.

1994-08-01

The time-resolved fluorescence decay of single tryptophan (Trp) proteins is typically described using either a distribution of lifetimes or a sum of two or more exponential terms. A possible interpretation for this fluorescence decay heterogeneity is the existence of different isomeric conformations of Trp about its (chi) +1) and (chi) +2) dihedral angles. Are multiple Trp conformations compatible with the remainder of the protein in its crystallographic configuration or do they require repacking of neighbor side chains? It is conceivable that isomers of the neighbor side chains interconvert slowly on the fluorescence timescale and contribute additional lifetime components to the fluorescence intensity. We have explored this possibility by performing minimum perturbation mapping simulations of Trp 28 and Trp 31 in thioredoxin (TRX) using CHARMm 22. Mappings of Trp 29 and Trp 31 give the TRX Trp residue energy landscape as a function of (chi) +1) and (chi) +2) dihedral angles. Time-resolved fluorescence intensity and anisotropy decay of mutant TRX (W28F and W31F) are measured and interpreted in light of the above simulations. Relevant observables, like order parameters and isomerization rates, can be derived from the minimum perturbation maps and compared with experiment.

Time resolved fluorescence of cow and goat milk powder

NASA Astrophysics Data System (ADS)

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

2017-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2006-10-01

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

Sub-Doppler two-photon absorption induced by the combination of a single-mode laser and a frequency comb

NASA Astrophysics Data System (ADS)

Moreno, Marco P.; Nogueira, Giovana T.; Felinto, Daniel; Vianna, Sandra S.

2017-08-01

The two-photon transition 5 S -5 P -5 D in rubidium vapor is investigated by detecting the fluorescence from the 6 P3 /2 state when the atomic system is excited by the combined action of a cw diode laser and a frequency comb. The cw laser plays a role as a velocity-selective filter and allows for sub-Doppler spectroscopy over a large spectral range including the 5 D3 /2 and 5 D5 /2 states. For a counterpropagating beam configuration, the response of each atomic velocity group is well characterized within the Doppler profile and the excited hyperfine levels are clearly resolved. The contribution of the optical pumping to the direct two-photon process is also revealed. The results are well described in a frequency domain picture by considering the interaction of each velocity group with the cw laser and the modes of the frequency comb.

Development of LEDs-based microplate reader for bioanalytical assay measurements

NASA Astrophysics Data System (ADS)

Alaruri, Sami D.; Katzlinger, Michael; Schinwald, Bernhard; Kronberger, Georg; Atzler, Joseph

2013-10-01

The optical design for an LEDs-based microplate reader that can perform fluorescence intensity (top and bottom), absorbance, luminescence and time-resolved fluorescence measurements is described. The microplate reader is the first microplate reader in the marketplace that incorporates LEDs as excitation light sources. Absorbance measurements over the 0-3.5 optical density range for caffeine solution are presented. Additionally, fluorescence intensity readings collected at 535 and 625 nm from a green and a red RediPlateTM are reported. Furthermore, fluorescence decay lifetime measurements obtained for Eu (europium) and Sm (samarium) standard solutions using 370 nm excitation are presented. The microplate reader detection limits for the fluorescence intensity top, fluorescence intensity bottom, fluorescence polarization and time-resolved fluorescence modes are 1.5 fmol 100 µL-1 fluorescein (384-well plate), 25 fmol 100 µL-1 fluorescein (384-well plate), 5 mP at 10 nM fluorescein (black 384-well plate) and 30 amol 100 µL-1 europium solution (white 384-well plate), respectively.

In vivo detection of macrophages in a rabbit atherosclerotic model by time-resolved laser-induced fluorescence spectroscopy

PubMed Central

Marcu, Laura; Fang, Qiyin; Jo, Javier A.; Papaioannou, Thanassis; Dorafshar, Amir; Reil, Todd; Qiao, Jian-Hua; Baker, J. Dennis; Freischlag, Julie A.; Fishbein, Michael C.

2007-01-01

Accumulation of numerous macrophages in the fibrous cap is a key identifying feature of plaque inflammation and vulnerability. This study investigates the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a potential tool for detection of macrophage foam cells in the intima of atherosclerotic plaques. Experiments were conducted in vivo on 14 New Zealand rabbits (6 control, 8 hypercholesterolemic) following aortotomy to expose the intimal luminal surface of the aorta. Tissue autofluorescence was induced with a nitrogen pulse laser (337 nm, 1 ns). Lesions were histologically classified by the percent of collagen or macrophage foam cells as well as thickness of the intima. Using parameters derived from the time-resolved fluorescence emission of plaques, we determined that intima rich in macrophage foam cells can be distinguished from intima rich in collagen with high sensitivity (>85%) and specificity (>95%). This study demonstrates, for the first time, that a time-resolved fluorescence-based technique can differentiate and demark macrophage content versus collagen content in vivo. Our results suggest that TR-LIFS technique can be used in clinical applications for identification of inflammatory cells important in plaque formation and rupture. PMID:16039283

In vivo detection of macrophages in a rabbit atherosclerotic model by time-resolved laser-induced fluorescence spectroscopy.

PubMed

Marcu, Laura; Fang, Qiyin; Jo, Javier A; Papaioannou, Thanassis; Dorafshar, Amir; Reil, Todd; Qiao, Jian-Hua; Baker, J Dennis; Freischlag, Julie A; Fishbein, Michael C

2005-08-01

Accumulation of numerous macrophages in the fibrous cap is a key identifying feature of plaque inflammation and vulnerability. This study investigates the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a potential tool for detection of macrophage foam cells in the intima of atherosclerotic plaques. Experiments were conducted in vivo on 14 New Zealand rabbits (6 control, 8 hypercholesterolemic) following aortotomy to expose the intimal luminal surface of the aorta. Tissue autofluorescence was induced with a nitrogen pulse laser (337 nm, 1 ns). Lesions were histologically classified by the percent of collagen or macrophage foam cells as well as thickness of the intima. Using parameters derived from the time-resolved fluorescence emission of plaques, we determined that intima rich in macrophage foam cells can be distinguished from intima rich in collagen with high sensitivity (>85%) and specificity (>95%). This study demonstrates, for the first time, that a time-resolved fluorescence-based technique can differentiate and demark macrophage content versus collagen content in vivo. Our results suggest that TR-LIFS technique can be used in clinical applications for identification of inflammatory cells important in plaque formation and rupture.

Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging

PubMed Central

Quinto-Su, Pedro A.; Lai, Hsuan-Hong; Yoon, Helen H.; Sims, Christopher E.; Allbritton, Nancy L.; Venugopalan, Vasan

2008-01-01

We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at λ = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications. PMID:18305858

Phase-sensitive flow cytometer

DOEpatents

Steinkamp, John A.

1993-01-01

A phase-sensitive flow cytometer (FCM) provides additional FCM capability to use the fluorescence lifetime of one or more fluorochromes bound to single cells to provide additional information regarding the cells. The resulting fluorescence emission can be resolved into individual fluorescence signals if two fluorochromes are present or can be converted directly to a decay lifetime from a single fluorochrome. The excitation light for the fluorochromes is modulated to produce an amplitude modulated fluorescence pulse as the fluorochrome is excited in the FCM. The modulation signal also forms a reference signal that is phase-shifted a selected amount for subsequent mixing with the output modulated fluorescence intensity signal in phase-sensitive detection circuitry. The output from the phase-sensitive circuitry is then an individual resolved fluorochrome signal or a single fluorochrome decay lifetime, depending on the applied phase shifts.

Depth resolved hyperspectral imaging spectrometer based on structured light illumination and Fourier transform interferometry

PubMed Central

Choi, Heejin; Wadduwage, Dushan; Matsudaira, Paul T.; So, Peter T.C.

2014-01-01

A depth resolved hyperspectral imaging spectrometer can provide depth resolved imaging both in the spatial and the spectral domain. Images acquired through a standard imaging Fourier transform spectrometer do not have the depth-resolution. By post processing the spectral cubes (x, y, λ) obtained through a Sagnac interferometer under uniform illumination and structured illumination, spectrally resolved images with depth resolution can be recovered using structured light illumination algorithms such as the HiLo method. The proposed scheme is validated with in vitro specimens including fluorescent solution and fluorescent beads with known spectra. The system is further demonstrated in quantifying spectra from 3D resolved features in biological specimens. The system has demonstrated depth resolution of 1.8 μm and spectral resolution of 7 nm respectively. PMID:25360367

Optical spectroscopy for quantitative sensing in human pancreatic tissues

NASA Astrophysics Data System (ADS)

Wilson, Robert H.; Chandra, Malavika; Lloyd, William; Chen, Leng-Chun; Scheiman, James; Simeone, Diane; McKenna, Barbara; Mycek, Mary-Ann

2011-07-01

Pancreatic adenocarcinoma has a five-year survival rate of only 6%, largely because current diagnostic methods cannot reliably detect the disease in its early stages. Reflectance and fluorescence spectroscopies have the potential to provide quantitative, minimally-invasive means of distinguishing pancreatic adenocarcinoma from normal pancreatic tissue and chronic pancreatitis. The first collection of wavelength-resolved reflectance and fluorescence spectra and time-resolved fluorescence decay curves from human pancreatic tissues was acquired with clinically-compatible instrumentation. Mathematical models of reflectance and fluorescence extracted parameters related to tissue morphology and biochemistry that were statistically significant for distinguishing between pancreatic tissue types. These results suggest that optical spectroscopy has the potential to detect pancreatic disease in a clinical setting.

Dynamic tissue analysis using time- and wavelength-resolved fluorescence spectroscopy for atherosclerosis diagnosis

PubMed Central

Sun, Yinghua; Sun, Yang; Stephens, Douglas; Xie, Hongtao; Phipps, Jennifer; Saroufeem, Ramez; Southard, Jeffrey; Elson, Daniel S.; Marcu, Laura

2011-01-01

Simultaneous time- and wavelength-resolved fluorescence spectroscopy (STWRFS) was developed and tested for the dynamic characterization of atherosclerotic tissue ex vivo and arterial vessels in vivo. Autofluorescence, induced by a 337 nm, 700 ps pulsed laser, was split to three wavelength sub-bands using dichroic filters, with each sub-band coupled into a different length of optical fiber for temporal separation. STWRFS allows for fast recording/analysis (few microseconds) of time-resolved fluorescence emission in these sub-bands and rapid scanning. Distinct compositions of excised human atherosclerotic aorta were clearly discriminated over scanning lengths of several centimeters based on fluorescence lifetime and the intensity ratio between 390 and 452 nm. Operation of STWRFS blood flow was further validated in pig femoral arteries in vivo using a single-fiber probe integrated with an ultrasound imaging catheter. Current results demonstrate the potential of STWRFS as a tool for real-time optical characterization of arterial tissue composition and for atherosclerosis research and diagnosis. PMID:21369214

Spatially resolved x-ray fluorescence spectroscopy of beryllium capsule implosions at the NIF

NASA Astrophysics Data System (ADS)

MacDonald, M. J.; Bishel, D. T.; Saunders, A. M.; Scott, H. A.; Kyrala, G.; Kline, J.; MacLaren, S.; Thorn, D. B.; Yi, S. A.; Zylstra, A. B.; Falcone, R. W.; Doeppner, T.

2017-10-01

Beryllium ablators used in indirectly driven inertial confinement fusion implosions are doped with copper to prevent preheat of the cryogenic hydrogen fuel. Here, we present analysis of spatially resolved copper K- α fluorescence spectra from the beryllium ablator layer. It has been shown that K- α fluorescence spectroscopy can be used to measure plasma conditions of partially ionized dopants in high energy density systems. In these experiments, K-shell vacancies in the copper dopant are created by the hotspot emission at stagnation, resulting in K-shell fluorescence at bang time. Spatially resolved copper K- α emission spectra are compared to atomic kinetics and radiation code simulations to infer density and temperature profiles. This work was supported by the US DOE under Grant No. DE-NA0001859, under the auspices of the US DOE by Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and by Los Alamos National Laboratory under contract DE-AC52-06NA52396.

A CTRW-based model of time-resolved fluorescence lifetime imaging in a turbid medium

NASA Astrophysics Data System (ADS)

Chernomordik, Victor; Gandjbakhche, Amir H.; Hassan, Moinuddin; Pajevic, Sinisa; Weiss, George H.

2010-12-01

We develop an analytic model of time-resolved fluorescent imaging of photons migrating through a semi-infinite turbid medium bounded by an infinite plane in the presence of a single stationary point fluorophore embedded in the medium. In contrast to earlier models of fluorescent imaging in which photon motion is assumed to be some form of continuous diffusion process, the present analysis is based on a continuous-time random walk (CTRW) on a simple cubic lattice, the objective being to estimate the position and lifetime of the fluorophore. This can provide information related to local variations in pH and temperature with potential medical significance. Aspects of the theory were tested using time-resolved measurements of the fluorescence from small inclusions inside tissue-like phantoms. The experimental results were found to be in good agreement with theoretical predictions provided that the fluorophore was not located too close to the planar boundary, a common problem in many diffusive systems.

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

PubMed

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

2014-04-07

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

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

Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Pian, Qi; Yao, Ruoyang; Sinsuebphon, Nattawut; Intes, Xavier

2017-07-01

Spectrally resolved fluorescence lifetime imaging and spatial multiplexing have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (∼40 ps temporal resolution) using fewer than N2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Förster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.

Time-resolved studies of energy transfer from meso-tetrakis(N-methylpyridinium-4-yl)- porphyrin to 3,3'-diethyl-2,2'-thiatricarbocyanine iodide along deoxyribonucleic acid Chain.

PubMed

Kakiuchi, Toshifumi; Ito, Fuyuki; Nagamura, Toshihiko

2008-04-03

The excitation energy transfer from meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) to 3,3'-diethyl-2,2'-thiatricarbocyanine iodide (DTTCI) along the deoxyribonucleic acid (DNA) double strand was investigated by the steady-state absorption and fluorescence measurements and time-resolved fluorescence measurements. The steady-state fluorescence spectra showed that the near-infrared fluorescence of DTTCI was strongly enhanced up to 86 times due to the energy transfer from the excited TMPyP molecule in DNA buffer solution. Furthermore, we elucidated the mechanism of fluorescence quenching and enhancement by the direct observation of energy transfer using the time-resolved measurements. The fluorescence quenching of TMPyP chiefly consists of a static component due to the formation of complex and dynamic components due to the excitation energy transfer. In a heterogeneous one-dimensional system such as a DNA chain, it was proved that the energy transfer process only carries out within the critical distance based on the Förster theory and within a threshold value estimated from the modified Stern-Volmer equation. The present results showed that DNA chain is one of the most powerful tools for nanoassemblies and will give a novel concepts of material design.

Time-resolved spectral studies of blue-green fluorescence of artichoke (Cynara cardunculus L. Var. Scolymus) leaves: identification of chlorogenic acid as one of the major fluorophores and age-mediated changes.

PubMed

Morales, Fermín; Cartelat, Aurélie; Alvarez-Fernández, Ana; Moya, Ismael; Cerovic, Zoran G

2005-12-14

Synchrotron radiation and the time-correlated single-photon counting technique were used to investigate the spectral and time-resolved characteristics of blue-green fluorescence (BGF) of artichoke leaves. Leaves emitted BGF under ultraviolet (UV) excitation; the abaxial side was much more fluorescent than the adaxial side, and in both cases, the youngest leaves were much more fluorescent than the oldest ones. The BGF of artichoke leaves was dominated by the presence of hydroxycinnamic acids. A decrease in the percentage of BGF attributable to the very short kinetic component (from 42 to 20%), in the shape of the BGF excitation spectra, and chlorogenic acid concentrations indicate that there is a loss of hydroxycinnamic acid with leaf age. Studies on excitation, emission, and synchronized fluorescence spectra of leaves and trichomes and chlorogenic acid contents indicate that chlorogenic acid is one of the main blue-green fluorophores in artichoke leaves. Results of the present study indicate that 20-42% (i.e., the very short kinetic component) of the overall BGF is emitted by chlorogenic acid. Time-resolved BGF measurements could be a means to extract information on chlorogenic acid fluorescence from the overall leaf BGF.

Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells

NASA Astrophysics Data System (ADS)

Rück, Angelika; Hauser, Carmen; Mosch, Simone; Kalinina, Sviatlana

2014-09-01

Fluorescence-guided diagnosis of tumor tissue is in many cases insufficient, because false positive results interfere with the outcome. Improvement through observation of cell metabolism might offer the solution, but needs a detailed understanding of the origin of autofluorescence. With respect to this, spectrally resolved multiphoton fluorescence lifetime imaging was investigated to analyze cell metabolism in metabolic phenotypes of malignant and nonmalignant oral mucosa cells. The time-resolved fluorescence characteristics of NADH were measured in cells of different origins. The fluorescence lifetime of bound and free NADH was calculated from biexponential fitting of the fluorescence intensity decay within different spectral regions. The mean lifetime was increased from nonmalignant oral mucosa cells to different squamous carcinoma cells, where the most aggressive cells showed the longest lifetime. In correlation with reports in the literature, the total amount of NADH seemed to be less for the carcinoma cells and the ratio of free/bound NADH was decreased from nonmalignant to squamous carcinoma cells. Moreover for squamous carcinoma cells a high concentration of bound NADH was found in cytoplasmic organelles (mainly mitochondria). This all together indicates that oxidative phosphorylation and a high redox potential play an important role in the energy metabolism of these cells.

Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

PubMed Central

Petersen, Karl J.; Peterson, Kurt C.; Muretta, Joseph M.; Higgins, Sutton E.; Gillispie, Gregory D.; Thomas, David D.

2014-01-01

We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5–10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications. PMID:25430092

Measurement of fluorophore concentrations and fluorescence quantum yield in tissue-simulating phantoms using three diffusion models of steady-state spatially resolved fluorescence.

PubMed

Diamond, Kevin R; Farrell, Thomas J; Patterson, Michael S

2003-12-21

Steady-state diffusion theory models of fluorescence in tissue have been investigated for recovering fluorophore concentrations and fluorescence quantum yield. Spatially resolved fluorescence, excitation and emission reflectance Carlo simulations, and measured using a multi-fibre probe on tissue-simulating phantoms containing either aluminium phthalocyanine tetrasulfonate (AlPcS4), Photofrin meso-tetra-(4-sulfonatophenyl)-porphine dihydrochloride The accuracy of the fluorophore concentration and fluorescence quantum yield recovered by three different models of spatially resolved fluorescence were compared. The models were based on: (a) weighted difference of the excitation and emission reflectance, (b) fluorescence due to a point excitation source or (c) fluorescence due to a pencil beam excitation source. When literature values for the fluorescence quantum yield were used for each of the fluorophores, the fluorophore absorption coefficient (and hence concentration) at the excitation wavelength (mu(a,x,f)) was recovered with a root-mean-square accuracy of 11.4% using the point source model of fluorescence and 8.0% using the more complicated pencil beam excitation model. The accuracy was calculated over a broad range of optical properties and fluorophore concentrations. The weighted difference of reflectance model performed poorly, with a root-mean-square error in concentration of about 50%. Monte Carlo simulations suggest that there are some situations where the weighted difference of reflectance is as accurate as the other two models, although this was not confirmed experimentally. Estimates of the fluorescence quantum yield in multiple scattering media were also made by determining mu(a,x,f) independently from the fitted absorption spectrum and applying the various diffusion theory models. The fluorescence quantum yields for AlPcS4 and TPPS4 were calculated to be 0.59 +/- 0.03 and 0.121 +/- 0.001 respectively using the point source model, and 0.63 +/- 0.03 and 0.129 +/- 0.002 using the pencil beam excitation model. These results are consistent with published values.

Thickness determination of thin solid films by angle-resolved X-ray fluorescence spectrometry using monochromatized synchrotron radiation

NASA Astrophysics Data System (ADS)

Schmitt, W.; Drotbohm, P.; Rothe, J.; Hormes, J.; Ottermann, C. R.; Bange, K.

1995-05-01

Thickness measurements by the method of angle-resolved, self-ratio X-ray fluorescence spectrometry (AR/SR/XFS) have been carried out on thin solid films using monochromatized synchrotron radiation at the Bonn storage ring ELSA. Synchrotron radiation was monochromatized by means of a double-crystal monochromator and fluorescence radiation was detected by a Si(Li) semiconductor detector. The results for sample systems consisting of Au on Si, Cr on SiO2 and TiO2 on alkali-free glass are very satisfactory and agree well with results obtained by other methods.

Detection of melanomas by digital imaging of spectrally resolved UV light-induced autofluorescence of human skin

NASA Astrophysics Data System (ADS)

Chwirot, B. W.; Chwirot, S.; Jedrzejczyk, W.; Redzinski, J.; Raczynska, A. M.; Telega, K.

2001-07-01

We studied spectral and spatial distributions of the intensity of the ultraviolet light-excited fluorescence of human skin. Our studied performed in situ in 162 patients with malignant and non-malignant skin lesions resulted in a new method of detecting melanomas in situ using digital imaging of the spectrally resolved fluorescence. With our diagnostic algorithm we could successfully detect 88.5% of the cases of melanoma in the group of patients subject to examinations with the fluorescence method. A patent application for the method has been submitted to the Patent Office in Warsaw.

Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP.

PubMed

Colletier, Jacques-Philippe; Sliwa, Michel; Gallat, François-Xavier; Sugahara, Michihiro; Guillon, Virginia; Schirò, Giorgio; Coquelle, Nicolas; Woodhouse, Joyce; Roux, Laure; Gotthard, Guillaume; Royant, Antoine; Uriarte, Lucas Martinez; Ruckebusch, Cyril; Joti, Yasumasa; Byrdin, Martin; Mizohata, Eiichi; Nango, Eriko; Tanaka, Tomoyuki; Tono, Kensuke; Yabashi, Makina; Adam, Virgile; Cammarata, Marco; Schlichting, Ilme; Bourgeois, Dominique; Weik, Martin

2016-03-03

Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins.

Time-resolved fluorescence and ultrafast energy transfer in a zinc (hydr)oxide-graphite oxide mesoporous composite

NASA Astrophysics Data System (ADS)

Secor, Jeff; Narinesingh, Veeshan; Seredych, Mykola; Giannakoudakis, Dimitrios A.; Bandosz, Teresa; Alfano, Robert R.

2015-01-01

Ultrafast energy decay kinetics of a zinc (hydr)oxide-graphite oxide (GO) composite is studied via time-resolved fluorescence spectroscopy. The time-resolved emission is spectrally decomposed into emission regions originating from the zinc (hydr)oxide optical gap, surface, and defect states of the composite material. The radiative lifetime of deep red emission becomes an order of magnitude longer than that of GO alone while the radiative lifetime of the zinc optical gap is shortened in the composite. An energy transfer scheme from the zinc (hydr)oxide to GO is considered.

Phase-sensitive flow cytometer

DOEpatents

Steinkamp, J.A.

1993-12-14

A phase-sensitive flow cytometer (FCM) provides additional FCM capability to use the fluorescence lifetime of one or more fluorochromes bound to single cells to provide additional information regarding the cells. The resulting fluorescence emission can be resolved into individual fluorescence signals if two fluorochromes are present or can be converted directly to a decay lifetime from a single fluorochrome. The excitation light for the fluorochromes is modulated to produce an amplitude modulated fluorescence pulse as the fluorochrome is excited in the FCM. The modulation signal also forms a reference signal that is phase-shifted a selected amount for subsequent mixing with the output modulated fluorescence intensity signal in phase-sensitive detection circuitry. The output from the phase-sensitive circuitry is then an individual resolved fluorochrome signal or a single fluorochrome decay lifetime, depending on the applied phase shifts. 15 figures.

Two-Photon Absorption Spectroscopy of Rubidium with a Dual-Comb Tequnique

NASA Astrophysics Data System (ADS)

Nishiyama, Akiko; Yoshida, Satoru; Hariki, Takuya; Nakajima, Yoshiaki; Minoshima, Kaoru

2017-06-01

Dual-comb spectroscopies have great potential for high-resolution molecular and atomic spectroscopies, thanks to the broadband comb spectrum consisting of dense narrow modes. In this study, we apply the dual-comb system to Doppler-free two-photon absorption spectroscopy. The outputs of two frequency combs excite several two-photon transitions of rubidium, and we obtained broadband Doppler-free spectra from dual-comb fluorescence signals. The fluorescence detection scheme circumvents the sensitivity limit which is effectively determined by the dynamic range of photodetectors in absorption-based dual-comb spectroscopies. Our system realized high-sensitive, Doppler-free high-resolution and broadband atomic spectroscopy. A part of observed spectra of 5S_{1/2} - 5D_{5/2} transition is shown in the figure. The hyperfine structures of the F" = 1 - F' = 3,2,1 transitions are fully-resolved and the spectral widths are approximately 5 MHz. The absolute frequency axis is precisely calibrated from comb mode frequencies which were stabilized to a GPS-disciplined clock. This work was supported by JST through the ERATO MINOSHIMA Intelligent Optical Synthesizer Project and Grant-in-Aid for JSPS Fellows (16J02345). A. Nishiyama, S. Yoshida, Y. Nakajima, H. Sasada, K. Nakagawa, A. Onae, K. and Minoshima, Opt. Express 24, 25894 (2016). A. Hipke, S. A. Meek, T. Ideguchi, T.W. Hänsch, and N. Picqué, Phys. Rev. A 90, 011805(R) (2014).

Drug/protein interactions studied by time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Gustavsson, Thomas; Markovitsi, Dimitra; Vayá, Ignacio; Bonancía, Paula; Jiménez, M. C.; Miranda, Miguel A.

2014-09-01

We report here on a recent time-resolved fluorescence study [1] of the interaction between flurbiprofen (FBP), a chiral non-steroidal anti-inflammatory drug, and human serum albumin (HSA), the main transport protein in the human body. We compare the results obtained for the drug-protein complex with those of various covalently linked flurbiprofentryptophan dyads having well-defined geometries. In all cases stereoselective dynamic fluorescence quenching is observed, varying greatly from one system to another. In addition, the fluorescence anisotropy decays also display a clear stereoselectivity. For the drug-protein complexes, this can be interpreted in terms of the protein microenvironment playing a significant role in the conformational relaxation of FBP, which is more restricted in the case of the (R)- enantiomer.

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.

Cucurbiturils: molecular nanocapsules for time-resolved fluorescence-based assays.

PubMed

Marquez, Cesar; Huang, Fang; Nau, Werner M

2004-03-01

A new fluorescent host-guest system based on the inclusion of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) into the cavity of the molecular container compound cucurbit[7]uril (CB7) has been designed which possesses an exceedingly long-lived emission (690 ns in aerated water). The large binding constant of (4 +/- 1) x 10(5) M(-1) along with the resistance of the CB7.DBO complex toward external fluorescence quenchers allow the use of CB7 as an enhancer in time-resolved fluorescence-based assays, e.g., to screen enzyme activity or inhibition by using DBO-labeled peptides as substrates. The response of CB7.DBO to different environmental conditions and possible quenchers are described.

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

PubMed Central

Tsuji, A; Sato, Y; Hirano, M; Suga, T; Koshimoto, H; Taguchi, T; Ohsuka, S

2001-01-01

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells. PMID:11423432

Impact of oxygen chemistry on the emission and fluorescence spectroscopy of laser ablation plumes

NASA Astrophysics Data System (ADS)

Hartig, K. C.; Brumfield, B. E.; Phillips, M. C.; Harilal, S. S.

2017-09-01

Oxygen present in the ambient gas medium may affect both laser-induced breakdown spectroscopy (LIBS) and laser-induced fluorescence (LIF) emission through a reduction of emission intensity and persistence. In this study, an evaluation is made on the role of oxygen in the ambient environment under atmospheric pressure conditions in LIBS and laser ablation (LA)-LIF emission. To generate plasmas, 1064 nm, 10 ns pulses were focused on an aluminum alloy sample. LIF was performed by frequency scanning a CW laser over the 396.15 nm (3s24s 2S1/2 → 3s23p 2P°3/2) Al I transition. Time-resolved emission and fluorescence signals were recorded to evaluate the variation in emission intensity caused by the presence of oxygen. The oxygen partial pressure (po) in the atmospheric pressure environment using N2 as the makeup gas was varied from 0 to 400 Torr O2. 2D-fluorescence spectroscopy images were obtained for various oxygen concentrations for simultaneous evaluation of the emission and excitation spectral features. Results showed that the presence of oxygen in the ambient environment reduces the persistence of the LIBS and LIF emission through an oxidation process that depletes the density of atomic species within the resulting laser-produced plasma (LPP) plume.

Dual lanthanide-doped complexes: the development of a time-resolved ratiometric fluorescent probe for anthrax biomarker and a paper-based visual sensor.

PubMed

Wang, Qi-Xian; Xue, Shi-Fan; Chen, Zi-Han; Ma, Shi-Hui; Zhang, Shengqiang; Shi, Guoyue; Zhang, Min

2017-08-15

In this work, a novel time-resolved ratiometric fluorescent probe based on dual lanthanide (Tb: terbium, and Eu: europium)-doped complexes (Tb/DPA@SiO 2 -Eu/GMP) has been designed for detecting anthrax biomarker (dipicolinic acid, DPA), a unique and major component of anthrax spores. In such complexes-based probe, Tb/DPA@SiO 2 can serve as a stable reference signal with green fluorescence and Eu/GMP act as a sensitive response signal with red fluorescence for ratiometric fluorescent sensing DPA. Additionally, the probe exhibits long fluorescence lifetime, which can significantly reduce the autofluorescence interferences from biological samples by using time-resolved fluorescence measurement. More significantly, a paper-based visual sensor for DPA has been devised by using filter paper embedded with Tb/DPA@SiO 2 -Eu/GMP, and we have proved its utility for fluorescent detection of DPA, in which only a handheld UV lamp is used. In the presence of DPA, the paper-based visual sensor, illuminated by a handheld UV lamp, would result in an obvious fluorescence color change from green to red, which can be easily observed with naked eyes. The paper-based visual sensor is stable, portable, disposable, cost-effective and easy-to-use. The feasibility of using a smartphone with easy-to-access color-scanning APP as the detection platform for quantitative scanometric assays has been also demonstrated by coupled with our proposed paper-based visual sensor. This work unveils an effective method for accurate, sensitive and selective monitoring anthrax biomarker with backgroud-free and self-calibrating properties. Copyright © 2017 Elsevier B.V. All rights reserved.

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 multi-analytical investigation of semi-conductor pigments with time-resolved spectroscopy and imaging

NASA Astrophysics Data System (ADS)

Nevin, A.; Cesaratto, A.; D'Andrea, C.; Valentini, Gianluca; Comelli, D.

2013-05-01

We present the non-invasive study of historical and modern Zn- and Cd-based pigments with time-resolved fluorescence spectroscopy, fluorescence multispectral imaging and fluorescence lifetime imaging (FLIM). Zinc oxide and Zinc sulphide are semiconductors which have been used as white pigments in paintings, and the luminescence of these pigments from trapped states is strongly dependent on the presence of impurities and crystal defects. Cadmium sulphoselenide pigments vary in hue from yellow to deep red based on their composition, and are another class of semiconductor pigments which emit both in the visible and the near infrared. The Fluorescence lifetime of historical and modern pigments has been measured using both an Optical Multichannel Analyser (OMA) coupled with a Nd:YAG nslaser, and a streak camera coupled with a ps-laser for spectrally-resolved fluorescence lifetime measurements. For Znbased pigments we have also employed Fluorescence Lifetime Imaging (FLIM) for the measurement of luminescence. A case study of FLIM applied to the analysis of the painting by Vincent Van Gogh on paper - "Les Bretonnes et le pardon de Pont-Aven" (1888) is presented. Through the integration of complementary, portable and non-invasive spectroscopic techniques, new insights into the optical properties of Zn- and Cd-based pigments have been gained which will inform future analysis of late 19th] and early 20th C. paintings.

Detecting beta-amyloid aggregation from time-resolved emission spectra

NASA Astrophysics Data System (ADS)

Alghamdi, A.; Vyshemirsky, V.; Birch, D. J. S.; Rolinski, O. J.

2018-04-01

The aggregation of beta-amyloids is one of the key processes responsible for the development of Alzheimer’s disease. Early molecular-level detection of beta-amyloid oligomers may help in early diagnosis and in the development of new intervention therapies. Our previous studies on the changes in beta-amyloid’s single tyrosine intrinsic fluorescence response during aggregation demonstrated a four-exponential fluorescence intensity decay, and the ratio of the pre-exponential factors indicated the extent of the aggregation in the early stages of the process before the beta-sheets were formed. Here we present a complementary approach based on the time-resolved emission spectra (TRES) of amyloid’s tyrosine excited at 279 nm and fluorescence in the window 240-450 nm. TRES have been used to demonstrate sturctural changes occuring on the nanosecond time scale after excitation which has significant advantages over using steady-state spectra. We demonstrate this by resolving the fluorescent species and revealing that beta-amyloid’s monomers show very fast dielectric relaxation, and its oligomers display a substantial spectral shift due to dielectric relaxation, which gradually decreases when the oligomers become larger.

Time-resolved laser-induced fluorescence system

NASA Astrophysics Data System (ADS)

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

2006-02-01

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

Femtosecond Fluorescence Upconversion Study of a Naphthalimide-Bithiophene-Triphenylamine Push-Pull Dye in Solution.

PubMed

Maffeis, Valentin; Brisse, Romain; Labet, Vanessa; Jousselme, Bruno; Gustavsson, Thomas

2018-06-13

There is a high interest in the development of new push-pull dyes for the use in dye sensitized solar cells. The pronounced charge transfer character of the directly photoexcited state is in principle favorable for a charge injection. Here, we report a time-resolved fluorescence study of a triphenylamine-bithiophene-naphthalimide dye in four solvents of varying polarity using fluorescence upconversion. The recording of femtosecond time-resolved fluorescence spectra corrected for the group velocity dispersion allows for a detailed analysis discriminating between spectral shifts and total intensity decays. After photoexcitation, the directly populated state (S 1 /FC) evolves toward a relaxed charge transfer state (S 1 /CT). This S 1 /CT state is characterized by a lower radiative transition moment and a higher nonradiative quenching. The fast dynamic shift of the fluorescence band is well described by solvation dynamics in polar solvents, but less so in nonpolar solvents, hinting that the excited-state relaxation process occurs on a free energy surface whose topology is strongly governed by the solvent polarity. This study underlines the influence of the environment on the intramolecular charge transfer (ICT) process, and the necessity to analyze time-resolved data in detail when solvation and ICT occur simultaneously.

Fluorescence Spectroscopic Properties of Normal and Abnormal Biomedical Materials

NASA Astrophysics Data System (ADS)

Pradhan, Asima

Steady state and time-resolved optical spectroscopy and native fluorescence is used to study the physical and optical properties occurring in diseased and non-diseased biological human tissue, in particular, cancer of the human breast, artery and the dynamics of a photosensitizer useful in photodynamic therapy. The main focus of the research is on the optical properties of cancer and atherosclerotic tissues as compared to their normal counterparts using the different luminescence based spectroscopic techniques such as steady state fluorescence, time-resolved fluorescence, excitation spectroscopy and phosphorescence. The excitation and steady-state spectroscopic fluorescence using visible excitation wavelength displays a difference between normal and malignant tissues. This difference is attributed to absorption of the emission by hemoglobin in normal tissues. This method using 488nm fails to distinguish neoplastic tissue such as benign tissues and tumors from malignant tumors. The time-resolved fluorescence at visible, near -uv and uv excitation wavelengths display non-exponential profiles which are significantly different for malignant tumors as compared to non-malignant tissues only with uv excitation. The differences observed with visible and near-uv excitation wavelengths are not as significant. The non-exponential profiles are interpreted as due to a combination of fluorophores along with the action of non-radiative processes. Low temperature luminescence studies confirm the occurrence of non-radiative decay processes while temporal studies of various relevant biomolecules indicate the probable fluorophores responsible for the observed signal in tissues. Phosphorescence from human tissues have been observed for the first time and lifetimes of a few hundred nanoseconds are measured for malignant and benign tissues. Time-resolved fluorescence studies of normal artery and atherosclerotic plaque have shown that a combination of two excitation wavelengths can distinguish fibrous and calcified atherosclerotic plaque from normal artery. A minor effort of the study involves the high intensity effects on the optical properties of the dye, doxycycline (a particular photosensitizer of the tetracycline group) occurring during relaxation when excited at different laser intensities. This study has been performed by observing the fluorescence lifetimes and quantum yields of DOTC at different excitation intensities. The results obtained support the sequential excited state absorption model.

Conformational Explosion: Understanding the Complexity of the Para-Dialkylbenzene Potential Energy Surfaces

NASA Astrophysics Data System (ADS)

Mishra, Piyush; Hewett, Daniel M.; Zwier, Timothy S.

2017-06-01

This talk focuses on the single-conformation spectroscopy of small-chain para-dialkylbenzenes. This work builds on previous studies from our group on long-chain n-alkylbenzenes that identified the first folded structure in octylbenzene. The dialkylbenzenes are representative of a class of molecules that are common components of coal and aviation fuel and are known to be present in vehicle exhaust. We bring the molecules para-diethylbenzene, para-dipropylbenzene and para-dibutylbenzene into the gas phase and cool the molecules in a supersonic expansion. The jet-cooled molecules are then interrogated using laser-induced fluorescence excitation, fluorescence dip IR spectroscopy (FDIRS) and dispersed fluorescence. The LIF spectra in the S_{0}-S_{1} origin region show dramatic increases in the number of resolved transitions with increasing length of alkyl chains, reflecting an explosion in the number of unique low-energy conformations formed when two independent alkyl chains are present. Since the barriers to isomerization of the alkyl chain are similar in size, this results in an 'egg carton' shape to the potential energy surface. We use a combination of electronic frequency shift and alkyl CH stretch infrared spectra to generate a consistent set of conformational assignments.

Screening of cardiomyocyte fluorescence during cell contraction by multi-dimensional TCSPC

NASA Astrophysics Data System (ADS)

Chorvat, D., Jr.; Abdulla, S.; Elzwiei, F.; Mateasik, A.; Chorvatova, A.

2008-02-01

Autofluorescence is one of the most versatile non-invasive tools for mapping the metabolic state of living tissues, such as the heart. We present a new approach to the investigation of changes in endogenous fluorescence during cardiomyocyte contraction - by spectrally-resolved, time correlated, single photon counting (TCSPC). Cell contraction is stimulated by external platinum electrodes, incorporated in a home-made bath and triggered by a pulse generator at a frequency of 0.5 Hz (to stabilize sarcoplasmic reticulum loading), or 5 Hz (the rat heart rate). Cell illumination by the laser is synchronized with cell contraction, using TTL logic pulses operated by a stimulator and delayed to study mitochondrial metabolism at maximum contraction (10-110 ms) and/or at steady state (1000-1100 ms at 0.5 Hz). To test the setup, we recorded calcium transients in cells loaded with the Fluo-3 fluorescent probe (excited by 475 nm pulsed picosecond diode laser). We then evaluated recordings of flavin AF (excited by 438 nm pulsed laser) at room and physiological temperatures. Application of the presented approach will shed new insight into metabolic changes in living, contracting myocytes and, therefore, regulation of excitation-contraction coupling and/or ionic homeostasis and, thus, heart excitability.

Time-resolved tryptophan fluorescence in photosynthetic reaction centers from Rhodobacter sphaeroides

NASA Technical Reports Server (NTRS)

Godik, V. I.; Blankenship, R. E.; Causgrove, T. P.; Woodbury, N.

1993-01-01

Tryptophan fluorescence of reaction centers isolated from Rhodobacter sphaeroides, both stationary and time-resolved, was studied. Fluorescence kinetics were found to fit best a sum of four discrete exponential components. Half of the initial amplitude was due to a component with a lifetime of congruent to 60 ps, belonging to Trp residues, capable of efficient transfer of excitation energy to bacteriochlorophyll molecules of the reaction center. The three other components seem to be emitted by Trp ground-state conformers, unable to participate in such a transfer. Under the influence of intense actinic light, photooxidizing the reaction centers, the yield of stationary fluorescence diminished by congruent to 1.5 times, while the number of the kinetic components and their life times remained practically unchanged. Possible implications of the observed effects for the primary photosynthesis events are considered.

Frequency domain technique for a two-dimensional mapping of optical tissue properties

NASA Astrophysics Data System (ADS)

Bocher, Thomas; Beuthan, Juergen; Minet, Olaf; Naber, Rolf-Dieter; Mueller, Gerhard J.

1995-12-01

Locally and individually varying optical tissue parameters (mu) a, (mu) s, and g are responsible for non-neglectible uncertainties in the interpretation of spectroscopic data in optical biopsy techniques. The intrinsic fluorescence signal for instance doesn't depend only on the fluorophore concentration but also on the amount of other background absorbers and on alterations of scattering properties. Therefore neither a correct relative nor an absolute mapping of the lateral fluorophore concentration can be derived from the intrinsic fluorescence signal alone. Using MC-simulations it can be shown that in time-resolved LIFS the simultaneously measured backscattered signal of the excitation wavelength (UV) can be used to develop a special, linearized rescaling algorithm to take into account the most dominant of these varying tissue parameters which is (mu) a,ex. In combination with biochemical calibration measurements we were able to perform fiberbased quantitative NADH- concentration measurements. In this paper a new rescaling method for VIS and IR light in the frequency domain is proposed. It can be applied within the validity range of the diffusion approximation and provides full (mu) a and (mu) s rescaling possibility in a 2- dimensional, non-contact mapping mode. The scanning device is planned to be used in combination with a standard operation microscope of ZEISS, Germany.

Simultaneous measurements of velocity, temperature, and pressure using rapid CW wavelength-modulation laser-induced fluorescence of OH

NASA Technical Reports Server (NTRS)

Chang, A. Y.; Battles, B. E.; Hanson, R. K.

1990-01-01

In high speed flows, laser induced fluorescence (LIF) on Doppler shifted transitions is an attractive technique for velocity measurement. LIF velocimetry was applied to combined single-point measurements of velocity, temperature, and pressure and 2-D imaging of velocity and pressure. Prior to recent research using NO, LIF velocimetry in combustion related flows relied largely on the use of seed molecules. Simultaneous, single-point LIF measurements is reported of velocity, temperature, and pressure using the naturally occurring combustion species OH. This experiment is an extension of earlier research in which a modified ring dye laser was used to make time resolved temperature measurements behind reflected shock waves by using OH absorption an in postflame gases by using OH LIF. A pair of fused-silica rhombs mounted on a single galvanonmeter in an intracavity-doubled Spectra-Physics 380 ring laser permit the UV output to be swept continuously over a few wave numbers at an effective frequency of 3kHz.

Three-photon excitation source at 1250 nm generated in a dual zero dispersion wavelength nonlinear fiber

DOE PAGES

Domingue, Scott R.; Bartels, Randy A.

2014-12-04

Here, we demonstrate 1250 nm pulses generated in dual-zero dispersion photonic crystal fiber capable of three-photon excitation fluorescence microscopy. The total power conversion efficiency from the 28 fs seed pulse centered at 1075 nm to pulses at 1250 nm, including coupling losses from the nonlinear fiber, is 35%, with up to 67% power conversion efficiency of the fiber coupled light. Frequency-resolved optical gating measurements characterize 1250 nm pulses at 0.6 nJ and 2 nJ, illustrating the change in nonlinear spectral phase accumulation with pulse energy even for nonlinear fiber lengths < 50 mm. The 0.6 nJ pulse has a 26more » fs duration and is the shortest nonlinear fiber derived 1250 nm pulse yet reported (to the best of our knowledge). The short pulse durations and energies make these pulses a viable route to producing light at 1250 nm for multiphoton microscopy, which we we demonstrate here, via a three-photon excitation fluorescence microscope.« less

Cellular Oxygen and Nutrient Sensing in Microgravity Using Time-Resolved Fluorescence Microscopy

NASA Technical Reports Server (NTRS)

Szmacinski, Henryk

2003-01-01

Oxygen and nutrient sensing is fundamental to the understanding of cell growth and metabolism. This requires identification of optical probes and suitable detection technology without complex calibration procedures. Under this project Microcosm developed an experimental technique that allows for simultaneous imaging of intra- and inter-cellular events. The technique consists of frequency-domain Fluorescence Lifetime Imaging Microscopy (FLIM), a set of identified oxygen and pH probes, and methods for fabrication of microsensors. Specifications for electronic and optical components of FLIM instrumentation are provided. Hardware and software were developed for data acquisition and analysis. Principles, procedures, and representative images are demonstrated. Suitable lifetime sensitive oxygen, pH, and glucose probes for intra- and extra-cellular measurements of analyte concentrations have been identified and tested. Lifetime sensing and imaging have been performed using PBS buffer, culture media, and yeast cells as a model systems. Spectral specifications, calibration curves, and probes availability are also provided in the report.

Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy.

PubMed

Zhao, Ming; Huang, Run; Peng, Leilei

2012-11-19

Förster resonant energy transfer (FRET) is extensively used to probe macromolecular interactions and conformation changes. The established FRET lifetime analysis method measures the FRET process through its effect on the donor lifetime. In this paper we present a method that directly probes the time-resolved FRET signal with frequency domain Fourier lifetime excitation-emission matrix (FLEEM) measurements. FLEEM separates fluorescent signals by their different phonon energy pathways from excitation to emission. The FRET process generates a unique signal channel that is initiated by donor excitation but ends with acceptor emission. Time-resolved analysis of the FRET EEM channel allows direct measurements on the FRET process, unaffected by free fluorophores that might be present in the sample. Together with time-resolved analysis on non-FRET channels, i.e. donor and acceptor EEM channels, time resolved EEM analysis allows precise quantification of FRET in the presence of free fluorophores. The method is extended to three-color FRET processes, where quantification with traditional methods remains challenging because of the significantly increased complexity in the three-way FRET interactions. We demonstrate the time-resolved EEM analysis method with quantification of three-color FRET in incompletely hybridized triple-labeled DNA oligonucleotides. Quantitative measurements of the three-color FRET process in triple-labeled dsDNA are obtained in the presence of free single-labeled ssDNA and double-labeled dsDNA. The results establish a quantification method for studying multi-color FRET between multiple macromolecules in biochemical equilibrium.

Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy

PubMed Central

Zhao, Ming; Huang, Run; Peng, Leilei

2012-01-01

Förster resonant energy transfer (FRET) is extensively used to probe macromolecular interactions and conformation changes. The established FRET lifetime analysis method measures the FRET process through its effect on the donor lifetime. In this paper we present a method that directly probes the time-resolved FRET signal with frequency domain Fourier lifetime excitation-emission matrix (FLEEM) measurements. FLEEM separates fluorescent signals by their different phonon energy pathways from excitation to emission. The FRET process generates a unique signal channel that is initiated by donor excitation but ends with acceptor emission. Time-resolved analysis of the FRET EEM channel allows direct measurements on the FRET process, unaffected by free fluorophores that might be present in the sample. Together with time-resolved analysis on non-FRET channels, i.e. donor and acceptor EEM channels, time resolved EEM analysis allows precise quantification of FRET in the presence of free fluorophores. The method is extended to three-color FRET processes, where quantification with traditional methods remains challenging because of the significantly increased complexity in the three-way FRET interactions. We demonstrate the time-resolved EEM analysis method with quantification of three-color FRET in incompletely hybridized triple-labeled DNA oligonucleotides. Quantitative measurements of the three-color FRET process in triple-labeled dsDNA are obtained in the presence of free single-labeled ssDNA and double-labeled dsDNA. The results establish a quantification method for studying multi-color FRET between multiple macromolecules in biochemical equilibrium. PMID:23187535

Quantization of bovine serum albumin by fluorescence enhancement effects and corresponding binding of macrocyclic host-protein assembly.

PubMed

Bardhan, Munmun; Misra, Tapas; Ganguly, Tapan

2012-01-05

The present paper reports the investigations on the spectroscopic behavior of the binary complexes of the dye aurintricarboxylic acid (ATA) with protein bovine serum albumin (BSA) and 18-crown 6 (CW) (ATA·BSA, ATA·CW) and the ternary complex ATA·CW·BSA by using UV-vis steady state and time resolved fluorescence spectroscopy. The primary aim of the work is to determine the protein (BSA) quantization by fluorescence enhancement method and investigate the 'enhancer' activity of crown ether (CW) on it to increase the resolution. Steady state and time resolved fluorescence measurements demonstrated how fluorescence intensity of ATA could be used for the determination of the protein BSA in aqueous solution. The binding of dye (probe/fluorescent medicinal molecule) with protein and the denaturing effect in the polar environment of acetonitrile of the dye protein complex act as drug binding as well as drug release activity. Apart from its basic research point of view, the present study also possesses significant importance and applications in the field of medicinal chemistry. Copyright © 2011 Elsevier B.V. All rights reserved.

Lateral microheterogeneity of diphenylhexatriene-labeled choline phospholipids in the erythrocyte ghost membrane as determined by time-resolved fluorescence spectroscopy.

PubMed

Prenner, E; Sommer, A; Maurer, N; Glatter, O; Gorges, R; Paltauf, F; Hermetter, A

2000-04-01

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane.

Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy.

PubMed Central

Buehler, C; Dong, C Y; So, P T; French, T; Gratton, E

2000-01-01

We report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-microm orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from approximately 30 to approximately 150 ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell. PMID:10866979

Wide-field spectrally resolved quantitative fluorescence imaging system: toward neurosurgical guidance in glioma resection

NASA Astrophysics Data System (ADS)

Xie, Yijing; Thom, Maria; Ebner, Michael; Wykes, Victoria; Desjardins, Adrien; Miserocchi, Anna; Ourselin, Sebastien; McEvoy, Andrew W.; Vercauteren, Tom

2017-11-01

In high-grade glioma surgery, tumor resection is often guided by intraoperative fluorescence imaging. 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) provides fluorescent contrast between normal brain tissue and glioma tissue, thus achieving improved tumor delineation and prolonged patient survival compared with conventional white-light-guided resection. However, commercially available fluorescence imaging systems rely solely on visual assessment of fluorescence patterns by the surgeon, which makes the resection more subjective than necessary. We developed a wide-field spectrally resolved fluorescence imaging system utilizing a Generation II scientific CMOS camera and an improved computational model for the precise reconstruction of the PpIX concentration map. In our model, the tissue's optical properties and illumination geometry, which distort the fluorescent emission spectra, are considered. We demonstrate that the CMOS-based system can detect low PpIX concentration at short camera exposure times, while providing high-pixel resolution wide-field images. We show that total variation regularization improves the contrast-to-noise ratio of the reconstructed quantitative concentration map by approximately twofold. Quantitative comparison between the estimated PpIX concentration and tumor histopathology was also investigated to further evaluate the system.

Flash lamp-excited time-resolved fluorescence microscope suppresses autofluorescence in water concentrates to deliver an 11-fold increase in signal-to-noise ratio.

PubMed

Connally, Russell; Veal, Duncan; Piper, James

2004-01-01

The ubiquity of naturally fluorescing components (autofluorophores) encountered in most biological samples hinders the detection and identification of labeled targets through fluorescence-based techniques. Time-resolved fluorescence (TRF) is a technique by which the effects of autofluorescence are reduced by using specific fluorescent labels with long fluorescence lifetimes (compared with autofluorophores) in conjunction with time-gated detection. A time-resolved fluorescence microscope (TRFM) is described that is based on a standard epifluorescence microscope modified by the addition of a pulsed excitation source and an image-intensified time-gateable CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flash lamp with rapid discharge characteristics was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, the flash output decayed with an approximate lifetime of 18 micros and the TRFM required a long-lived lanthanide chelate label to ensure that probe fluorescence was visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the waterborne parasite Giardia lamblia. For a 600-nm bandpass filter set and a gate delay of 60 micros, the TRFM provided an 11.3-fold improvement in the signal-to-noise ratio (S/N) of labeled Giardia over background. A smaller gain in an SNR of 9.69-fold was achieved with a 420-nm longpass filter set; however, the final contrast ratio between labeled cyst and background was higher (11.3 versus 8.5). Despite the decay characteristics of the light pulse, flash lamps have many practical advantages compared with optical chopper wheels and modulated lasers for applications in TRFM.

Fluorescence lifetimes of anthracycline drugs in phospholipid bilayers determined by frequency-domain fluorometry

NASA Astrophysics Data System (ADS)

Burke, Thomas G.; Malak, Henryk M.; Doroshow, James H.

1990-05-01

Time-resolved fluorescence intensity decay data from anthracycline anticancer drugs present in model membranes were obtained using a gigahertz frequency-domain fluorometer [Lakowicz et al. (1986) Rev. Sci. Instrum. 57, 2499-2506]. Exciting light of 290 nm, modulated at multiple frequencies from 8 MHz to 400 MHz, was used to study the interactions of Adriamycin, daunomycin and related antibiotics with small unilamellar vesicles composed of dimyristoylphosphatidylcholine (DMPC) at 28°C. Fluorescence decay data for drug molecules free in solution as well as bound to membranes were best fit by exponentials requiring two terms rather than by single exponential decays. For example, one-component analysis of the decay data for Adriamycin free in phosphate buffered saline (PBS) solution resulted in a reduced x2 value of 140 ((tau) = 0.88 ns), while a two-component fit resulted in a substantially smaller reduced x2 value of 2.6 ((tau)1 = 1.13 ns, (alpha)1 = 0.60, (tau)2 = 0.30 ns). Upon association with membranes, each of the anthracyclines studied displayed a larger r1 value while the r2 value remained the same or increased (for example, DMPC-bound Adriamycin showed r1 = 1.68 ns , a1 = 0 . 64 , r2 = 0 . 33 ns) . Analyses of the fluorescence emission decays of anthracyclines were also made assuming each decay is composed of a single Lorentzian distribution of lifetimes. Data taken on Adriamycin in PBS, when fit using one continuous component, displayed (tau), (alpha), w, and reduced x2 values of 0.68 ns, 1, 0.60 ns, and 9.1, respectively. The distribution became quite broad upon drug association with membrane (DMPCbound Adriamycin: (tau) = 0.75 ns, (alpha) = 1, w = 2.24 ns, x2 = 13). For each anthracycline studied, continuous component fits showed significant broadening in the distributions upon drug association with membrane. Relatively large shifts in lifetime values were observed for the carminomycin and 4-demethoxydaunomycin analogues upon binding model lipid membranes, making these agents good candidates to employ in future studies on anthracycline interactions with more environmentally-complex biological membranes.

Analysis of hydrocarbon-bearing fluid inclusions (HCFI) using time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Przyjalgowski, Milosz A.; Ryder, Alan G.; Feely, Martin; Glynn, Thomas J.

2005-06-01

Hydrocarbon-bearing fluid inclusions (HCFI) are microscopic cavities within rocks that are filled with petroleum oil, the composition of which may not have changed since the trapping event. Thus, the composition of that entrapped oil can provide information about the formation and evolution of the oil reservoir. This type of information is important to the petroleum production and exploration industries. Crude oil fluorescence originates from the presence of cyclic aromatic compounds and the nature of the emission is governed by the chemical composition of the oil. Fluorescence based methods are widely used for analysis of crude oil because they offer robust, non-contact and non-destructive measurement options. The goal of our group is the development of a non-destructive analytical method for HCFI using time-resolved fluorescence methods. In broad terms, crude oil fluorescence behavior is governed by the concentration of quenching species and the distribution of fluorophores. For the intensity averaged fluorescence lifetime, the best correlations have been found between polar or alkane concentrations, but these are not suitable for robust, quantitative analysis. We have recently started to investigate another approach for characterizing oils by looking at Time-resolved Emission Spectra (TRES). TRES are constructed from intensities sampled at discrete times during the fluorescence decay of the sample. In this study, TRES, from a series of 10 crude oils from the Middle East, have been measured at discrete time gates (0.5 ns, 1 ns, 2 ns, 4 ns) over the 450-700 nm wavelength range. The spectral changes in TRES, such as time gate dependent Stokes' shift and spectral broadening, are analyzed in the context of energy transfer rates. In this work, the efficacy of using TRES for fingerprinting individual oils and HCFI is also demonstrated.

Wavelength dispersive analysis with the synchrotron x ray fluorescence microprobe

NASA Technical Reports Server (NTRS)

Rivers, M. L.; Thorn, K. S.; Sutton, S. R.; Jones, K. W.; Bajt, S.

1993-01-01

A wavelength dispersive spectrometer (WDS) was tested on the synchrotron x ray fluorescence microprobe at Brookhaven National Laboratory. Compared to WDS spectra using an electron microprobe, the synchrotron WDS spectra have much better sensitivity and, due to the absence of bremsstrahlung radiation, lower backgrounds. The WDS spectrometer was successfully used to resolve REE L fluorescence spectra from standard glasses and transition metal K fluorescence spectra from kamacite.

Hydrogen bonding-assisted interaction between amitriptyline hydrochloride and hemoglobin: spectroscopic and molecular dynamics studies.

PubMed

Maurya, Neha; Maurya, Jitendra Kumar; Kumari, Meena; Khan, Abbul Bashar; Dohare, Ravins; Patel, Rajan

2017-05-01

Herein, we have explored the interaction between amitriptyline hydrochloride (AMT) and hemoglobin (Hb), using steady-state and time-resolved fluorescence spectroscopy, UV-visible spectroscopy, and circular dichroism spectroscopy, in combination with molecular docking and molecular dynamic (MD) simulation methods. The steady-state fluorescence reveals the static quenching mechanism in the interaction system, which was further confirmed by UV-visible and time-resolved fluorescence spectroscopy. The binding constant, number of binding sites, and thermodynamic parameters viz. ΔG, ΔH, ΔS are also considered; result confirms that the binding of the AMT with Hb is a spontaneous process, involving hydrogen bonding and van der Waals interactions with a single binding site, as also confirmed by molecular docking study. Synchronous fluorescence, CD data, and MD simulation results contribute toward understanding the effect of AMT on Hb to interpret the conformational change in Hb upon binding in aqueous solution.

Probing Membrane Order and Topography in Supported Lipid Bilayers by Combined Polarized Total Internal Reflection Fluorescence-Atomic Force Microscopy

PubMed Central

Oreopoulos, John; Yip, Christopher M.

2009-01-01

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C20 or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions. PMID:19254557

Frequency domain, time-resolved and spectroscopic investigations of photosensitizers encapsulated in liposomal phantoms

NASA Astrophysics Data System (ADS)

Mermut, Ozzy; Bouchard, Jean-Pierre; Cormier, Jean-Francois; Diamond, Kevin R.; Noiseux, Isabelle; Vernon, Marcia L.; Patterson, Michael S.

2007-07-01

A broadband frequency domain fluorescence lifetime system (from ns to ms time scale) has been developed to study the photochemical and photodynamic behavior of model, well-controlled photosensitizer-encapsulating liposomes. Liposomes are known to be efficient and selective photosensitizer (PS) drug delivery vesicles, however, their chemical and physical effects on the photochemical properties of the photosensitizer have not been well characterized. The liposomes employed in this study (both blank and photosensitizer-complexed) were characterized to determine their: a) size distribution (dynamic light scattering), b) image (scanning electron microscope, confocal fluorescence microscopy), c) concentration of particles (flow cytometry), d) temperature-dependant phase transition behavior (differential scanning calorimetry, and e) spectrofluorescent spectrophotometric properties, e.g. aggregation, in the confined environment. The fluorescence decay behavior of two families of encapsulated photosensitizers, di-and tetrasulfonated metallophthalocyanines, and 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH), has been examined as a function of the liposome's physical properties (size-scale, distribution and concentration of scatterer) and the impact of the photosensitizer spatial confinement determined. It is found that the achievable size range and distribution of the PS-liposomes is controlled by the chemical nature of the PS for large liposomes (1000 nm), and is PS independent for small PS-liposomes (~140nm). The lifetime decay behavior was studied for all three photosensitizer-liposome systems and compared before and after confinement. We found the nature of the decay to be similar before and after encapsulation for the sulfonated phthalocyanines containing ionic moieties (primarily monoexponential) but not for HPPH. In the latter, the decay transitioned from multi- to monoexponential decay upon localizing lypophilic HPPH to the liposomal membrane. This behavior was confirmed by obtaining a similar change in lifetime response with an independent timedomain system. We also varied the environment in temperature and oxygen content to examine the effects on the fluorescent lifetimes of the liposomal complexes. The fluorescence decay of all three PS-containing liposomes showed that the local spatial confinement of PS (dictated by the PS chemistry) into different domains within the liposome directly controls the temperature-response. Membrane-bound photosensitizers were less sensitive to temperature effects as illustrated by the decay dynamics observed in solu, that is, they developed a unique decay behavior that correlated with the phase transition of the membrane. The fluorescent lifetime of PS-encapsulated liposomes in deoxygenated environments, relevant to oxygen independent type I phototoxicity, was also probed in the frequency-domain revealing that liposome-confined PS display very different trends than those observed in solu.

Fluorescence lifetime imaging of skin cancer

NASA Astrophysics Data System (ADS)

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

2011-03-01

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

Glucose Sensing by Time-Resolved Fluorescence of Sol-Gel Immobilized Glucose Oxidase

PubMed Central

Esposito, Rosario; Ventura, Bartolomeo Della; De Nicola, Sergio; Altucci, Carlo; Velotta, Raffaele; Mita, Damiano Gustavo; Lepore, Maria

2011-01-01

A monolithic silica gel matrix with entrapped glucose oxidase (GOD) was constructed as a bioactive element in an optical biosensor for glucose determination. Intrinsic fluorescence of free and immobilised GOD was investigated in the visible range in presence of different glucose concentrations by time-resolved spectroscopy with time-correlated single-photon counting detector. A three-exponential model was used for analysing the fluorescence transients. Fractional intensities and mean lifetime were shown to be sensitive to the enzymatic reaction and were used for obtaining calibration curve for glucose concentration determination. The sensing system proposed achieved high resolution (up to 0.17 mM) glucose determination with a detection range from 0.4 mM to 5 mM. PMID:22163807

The supercontinuum laser as a flexible source for quasi-steady state and time resolved fluorescence studies

NASA Astrophysics Data System (ADS)

Fenske, Roger; Näther, Dirk U.; Dennis, Richard B.; Smith, S. Desmond

2010-02-01

Commercial Fluorescence Lifetime Spectrometers have long suffered from the lack of a simple, compact and relatively inexpensive broad spectral band light source that can be flexibly employed for both quasi-steady state and time resolved measurements (using Time Correlated Single Photon Counting [TCSPC]). This paper reports the integration of an optically pumped photonic crystal fibre, supercontinuum source1 (Fianium model SC400PP) as a light source in Fluorescence Lifetime Spectrometers (Edinburgh Instruments FLS920 and Lifespec II), with single photon counting detectors (micro-channel plate photomultiplier and a near-infrared photomultiplier) covering the UV to NIR range. An innovative method of spectral selection of the supercontinuum source involving wedge interference filters is also discussed.

Introduction to Time-Resolved Spectroscopy: Nanosecond Transient Absorption and Time-Resolved Fluorescence of Eosin B

ERIC Educational Resources Information Center

Farr, Erik P.; Quintana, Jason C.; Reynoso, Vanessa; Ruberry, Josiah D.; Shin, Wook R.; Swartz, Kevin R.

2018-01-01

Here we present a new undergraduate laboratory that will introduce the concepts of time-resolved spectroscopy and provide insight into the natural time scales on which chemical dynamics occur through direct measurement. A quantitative treatment of the acquired data will provide a deeper understanding of the role of quantum mechanics and various…

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

NASA Astrophysics Data System (ADS)

Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

2011-07-01

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

Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

PubMed Central

Chan, Jacky C. K.; Diebold, Eric D.; Buckley, Brandon W.; Mao, Sien; Akbari, Najva; Jalali, Bahram

2014-01-01

Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat’s-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures. PMID:25574449

Silicon drift detectors as a tool for time-resolved fluorescence XAFS on low-concentrated samples in catalysis.

PubMed

Kappen, Peter; Tröger, Larc; Materlik, Gerhard; Reckleben, Christian; Hansen, Karsten; Grunwaldt, Jan-Dierk; Clausen, Bjerne S

2002-07-01

A silicon drift detector (SDD) was used for ex situ and time-resolved in situ fluorescence X-ray absorption fine structure (XAFS) on low-concentrated catalyst samples. For a single-element and a seven-element SDD the energy resolution and the peak-to-background ratio were verified at high count rates, sufficient for fluorescence XAFS. An experimental set-up including the seven-element SDD without any cooling and an in situ cell with gas supply and on-line gas analysis was developed. With this set-up the reduction and oxidation of a zeolite supported catalyst containing 0.3 wt% platinum was followed by fluorescence near-edge scans with a time resolution of 10 min each. From ex situ experiments on low-concentrated platinum- and gold-based catalysts fluorescence XAFS scans could be obtained with sufficient statistical quality for a quantitative analysis. Structural information on the gold and platinum particles could be extracted by both the Fourier transforms and the near-edge region of the XAFS spectra. Moreover, it was found that with the seven-element SDD concentrations of the element of interest as low as 100 ppm can be examined by fluorescence XAFS.

Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

PubMed Central

Fu, Henry L.; Mueller, Jenna L.; Javid, Melodi P.; Mito, Jeffrey K.; Kirsch, David G.; Ramanujam, Nimmi; Brown, J. Quincy

2013-01-01

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy. PMID:23894357

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

PubMed

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

2016-06-20

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

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

NASA Astrophysics Data System (ADS)

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

2018-03-01

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

Excited-state proton transfer dynamics of firefly's chromophore D-luciferin in DMSO-water binary mixture.

PubMed

Kuchlyan, Jagannath; Banik, Debasis; Roy, Arpita; Kundu, Niloy; Sarkar, Nilmoni

2014-12-04

In this article we have investigated intermolecular excited-state proton transfer (ESPT) of firefly's chromophore D-luciferin in DMSO-water binary mixtures using steady-state and time-resolved fluorescence spectroscopy. The unusual behavior of DMSO-water binary mixture as reported by Bagchi et al. (J. Phys. Chem. B 2010, 114, 12875-12882) was also found using D-luciferin as intermolecular ESPT probe. The binary mixture has given evidence of its anomalous nature at low mole fractions of DMSO (below XD = 0.4) in our systematic investigation. Upon excitation of neutral D-luciferin molecule, dual fluorescence emissions (protonated and deprotonated form) are observed in DMSO-water binary mixture. A clear isoemissive point in the time-resolved area normalized emission spectra further indicates two emissive species in the excited state of D-luciferin in DMSO-water binary mixture. DMSO-water binary mixtures of different compositions are fascinating hydrogen bonding systems. Therefore, we have observed unusual changes in the fluorescence emission intensity, fluorescence quantum yield, and fluorescence lifetime of more hydrogen bonding sensitive anionic form of D-luciferin in low DMSO content of DMSO-water binary mixture.

Terahertz-Radiation-Enhanced Emission of Fluorescence from Gas Plasma

NASA Astrophysics Data System (ADS)

Liu, Jingle; Zhang, X.-C.

2009-12-01

We report the study of femtosecond laser-induced air plasma fluorescence under the illumination of terahertz (THz) pulses. Semiclassical modeling and experimental verification indicate that time-resolved THz radiation-enhanced emission of fluorescence is dominated by the electron kinetics and the electron-impact excitation of gas molecules or ions. We demonstrate that the temporal waveform of the THz field could be retrieved from the transient enhanced fluorescence, making omnidirectional, coherent detection available for THz time-domain spectroscopy.

Fluorescence lifetime in cardiovascular diagnostics

NASA Astrophysics Data System (ADS)

Marcu, Laura

2010-01-01

We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications.

Fluorescence lifetime in cardiovascular diagnostics.

PubMed

Marcu, Laura

2010-01-01

We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications.

Fluorescence spectroscopy and imaging for noninvasive diagnostics: applications to early cancer detection in the lung

NASA Astrophysics Data System (ADS)

Mycek, Mary-Ann; Urayama, Paul; Zhong, Wei; Sloboda, Roger D.; Dragnev, Konstantin H.; Dmitrovsky, Ethan

2003-10-01

Tissue fluorescence spectroscopy and imaging are being investigated as potential methods for non-invasive detection of pre-neoplastic change in the lung and other organ systems. A substantial contribution to tissue fluorescence is known to arise from endogenous cellular fluorophores. Using steady-state and time-resolved fluorescence spectroscopy and imaging, we characterized the endogenous fluorescence properties of immortalized and carcinogen-transformed human bronchial epithelial cells. Non-invasive sensing of endogenous molecular biomarkers associated with human bronchial pre-neoplasia will be discussed.

Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging

PubMed Central

Spagnol, Stephen T.; Dahl, Kris Noel

2016-01-01

The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes. PMID:26765322

Spectrally resolving and scattering-compensated x-ray luminescence/fluorescence computed tomography

PubMed Central

Cong, Wenxiang; Shen, Haiou; Wang, Ge

2011-01-01

The nanophosphors, or other similar materials, emit near-infrared (NIR) light upon x-ray excitation. They were designed as optical probes for in vivo visualization and analysis of molecular and cellular targets, pathways, and responses. Based on the previous work on x-ray fluorescence computed tomography (XFCT) and x-ray luminescence computed tomography (XLCT), here we propose a spectrally-resolving and scattering-compensated x-ray luminescence/fluorescence computed tomography (SXLCT or SXFCT) approach to quantify a spatial distribution of nanophosphors (other similar materials or chemical elements) within a biological object. In this paper, the x-ray scattering is taken into account in the reconstruction algorithm. The NIR scattering is described in the diffusion approximation model. Then, x-ray excitations are applied with different spectra, and NIR signals are measured in a spectrally resolving fashion. Finally, a linear relationship is established between the nanophosphor distribution and measured NIR data using the finite element method and inverted using the compressive sensing technique. The numerical simulation results demonstrate the feasibility and merits of the proposed approach. PMID:21721815

Development of 2D imaging of SXR plasma radiation by means of GEM detectors

NASA Astrophysics Data System (ADS)

Chernyshova, M.; Czarski, T.; Jabłoński, S.; Kowalska-Strzeciwilk, E.; Poźniak, K.; Kasprowicz, G.; Zabołotny, W.; Wojeński, A.; Byszuk, A.; Burza, M.; Juszczyk, B.; Zienkiewicz, P.

2014-11-01

Presented 2D gaseous detector system has been developed and designed to provide energy resolved fast dynamic plasma radiation imaging in the soft X-Ray region with 0.1 kHz exposure frequency for online, made in real time, data acquisition (DAQ) mode. The detection structure is based on triple Gas Electron Multiplier (GEM) amplification structure followed by the pixel readout electrode. The efficiency of detecting unit was adjusted for the radiation energy region of tungsten in high-temperature plasma, the main candidate for the plasma facing material for future thermonuclear reactors. Here we present preliminary laboratory results and detector parameters obtained for the developed system. The operational characteristics and conditions of the detector were designed to work in the X-Ray range of 2-17 keV. The detector linearity was checked using the fluorescence lines of different elements and was found to be sufficient for good photon energy reconstruction. Images of two sources through various screens were performed with an X-Ray laboratory source and 55Fe source showing a good imaging capability. Finally offline stream-handling data acquisition mode has been developed for the detecting system with timing down to the ADC sampling frequency rate (~13 ns), up to 2.5 MHz of exposure frequency, which could pave the way to invaluable physics information about plasma dynamics due to very good time resolving ability. Here we present results of studied spatial resolution and imaging properties of the detector for conditions of laboratory moderate counting rates and high gain.

Experimental Study of the NaK 3(1)Pi State.

PubMed

Laub; Mazsa; Webb; La Civita J; Prodan; Jabbour; Namiotka; Huennekens

1999-02-01

We report the results of an optical-optical double resonance experiment to determine the NaK 3(1)Pi state potential energy curve. In the first step, a narrow band cw dye laser (PUMP) is tuned to line center of a particular 2(A)1Sigma+(v', J') <-- 1(X)1Sigma+(v", J") transition, and its frequency is then fixed. A second narrowband tunable cw Ti:Sapphirelaser (PROBE) is then scanned, while 3(1)Pi --> 1(X)1Sigma+ violet fluorescence is monitored. The Doppler-free signals accurately map the 3(1)Pi(v, J) ro-vibrational energy levels. These energy levels are then fit to a Dunham expansion to provide a set of molecular constants. The Dunham constants, in turn, are used to construct an RKR potential curve. Resolved 3(1)Pi(v, J) --> 1(X)1Sigma+(v", J") fluorescence scans are also recorded with both PUMP and PROBE laser frequencies fixed. Comparison between observed and calculated Franck-Condon factors is used to determine the absolute vibrational numbering of the 3(1)Pi state levels and to determine the variation of the 3(1)Pi --> 1(X)1Sigma+ transitiondipole moment with internuclear separation. The recent theoretical calculation of the NaK 3(1)Pi state potential reported by Magnier and Millié (1996, Phys. Rev. A 54, 204) is in excellent agreement with the present experimental RKR curve. Copyright 1999 Academic Press.

Experimental Study of the NaK 3 1Π State

NASA Astrophysics Data System (ADS)

Laub, E.; Mazsa, I.; Webb, S. C.; La Civita, J.; Prodan, I.; Jabbour, Z. J.; Namiotka, R. K.; Huennekens, J.

1999-02-01

We report the results of an optical-optical double resonance experiment to determine the NaK 31Π state potential energy curve. In the first step, a narrow band cw dye laser (PUMP) is tuned to line center of a particular 2(A)1Σ+(v‧,J‧) ← 1(X)1Σ+(v",J") transition, and its frequency is then fixed. A second narrowband tunable cw Ti:Sapphirelaser (PROBE) is then scanned, while 31Π → 1(X)1Σ+violet fluorescence is monitored. The Doppler-free signals accurately map the 31Π(v,J) ro-vibrational energy levels. These energy levels are then fit to a Dunham expansion to provide a set of molecular constants. The Dunham constants, in turn, are used to construct an RKR potential curve. Resolved 31Π(v,J) → 1(X)1Σ+(v",J") fluorescence scans are also recorded with both PUMP and PROBE laser frequencies fixed. Comparison between observed and calculated Franck-Condon factors is used to determine the absolute vibrational numbering of the 31Π state levels and to determine the variation of the 31Π → 1(X)1Σ+transitiondipole moment with internuclear separation. The recent theoretical calculation of the NaK 31Π state potential reported by Magnier and Millié (1996,Phys. Rev. A54, 204) is in excellent agreement with the present experimental RKR curve.

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma.

PubMed

Meier, Jeremy D; Xie, Hongtao; Sun, Yang; Sun, Yinghua; Hatami, Nisa; Poirier, Brian; Marcu, Laura; Farwell, D Gregory

2010-06-01

The objectives of this study were to 1) determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract, and 2) evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). Cross-sectional study. University-based medical center. Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700-picosecond pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared with the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than that of normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360 to approximately 660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7 +/- 0.06 ns [not significant] for normal and 1.3 +/- 0.06 ns for tumor, P = 0.0025) and the second-order Laguerre expansion coefficient (LEC-2) (i.e., at 460 nm: 0.135 +/- 0.001 for normal and 0.155 +/- 0.007 for tumor, P < 0.05). These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a noninvasive diagnostic technique for HNSCC. Copyright 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma

PubMed Central

Meier, Jeremy D.; Xie, Hongtao; Sun, Yang; Sun, Yinghua; Hatami, Nisa; Poirier, Brian; Marcu, Laura; Farwell, D. Gregory

2011-01-01

OBJECTIVE 1) Determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract. 2) Evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN Cross-sectional study. SETTING University-based medical center. SUBJECTS AND METHODS Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700 ps pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared to the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. RESULTS Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than the normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360~660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7±0.06 ns for normal and 1.3±0.06 ns for tumor, P=0.0025), and the Laguerre coefficients, LEC-2 (i.e., at 460 nm: 0.135±0.001 for normal and 0.155±0.007 for tumor, P<0.05). CONCLUSION These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a non-invasive diagnostic technique for HNSCC. PMID:20493355

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

PubMed Central

Marriott, G.; Heidecker, M.; Diamandis, E. P.; Yan-Marriott, Y.

1994-01-01

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found. Through the coupling of SBMC to streptavidin,a plethora of biotin-based tracer molecules are available for immunocytochemical studies. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:7811952

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.

Time-resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications.

PubMed

Lassiter, S J; Stryjewski, W; Legendre, B L; Erdmann, R; Wahl, M; Wurm, J; Peterson, R; Middendorf, L; Soper, S A

2000-11-01

A compact time-resolved near-IR fluorescence imager was constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 microns) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnano-second lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in approximately 6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of approximately 0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used to construct the decay profiles. Comparison of the lifetime base calling to a single-dye/four-lane sequencing strategy indicated similar results in terms of miscalls, but reduced insertion and deletion errors using lifetime identification methods, improving the overall read accuracy.

Site-Dependent Fluorescence Decay of Malachite Green Doped in Onion Cell

NASA Astrophysics Data System (ADS)

Nakatsuka, Hiroki; Sekine, Masaya; Suzuki, Yuji; Hattori, Toshiaki

1999-03-01

Time-resolved fluorescence measurements of malachite green dye moleculesdoped in onion cells were carried out.The fluorescence decay time was dependent on the individual cell and on theposition of the dye in a cell, which reflect the microscopic dynamics of each boundsite.Upon cooling, the decay time increased and this increase was accelerated ataround the freezing point of the onion cell.

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

PubMed

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

2007-01-15

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

Spatially resolved density and ionization measurements of shocked foams using x-ray fluorescence

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

MacDonald, M. J.; Keiter, P. A.; Montgomery, D. S.

2016-09-28

We present experiments at the Trident laser facility demonstrating the use of x-ray fluorescence (XRF) to simultaneously measure density, ionization state populations, and electron temperature in shocked foams. An imaging x-ray spectrometer obtained spatially resolved measurements of Ti K-α emission. Density profiles were measured from K-α intensity. Ti ionization state distributions and electron temperatures were inferred by fitting K-α spectra to spectra from CRETIN simulations. This work shows that XRF provides a powerful tool to complement other diagnostics to make equation of state measurements of shocked materials containing a suitable tracer element.

Thermal and Denaturation Studies of the Time-Resolved Fluorescence Decay of Human Superoxide Dismutase

NASA Astrophysics Data System (ADS)

Silva, Norberto De Jesus

Previous studies have shown that time-resolved fluorescence decay of various single tryptophan proteins is best described by a distribution of fluorescence lifetimes rather than one or two lifetimes. The thermal dependence of the lifetime distributions is consistent with the hypothesis that proteins fluctuate between a hierarchy of many conformational substates. With this scenario as a theoretical framework, the correlations between protein dynamic and structure are investigated by studying the time-resolved fluorescence and anisotropy decay of the single tryptophan (Trp) residue of human superoxide dismutase (HSOD) over a wide range of temperatures and at different denaturant concentrations. First, it is demonstrated that the center of the lifetime distribution can characterize the average deactivation environment of the excited Trp-protein system. A qualitative model is introduced to explain the time-resolved fluorescence decay of HSOD in 80% glycerol over a wide range of temperatures. The dynamical model features isoenergetic conformational substates separated by a hierarchy of energy barriers. The HSOD system is also investigated as a function of denaturant concentration in aqueous solution. As a function of guanidine hydrochloride (GdHCl), the width of the fluorescence lifetime distribution of HSOD displays a maximum which is not coincident with the fully denatured form of HSOD at 6.5M GdHCl. Furthermore, the width for the fully denatured form of HSOD is greater than that of the native form. This is consistent with the scenario that more conformational substates are being created upon denaturation of HSOD. HSOD is a dimeric protein and it was observed that the width of the lifetime distribution of HSOD at intermediate GdHCl concentrations increased with decreasing protein concentration. In addition, the secondary structure of HSOD at intermediate GdHCl concentration does not change with protein concentration. These results suggest that HSOD display structural microheterogeneity which is consistent with the hypothesis of conformational substates. Further analysis show that, during denaturation, the monomeric form of HSOD is an intermediate which displays native-like secondary structure and fluctuating tertiary structure; i.e., the monomeric form of HSOD is a molten globule.

Combining Graphical and Analytical Methods with Molecular Simulations To Analyze Time-Resolved FRET Measurements of Labeled Macromolecules Accurately

PubMed Central

2017-01-01

Förster resonance energy transfer (FRET) measurements from a donor, D, to an acceptor, A, fluorophore are frequently used in vitro and in live cells to reveal information on the structure and dynamics of DA labeled macromolecules. Accurate descriptions of FRET measurements by molecular models are complicated because the fluorophores are usually coupled to the macromolecule via flexible long linkers allowing for diffusional exchange between multiple states with different fluorescence properties caused by distinct environmental quenching, dye mobilities, and variable DA distances. It is often assumed for the analysis of fluorescence intensity decays that DA distances and D quenching are uncorrelated (homogeneous quenching by FRET) and that the exchange between distinct fluorophore states is slow (quasistatic). This allows us to introduce the FRET-induced donor decay, εD(t), a function solely depending on the species fraction distribution of the rate constants of energy transfer by FRET, for a convenient joint analysis of fluorescence decays of FRET and reference samples by integrated graphical and analytical procedures. Additionally, we developed a simulation toolkit to model dye diffusion, fluorescence quenching by the protein surface, and FRET. A benchmark study with simulated fluorescence decays of 500 protein structures demonstrates that the quasistatic homogeneous model works very well and recovers for single conformations the average DA distances with an accuracy of < 2%. For more complex cases, where proteins adopt multiple conformations with significantly different dye environments (heterogeneous case), we introduce a general analysis framework and evaluate its power in resolving heterogeneities in DA distances. The developed fast simulation methods, relying on Brownian dynamics of a coarse-grained dye in its sterically accessible volume, allow us to incorporate structural information in the decay analysis for heterogeneous cases by relating dye states with protein conformations to pave the way for fluorescence and FRET-based dynamic structural biology. Finally, we present theories and simulations to assess the accuracy and precision of steady-state and time-resolved FRET measurements in resolving DA distances on the single-molecule and ensemble level and provide a rigorous framework for estimating approximation, systematic, and statistical errors. PMID:28709377

Global and time-resolved monitoring of crop photosynthesis with chlorophyll fluorescence

USDA-ARS?s Scientific Manuscript database

Global monitoring of agricultural productivity is critical in a world under a continuous increase of food demand. Here we have used new spaceborne retrievals of chlorophyll fluorescence, an emission quantity intrinsically linked to photosynthesis, to derive spatially explicit photosynthetic uptake r...

Selective time-resolved binding of copper(II) by pyropheophorbide-a methyl ester.

PubMed

Ghosh, Indrajit; Saleh, Na'il; Nau, Werner M

2010-05-01

The complexation behavior of pyropheophorbide-a methyl ester (PPME) with transition metal ions as well as other biologically relevant metal ions has been investigated in water-DMF (2 : 1 v/v) solution. PPME was found to selectively complex Cu(2+) ions, which leads to a distinct change in its absorption spectrum as well as efficient fluorescence quenching. The degree of fluorescence quenching by Cu(2+) depended on concentration and time. Upon addition of Cu(2+), the fluorescence showed a time-resolved decay on the time scale of minutes to hours, with the decay rate being dependent on the cation concentration. Fitting according to a bimolecular reaction rate law provided a rate constant of 650 +/- 90 M(-1) s(-1) at 298 K for metallochlorin formation. The potential implications of Cu(2+) binding for the use of PPME in photodynamic therapy are discussed, along with its use as a fluorescent sensor for detection of micromolar concentrations of Cu(2+).

Sizing protein-templated gold nanoclusters by time resolved fluorescence anisotropy decay measurements

NASA Astrophysics Data System (ADS)

Soleilhac, Antonin; Bertorelle, Franck; Antoine, Rodolphe

2018-03-01

Protein-templated gold nanoclusters (AuNCs) are very attractive due to their unique fluorescence properties. A major problem however may arise due to protein structure changes upon the nucleation of an AuNC within the protein for any future use as in vivo probes, for instance. In this work, we propose a simple and reliable fluorescence based technique measuring the hydrodynamic size of protein-templated gold nanoclusters. This technique uses the relation between the time resolved fluorescence anisotropy decay and the hydrodynamic volume, through the rotational correlation time. We determine the molecular size of protein-directed AuNCs, with protein templates of increasing sizes, e.g. insulin, lysozyme, and bovine serum albumin (BSA). The comparison of sizes obtained by other techniques (e.g. dynamic light scattering and small-angle X-ray scattering) between bare and gold clusters containing proteins allows us to address the volume changes induced either by conformational changes (for BSA) or the formation of protein dimers (for insulin and lysozyme) during cluster formation and incorporation.

Chain-Length-Dependent Exciton Dynamics in Linear Oligothiophenes Probed Using Ensemble and Single-Molecule Spectroscopy.

PubMed

Kim, Tae-Woo; Kim, Woojae; Park, Kyu Hyung; Kim, Pyosang; Cho, Jae-Won; Shimizu, Hideyuki; Iyoda, Masahiko; Kim, Dongho

2016-02-04

Exciton dynamics in π-conjugated molecular systems is highly susceptible to conformational disorder. Using time-resolved and single-molecule spectroscopic techniques, the effect of chain length on the exciton dynamics in a series of linear oligothiophenes, for which the conformational disorder increased with increasing chain length, was investigated. As a result, extraordinary features of the exciton dynamics in longer-chain oligothiophene were revealed. Ultrafast fluorescence depolarization processes were observed due to exciton self-trapping in longer and bent chains. Increase in exciton delocalization during dynamic planarization processes was also observed in the linear oligothiophenes via time-resolved fluorescence spectra but was restricted in L-10T because of its considerable conformational disorder. Exciton delocalization was also unexpectedly observed in a bent chain using single-molecule fluorescence spectroscopy. Such delocalization modulates the fluorescence spectral shape by attenuating the 0-0 peak intensity. Collectively, these results provide significant insights into the exciton dynamics in conjugated polymers.

Distance distributions of short polypeptides recovered by fluorescence resonance energy transfer in the 10 A domain.

PubMed

Sahoo, Harekrushna; Roccatano, Danilo; Zacharias, Martin; Nau, Werner M

2006-06-28

Fluorescence resonance energy transfer (FRET) between tryptophan (Trp) as donor and 2,3-diazabicyclo[2.2.2]oct-2-ene (Dbo) as acceptor was studied by steady-state and time-resolved fluorescence spectroscopy. The unique feature of this FRET pair is its exceptionally short Förster radius (10 A), which allows one to recover distance distributions in very short structureless peptides. The technique was applied to Trp-(GlySer)n-Dbo-NH2 peptides with n = 0-10, for which the average probe/quencher distance ranged between 8.7 and 13.7 A experimentally (in propylene glycol, analysis according to wormlike chain model) and 8.6-10.2 A theoretically (for n = 0-6, GROMOS96 molecular dynamics simulations). The larger FRET efficiency in steady-state compared to time-resolved fluorescence experiments was attributed to a static quenching component, suggesting that a small but significant part (ca. 10%) of the conformations are already in van der Waals contact when excitation occurs.

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

A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy.

PubMed

Tiwari, Dhermendra K; Arai, Yoshiyuki; Yamanaka, Masahito; Matsuda, Tomoki; Agetsuma, Masakazu; Nakano, Masahiro; Fujita, Katsumasa; Nagai, Takeharu

2015-06-01

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

Using resolvent analysis for the design of separation control on a NACA 0012 airfoil

NASA Astrophysics Data System (ADS)

Yeh, Chi-An; Taira, Kunihiko

2017-11-01

A combined effort based on large-eddy simulation and resolvent analysis on the separated flow over a NACA 0012 airfoil is conducted to design active flow control for suppression of separation. This study considers the the airfoil at 6 deg. angle-of-attack and Reynolds number of 23000. The response mode obtained from the resolvent analysis about the baseline turbulent mean flow reveals modal structures that can be categorized into three families when sweeping through the resonant frequency: (1) von Karman wake structure for low frequency; (2) Kelvin-Helmholtz structure in the separation bubble for high frequency; (3) blended structure of (1) and (2) for the intermediate frequency. Leveraging the insights from resolvent analysis, unsteady thermal actuation is introduced to the flow near the leading-edge to examine the use of the frequencies from three families for separation control in LES. As indicated by the resolvent response modes, we find that the use of intermediate frequencies are most effective in suppressing the flow separation, since the shear layer over the separation bubble and the wake are both receptive to the perturbation at the these frequencies. The resolvent-analysis-based control strategy achieves 35% drag reduction and 9% lift increase with effective frequency. This work was supported by Office of Naval Research (N00014-15-R-FO13) and Army Research Office (W911NF-14-1-0224).

Atomic oxygen production scaling in a nanosecond-pulsed externally grounded dielectric barrier plasma jet

NASA Astrophysics Data System (ADS)

Sands, Brian; Schmidt, Jacob; Ganguly, Biswa; Scofield, James

2014-10-01

Atomic oxygen production is studied in a capillary dielectric barrier plasma jet that is externally grounded and driven with a 20-ns risetime positive unipolar pulsed voltage at pulse repetition rates up to 25 kHz. The power coupled to the discharge can be easily increased by increasing the pulse repetition rate. At a critical turnover frequency, determined by the net energy density coupled to the discharge, the plasma chemistry abruptly changes. This is indicated by increased plasma conductance and a transition in reactive oxygen species production from an ozone-dominated production regime below the turnover frequency to atomic-oxygen-dominated production at higher pulse rates. Here, we characterize atomic oxygen production scaling using spatially- and temporally-resolved two-photon absorption laser-induced-fluorescence (TALIF). Quantitative results are obtained via calibration with xenon using a similar laser excitation and collection system. These results are compared with quantitative ozone and discharge power measurements using a helium gas flow with oxygen admixtures up to 3%.

Study on the conformation changes of Lysozyme induced by Hypocrellin A: The mechanism investigation

NASA Astrophysics Data System (ADS)

Ma, Fei; Huang, He-Yong; Zhou, Lin; Yang, Chao; Zhou, Jia-Hong; Liu, Zheng-Ming

2012-11-01

The interactions between Lysozyme and Hypocrellin A are investigated in details using time-resolved fluorescence, fourier transform infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), three-dimensional fluorescence spectra, and thermal gravimetric analysis (TGA) techniques. The results of time-resolved fluorescence suggest that the quenching mechanism is static quenching. FTIR and CD spectroscopy provide evidences of the reducing of α-helix after interaction. Hypocrellin A could change the micro-environmental of Lysozyme according to hydrophobic interaction between the aromatic ring and the hydrophobic amino acid residues, and the altered polypeptide backbone structures induce the reduction of α-helical structures. Moreover, TGA study further demonstrates the structure changes of Lysozyme on the effect of Hypocrellin A. This study could provide some important information for the derivatives of HA in pharmacy, pharmacology and biochemistry.

Rotationally resolved fluorescence spectroscopy of molecular iodine

NASA Astrophysics Data System (ADS)

Lemon, Christopher; Canagaratna, Sebastian; Gray, Jeffrey

2008-03-01

Vibration-electronic spectroscopy of I2 vapor is a common, important experiment in physical chemistry lab courses. We use narrow bandwidth diode-pumped solid state (DPSS) lasers to excite specific rotational levels; these lasers are surprisingly stable and are now available at low cost. We also use efficient miniature fiber-optic spectrometers to resolve rotational fluorescence patterns in a vibrational progression. The resolution enables thorough and accurate analysis of spectroscopic constants for the ground electronic state. The high signal-to-noise ratio, which is easily achieved, also enables students to precisely measure fluorescence band intensities, providing further insight into vibrational wavefunctions and the molecular potential function. We will provide a detailed list of parts for the apparatus as well as modeling algorithms with statistical evaluation to facilitate widespread adoption of these experimental improvements by instructors of intermediate and advanced lab courses.

Time-resolved stimulated emission depletion and energy transfer dynamics in two-photon excited EGFP.

PubMed

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

2018-04-07

Time and polarization-resolved stimulated emission depletion (STED) measurements are used to investigate excited state evolution following the two-photon excitation of enhanced green fluorescent protein (EGFP). We employ a new approach for the accurate STED measurement of the hitherto unmeasured degree of hexadecapolar transition dipole moment alignment α 40 present at a given excitation-depletion (pump-dump) pulse separation. Time-resolved polarized fluorescence measurements as a function of pump-dump delay reveal the time evolution of α 40 to be considerably more rapid than predicted for isotropic rotational diffusion in EGFP. Additional depolarization by homo-Förster resonance energy transfer is investigated for both α 20 (quadrupolar) and α 40 transition dipole alignments. These results point to the utility of higher order dipole correlation measurements in the investigation of resonance energy transfer processes.

340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays.

PubMed

Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Petersen, Paul Michael; Pedersen, Christian

2016-09-19

We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng/L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flash lamp and the LED based system and discussed temporal, spatial, and spectral features of the LED excitation for time-resolved fluorimetry. Optimization of the suggested key parameters of the LED promises significant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems.

Photophysical studies on the interaction of acridinedione dyes with universal protein denaturant: guanidine hydrochloride.

PubMed

Kumaran, R; Varalakshmi, T; Malar, E J Padma; Ramamurthy, P

2010-09-01

Photophysical studies of photoinduced electron transfer (PET) and non-PET based acridinedione dyes with guanidine hydrochloride (GuHCl) were carried out in water and methanol. Addition of GuHCl to photoinduced electron transfer (PET) based acridinedione dye (ADR 1) results in a fluorescence enhancement, whereas a non-PET based dye (ADR 2) shows no significant change in the fluorescence intensity and lifetime. Addition of GuHCl to ADR 1 dye in methanol results in single exponential decay behaviour, on the contrary a biexponential decay pattern was observed on the addition of GuHCl in water. Absorption and emission spectral studies of ADR 1 dye interaction with GuHCl reveals that the dye molecule is not in the protonated form in aqueous GuHCl solution, and the dye is confined to two distinguishable microenvironment in the aqueous phase. A large variation in the microenvironment around the dye molecule is created on the addition of GuHCl and this was ascertained by time-resolved area normalized emission spectroscopy (TRANES) and time-resolved emission spectroscopy (TRES). The dye molecule prefers to reside in the hydrophobic microenvironment, rather in the hydrophilic aqueous phase is well emphasized by time-resolved fluorescence lifetime studies. The mechanism of fluorescence enhancement of ADR 1 dye by GuHCl is attributed to the suppression of the PET process occurring through space.

An integrated logic system for time-resolved fluorescent "turn-on" detection of cysteine and histidine base on terbium (III) coordination polymer-copper (II) ensemble.

PubMed

Xue, Shi-Fan; Lu, Ling-Fei; Wang, Qi-Xian; Zhang, Shengqiang; Zhang, Min; Shi, Guoyue

2016-09-01

Cysteine (Cys) and histidine (His) both play indispensable roles in many important biological activities. An enhanced Cys level can result in Alzheimer's and cardiovascular diseases. Likewise, His plays a significant role in the growth and repair of tissues as well as in controlling the transmission of metal elements in biological bases. Therefore, it is meaningful to detect Cys and His simultaneously. In this work, a novel terbium (III) coordination polymer-Cu (II) ensemble (Tb(3+)/GMP-Cu(2+)) was proposed. Guanosine monophosphate (GMP) can self-assemble with Tb(3+) to form a supramolecular Tb(3+) coordination polymer (Tb(3+)/GMP), which can be suited as a time-resolved probe. The fluorescence of Tb(3+)/GMP would be quenched upon the addition of Cu(2+), and then the fluorescence of the as-prepared Tb(3+)/GMP-Cu(2+) ensemble would be restored again in the presence of Cys or His. By incorporating N-Ethylmaleimide and Ni(2+) as masking agents, Tb(3+)/GMP-Cu(2+) was further exploited as an integrated logic system and a specific time-resolved fluorescent "turn-on" assay for simultaneously sensing His and Cys was designed. Meanwhile it can also be used in plasma samples, showing great potential to meet the need of practical application. Copyright © 2016 Elsevier B.V. All rights reserved.

Two-site ionic labeling with pyranine: implications for structural dynamics studies of polymers and polypeptides by time-resolved fluorescence anisotropy.

PubMed

Sharma, Jai; Tleugabulova, Dina; Czardybon, Wojciech; Brennan, John D

2006-04-26

Time-resolved fluorescence anisotropy (TRFA) is widely used to study dynamic motions of biomolecules in a variety of environments. However, depolarization due to rapid side chain motions often complicates the interpretation of anisotropy decay data and interferes with the accurate observation of segmental motions. Here, we demonstrate a new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene (pyranine). TRFA analysis shows that such labeling provides a more rigid attachment of the fluorophore to the macromolecule than the covalent or single-point ionic labeling of amino groups, leading to time-resolved anisotropy decays that better reflect the backbone motion of the labeled polymer segment. Optimal coupling of pyranine to biomolecule dynamics is shown to be obtained for appropriately spaced Arg groups, and in such cases the ionic binding is stable up to 150 mM ionic strength. TRFA was used to monitor the behavior of pyranine-labeled poly(allylamine) (PAM) and poly-d-lysine (PL) in sodium silicate derived sol-gel materials and revealed significant restriction of backbone motion upon entrapment for both polymers, an observation that was not readily apparent in a previous study with entrapped fluorescein-labeled PAM and PL. The implications of these findings for fluorescence studies of polymer and biomolecule dynamics are discussed.

Comparison between two time-resolved approaches for prostate cancer diagnosis: high rate imager vs. photon counting system

NASA Astrophysics Data System (ADS)

Boutet, J.; Debourdeau, M.; Laidevant, A.; Hervé, L.; Dinten, J.-M.

2010-02-01

Finding a way to combine ultrasound and fluorescence optical imaging on an endorectal probe may improve early detection of prostate cancer. A trans-rectal probe adapted to fluorescence diffuse optical tomography measurements was developed by our team. This probe is based on a pulsed NIR laser source, an optical fiber network and a time-resolved detection system. A reconstruction algorithm was used to help locate and quantify fluorescent prostate tumors. In this study, two different kinds of time-resolved detectors are compared: High Rate Imaging system (HRI) and a photon counting system. The HRI is based on an intensified multichannel plate and a CCD Camera. The temporal resolution is obtained through a gating of the HRI. Despite a low temporal resolution (300ps), this system allows a simultaneous acquisition of the signal from a large number of detection fibers. In the photon counting setup, 4 photomultipliers are connected to a Time Correlated Single Photon Counting (TCSPC) board, providing a better temporal resolution (0.1 ps) at the expense of a limited number of detection fibers (4). At last, we show that the limited number of detection fibers of the photon counting setup is enough for a good localization and dramatically improves the overall acquisition time. The photon counting approach is then validated through the localization of fluorescent inclusions in a prostate-mimicking phantom.

Novel flashlamp-based time-resolved fluorescence microscope reduces autofluorescence for 30-fold contrast enhancement in environmental samples

NASA Astrophysics Data System (ADS)

Connally, Russell; Veal, Duncan; Piper, James A.

2003-07-01

The abundance of naturally fluorescing components (autofluorophors) encountered in environmentally sourced samples can greatly hinder the detection and identification of fluorescently labeled target using fluorescence microscopy. Time-resolved fluorescence microscopy (TRFM) is a technique that reduces the effects of autofluorescence through precisely controlled time delays. Lanthanide chelates have fluorescence lifetimes many orders of magnitude greater than typical autofluorophors, and persist in their luminescence long after autofluorescence has ceased. An intense short pulse of (UV) light is used to excite fluorescence in the sample and after a short delay period the longer persisting fluorescence from the chelate is captured with an image-intensified CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flashlamp with a short pulse duration was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, flash output decays with an approximate lifetime of 18μs and the TRFM requires a long-lived chelate to ensure probe fluorescence is still visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the water-borne parasite Giardia lamblia. Fluorescence lifetime of the construct was determined to be 339μs +/- 14μs and provided a 45-fold enhancement of labeled Giardia over background using a gate delay of 100μs. Despite the sub-optimal decay characteristics of the light pulse, flashlamps have many advantages compared to optical chopper wheels and modulated lasers. Their low cost, lack of vibration, ease of interface and small footprint are important factors to consider in TRFM design.

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.

Studies of Minerals, Organic and Biogenic Materials through Time-Resolved Raman Spectroscopy

NASA Technical Reports Server (NTRS)

Garcia, Christopher S.; Abedin, M. Nurul; Ismail, Syed; Sharma, Shiv K.; Misra, Anupam K.; Nyugen, Trac; Elsayed-Ali, hani

2009-01-01

A compact remote Raman spectroscopy system was developed at NASA Langley Research center and was previously demonstrated for its ability to identify chemical composition of various rocks and minerals. In this study, the Raman sensor was utilized to perform time-resolved Raman studies of various samples such as minerals and rocks, Azalea leaves and a few fossil samples. The Raman sensor utilizes a pulsed 532 nm Nd:YAG laser as excitation source, a 4-inch telescope to collect the Raman-scattered signal from a sample several meters away, a spectrograph equipped with a holographic grating, and a gated intensified CCD (ICCD) camera system. Time resolved Raman measurements were carried out by varying the gate delay with fixed short gate width of the ICCD camera, allowing measurement of both Raman signals and fluorescence signals. Rocks and mineral samples were characterized including marble, which contain CaCO3. Analysis of the results reveals the short (approx.10-13 s) lifetime of the Raman process, and shows that Raman spectra of some mineral samples contain fluorescence emission due to organic impurities. Also analyzed were a green (pristine) and a yellow (decayed) sample of Gardenia leaves. It was observed that the fluorescence signals from the green and yellow leaf samples showed stronger signals compared to the Raman lines. Moreover, it was also observed that the fluorescence of the green leaf was more intense and had a shorter lifetime than that of the yellow leaf. For the fossil samples, Raman shifted lines could not be observed due the presence of very strong short-lived fluorescence.

Hyperspectral imaging of endogenous fluorescent metabolic molecules to identify pain states in central nervous system tissue

NASA Astrophysics Data System (ADS)

Staikopoulos, Vasiliki; Gosnell, Martin E.; Anwer, Ayad G.; Mustafa, Sanam; Hutchinson, Mark R.; Goldys, Ewa M.

2016-12-01

Fluorescence-based bio-imaging methods have been extensively used to identify molecular changes occurring in biological samples in various pathological adaptations. Auto-fluorescence generated by endogenous fluorescent molecules within these samples can interfere with signal to background noise making positive antibody based fluorescent staining difficult to resolve. Hyperspectral imaging uses spectral and spatial imaging information for target detection and classification, and can be used to resolve changes in endogenous fluorescent molecules such as flavins, bound and free NADH and retinoids that are involved in cell metabolism. Hyperspectral auto-fluorescence imaging of spinal cord slices was used in this study to detect metabolic differences within pain processing regions of non-pain versus sciatic chronic constriction injury (CCI) animals, an established animal model of peripheral neuropathy. By using an endogenous source of contrast, subtle metabolic variations were detected between tissue samples, making it possible to distinguish between animals from non-injured and injured groups. Tissue maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant tissue regions with compromised mitochondrial function. Taken together, our results demonstrate that hyperspectral imaging provides a new non-invasive method to investigate central changes of peripheral neuropathic injury and other neurodegenerative disease models, and paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.

Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection.

PubMed

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

2007-12-26

The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

Heterogenous fluorescence decay of (4→6)-and (4→8)-linked dimers of (+)-catechin and (-) epicatechin as a result of rotational isomerism.

Treesearch

Wolfgang R. Bergmann; Mary D. Barkley; Richard W. Hemingway; Wayne Mattice

1987-01-01

The time-resolved fluorescence of (+)-catechin and ( -)-epicatechin decays as a single exponential. In contrast dimers formed from (+)-catechin and (-)-epicatechin have more complex decays unless rotation about the interflavan bond is constrained by the introduction of a new ring. The fluorescence decay in unconstrained dimers is adequately described by the sum of two...

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.

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

NASA Astrophysics Data System (ADS)

Rolinski, Olaf J.; McLaughlin, Damien; Birch, David J. S.; Vyshemirsky, Vladislav

2016-09-01

The fluorescence intensity decay of protein is easily measurable and reports on the intrinsic fluorophore-local environment interactions on the sub-nm spatial and sub-ns temporal scales, which are consistent with protein activity in numerous biomedical and industrial processes. This makes time-resolved fluorescence a perfect tool for understanding, monitoring and controlling these processes at the molecular level, but the complexity of the decay, which has been traditionally fitted to multi-exponential functions, has hampered the development of this technique over the last few decades. Using the example of tryptophan in HSA we present the alternative to the conventional approach to modelling intrinsic florescence intensity decay in protein where the key factors determining fluorescence decay, i.e. the excited-state depopulation and the dielectric relaxation (Toptygin and Brand 2000 Chem. Phys. Lett. 322 496-502), are represented by the individual relaxation functions. This allows quantification of both effects separately by determining their parameters from the global analysis of a series of fluorescence intensity decays measured at different detection wavelengths. Moreover, certain pairs of the recovered parameters of tryptophan were found to be correlated, indicating the influence of the dielectric relaxation on the transient rate of the electronic transitions. In this context the potential for the dual excited state depopulation /dielectric relaxation fluorescence lifetime sensing is discussed.

Time-resolved fluorescence polarization spectroscopy of visible and near infrared dyes in picosecond dynamics

NASA Astrophysics Data System (ADS)

Pu, Yang; Alfano, Robert R.

2015-03-01

Near-infrared (NIR) dyes absorb and emit light within the range from 700 to 900 nm have several benefits in biological studies for one- and/or two-photon excitation for deeper penetration of tissues. These molecules undergo vibrational and rotational motion in the relaxation of the excited electronic states, Due to the less than ideal anisotropy behavior of NIR dyes stemming from the fluorophores elongated structures and short fluorescence lifetime in picosecond range, no significant efforts have been made to recognize the theory of these dyes in time-resolved polarization dynamics. In this study, the depolarization of the fluorescence due to emission from rotational deactivation in solution will be measured with the excitation of a linearly polarized femtosecond laser pulse and a streak camera. The theory, experiment and application of the ultrafast fluorescence polarization dynamics and anisotropy are illustrated with examples of two of the most important medical based dyes. One is NIR dye, namely Indocyanine Green (ICG) and is compared with Fluorescein which is in visible range with much longer lifetime. A set of first-order linear differential equations was developed to model fluorescence polarization dynamics of NIR dye in picosecond range. Using this model, the important parameters of ultrafast polarization spectroscopy were identified: risetime, initial time, fluorescence lifetime, and rotation times.

Fluorescence lifetime in cardiovascular diagnostics

PubMed Central

Marcu, Laura

2010-01-01

We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications. PMID:20210432

Fluorescence diffuse tomography of small animals with DsRed2 fluorescent protein

NASA Astrophysics Data System (ADS)

Turchin, I. V.; Plehanov, V. I.; Orlova, A. G.; Kamenskiy, V. A.; Kleshnin, M. S.; Shirmanova, M. V.; Shakhova, N. M.; Balalaeva, I. V.; Savitskiy, A. P.

2006-05-01

Fluorescent compounds are used as markers to diagnose oncological diseases, to study molecular processes typical for carcinogenesis, and to investigate metastasis formation and tumor regress under the influence of therapeutics. Different types of tomography, such as continuous wave (CW), frequency-domain (FD), and time-domain (TD) tomography, allow fluorescence imaging of tumors located deep in human or animal tissue. In this work, preliminary results of the frequency domain fluorescent diffuse tomography (FDT) method in application to DsRed2 protein as a fluorescent agent are presented. For the first step of our experiments, we utilized low-frequency amplitude modulation (1 kHz) of second harmonic of Nd: YAG (532 nm). The transilluminative configuration was used in the setup. The results of post mortem experiments with capsules containing DsRed2 inserted inside the esophagus of a 3-day-old hairless rat to simulate tumor are shown. An algorithm of processing fluorescent images based on calculating the zero of maximum curvature has been applied to detect fluorescent inclusion boundaries in the image. This work demonstrates the potential capability of the FDT method for imaging deep fluorescent tumors in human tissue or animal models of human cancer. Improvement of the setup can be accomplished by using high-frequency modulation (using a 110-MHz acoustooptical modulator).

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

PubMed

Siegel, Nisan; Brooker, Gary

2014-09-22

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

Do fluorescence and transient absorption probe the same intramolecular charge transfer state of 4-(dimethylamino)benzonitrile?

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

Gustavsson, Thomas; Coto, Pedro B.; Serrano-Andres, Luis

2009-07-21

We present here the results of time-resolved absorption and emission experiments for 4-(dimethylamino)benzonitrile in solution, which suggest that the fluorescent intramolecular charge transfer (ICT) state may differ from the twisted ICT (TICT) state observed in transient absorption.

Portable, battery-operated, fluorescence field microscope for the developing world

NASA Astrophysics Data System (ADS)

Miller, Andrew R.; Davis, Gregory; Pierce, Mark; Oden, Z. Maria; Richards-Kortum, Rebecca

2010-02-01

In many areas of the world, current methods for diagnosis of infectious diseases such as malaria and tuberculosis involve microscopic evaluation of a patient specimen. Advances in fluorescence microscopy can improve diagnostic sensitivity and reduce time and expertise necessary to interpret diagnostic results. However, modern research-grade microscopes are neither available nor appropriate for use in many settings in the developing world. To address this need, we designed, fabricated, and tested a portable, battery-powered, bright field and fluorescence inverted field microscope, optimized for infrastructural constraints of the developing world. We characterized an initial prototype constructed with rapidprototyping techniques, which utilized low-cost, over-the-counter components such as a battery-powered LED flashlight as the light source. The microscope exhibited suitable spatial resolution (0.8 μm) in fluorescence mode to resolve M. tuberculosis bacilli. In bright field mode, malaria parasites were resolvable at 1000x magnification. The initial prototype cost 480 USD and we estimate that the microscope can be manufactured for 230 USD. While future studies are planned to evaluate ease-of-use and reliability, our current system serves as a proof of concept that combined fluorescence and bright field microscopy is possible in a low-cost and portable system.

Development of a Multi-modal Tissue Diagnostic System Combining High Frequency Ultrasound and Photoacoustic Imaging with Lifetime Fluorescence Spectroscopy

PubMed Central

Sun, Yang; Stephens, Douglas N.; Park, Jesung; Sun, Yinghua; Marcu, Laura; Cannata, Jonathan M.; Shung, K. Kirk

2010-01-01

We report the development and validate a multi-modal tissue diagnostic technology, which combines three complementary techniques into one system including ultrasound backscatter microscopy (UBM), photoacoustic imaging (PAI), and time-resolved laser-induced fluorescence spectroscopy (TR-LIFS). UBM enables the reconstruction of the tissue microanatomy. PAI maps the optical absorption heterogeneity of the tissue associated with structure information and has the potential to provide functional imaging of the tissue. Examination of the UBM and PAI images allows for localization of regions of interest for TR-LIFS evaluation of the tissue composition. The hybrid probe consists of a single element ring transducer with concentric fiber optics for multi-modal data acquisition. Validation and characterization of the multi-modal system and ultrasonic, photoacoustic, and spectroscopic data coregistration were conducted in a physical phantom with properties of ultrasound scattering, optical absorption, and fluorescence. The UBM system with the 41 MHz ring transducer can reach the axial and lateral resolution of 30 and 65 μm, respectively. The PAI system with 532 nm excitation light from a Nd:YAG laser shows great contrast for the distribution of optical absorbers. The TR-LIFS system records the fluorescence decay with the time resolution of ~300 ps and a high sensitivity of nM concentration range. Biological phantom constructed with different types of tissues (tendon and fat) was used to demonstrate the complementary information provided by the three modalities. Fluorescence spectra and lifetimes were compared to differentiate chemical composition of tissues at the regions of interest determined by the coregistered high resolution UBM and PAI image. Current results demonstrate that the fusion of these techniques enables sequentially detection of functional, morphological, and compositional features of biological tissue, suggesting potential applications in diagnosis of tumors and atherosclerotic plaques. PMID:21894259

Development of a Multi-modal Tissue Diagnostic System Combining High Frequency Ultrasound and Photoacoustic Imaging with Lifetime Fluorescence Spectroscopy.

PubMed

Sun, Yang; Stephens, Douglas N; Park, Jesung; Sun, Yinghua; Marcu, Laura; Cannata, Jonathan M; Shung, K Kirk

2008-01-01

We report the development and validate a multi-modal tissue diagnostic technology, which combines three complementary techniques into one system including ultrasound backscatter microscopy (UBM), photoacoustic imaging (PAI), and time-resolved laser-induced fluorescence spectroscopy (TR-LIFS). UBM enables the reconstruction of the tissue microanatomy. PAI maps the optical absorption heterogeneity of the tissue associated with structure information and has the potential to provide functional imaging of the tissue. Examination of the UBM and PAI images allows for localization of regions of interest for TR-LIFS evaluation of the tissue composition. The hybrid probe consists of a single element ring transducer with concentric fiber optics for multi-modal data acquisition. Validation and characterization of the multi-modal system and ultrasonic, photoacoustic, and spectroscopic data coregistration were conducted in a physical phantom with properties of ultrasound scattering, optical absorption, and fluorescence. The UBM system with the 41 MHz ring transducer can reach the axial and lateral resolution of 30 and 65 μm, respectively. The PAI system with 532 nm excitation light from a Nd:YAG laser shows great contrast for the distribution of optical absorbers. The TR-LIFS system records the fluorescence decay with the time resolution of ~300 ps and a high sensitivity of nM concentration range. Biological phantom constructed with different types of tissues (tendon and fat) was used to demonstrate the complementary information provided by the three modalities. Fluorescence spectra and lifetimes were compared to differentiate chemical composition of tissues at the regions of interest determined by the coregistered high resolution UBM and PAI image. Current results demonstrate that the fusion of these techniques enables sequentially detection of functional, morphological, and compositional features of biological tissue, suggesting potential applications in diagnosis of tumors and atherosclerotic plaques.

Image-guided intervention in the human bile duct using scanning fiber endoscope system

NASA Astrophysics Data System (ADS)

Seibel, Eric J.; Jo, Javier A.; Melville, C. David; Johnston, Richard S.; Naumann, Christopher R.; Saunders, Michael D.

2012-01-01

Bile duct cancers are increasing in frequency while being difficult to diagnose. Currently available endoscopic imaging devices used in the biliary tree are low resolution with poor image quality, leading to inadequate evaluation of indeterminate biliary strictures. However, a new ultrathin and flexible cholangioscope system has been successfully demonstrated in a human subject. This mini-cholangioscope system uses a scanning fiber endoscope (SFE) as a forward-imaging guidewire, dimensions of 1.2-mm diameter and 3-m length. Full color video (500-line resolution at 30Hz) is the standard SFE imaging mode using spiral scanning of red, green, and blue laser light at low power. Image-guided operation of the biopsy forceps was demonstrated in healthy human bile ducts with and without saline flushing. The laser-based video imaging can be switched to various modes to enhance tissue markers of disease, such as widefield fluorescence and enhanced spectral imaging. In parallel work, biochemical discrimination of tissue health in pig bile duct has been accomplished using fiberoptic delivery of pulsed UV illumination and time-resolved autofluorescence spectroscopic measurements. Implementation of time-resolved fluorescence spectroscopy for biochemical assessment of the bile duct wall is being done through a secondary endoscopic channel. Preliminary results indicate that adequate SNR levels (> 30 dB) can be achieved through a 50 micron fiber, which could serve as an optical biopsy probe. The SFE is an ideal mini-cholangioscope for integration of both tissue and molecular specific image contrast in the future. This will provide the physician with unprecedented abilities to target biopsy locations and perform endoscopically-guided therapies.

Spatially resolved density and ionization measurements of shocked foams using x-ray fluorescence

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

MacDonald, M. J.; Keiter, P. A.; Montgomery, D. S.

2016-09-22

We present experiments at the Trident laser facility demonstrating the use of x-ray fluorescence (XRF) to simultaneously measure density, ionization state populations, and electron temperature in shocked foams. An imaging x-ray spectrometer was used to obtain spatially-resolved measurements of Ti K-more » $$\\alpha$$ emission. Density profiles were measured from K-$$\\alpha$$ intensity. Ti ionization state distributions and electron temperatures were inferred by fitting K-$$\\alpha$$ spectra to spectra from CRETIN simulations. This study shows that XRF provides a powerful tool to complement other diagnostics to make equation of state measurements of shocked materials containing a suitable tracer element.« less

Investigations on the interactions of aurintricarboxylic acid with bovine serum albumin: Steady state/time resolved spectroscopic and docking studies.

PubMed

Bardhan, Munmun; Chowdhury, Joydeep; Ganguly, Tapan

2011-01-10

In this paper, the nature of the interactions between bovine serum albumin (BSA) and aurintricarboxylic acid (ATA) has been investigated by measuring steady state and time-resolved fluorescence, circular dichroism (CD), FT-IR and fluorescence anisotropy in protein environment under physiological conditions. From the analysis of the steady state and time-resolved fluorescence quenching of BSA in aqueous solution in presence of ATA it has been inferred that the nature of the quenching originates from the combined effect of static and dynamic modes. From the determination of the thermodynamic parameters obtained from temperature-dependent changes in K(b) (binding constant) it was apparent that the combined effect of hydrophobic association and electrostatic attraction is responsible for the interaction of ATA with BSA. The effect of ATA on the conformation of BSA has been examined by analyzing CD spectrum. Though the observed results demonstrate some conformational changes in BSA in presence of ATA but the secondary structure of BSA, predominantly of α-helix, is found to retain its identity. Molecular docking of ATA with BSA also indicates that ATA docks through hydrophobic interaction. Copyright © 2010 Elsevier B.V. All rights reserved.

Frequency-domain photoacoustic and fluorescence microscopy: application on labeled and unlabeled cells

NASA Astrophysics Data System (ADS)

Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Pfeffer, Karoline; Wohlfarth, Sven; Hannesschläger, Günther; Klar, Thomas A.; Berer, Thomas

2018-02-01

In this paper, multimodal optical-resolution frequency-domain photoacoustic and fluorescence scanning microscopy is presented on labeled and unlabeled cells. In many molecules, excited electrons relax radiatively and non-radiatively, leading to fluorescence and photoacoustic signals, respectively. Both signals can then be detected simultaneously. There also exist molecules, e.g. hemoglobin, which do not exhibit fluorescence, but provide photoacoustic signals solely. Other molecules, especially fluorescent dyes, preferentially exhibit fluorescence. The fluorescence quantum yield of a molecule and with it the strength of photoacoustic and fluorescence signals depends on the local environment, e.g. on the pH. Therefore, the local distribution of the simultaneously recorded photoacoustic and fluorescence signals may be used in order to obtain information about the local chemistry.

Rapid and economical data acquisition in ultrafast frequency-resolved spectroscopy using choppers and a microcontroller.

PubMed

Guo, Liang; Monahan, Daniele M; Fleming, Graham

2016-08-08

Spectrometers and cameras are used in ultrafast spectroscopy to achieve high resolution in both time and frequency domains. Frequency-resolved signals from the camera pixels cannot be processed by common lock-in amplifiers, which have only a limited number of input channels. Here we demonstrate a rapid and economical method that achieves the function of a lock-in amplifier using mechanical choppers and a programmable microcontroller. We demonstrate the method's effectiveness by performing a frequency-resolved pump-probe measurement on the dye Nile Blue in solution.

Time-resolved fluorescence study of exciplex formation in diastereomeric naproxen-pyrrolidine dyads.

PubMed

Khramtsova, Ekaterina A; Plyusnin, Viktor F; Magin, Ilya M; Kruppa, Alexander I; Polyakov, Nikolay E; Leshina, Tatyana V; Nuin, Edurne; Marin, M Luisa; Miranda, Miguel A

2013-12-19

The influence of chirality on the elementary processes triggered by excitation of the (S,S)- and (R,S)- diastereoisomers of naproxen-pyrrolidine (NPX-Pyr) dyads has been studied by time-resolved fluorescence in acetonitrile-benzene mixtures. In these systems, the quenching of the (1)NPX*-Pyr singlet excited state occurs through electron transfer and exciplex formation. Fluorescence lifetimes and quantum yields revealed a significant difference (around 20%) between the (S,S)- and (R,S)- diastereomers. In addition, the quantum yields of exciplexes differed by a factor of 2 regardless of solvent polarity. This allows us to suggest a similar influence of the chiral centers on the local charge transfer resulting in exciplex and full charge separation that leads to ion-biradicals. A simplified scheme is proposed to estimate a set of rate constant values (k1-k5) for the elementary stages in each solvent system.

In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Gray, Daniel C.; Merigan, William; Wolfing, Jessica I.; Gee, Bernard P.; Porter, Jason; Dubra, Alfredo; Twietmeyer, Ted H.; Ahamd, Kamran; Tumbar, Remy; Reinholz, Fred; Williams, David R.

2006-08-01

The ability to resolve single cells noninvasively in the living retina has important applications for the study of normal retina, diseased retina, and the efficacy of therapies for retinal disease. We describe a new instrument for high-resolution, in vivo imaging of the mammalian retina that combines the benefits of confocal detection, adaptive optics, multispectral, and fluorescence imaging. The instrument is capable of imaging single ganglion cells and their axons through retrograde transport in ganglion cells of fluorescent dyes injected into the monkey lateral geniculate nucleus (LGN). In addition, we demonstrate a method involving simultaneous imaging in two spectral bands that allows the integration of very weak signals across many frames despite inter-frame movement of the eye. With this method, we are also able to resolve the smallest retinal capillaries in fluorescein angiography and the mosaic of retinal pigment epithelium (RPE) cells with lipofuscin autofluorescence.

Multimodal imaging of vascular grafts using time-resolved fluorescence and ultrasound

NASA Astrophysics Data System (ADS)

Fatakdawala, Hussain; Griffiths, Leigh G.; Wong, Maelene L.; Humphrey, Sterling; Marcu, Laura

2015-02-01

The translation of engineered tissues into clinic requires robust monitoring of tissue development, both in vitro and in vivo. Traditional methods for the same are destructive, inefficient in time and cost and do not allow time-lapse measurements from the same sample or animal. This study reports on the ability of time-resolved fluorescence and ultrasound measurements for non-destructive characterization of explanted tissue engineered vascular grafts. Results show that TRFS and FLIm are able to assess alterations in luminal composition namely elastin, collagen and cellular (hyperplasia) content via changes in fluorescence lifetime values between normal and grafted tissue. These observations are complemented by structural changes observed in UBM pertaining to graft integration and intimal thickness over the grafted region. These results encourage the future application of a catheter-based technique that combines these imaging modalities for non-destructive characterization of vascular grafts in vivo.

Experimental Potential Energy Curve for the 43 Π Electronic State of NaCs

NASA Astrophysics Data System (ADS)

Steely, Andrew; Cooper, Hannah; Zain, Hareem; Whipp, Ciara; Faust, Carl; Kortyna, Andrew; Huennekens, John

2017-04-01

We present results from experimental studies of the 43 Π electronic state of the NaCs molecule. This electronic state is interesting in that its potential energy curve likely exhibits a double minimum. As a result, interference effects are observed in the resolved bound-free fluorescence spectra. The optical-optical double resonance method was used to obtain Doppler-free excitation spectra for the 43 Π state. This dataset of measured level energies was expanded largely by observing fluorescence from levels populated by collisions. To aid in level assignments, simulations of resolved bound-free fluorescence spectra were calculated using the BCONT program (R. J. Le Roy, University of Waterloo). Spectroscopic constants were determined to summarize data belonging to inner well, outer well, and above barrier regions of the electronic state. Current work focuses on using the IPA method to construct an experimental potential energy curve. Work supported by NSF and Susquehanna University.

Wide-field depth-sectioning fluorescence microscopy using projector-generated patterned illumination

NASA Astrophysics Data System (ADS)

Delica, Serafin; Mar Blanca, Carlo

2007-10-01

We present a simple and cost-effective wide-field, depth-sectioning, fluorescence microscope utilizing a commercial multimedia projector to generate excitation patterns on the sample. Highly resolved optical sections of fluorescent pollen grains at 1.9 μm axial resolution are constructed using the structured illumination technique. This requires grid excitation patterns to be scanned across the sample, which is straightforwardly implemented by creating slideshows of gratings at different phases, projecting them onto the sample, and synchronizing camera acquisition with slide transition. In addition to rapid dynamic pattern generation, the projector provides high illumination power and spectral excitation selectivity. We exploit these properties by imaging mouse neural cells in cultures multistained with Alexa 488 and Cy3. The spectral and structural neural information is effectively resolved in three dimensions. The flexibility and commercial availability of this light source is envisioned to open multidimensional imaging to a broader user base.

Spectrally resolved visualization of fluorescent dyes permeating into skin

NASA Astrophysics Data System (ADS)

Maeder, Ulf; Bergmann, Thorsten; Beer, Sebastian; Burg, Jan Michael; Schmidts, Thomas; Runkel, Frank; Fiebich, Martin

2012-03-01

We present a spectrally resolved confocal imaging approach to qualitatively asses the overall uptake and the penetration depth of fluorescent dyes into biological tissue. We use a confocal microscope with a spectral resolution of 5 nm to measure porcine skin tissue after performing a Franz-Diffusion experiment with a submicron emulsion enriched with the fluorescent dye Nile Red. The evaluation uses linear unmixing of the dye and the tissue autofluorescence spectra. The results are combined with a manual segmentation of the skin's epidermis and dermis layers to assess the penetration behavior additionally to the overall uptake. The diffusion experiments, performed for 3h and 24h, show a 3-fold increased dye uptake in the epidermis and dermis for the 24h samples. As the method is based on spectral information it does not face the problem of superimposed dye and tissue spectra and therefore is more precise compared to intensity based evaluation methods.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest

NASA Astrophysics Data System (ADS)

Dahlberg, Peter D.; Boughter, Christopher T.; Faruk, Nabil F.; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A.; Shaiber, Alon; Sherani, Aiman; Zhang, Jiacheng; Jureller, Justin E.; Hammond, Adam T.

2016-11-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest.

PubMed

Dahlberg, Peter D; Boughter, Christopher T; Faruk, Nabil F; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A; Shaiber, Alon; Sherani, Aiman; Zhang, Jiacheng; Jureller, Justin E; Hammond, Adam T

2016-11-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH 3 NH 3 PbBr 3 perovskites and measure differences between nanocrystal films and micron scale crystals.

Time-resolved stimulated emission depletion and energy transfer dynamics in two-photon excited EGFP

NASA Astrophysics Data System (ADS)

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

2018-04-01

Time and polarization-resolved stimulated emission depletion (STED) measurements are used to investigate excited state evolution following the two-photon excitation of enhanced green fluorescent protein (EGFP). We employ a new approach for the accurate STED measurement of the hitherto unmeasured degree of hexadecapolar transition dipole moment alignment ⟨α40 ⟩ present at a given excitation-depletion (pump-dump) pulse separation. Time-resolved polarized fluorescence measurements as a function of pump-dump delay reveal the time evolution of ⟨α40 ⟩ to be considerably more rapid than predicted for isotropic rotational diffusion in EGFP. Additional depolarization by homo-Förster resonance energy transfer is investigated for both ⟨α20 ⟩ (quadrupolar) and ⟨α40 ⟩ transition dipole alignments. These results point to the utility of higher order dipole correlation measurements in the investigation of resonance energy transfer processes.

Confocal depth-resolved fluorescence micro-X-ray absorption spectroscopy for the study of cultural heritage materials: a new mobile endstation at the Beijing Synchrotron Radiation Facility

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

Chen, Guang; Chu, Shengqi; Sun, Tianxi

A confocal fluorescence endstation for depth-resolved micro-X-ray absorption spectroscopy is described. A polycapillary half-lens defines the incident beam path and a second polycapillary half-lens at 90° defines the probe sample volume. An automatic alignment program based on an evolutionary algorithm is employed to make the alignment procedure efficient. This depth-resolved system was examined on a general X-ray absorption spectroscopy (XAS) beamline at the Beijing Synchrotron Radiation Facility. Sacrificial red glaze (AD 1368–1644) china was studied to show the capability of the instrument. As a mobile endstation to be applied on multiple beamlines, the confocal system can improve the function andmore » flexibility of general XAS beamlines, and extend their capabilities to a wider user community.« less

Key Physiological Parameters Dictate Triggering of Activity-Dependent Bulk Endocytosis in Hippocampal Synapses

PubMed Central

Wenzel, Eva M.; Morton, Andrew; Ebert, Katrin; Welzel, Oliver; Kornhuber, Johannes; Cousin, Michael A.; Groemer, Teja W.

2012-01-01

To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity. PMID:22675521

Improved axial point spread function in a two-frequency laser scanning confocal fluorescence microscope

NASA Astrophysics Data System (ADS)

Wu, Jheng-Syong; Chung, Yung-Chin; Chien, Jun-Jei; Chou, Chien

2018-01-01

A two-frequency laser scanning confocal fluorescence microscope (TF-LSCFM) based on intensity modulated fluorescence signal detection was proposed. The specimen-induced spherical aberration and scattering effect were suppressed intrinsically, and high image contrast was presented due to heterodyne interference. An improved axial point spread function in a TF-LSCFM compared with a conventional laser scanning confocal fluorescence microscope was demonstrated and discussed.

Capture of Fluorescence Decay Times by Flow Cytometry

PubMed Central

Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

2012-01-01

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy

PubMed Central

Siegel, Nisan; Brooker, Gary

2014-01-01

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called “CINCH”. PMID:25321701

Sizing protein-templated gold nanoclusters by time resolved fluorescence anisotropy decay measurements.

PubMed

Soleilhac, Antonin; Bertorelle, Franck; Antoine, Rodolphe

2018-03-15

Protein-templated gold nanoclusters (AuNCs) are very attractive due to their unique fluorescence properties. A major problem however may arise due to protein structure changes upon the nucleation of an AuNC within the protein for any future use as in vivo probes, for instance. In this work, we propose a simple and reliable fluorescence based technique measuring the hydrodynamic size of protein-templated gold nanoclusters. This technique uses the relation between the time resolved fluorescence anisotropy decay and the hydrodynamic volume, through the rotational correlation time. We determine the molecular size of protein-directed AuNCs, with protein templates of increasing sizes, e.g. insulin, lysozyme, and bovine serum albumin (BSA). The comparison of sizes obtained by other techniques (e.g. dynamic light scattering and small-angle X-ray scattering) between bare and gold clusters containing proteins allows us to address the volume changes induced either by conformational changes (for BSA) or the formation of protein dimers (for insulin and lysozyme) during cluster formation and incorporation. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiwavelength FLIM: new applications and algorithms

NASA Astrophysics Data System (ADS)

Rück, A.; Strat, D.; Dolp, F.; von Einem, B.; von Arnim, C. A. F.

2011-03-01

The combination of time-resolved and spectral resolved techniques as achieved by SLIM (spectrally resolved fluorescence lifetime imaging) improves the analysis of complex situations, when different fluorophores have to be distinguished. This could be the case when endogenous fluorophores of living cells and tissues are observed to identify the redox state and oxidative metabolic changes of the mitochondria. Other examples are FRET (resonant energy transfer) measurements, when different donor/acceptor pairs are observed simultaneously. SLIM is working in the time domain employing excitation with short light pulses and detection of the fluorescence intensity decay in many cases with time-correlated single photon counting (TCSPC). Spectral resolved detection is achieved by a polychromator in the detection path and a 16-channel multianode photomultiplier tube with the appropriate routing electronics. Within this paper special attention will be focused on FRET measurements with respect to protein interactions in Alzheimers disease. Using global analysis as the phasor plot approach or integration of the kinetic equations taking into account the multidimensional datasets in every spectral channel we could demonstrate considerable improvement of our calculations.

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.

Synthesis and characterization of a fluorescent water-soluble paclitaxel prodrug.

PubMed

Sohn, Jeong-Sun; Choi, Eun-Sun; Jo, Byung-Wook; Hess, Michael; Han, Song-Hee

2010-05-01

A fluorescence susceptible water-soluble paclitaxel was synthesized by a condensation reaction between PEGylated paclitaxel (namely, PP7) and 1-pyrene butyric acid (PBA) in order to obtain a better understanding of the mechanism of action of paclitaxel as well as of the environment of the paclitaxel-binding site. The reaction was performed successfully and the resulting paclitaxel was characterized by FT-NMR, analytical-HPLC, UV spectro photometry, and fluorescence spectrometry. The synthesized paclitaxel analogue showed a high susceptibility to fluorescence in both excitation and emission spectra. And we have investigated the time-resolved fluorescence behavior of them in different solvents and at different excitation wavelengths.

An x-ray fluorescence imaging system for gold nanoparticle detection.

PubMed

Ricketts, K; Guazzoni, C; Castoldi, A; Gibson, A P; Royle, G J

2013-11-07

Gold nanoparticles (GNPs) may be used as a contrast agent to identify tumour location and can be modified to target and image specific tumour biological parameters. There are currently no imaging systems in the literature that have sufficient sensitivity to GNP concentration and distribution measurement at sufficient tissue depth for use in in vivo and in vitro studies. We have demonstrated that high detecting sensitivity of GNPs can be achieved using x-ray fluorescence; furthermore this technique enables greater depth imaging in comparison to optical modalities. Two x-ray fluorescence systems were developed and used to image a range of GNP imaging phantoms. The first system consisted of a 10 mm(2) silicon drift detector coupled to a slightly focusing polycapillary optic which allowed 2D energy resolved imaging in step and scan mode. The system has sensitivity to GNP concentrations as low as 1 ppm. GNP concentrations different by a factor of 5 could be resolved, offering potential to distinguish tumour from non-tumour. The second system was designed to avoid slow step and scan image acquisition; the feasibility of excitation of the whole specimen with a wide beam and detection of the fluorescent x-rays with a pixellated controlled drift energy resolving detector without scanning was investigated. A parallel polycapillary optic coupled to the detector was successfully used to ascertain the position where fluorescence was emitted. The tissue penetration of the technique was demonstrated to be sufficient for near-surface small-animal studies, and for imaging 3D in vitro cellular constructs. Previous work demonstrates strong potential for both imaging systems to form quantitative images of GNP concentration.

Novel physical chemistry approaches in biophysical researches with advanced application of lasers: Detection and manipulation.

PubMed

Iwata, Koichi; Terazima, Masahide; Masuhara, Hiroshi

2018-02-01

Novel methodologies utilizing pulsed or intense CW irradiation obtained from lasers have a major impact on biological sciences. In this article, recent development in biophysical researches fully utilizing the laser irradiation is described for three topics, time-resolved fluorescence spectroscopy, time-resolved thermodynamics, and manipulation of the biological assemblies by intense laser irradiation. First, experimental techniques for time-resolved fluorescence spectroscopy are concisely explained in Section 2. As an example of the recent application of time-resolved fluorescence spectroscopy to biological systems, evaluation of the viscosity of lipid bilayer membranes is described. The results of the spectroscopic experiments strongly suggest the presence of heterogeneous membrane structure with two different viscosity values in liposomes formed by a single phospholipid. Section 3 covers the time-resolved thermodynamics. Thermodynamical properties are important to characterize biomolecules. However, measurement of these quantities for short-lived intermediate species has been impossible by traditional thermodynamical techniques. Recently, development of a spectroscopic method based on the transient grating method enables us to measure these quantities and also to elucidate reaction kinetics which cannot be detected by other spectroscopic methods. The principle of the measurements and applications to some protein reactions are reviewed. Manipulation and fabrication of supramolecues, amino acids, proteins, and living cells by intense laser irradiation are described in Section 4. Unconventional assembly, crystallization and growth, amyloid fibril formation, and living cell manipulation are achieved by CW laser trapping and femtosecond laser-induced cavitation bubbling. Their spatio-temporal controllability is opening a new avenue in the relevant molecular and bioscience research fields. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017. Published by Elsevier B.V.

Interaction and energy transfer studies between bovine serum albumin and CdTe quantum dots conjugates: CdTe QDs as energy acceptor probes.

PubMed

Kotresh, M G; Inamdar, L S; Shivkumar, M A; Adarsh, K S; Jagatap, B N; Mulimani, B G; Advirao, G M; Inamdar, S R

2017-06-01

In this paper, a systematic investigation of the interaction of bovine serum albumin (BSA) with water-soluble CdTe quantum dots (QDs) of two different sizes capped with carboxylic thiols is presented based on steady-state and time-resolved fluorescence measurements. Efficient Förster resonance energy transfer (FRET) was observed to occur from BSA donor to CdTe acceptor as noted from reduction in the fluorescence of BSA and enhanced fluorescence from CdTe QDs. FRET parameters such as Förster distance, spectral overlap integral, FRET rate constant and efficiency were determined. The quenching of BSA fluorescence in aqueous solution observed in the presence of CdTe QDs infers that fluorescence resonance energy transfer is primarily responsible for the quenching phenomenon. Bimolecular quenching constant (k q ) determined at different temperatures and the time-resolved fluorescence data provide additional evidence for this. The binding stoichiometry and various thermodynamic parameters are evaluated by using the van 't Hoff equation. The analysis of the results suggests that the interaction between BSA and CdTe QDs is entropy driven and hydrophobic forces play a key role in the interaction. Binding of QDs significantly shortened the fluorescence lifetime of BSA which is one of the hallmarks of FRET. The effect of size of the QDs on the FRET parameters are discussed in the light of FRET parameters obtained. Copyright © 2016 John Wiley & Sons, Ltd.

Excitation wavelength dependence of excited state intramolecular proton transfer reaction of 4'-N,N-diethylamino-3-hydroxyflavone in room temperature ionic liquids studied by optical Kerr gate fluorescence measurement.

PubMed

Suda, Kayo; Terazima, Masahide; Sato, Hirofumi; Kimura, Yoshifumi

2013-10-17

Excited state intramolecular proton transfer reactions (ESIPT) of 4'-N,N-diethylamino-3-hydroxyflavone (DEAHF) in ionic liquids have been studied by steady-state and time-resolved fluorescence measurements at different excitation wavelengths. Steady-state measurements show the relative yield of the tautomeric form to the normal form of DEAHF decreases as excitation wavelength is increased from 380 to 450 nm. The decrease in yield is significant in ionic liquids that have cations with long alkyl chains. The extent of the decrease is correlated with the number of carbon atoms in the alkyl chains. Time-resolved fluorescence measurements using optical Kerr gate spectroscopy show that ESIPT rate has a strong excitation wavelength dependence. There is a large difference between the spectra at a 200 ps delay from different excitation wavelengths in each ionic liquid. The difference is pronounced in ionic liquids having a long alkyl chain. The equilibrium constant in the electronic excited state obtained at a 200 ps delay and the average reaction rate are also correlated with the alkyl chain length. Considering the results of the steady-state fluorescence and time-resolved measurements, the excitation wavelength dependence of ESIPT is explained by state selective excitation due to the difference of the solvation, and the number of alkyl chain carbon atoms is found to be a good indicator of the effect of inhomogeneity for this reaction.

Silver nanoparticles-enhanced time-resolved fluorescence sensor for VEGF(165) based on Mn-doped ZnS quantum dots.

PubMed

Zhu, Dong; Li, Wei; Wen, Hong-Mei; Yu, Sheng; Miao, Zhao-Yi; Kang, An; Zhang, Aihua

2015-12-15

A silver nanoparticles (AgNPs)-enhanced time-resolved fluorescence (TR-FL) sensor based on long-lived fluorescent Mn-doped ZnS quantum dots (QDs) is developed for the sensitive detection of vascular endothelial growth factor-165 (VEGF165), a predominant cancer biomarker in cancer angiogenesis. The aptamers bond with the Mn-doped ZnS QDs and the BHQ-2 quencher-labelling strands hybridized in duplex are coupled with streptavidin (SA)-functionalized AgNPs to form the AgNPs-enhanced TR-FL sensor, showing lower fluorescence intensity in the duplex state due to the fluorescence resonance energy transfer (FRET) between the Mn-doped ZnS QDs and quenchers. Upon the addition of VEGF165, the BHQ-2 quencher-labelling strands of the duplex are displaced, leading to the disruption of the FRET. As a result, the fluorescence of the Mn-doped QDs within the proximity of the AgNPs is recovered. The FL signal can be measured free of the interference of short-lived background by setting appropriate delay time and gate time, which offers a signal with high signal-to-noise ratio in photoluminescent biodetection. Compared with the bare TR-FL sensor, the AgNPs-based TR-FL sensor showed a huge improvement in fluorescence based on metal-enhanced fluorescence (MEF) effect, and the sensitivity increased 11-fold with the detection limit of 0.08 nM. In addition, the sensor provided a wide range of linear detection from 0.1 nM to 16 nM. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

Lamb, D C; Müller, B K; Bräuchle, C

2005-10-01

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

The visible spectrum of zirconium dioxide, ZrO2

NASA Astrophysics Data System (ADS)

Le, Anh; Steimle, Timothy C.; Gupta, Varun; Rice, Corey A.; Maier, John P.; Lin, Sheng H.; Lin, Chih-Kai

2011-09-01

The electronic spectrum of a cold molecular beam of zirconium dioxide, ZrO2, has been investigated using laser induced fluorescence (LIF) in the region from 17 000 cm-1 to 18 800 cm-1 and by mass-resolved resonance enhanced multi-photon ionization (REMPI) spectroscopy from 17 000 cm-1-21 000 cm-1. The LIF and REMPI spectra are assigned to progressions in the tilde A{^1}B_2(ν1, ν2, ν3) ← tilde X{^1}A_1(0, 0, 0) transitions. Dispersed fluorescence from 13 bands was recorded and analyzed to produce harmonic vibrational parameters for the tilde X{^1}A_1 state of ω1 = 898(1) cm-1, ω2 = 287(2) cm-1, and ω3 = 808(3) cm-1. The observed transition frequencies of 45 bands in the LIF and REMPI spectra produce origin and harmonic vibrational parameters for the tilde A{^1}B_2 state of Te = 16 307(8) cm-1, ω1 = 819(3) cm-1, ω2 = 149(3) cm-1, and ω3 = 518(4) cm-1. The spectra were modeled using a normal coordinate analysis and Franck-Condon factor predictions. The structures, harmonic vibrational frequencies, and the potential energies as a function of bending angle for the tilde A{^1}B_2 and tilde X{^1}A_1 states are predicted using time-dependent density functional theory, complete active space self-consistent field, and related first-principle calculations. A comparison with isovalent TiO2 is made.

Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy.

PubMed

Fang, Chong; Frontiera, Renee R; Tran, Rosalie; Mathies, Richard A

2009-11-12

Tracing the transient atomic motions that lie at the heart of chemical reactions requires high-resolution multidimensional structural information on the timescale of molecular vibrations, which commonly range from 10 fs to 1 ps. For simple chemical systems, it has been possible to map out in considerable detail the reactive potential-energy surfaces describing atomic motions and resultant reaction dynamics, but such studies remain challenging for complex chemical and biological transformations. A case in point is the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, which is a widely used gene expression marker owing to its efficient bioluminescence. This feature is known to arise from excited-state proton transfer (ESPT), yet the atomistic details of the process are still not fully understood. Here we show that femtosecond stimulated Raman spectroscopy provides sufficiently detailed and time-resolved vibrational spectra of the electronically excited chromophore of GFP to reveal skeletal motions involved in the proton transfer that produces the fluorescent form of the protein. In particular, we observe that the frequencies and intensities of two marker bands, the C-O and C = N stretching modes at opposite ends of the conjugated chromophore, oscillate out of phase with a period of 280 fs; we attribute these oscillations to impulsively excited low-frequency phenoxyl-ring motions, which optimize the geometry of the chromophore for ESPT. Our findings illustrate that femtosecond simulated Raman spectroscopy is a powerful approach to revealing the real-time nuclear dynamics that make up a multidimensional polyatomic reaction coordinate.

Ultrafast fluorescence spectroscopy via upconversion applications to biophysics.

PubMed

Xu, Jianhua; Knutson, Jay R

2008-01-01

This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed.

Advanced fluorescence microscopy techniques for the life sciences

PubMed Central

Aguib, Yasmine; Yacoub, Magdi H.

The development of super-resolved fluorescence microscopy, for which the Nobel Prize was awarded in 2014, has been a topic of interest to physicists and biologists alike. It is inevitable that numerous questions in biomedical research cannot be answered by means other than direct observation. In this review, advances to fluorescence microscopy are covered in a widely accessible fashion to facilitate its use in decisions related to its acquisition and utilization in biomedical research. PMID:29043264

Fluorescence lifetime spectroscopy for guided therapy of brain tumors.

PubMed

Butte, Pramod V; Mamelak, Adam N; Nuno, Miriam; Bannykh, Serguei I; Black, Keith L; Marcu, Laura

2011-01-01

This study evaluates the potential of time-resolved laser induced fluorescence spectroscopy (TR-LIFS) as intra-operative tool for the delineation of brain tumor from normal brain. Forty two patients undergoing glioma (WHO grade I-IV) surgery were enrolled in this study. A TR-LIFS prototype apparatus (gated detection, fast digitizer) was used to induce in-vivo fluorescence using a pulsed N2 laser (337 nm excitation, 0.7 ns pulse width) and to record the time-resolved spectrum (360-550 nm range, 10 nm interval). The sites of TR-LIFS measurement were validated by conventional histopathology (H&E staining). Parameters derived from the TR-LIFS data including intensity values and time-resolved intensity decay features (average fluorescence lifetime and Laguerre coefficients values) were used for tissue characterization and classification. 71 areas of tumor and normal brain were analyzed. Several parameters allowed for the differentiation of distinct tissue types. For example, normal cortex (N=35) and normal white matter (N=12) exhibit a longer-lasting fluorescence emission at 390 nm (τ390=2.12±0.10 ns) when compared with 460 nm (τ460=1.16±0.08 ns). High grade glioma (grades III and IV) samples (N=17) demonstrate emission peaks at 460 nm, with large variation at 390 nm while low grade glioma (I and II) samples (N=7) demonstrated a peak fluorescence emission at 460 nm. A linear discriminant algorithm allowed for the classification of low-grade gliomas with 100% sensitivity and 98% specificity. High-grade glioma demonstrated a high degree of heterogeneity thus reducing the discrimination accuracy of these tumors to 47% sensitivity and 94% specificity. Current findings demonstrate that TR-LIFS holds the potential to diagnose brain tumors intra-operatively and to provide a valuable tool for aiding the neurosurgeon-neuropathologist team in to rapidly distinguish between tumor and normal brain during surgery. Copyright © 2010 Elsevier Inc. All rights reserved.

Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

NASA Astrophysics Data System (ADS)

Yankelevich, Diego R.; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Elson, Daniel S.; Marcu, Laura

2014-03-01

The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

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.

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

PubMed

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

2015-08-01

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

Frequency domain fluorescent diffuse tomography of small animals with DsRed2-expressed tumors

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Orlova, Anna G.; Sergeeva, Ekaterina A.; Kleshnin, Mikhail S.; Shirmanova, Marina V.

2006-02-01

The main applications of fluorescent proteins (FPs) are monitoring tumor growth, angiogenesis, metastases formation and effects of new classes of drugs. Different types of tomography allow fluorescence imaging of tumors located deep in human or animal tissue. These techniques were used for investigation of the distribution of near-infrared fluorescent probes, but only a few works are devoted to fluorescence tomography in visible light. In this work, preliminary results of the frequency domain fluorescent diffuse tomography (FD FDT) method in application to DsRed2 protein as a fluorescent agent are presented. For the first step of our experiments we utilized second harmonic generation of Nd:YAG laser (532 nm) modulated by low frequency (1 kHz) in the experimental setup. The transilluminative planar configuration was used in the setup. A series of model experiments has been conducted and show good agreement between theoretical and experimental fluorescence intensity. Post mortem experiments with capsules containing DsRed2 and scattering solution introduced into esophagus of rats to simulate tumor formation have been conducted. The results of these experiments show that sensitivity of the setup is sufficient to detect DsRed2 in concentrations similar to those in FP-expressed tumor, but the contrast is not enough high to separate fluorescence of DsRed2 and surrounding tissues. The setup can be significantly improved by utilizing high-frequency modulation (110 MHz using acousto-optical modulator) of the excitation light and precise phase measurements due to difference in fluorescence life-time of FPs and surrounding tissues. An algorithm of processing a fluorescent image based on calculating zero of maximum curvature was employed for detection of fluorescent inclusions boundaries in the image.

Open-air multispectral fluorescence-guided surgery platform for intraoperative detection of malignant tissue under ambient lighting conditions

NASA Astrophysics Data System (ADS)

Behrooz, Ali; Vasquez, Kristine O.; Waterman, Peter; Meganck, Jeff; Peterson, Jeffrey D.; Miller, Peter; Kempner, Joshua

2017-02-01

Intraoperative resection of tumors currently relies upon the surgeon's ability to visually locate and palpate tumor nodules. Undetected residual malignant tissue often results in the need for additional treatment or surgical intervention. The Solaris platform is a multispectral open-air fluorescence imaging system designed for translational fluorescence-guided surgery. Solaris supports video-rate imaging in four fixed fluorescence channels ranging from visible to near infrared, and a multispectral channel equipped with a liquid crystal tunable filter (LCTF) for multispectral image acquisition (520-620 nm). Identification of tumor margins using reagents emitting in the visible spectrum (400-650 nm), such as fluorescein isothiocyanate (FITC), present challenges considering the presence of auto-fluorescence from tissue and food in the gastrointestinal (GI) tract. To overcome this, Solaris acquires LCTF-based multispectral images, and by applying an automated spectral unmixing algorithm to the data, separates reagent fluorescence from tissue and food auto-fluorescence. The unmixing algorithm uses vertex component analysis to automatically extract the primary pure spectra, and resolves the reagent fluorescent signal using non-negative least squares. For validation, intraoperative in vivo studies were carried out in tumor-bearing rodents injected with FITC-dextran reagent that is primarily residing in malignant tissue 24 hours post injection. In the absence of unmixing, fluorescence from tumors is not distinguishable from that of surrounding tissue. Upon spectral unmixing, the FITC-labeled malignant regions become well defined and detectable. The results of these studies substantiate the multispectral power of Solaris in resolving FITC-based agent signal in deep tumor masses, under ambient and surgical light, and enhancing the ability to surgically resect them.

Planetary Surface Exploration Using Time-Resolved Laser Spectroscopy on Rovers and Landers

NASA Astrophysics Data System (ADS)

Blacksberg, Jordana; Alerstam, Erik; Maruyama, Yuki; Charbon, Edoardo; Rossman, George

2013-04-01

Planetary surface exploration using laser spectroscopy has become increasingly relevant as these techniques become a reality on Mars surface missions. The ChemCam instrument onboard the Curiosity rover is currently using laser induced breakdown spectroscopy (LIBS) on a mast-mounted platform to measure elemental composition of target rocks. The RLS Raman Spectrometer is included on the payload for the ExoMars mission to be launched in 2018 and will identify minerals and organics on the Martian surface. We present a next-generation instrument that builds on these widely used techniques to provide a means for performing both Raman spectroscopy and LIBS in conjunction with microscopic imaging. Microscopic Raman spectroscopy with a laser spot size smaller than the grains of interest can provide surface mapping of mineralogy while preserving morphology. A very small laser spot size (~ 1 µm) is often necessary to identify minor phases that are often of greater interest than the matrix phases. In addition to the difficulties that can be posed by fine-grained material, fluorescence interference from the very same material is often problematic. This is particularly true for many of the minerals of interest that form in environments of aqueous alteration and can be highly fluorescent. We use time-resolved laser spectroscopy to eliminate fluorescence interference that can often make it difficult or impossible to obtain Raman spectra. As an added benefit, we have found that with small changes in operating parameters we can include microscopic LIBS using the same hardware. This new technique relies on sub-ns, high rep-rate lasers with relatively low pulse energy and compact solid state detectors with sub-ns time resolution. The detector technology that makes this instrument possible is a newly developed Single-Photon Avalanche Diode (SPAD) sensor array based on Complementary Metal-Oxide Semiconductor (CMOS) technology. The use of this solid state time-resolved detector offers a significant reduction in size, weight, power, and overall complexity - making time resolved detection feasible for planetary applications. We will discuss significant advances leading to the feasibility of a compact time-resolved spectrometer. We will present results on planetary analog minerals to demonstrate the instrument performance including fluorescence rejection and combined Raman-LIBS capability.

Liquid nitrogen-assisted synthesis of fluorescent carbon dots from Blueberry and their performance in Fe3+ detection

NASA Astrophysics Data System (ADS)

Aslandaş, Ayşe Merve; Balcı, Neslihan; Arık, Mustafa; Şakiroğlu, Halis; Onganer, Yavuz; Meral, Kadem

2015-11-01

Fluorescent carbon dots (C-dots) were synthesized by a facile method containing liquid N2 treatment and centrifuge processes. The photophysical properties of the C-dots in an aqueous solution were examined at various conditions such as concentration, temperature, pH and excitation wavelength by using UV-vis absorption, fluorescence and time-resolved fluorescence spectroscopies. The C-dots emitted a broad fluorescence between approximately 350-550 nm and their fluorescence was tuned by changing excitation wavelength. The as-prepared C-dots were applied to Fe3+ detection from aqueous solution. Spectroscopic data revealed that the as-prepared C-dots were used to detect Fe3+ in the range of 12.5 μM to 100 μM as a fluorescence sensor.

Inkjet printed fluorescent nanorod layers exhibit superior optical performance over quantum dots

NASA Astrophysics Data System (ADS)

Halivni, Shira; Shemesh, Shay; Waiskopf, Nir; Vinetsky, Yelena; Magdassi, Shlomo; Banin, Uri

2015-11-01

Semiconductor nanocrystals exhibit unique fluorescence properties which are tunable in size, shape and composition. The high quantum yield and enhanced stability have led to their use in biomedical imaging and flat panel displays. Here, semiconductor nanorod based inkjet inks are presented, overcoming limitations of the commonly reported quantum dots in printing applications. Fluorescent seeded nanorods were found to be outstanding candidates for fluorescent inks, due to their low particle-particle interactions and negligible self-absorption. This is manifested by insignificant emission shifts upon printing, even in highly concentrated printed layers and by maintenance of a high fluorescence quantum yield, unlike quantum dots which exhibit fluorescence wavelength shifts and quenching effects. This behavior results from the reduced absorption/emission overlap, accompanied by low energy transfer efficiencies between the nanorods as supported by steady state and time resolved fluorescence measurements. The new seeded nanorod inks enable patterning of thin fluorescent layers, for demanding light emission applications such as signage and displays.Semiconductor nanocrystals exhibit unique fluorescence properties which are tunable in size, shape and composition. The high quantum yield and enhanced stability have led to their use in biomedical imaging and flat panel displays. Here, semiconductor nanorod based inkjet inks are presented, overcoming limitations of the commonly reported quantum dots in printing applications. Fluorescent seeded nanorods were found to be outstanding candidates for fluorescent inks, due to their low particle-particle interactions and negligible self-absorption. This is manifested by insignificant emission shifts upon printing, even in highly concentrated printed layers and by maintenance of a high fluorescence quantum yield, unlike quantum dots which exhibit fluorescence wavelength shifts and quenching effects. This behavior results from the reduced absorption/emission overlap, accompanied by low energy transfer efficiencies between the nanorods as supported by steady state and time resolved fluorescence measurements. The new seeded nanorod inks enable patterning of thin fluorescent layers, for demanding light emission applications such as signage and displays. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06248a

Application of a high-repetition-rate laser diagnostic system for single-cycle-resolved imaging in internal combustion engines.

PubMed

Hult, Johan; Richter, Mattias; Nygren, Jenny; Aldén, Marcus; Hultqvist, Anders; Christensen, Magnus; Johansson, Bengt

2002-08-20

High-repetition-rate laser-induced fluorescence measurements of fuel and OH concentrations in internal combustion engines are demonstrated. Series of as many as eight fluorescence images, with a temporal resolution ranging from 10 micros to 1 ms, are acquired within one engine cycle. A multiple-laser system in combination with a multiple-CCD camera is used for cycle-resolved imaging in spark-ignition, direct-injection stratified-charge, and homogeneous-charge compression-ignition engines. The recorded data reveal unique information on cycle-to-cycle variations in fuel transport and combustion. Moreover, the imaging system in combination with a scanning mirror is used to perform instantaneous three-dimensional fuel-concentration measurements.

Time-resolved wide-field optically sectioned fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dupuis, Guillaume; Benabdallah, Nadia; Chopinaud, Aurélien; Mayet, Céline; Lévêque-Fort, Sandrine

2013-02-01

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

Theoretical Interpretation of the Fluorescence Spectra of Toluene and P- Cresol

DTIC Science & Technology

1994-07-01

NUMBER OF PAGES Toluene Geometrica 25 p-Cresol Fluorescence Is. PRICE CODE Spectra 17. SECURITY CLASSIFICATION 13. SECURITY CLASSIFICATION 19...State Frequencies of Toluene ................ 19 6 Computed and exp" Ground State Frequencies of p-Cresol ............... 20 7 Correction Factors for...Computed Ground State Vibrational Frequencies ....... 21 8 Computed and Corrected Excited State Frequencies of Toluene ............. 22 9 Computed and

Human serum albumin stability and toxicity of anthraquinone dye alizarin complexone: an albumin-dye model.

PubMed

Ding, Fei; Zhang, Li; Diao, Jian-Xiong; Li, Xiu-Nan; Ma, Lin; Sun, Ying

2012-05-01

The complexation between the primary vector of ligands in blood plasma, human serum albumin (HSA) and a toxic anthraquinone dye alizarin complexone, was unmasked by means of circular dichroism (CD), molecular modeling, steady state and time-resolved fluorescence, and UV/vis absorption measurements. The structural investigation of the complexed HSA through far-UV CD, three-dimensional and synchronous fluorescence shown the polypeptide chain of HSA partially destabilizing with a reduction of α-helix upon conjugation. From molecular modeling and competitive ligand binding results, Sudlow's site I, which was the same as that of warfarin-azapropazone site, was appointed to retain high-affinity for alizarin complexone. Moreover, steady state fluorescence displayed that static type and Förster energy transfer is the operational mechanism for the vanish in the tryptophan (Trp)-214 fluorescence, this corroborates time-resolved fluorescence that HSA-alizarin complexone adduct formation has an affinity of 10(5) M(-1), and the driving forces were found to be chiefly π-π, hydrophobic, and hydrogen bonds, associated with an exothermic free energy change. These data should be utilized to illustrate the mechanism by which the toxicological action of anthraquinone dyes is mitigated by transporter HSA. Copyright © 2012 Elsevier Inc. All rights reserved.

Extended wavelength anisotropy resolved multidimensional emission spectroscopy (ARMES) measurements: better filters, validation standards, and Rayleigh scatter removal methods

NASA Astrophysics Data System (ADS)

Casamayou-Boucau, Yannick; Ryder, Alan G.

2017-09-01

Anisotropy resolved multidimensional emission spectroscopy (ARMES) provides valuable insights into multi-fluorophore proteins (Groza et al 2015 Anal. Chim. Acta 886 133-42). Fluorescence anisotropy adds to the multidimensional fluorescence dataset information about the physical size of the fluorophores and/or the rigidity of the surrounding micro-environment. The first ARMES studies used standard thin film polarizers (TFP) that had negligible transmission between 250 and 290 nm, preventing accurate measurement of intrinsic protein fluorescence from tyrosine and tryptophan. Replacing TFP with pairs of broadband wire grid polarizers enabled standard fluorescence spectrometers to accurately measure anisotropies between 250 and 300 nm, which was validated with solutions of perylene in the UV and Erythrosin B and Phloxine B in the visible. In all cases, anisotropies were accurate to better than ±1% when compared to literature measurements made with Glan Thompson or TFP polarizers. Better dual wire grid polarizer UV transmittance and the use of excitation-emission matrix measurements for ARMES required complete Rayleigh scatter elimination. This was achieved by chemometric modelling rather than classical interpolation, which enabled the acquisition of pure anisotropy patterns over wider spectral ranges. In combination, these three improvements permit the accurate implementation of ARMES for studying intrinsic protein fluorescence.

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

PubMed

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

2013-07-02

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

Studies on the binding behavior of prodigiosin with bovine hemoglobin by multi-spectroscopic techniques

NASA Astrophysics Data System (ADS)

Tang, Jing; Yang, Chao; Zhou, Lin; Ma, Fei; Liu, Shuchao; Wei, Shaohua; Zhou, Jiahong; Zhou, Yanhuai

2012-10-01

In this article, the interaction mechanism of prodigiosin (PG) with bovine hemoglobin (BHb) is studied in detail using various spectroscopic technologies. UV-vis absorption and fluorescence spectra demonstrate the interaction process. The Stern-Volmer plot and the time-resolved fluorescence study suggest the quenching mechanism of fluorescence of BHb by PG is a static quenching procedure, and the hydrophobic interactions play a major role in binding of PG to BHb. Furthermore, synchronous fluorescence studies, Fourier transform infrared (FTIR) and circular dichroism (CD) spectra reveal that the conformation of BHb is changed after conjugation with PG.

The nature of multiphoton fluorescence from red blood cells

NASA Astrophysics Data System (ADS)

Saytashev, Ilyas; Murphy, Michael; Osseiran, Sam; Spence, Dana M.; Evans, Conor L.; Dantus, Marcos

2016-03-01

We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC's, but longer more intense pulses lead to their destruction.

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

NASA Astrophysics Data System (ADS)

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

2017-09-01

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

Ultrafast Fluorescence Spectroscopy via Upconversion: Applications to Biophysics

PubMed Central

Xu, Jianhua; Knutson, Jay R.

2012-01-01

This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed. PMID:19152860

High-resolution time series of Pseudomonas aeruginosa gene expression and rhamnolipid secretion through growth curve synchronization.

PubMed

van Ditmarsch, Dave; Xavier, João B

2011-06-17

Online spectrophotometric measurements allow monitoring dynamic biological processes with high-time resolution. Contrastingly, numerous other methods require laborious treatment of samples and can only be carried out offline. Integrating both types of measurement would allow analyzing biological processes more comprehensively. A typical example of this problem is acquiring quantitative data on rhamnolipid secretion by the opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa cell growth can be measured by optical density (OD600) and gene expression can be measured using reporter fusions with a fluorescent protein, allowing high time resolution monitoring. However, measuring the secreted rhamnolipid biosurfactants requires laborious sample processing, which makes this an offline measurement. Here, we propose a method to integrate growth curve data with endpoint measurements of secreted metabolites that is inspired by a model of exponential cell growth. If serial diluting an inoculum gives reproducible time series shifted in time, then time series of endpoint measurements can be reconstructed using calculated time shifts between dilutions. We illustrate the method using measured rhamnolipid secretion by P. aeruginosa as endpoint measurements and we integrate these measurements with high-resolution growth curves measured by OD600 and expression of rhamnolipid synthesis genes monitored using a reporter fusion. Two-fold serial dilution allowed integrating rhamnolipid measurements at a ~0.4 h-1 frequency with high-time resolved data measured at a 6 h-1 frequency. We show how this simple method can be used in combination with mutants lacking specific genes in the rhamnolipid synthesis or quorum sensing regulation to acquire rich dynamic data on P. aeruginosa virulence regulation. Additionally, the linear relation between the ratio of inocula and the time-shift between curves produces high-precision measurements of maximum specific growth rates, which were determined with a precision of ~5.4%. Growth curve synchronization allows integration of rich time-resolved data with endpoint measurements to produce time-resolved quantitative measurements. Such data can be valuable to unveil the dynamic regulation of virulence in P. aeruginosa. More generally, growth curve synchronization can be applied to many biological systems thus helping to overcome a key obstacle in dynamic regulation: the scarceness of quantitative time-resolved data.

Diagnosis of meningioma by time-resolved fluorescence spectroscopy.

PubMed

Butte, Pramod V; Pikul, Brian K; Hever, Aviv; Yong, William H; Black, Keith L; Marcu, Laura

2005-01-01

We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors.

Diagnosis of meningioma by time-resolved fluorescence spectroscopy

PubMed Central

Butte, Pramod V.; Pikul, Brian K.; Hever, Aviv; Yong, William H.; Black, Keith L.; Marcu, Laura

2010-01-01

We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors. PMID:16409091

Site-specific structural dynamics of α-Synuclein revealed by time-resolved fluorescence spectroscopy: a review

NASA Astrophysics Data System (ADS)

Sahay, Shruti; Krishnamoorthy, G.; Maji, Samir K.

2016-12-01

Aggregation of α-Synuclein (α-Syn) into amyloid fibrils is known to be associated with the pathogenesis of Parkinson’s disease (PD). Several missense mutations of the α-Syn gene have been associated with rare, early onset familial forms of PD. Despite several studies done so far, the local/residue-level structure and dynamics of α-Syn in its soluble and aggregated fibril form and how these are affected by the familial PD associated mutations are still not clearly understood. Here, we review studies performed by our group as well as other research groups, where time-resolved fluorescence spectroscopy has been used to understand the site-specific structure and dynamics of α-Syn under physiological conditions as well as under conditions that alter the aggregation properties of the protein such as low pH, high temperature, presence of membrane mimics and familial PD associated mutations. These studies have provided important insights into the critical structural properties of α-Syn that may govern its aggregation. The review also highlights time-resolved fluorescence as a promising tool to study the critical conformational transitions associated with early oligomerization of α-Syn, which are otherwise not accessible using other commonly used techniques such as thioflavin T (ThT) binding assay.

Progress on laser technology for proposed space-based sodium lidar

NASA Astrophysics Data System (ADS)

Krainak, Michael A.; Yu, Anthony W.; Li, Steven X.; Bai, Yingxin; Numata, Kenji; Chen, Jeffrey R.; Fahey, Molly E.; Micalizzi, Frankie; Konoplev, Oleg A.; Janches, Diego; Gardner, Chester S.; Allan, Graham R.

2018-02-01

We propose a nadir-pointing space-based Na Doppler resonance fluorescence LIDAR on board of the International Space Station (ISS). The science instrument goal is temperature and vertical wind measurements of the Earth Mesosphere Lower Thermosphere (MLT) 75-115 km region using atomic sodium as a tracer. Our instrument concept uses a high-energy laser transmitter at 589 nm and highly sensitive photon counting detectors that permit range-resolved atmospheric-sodium-temperature profiles. The atmospheric temperature is deduced from the linewidth of the resonant fluorescence from the atomic sodium vapor D2 line as measured by our tunable laser. We are pursuing high power laser architectures that permit limited day time sodium lidar observations with the help of a narrow bandpass etalon filter. We discuss technology, prototypes, risks and trades for two 589 nm wavelength laser architectures: 1) Raman laser 2) Sum Frequency Generation. Laser-induced saturation of atomic sodium in the MLT region affects both sodium density and temperature measurements. We discuss the saturation impact on the laser parameters, laser architecture and instrument trades. Off-nadir pointing from the ISS causes Doppler shifts that effect the sodium spectroscopy. We discuss laser wavelength locking, tuning and spectroscopic-line sampling strategy.

Nonradiative transport of atomic excitation in Na vapor

NASA Astrophysics Data System (ADS)

Zajonc, Arthur G.; Phelps, A. V.

1981-05-01

Measurements are reported which show the effect of nonradiative losses at a gas-window interface on the backscattered fluorescence intensity for Na vapor at frequencies in the vicinity of the resonance lines near 589 nm. The Na 3P12,32 states are excited with a low-intensity single-mode tunable dye laser at high Na densities and the frequency integral of the backscattered fluorescence intensity in the D1 and D2 lines is measured. As the laser is tuned through resonance, the loss of atomic excitation to the window appears as a sharp decrease in the frequency-integrated fluorescence intensity. For example, at 7×1020 atoms m-3 the fluorescence intensity decreases by a factor of 4 in a frequency interval of 4 GHz. Measured absolute fluorescence intensities versus laser frequency are compared with predictions made using the theory of Hummer and Kunasz which includes both radiative and nonradiative transport processes. The agreement between theory and experiment is remarkably good when one considers that the theory contains only one unknown coefficient, i.e., the reflection coefficient for excited atoms at the windows. In our case the excited atoms are assumed to be completely destroyed at the window.

pH and chloride recordings in living cells using two-photon fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Lahn, Mattes; Hille, Carsten; Koberling, Felix; Kapusta, Peter; Dosche, Carsten

2010-02-01

Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences. The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a combination with two-photon (2P) excitation is quite promising. Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique. We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values. However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range. Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated pharmacologically. In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time measurements in combination with reduced cell and dye damages.

High-speed digital imaging of cytosolic Ca2+ and contraction in single cardiomyocytes.

PubMed

O'Rourke, B; Reibel, D K; Thomas, A P

1990-07-01

A charge-coupled device (CCD) camera, with the capacity for simultaneous spatially resolved photon counting and rapid frame transfer, was utilized for high-speed digital image collection from an inverted epifluorescence microscope. The unique properties of the CCD detector were applied to an analysis of cell shortening and the Ca2+ transient from fluorescence images of fura-2-loaded [corrected] cardiomyocytes. On electrical stimulation of the cell, a series of sequential subimages was collected and used to create images of Ca2+ within the cell during contraction. The high photosensitivity of the camera, combined with a detector-based frame storage technique, permitted collection of fluorescence images 10 ms apart. This rate of image collection was sufficient to resolve the rapid events of contraction, e.g., the upstroke of the Ca2+ transient (less than 40 ms) and the time to peak shortening (less than 80 ms). The technique was used to examine the effects of beta-adrenoceptor activation, fura-2 load, and stimulus frequency on cytosolic Ca2+ transients and contractions of single cardiomyocytes. beta-Adrenoceptor stimulation resulted in pronounced increases in peak Ca2+, maximal rates of rise and decay of Ca2+, extent of shortening, and maximal velocities of shortening and relaxation. Raising the intracellular load of fura-2 had little effect on the rising phase of Ca2+ or the extent of shortening but extended the duration of the Ca2+ transient and contraction. In related experiments utilizing differential-interference contrast microscopy, the same technique was applied to visualize sarcomere dynamics in contracting cells. This newly developed technique is a versatile tool for analyzing the Ca2+ transient and mechanical events in studies of excitation-contraction coupling in cardiomyocytes.

Whole-body multicolor spectrally resolved fluorescence imaging for development of target-specific optical contrast agents using genetically engineered probes

NASA Astrophysics Data System (ADS)

Kobayashi, Hisataka; Hama, Yukihiro; Koyama, Yoshinori; Barrett, Tristan; Urano, Yasuteru; Choyke, Peter L.

2007-02-01

Target-specific contrast agents are being developed for the molecular imaging of cancer. Optically detectable target-specific agents are promising for clinical applications because of their high sensitivity and specificity. Pre clinical testing is needed, however, to validate the actual sensitivity and specificity of these agents in animal models, and involves both conventional histology and immunohistochemistry, which requires large numbers of animals and samples with costly handling. However, a superior validation tool takes advantage of genetic engineering technology whereby cell lines are transfected with genes that induce the target cell to produce fluorescent proteins with characteristic emission spectra thus, identifying them as cancer cells. Multicolor fluorescence imaging of these genetically engineered probes can provide rapid validation of newly developed exogenous probes that fluoresce at different wavelengths. For example, the plasmid containing the gene encoding red fluorescent protein (RFP) was transfected into cell lines previously developed to either express or not-express specific cell surface receptors. Various antibody-based or receptor ligand-based optical contrast agents with either green or near infrared fluorophores were developed to concurrently target and validate cancer cells and their positive and negative controls, such as β-D-galactose receptor, HER1 and HER2 in a single animal/organ. Spectrally resolved fluorescence multicolor imaging was used to detect separate fluorescent emission spectra from the exogenous agents and RFP. Therefore, using this in vivo imaging technique, we were able to demonstrate the sensitivity and specificity of the target-specific optical contrast agents, thus reducing the number of animals needed to conduct these experiments.

Three-dimensional online surface reconstruction of augmented fluorescence lifetime maps using photometric stereo (Conference Presentation)

NASA Astrophysics Data System (ADS)

Unger, Jakob; Lagarto, Joao; Phipps, Jennifer; Ma, Dinglong; Bec, Julien; Sorger, Jonathan; Farwell, Gregory; Bold, Richard; Marcu, Laura

2017-02-01

Multi-Spectral Time-Resolved Fluorescence Spectroscopy (ms-TRFS) can provide label-free real-time feedback on tissue composition and pathology during surgical procedures by resolving the fluorescence decay dynamics of the tissue. Recently, an ms-TRFS system has been developed in our group, allowing for either point-spectroscopy fluorescence lifetime measurements or dynamic raster tissue scanning by merging a 450 nm aiming beam with the pulsed fluorescence excitation light in a single fiber collection. In order to facilitate an augmented real-time display of fluorescence decay parameters, the lifetime values are back projected to the white light video. The goal of this study is to develop a 3D real-time surface reconstruction aiming for a comprehensive visualization of the decay parameters and providing an enhanced navigation for the surgeon. Using a stereo camera setup, we use a combination of image feature matching and aiming beam stereo segmentation to establish a 3D surface model of the decay parameters. After camera calibration, texture-related features are extracted for both camera images and matched providing a rough estimation of the surface. During the raster scanning, the rough estimation is successively refined in real-time by tracking the aiming beam positions using an advanced segmentation algorithm. The method is evaluated for excised breast tissue specimens showing a high precision and running in real-time with approximately 20 frames per second. The proposed method shows promising potential for intraoperative navigation, i.e. tumor margin assessment. Furthermore, it provides the basis for registering the fluorescence lifetime maps to the tissue surface adapting it to possible tissue deformations.

Photodynamic tumor therapy and on-line fluorescence spectroscopy after aminolevulinic acid administration using 633-nm light as therapeutic and fluorescence excitation radiation

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Kienle, Alwin; Boehncke, Wolf-Henning; Kaufmann, Roland; Rueck, Angelika C.; Meier, Thomas H.; Steiner, Rudolf W.

1994-03-01

PDT and on-line fluorescence spectroscopy were carried out on human tumors after ALA- administration using 633 nm-light of a dye laser as therapeutic radiation and as fluorescence excitation radiation. This has the following advantages: (1) use of one laser for PDT and fluorescence diagnosis only, (2) the possibility of on-line fluorescence measurements, and (3) excitation of protoporphyrin molecules in deep tissue layers. Monte Carlo calculations were carried out to determine the excitation and fluorescence photon distribution in the case of red and violet excitation radiation. The results show the possibility of depth-resolved measurements on the fluorophore distribution by variation of the excitation wavelength. The influence of remitted excitation light and of the spontaneous radiation from the laser as well as the possible excitation of food-based degradation products of chlorophyll has to be considered in high-sensitive fluorescence measurements.

Confocal fluorescence techniques in industrial application

NASA Astrophysics Data System (ADS)

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

2003-06-01

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

Fluorescence properties of Schiff base - N,N‧-bis(salicylidene) - 1,2-Phenylenediamine in presence of bile acid host

NASA Astrophysics Data System (ADS)

Roy, Nayan; Paul, Pradip C.; Singh, T. Sanjoy

2015-05-01

Fluorescence properties of Schiff base - N,N‧-bis(salicylidene) - 1,2-phenylenediamine (LH2) is used to study the micelles formed by aggregation of different important bile acids like cholic acid, deoxycholic acid, chenodeoxycholic acid and glycocholic acid by steady state and picosecond time-resolved fluorescence spectroscopy. The fluorescence band intensity was found out to increase with concomitant red shift with gradual addition of different bile acids. Binding constant of the probe with different bile acids as well as critical micelle concentration was obtained from the variation of fluorescence intensity on increasing concentration of bile acids in the medium. The increase in fluorescence quantum yields, fluorescence decay times and substantial decrease in nonradiative decay rate constants in bile acids micellar environment points to the restricted motion of the fluorophore inside the micellar subdomains.

Confocal Microscopy Imaging with an Optical Transition Edge Sensor

NASA Astrophysics Data System (ADS)

Fukuda, D.; Niwa, K.; Hattori, K.; Inoue, S.; Kobayashi, R.; Numata, T.

2018-05-01

Fluorescence color imaging at an extremely low excitation intensity was performed using an optical transition edge sensor (TES) embedded in a confocal microscope for the first time. Optical TES has the ability to resolve incident single photon energy; therefore, the wavelength of each photon can be measured without spectroscopic elements such as diffraction gratings. As target objects, animal cells labeled with two fluorescent dyes were irradiated with an excitation laser at an intensity below 1 μW. In our confocal system, an optical fiber-coupled TES device is used to detect photons instead of the pinhole and photomultiplier tube used in typical confocal microscopes. Photons emitted from the dyes were collected by the objective lens, and sent to the optical TES via the fiber. The TES measures the wavelength of each photon arriving in an exposure time of 70 ms, and a fluorescent photon spectrum is constructed. This measurement is repeated by scanning the target sample, and finally a two-dimensional RGB-color image is obtained. The obtained image showed that the photons emitted from the dyes of mitochondria and cytoskeletons were clearly resolved at a detection intensity level of tens of photons. TES exhibits ideal performance as a photon detector with a low dark count rate (< 1 Hz) and wavelength resolving power. In the single-mode fiber-coupled system, the confocal microscope can be operated in the super-resolution mode. These features are very promising to realize high-sensitivity and high-resolution photon spectral imaging, and would help avoid cell damage and photobleaching of fluorescence dyes.

Isomers in the excited state of electron-transferring flavoprotein from Megasphaera elsdenii: spectral resolution from the time-resolved fluorescence spectra.

PubMed

Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi; Tanaka, Fumio

2008-02-27

Electron-transferring flavoprotein (Holo-ETF) from Megasphaera elsdenii contains two FAD's, one of which easily dissociates to form Iso-ETF (contains one FAD). Time-resolved fluorescence of FAD in Iso-ETF, and Holo-ETF were measured at 5 degrees C and 25 degrees C. Wavelength-dependent fluorescence decays of the both ETF at 5 degrees C and 25 degrees C were analyzed to resolve them into two independent spectra. It was found that Iso-ETF displayed two spectra with lifetime of 0.605 ns (emission peak, 508 nm) and with lifetime of 1.70 ns (emission peak, 540 nm) at 5 degrees C, and with lifetime of 0.693 ns (emission peak, 508 nm) and with lifetime of 2.75 ns (emission peak, 540 nm) at 25 degrees C. Holo-ETF displayed two spectra with lifetime of 0.739 ns (emission peak, 508 nm) and with lifetime of 2.06 ns (emission peak, 545 nm) at 5 degrees C, and with lifetime of 0.711 ns (emission peak, 527 nm) and with lifetime of 3.08 ns (emission peak, 540 nm) at 25 degrees C. Thus fluorescence lifetimes of every spectrum increased upon elevating temperature. Emission peaks Iso-ETF did not change much upon elevating temperature. Activation enthalpy changes, activation entropy changes and activation Gibbs energy changes of quenching rates all displayed negative. Two emission species in the both ETF may be hydrogen-bonding isomers, because isoalloxazine ring of FAD contains four hydrogen acceptors and one donor.

Novel Insight for Organic Matter Sourcing: Interest of Time Resolved Fluorescence to Qualify and Quantify PAH Content of Solid Matrix at High Resolution

NASA Astrophysics Data System (ADS)

Quiers, M.; Perrette, Y.; Jacq, K.; Pousset, E.; Plassart, G.

2017-12-01

OM fluorescence is today a well-developed tool used to characterize and quantify organic matter (OM), but also to evaluate and discriminate OM fate and changes related to climate and environmental modifications. While fluorescence measurements on water and soils extracts provide information about organic fluxes today, solid phase fluorescence using natural archives allows to obtain high resolution records of OM evolution during time. These evolutions can be discussed in regards of climate and environmental perturbations detected in archives using different proxies, and thus provide keys for understanding factors driving carbon fluxes mechanisms. Among fluorescent organic species, Polycyclic Aromatic Hydrocarbons (PAH) have been used as probe molecules for organic contamination tracking. Moreover, monitoring studies have shown that PAH could also be used as markers to discriminates atmospheric and erosion factors leading to PAH and organic matter fluxes to the aquifer. PAH records in soils and natural archives appear as a promising proxy to follow both past atmospheric contamination and soil erosion. But, PAH fluorescence is difficult to discriminate from bulk OM fluorescence using steady-state fluorescence (SSF) technics as their fluorescence domains recover. Time resolved emission spectroscopy (TRES) increases the information provided by SSF technic, adding a time dimension to measurements and allowing to discriminate PAH fluorescence. We report here a first application of this technic on natural archives. The challenge is to obtain TRES signature along the sample, including for low PAH concentrations. This study aims to evaluate the reliability of high resolution TRES measurement as PAH carbon fluxes sources. Method is based on LIF instrument for solid phase fluorescence measurement. An instrument coupling an excitation system constituting by 2 pulsed lasers (266 and 355 nm) and a detection system was developed. This measurement provides high resolution record of PAH fluorescence. Preliminary results on stalagmite samples, lake sediments and soils will be reported. PAH content variations along the sample were compared with PAH concentration and with bulk OM content deduced from SSF records. The accuracy of the PAH fluorescence as source marker of fluxes will be discussed for each type of sample.

The dependence of the ultrafast relaxation kinetics of the S2 and S1 states in β-carotene homologs and lycopene on conjugation length studied by femtosecond time-resolved absorption and Kerr-gate fluorescence spectroscopies

NASA Astrophysics Data System (ADS)

Kosumi, Daisuke; Fujiwara, Masazumi; Fujii, Ritsuko; Cogdell, Richard J.; Hashimoto, Hideki; Yoshizawa, Masayuki

2009-06-01

The ultrafast relaxation kinetics of all-trans-β-carotene homologs with varying numbers of conjugated double bonds n(n =7-15) and lycopene (n =11) has been investigated using femtosecond time-resolved absorption and Kerr-gate fluorescence spectroscopies, both carried out under identical excitation conditions. The nonradiative relaxation rates of the optically allowed S2(1Bu+1) state were precisely determined by the time-resolved fluorescence. The kinetics of the optically forbidden S1(2Ag-1) state were observed by the time-resolved absorption measurements. The dependence of the S1 relaxation rates upon the conjugation length is adequately described by application of the energy gap law. In contrast to this, the nonradiative relaxation rates of S2 have a minimum at n =9 and show a reverse energy gap law dependence for values of n above 11. This anomalous behavior of the S2 relaxation rates can be explained by the presence of an intermediate state (here called the Sx state) located between the S2 and S1 states at large values of n (such as n =11). The presence of such an intermediate state would then result in the following sequential relaxation pathway S2→Sx→S1→S0. A model based on conical intersections between the potential energy curves of these excited singlet states can readily explain the measured relationships between the decay rates and the energy gaps.

Interaction of promethazine and adiphenine to human hemoglobin: A comparative spectroscopic and computational analysis

NASA Astrophysics Data System (ADS)

Maurya, Neha; ud din Parray, Mehraj; Maurya, Jitendra Kumar; Kumar, Amit; Patel, Rajan

2018-06-01

The binding nature of amphiphilic drugs viz. promethazine hydrochloride (PMT) and adiphenine hydrochloride (ADP), with human hemoglobin (Hb) was unraveled by fluorescence, absorbance, time resolved fluorescence, fluorescence resonance energy transfer (FRET) and circular dichroism (CD) spectral techniques in combination with molecular docking and molecular dynamic simulation methods. The steady state fluorescence spectra indicated that both PMT and ADP quenches the fluorescence of Hb through static quenching mechanism which was further confirmed by time resolved fluorescence spectra. The UV-Vis spectroscopy suggested ground state complex formation. The activation energy (Ea) was observed more in the case of Hb-ADP than Hb-PMT interaction system. The FRET result indicates the high probability of energy transfer from β Trp37 residue of Hb to the PMT (r = 2.02 nm) and ADP (r = 2.33 nm). The thermodynamic data reveal that binding of PMT with Hb are exothermic in nature involving hydrogen bonding and van der Waal interaction whereas in the case of ADP hydrophobic forces play the major role and binding process is endothermic in nature. The CD results show that both PMT and ADP, induced secondary structural changes of Hb and unfold the protein by losing a large helical content while the effect is more pronounced with ADP. Additionally, we also utilized computational approaches for deep insight into the binding of these drugs with Hb and the results are well matched with our experimental results.

Interaction of promethazine and adiphenine to human hemoglobin: A comparative spectroscopic and computational analysis.

PubMed

Maurya, Neha; Ud Din Parray, Mehraj; Maurya, Jitendra Kumar; Kumar, Amit; Patel, Rajan

2018-06-15

The binding nature of amphiphilic drugs viz. promethazine hydrochloride (PMT) and adiphenine hydrochloride (ADP), with human hemoglobin (Hb) was unraveled by fluorescence, absorbance, time resolved fluorescence, fluorescence resonance energy transfer (FRET) and circular dichroism (CD) spectral techniques in combination with molecular docking and molecular dynamic simulation methods. The steady state fluorescence spectra indicated that both PMT and ADP quenches the fluorescence of Hb through static quenching mechanism which was further confirmed by time resolved fluorescence spectra. The UV-Vis spectroscopy suggested ground state complex formation. The activation energy (E a ) was observed more in the case of Hb-ADP than Hb-PMT interaction system. The FRET result indicates the high probability of energy transfer from β Trp37 residue of Hb to the PMT (r=2.02nm) and ADP (r=2.33nm). The thermodynamic data reveal that binding of PMT with Hb are exothermic in nature involving hydrogen bonding and van der Waal interaction whereas in the case of ADP hydrophobic forces play the major role and binding process is endothermic in nature. The CD results show that both PMT and ADP, induced secondary structural changes of Hb and unfold the protein by losing a large helical content while the effect is more pronounced with ADP. Additionally, we also utilized computational approaches for deep insight into the binding of these drugs with Hb and the results are well matched with our experimental results. Copyright © 2018 Elsevier B.V. All rights reserved.

Solvent and solute ingress into hydrogels resolved by a combination of imaging techniques

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

Wagner, D.; Burbach, J.; Egelhaaf, S. U.

2016-05-28

Using simultaneous neutron, fluorescence, and optical brightfield transmission imaging, the diffusion of solvent, fluorescent dyes, and macromolecules into a crosslinked polyacrylamide hydrogel was investigated. This novel combination of different imaging techniques enables us to distinguish the movements of the solvent and fluorescent molecules. Additionally, the swelling or deswelling of the hydrogels can be monitored. From the sequence of images, dye and solvent concentrations were extracted spatially and temporally resolved. Diffusion equations and different boundary conditions, represented by different models, were used to quantitatively analyze the temporal evolution of these concentration profiles and to determine the diffusion coefficients of solvent andmore » solutes. Solute size and network properties were varied and their effect was investigated. Increasing the crosslinking ratio or partially drying the hydrogel was found to hinder solute diffusion due to the reduced pore size. By contrast, solvent diffusion seemed to be slightly faster if the hydrogel was only partially swollen and hence solvent uptake enhanced.« less

A versatile fiber-optic coupled system for sensitive optical spectroscopy in strong ambient light

NASA Astrophysics Data System (ADS)

Sinha, Sudarson Sekhar; Verma, Pramod Kumar; Makhal, Abhinandan; Pal, Samir Kumar

2009-05-01

In this work we describe design and use of a fiber-optic based optical system for the spectroscopic studies on the samples under the presence of strong ambient light. The system is tested to monitor absorption, emission, and picosecond-resolved fluorescence transients simultaneously with a time interval of 500 ms for several hours on a biologically important sample (vitamin B2) under strong UV light. An efficient stray-light rejection ratio of the setup is achieved by the confocal geometry of the excitation and detection channels. It is demonstrated using this setup that even low optical signal from a liquid sample under strong UV-exposure for the picosecond-resolved fluorescence transient measurement can reliably be detected by ultrasensitive microchannel plate photomultiplier tube solid state detector. The kinetics of photodeterioration of vitamin B2 measured using our setup is consistent with that reported in the literature. Our present studies also justify the usage of tungsten light than the fluorescent light for the healthy preservation of food with vitamin B2.

Highly luminescent, biocompatible ytterbium(iii) complexes as near-infrared fluorophores for living cell imaging.

PubMed

Ning, Yingying; Tang, Juan; Liu, Yi-Wei; Jing, Jing; Sun, Yuansheng; Zhang, Jun-Long

2018-04-21

Herein, we report the design and synthesis of biocompatible Yb 3+ complexes for near-infrared (NIR) living cell imaging. Upon excitation at either the visible (Soret band) or red region (Q band), these β-fluorinated Yb 3+ complexes display high NIR luminescence (quantum yields up to 23% and 13% in dimethyl sulfoxide and water, respectively) and have higher stabilities and prolonged decay lifetimes (up to 249 μs) compared to the β-non-fluorinated counterparts. This renders the β-fluorinated Yb 3+ complexes as a new class of biological optical probes in both steady-state imaging and time-resolved fluorescence lifetime imaging (FLIM). NIR confocal fluorescence images showed strong and specific intracellular Yb 3+ luminescence signals when the biocompatible Yb 3+ complexes were uptaken into the living cells. Importantly, FLIM measurements showed an intracellular lifetime distribution between 100 and 200 μs, allowing an effective discrimination from cell autofluorescence, and afforded high signal-to-noise ratios as firstly demonstrated in the NIR region. These results demonstrated the prospects of NIR lanthanide complexes as biological probes for NIR steady-state fluorescence and time-resolved fluorescence lifetime imaging.

Noninvasive tumor detection using spectrally-resolved in-vivo imaging

NASA Astrophysics Data System (ADS)

Kostenich, Gennady; Kimel, Sol; Malik, Zvi; Orenstein, Arie

2000-11-01

A novel spectral image-analysis system was used for tumor fluorescence and reflectance imaging in an animal model and in patients. Transcutaneous fluorescence imaging was carried out on Balb/c mice bearing subcutaneous C26 colon carcinoma after intraperitoneal (i.p.) administration of 5-aminolevulinic acid (ALA), a metabolic precursor of protoporphyrin-IX (PP), and of a novel photosensitizer tetrahydroporphyrin (THP). Tumors were clearly observable by fluorescence detection using green light excitation. Tumor versus normal tissue uptake of the photosensitizing agents was determined by monitoring fluorescence intensity. Maximal PP accumulation in tumor was observed 3 h after i.p. injection of ALA, whereas THP showed selective accumulation in tumor 24 h after administration. Reflectance spectroscopy was employed to study pigmented human skin lesions (nevus, pigmented BCC and pigmented melanoma). In the near-infrared region (800-880 nm) pigmented BCC and melanoma exhibited a differently shaped reflectance spectrum compared to normal skin and nevus. Spatially and spectrally resolved imaging, in combination with mathematical algorithms (such as normalization, spectral similarity mapping and division) allowed unambiguous detection of malignancies. Optical biopsy results from a total of 51 patients showed 45 benign nevi, 3 pigmented BCC and 3 malignant melanomas, as confirmed by histology.

On-line and in-situ detection of polycyclic aromatic hydrocarbons (PAH) on aerosols via thermodesorption and laser-induced fluorescence spectroscopy.

PubMed

Panne, U; Knöller, A; Kotzick, R; Niessner, R

2000-02-01

A fiber optical sensor system for the determination of polycyclic aromatic hydrocarbons (PAH) on aerosols by laser-induced, time-resolved fluorescence is combined with a thermodesorption device. The sensor system is based on an aerosol flow cell, which is fibre-optically coupled to a pulsed nitrogen laser for excitation and the detection system. Time-resolved fluorescence emission spectra are detected by a monochromator equipped with a photomultiplier and a fast digital storage oscilloscope. The analytical figures of merit of the thermodenuder are reported for benzo[a]pyrene, benzo[b]fluoranthene, and benzo[ghi]-perylene on ultrafine soot and NaCl aerosols. By thermodesorption of the PAH, problems due to quenching of the PAH fluorescence by the bulk aerosol material or excimer formation on the aerosol surface were avoided. For the PAH under study, the sensitivity was improved considerably and detection limits between 110 and 850 ng m(-3) were attained, while a response time of 2-3 min was achieved with the thermodenuder. A calibration for PAH on ultrafine soot and NaCl aerosols was established independent of the aerosol substrate.

Simultaneous confocal fluorescence microscopy and optical coherence tomography for drug distribution and tissue integrity assessment

NASA Astrophysics Data System (ADS)

Rinehart, Matthew T.; LaCroix, Jeffrey; Henderson, Marcus; Katz, David; Wax, Adam

2011-03-01

The effectiveness of microbicidal gels, topical products developed to prevent infection by sexually transmitted diseases including HIV/AIDS, is governed by extent of gel coverage, pharmacokinetics of active pharmaceutical ingredients (APIs), and integrity of vaginal epithelium. While biopsies provide localized information about drug delivery and tissue structure, in vivo measurements are preferable in providing objective data on API and gel coating distribution as well as tissue integrity. We are developing a system combining confocal fluorescence microscopy with optical coherence tomography (OCT) to simultaneously measure local concentrations and diffusion coefficients of APIs during transport from microbicidal gels into tissue, while assessing tissue integrity. The confocal module acquires 2-D images of fluorescent APIs multiple times per second allowing analysis of lateral diffusion kinetics. The custom Fourier domain OCT module has a maximum a-scan rate of 54 kHz and provides depth-resolved tissue integrity information coregistered with the confocal fluorescence measurements. The combined system is validated by imaging phantoms with a surrogate fluorophore. Time-resolved API concentration measured at fixed depths is analyzed for diffusion kinetics. This multimodal system will eventually be implemented in vivo for objective evaluation of microbicide product performance.

Ultrafast time-resolved spectroscopy of the light-harvesting complex 2 (LH2) from the photosynthetic bacterium Thermochromatium tepidum.

PubMed

Niedzwiedzki, Dariusz M; Fuciman, Marcel; Kobayashi, Masayuki; Frank, Harry A; Blankenship, Robert E

2011-10-01

The light-harvesting complex 2 from the thermophilic purple bacterium Thermochromatium tepidum was purified and studied by steady-state absorption and fluorescence, sub-nanosecond-time-resolved fluorescence and femtosecond time-resolved transient absorption spectroscopy. The measurements were performed at room temperature and at 10 K. The combination of both ultrafast and steady-state optical spectroscopy methods at ambient and cryogenic temperatures allowed the detailed study of carotenoid (Car)-to-bacteriochlorophyll (BChl) as well BChl-to-BChl excitation energy transfer in the complex. The studies show that the dominant Cars rhodopin (N=11) and spirilloxanthin (N=13) do not play a significant role as supportive energy donors for BChl a. This is related with their photophysical properties regulated by long π-electron conjugation. On the other hand, such properties favor some of the Cars, particularly spirilloxanthin (N=13) to play the role of the direct quencher of the excited singlet state of BChl. © Springer Science+Business Media B.V. 2011

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest

PubMed Central

Dahlberg, Peter D.; Boughter, Christopher T.; Faruk, Nabil F.; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A.; Sherani, Aiman; Hammond, Adam T.

2016-01-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals. PMID:27910631

Time-to-digital converter card for multichannel time-resolved single-photon counting applications

NASA Astrophysics Data System (ADS)

Tamborini, Davide; Portaluppi, Davide; Tisa, Simone; Tosi, Alberto

2015-03-01

We present a high performance Time-to-Digital Converter (TDC) card that provides 10 ps timing resolution and 20 ps (rms) timing precision with a programmable full-scale-range from 160 ns to 10 μs. Differential Non-Linearity (DNL) is better than 1.3% LSB (rms) and Integral Non-Linearity (INL) is 5 ps rms. Thanks to the low power consumption (400 mW) and the compact size (78 mm x 28 mm x 10 mm), this card is the building block for developing compact multichannel time-resolved instrumentation for Time-Correlated Single-Photon Counting (TCSPC). The TDC-card outputs the time measurement results together with the rates of START and STOP signals and the number of valid TDC conversions. These additional information are needed by many TCSPC-based applications, such as: Fluorescence Lifetime Imaging (FLIM), Time-of-Flight (TOF) ranging measurements, time-resolved Positron Emission Tomography (PET), single-molecule spectroscopy, Fluorescence Correlation Spectroscopy (FCS), Diffuse Optical Tomography (DOT), Optical Time-Domain Reflectometry (OTDR), quantum optics, etc.

A Photoluminescence-Based Field Method for Detection of Traces of Explosives

PubMed Central

Menzel, E. Roland; Menzel, Laird W.; Schwierking, Jake R.

2004-01-01

We report a photoluminescence-based field method for detecting traces of explosives. In its standard version, the method utilizes a commercially available color spot test kit for treating explosive traces on filter paper after swabbing. The colored products are fluorescent under illumination with a laser that operates on three C-size flashlight batteries and delivers light at 532 nm. In the fluorescence detection mode, by visual inspection, the typical sensitivity gain is a factor of 100. The method is applicable to a wide variety of explosives. In its time-resolved version, intended for in situ work, explosives are tagged with europium complexes. Instrumentation-wise, the time-resolved detection, again visual, can be accomplished in facile fashion. The europium luminescence excitation utilizes a laser operating at 355 nm. We demonstrate the feasibility of CdSe quantum dot sensitization of europium luminescence for time-resolved purposes. This would allow the use of the above 532 nm laser. PMID:15349512

Single-molecule detection by two-photon excitation of fluorescence

NASA Astrophysics Data System (ADS)

Zander, Christoph; Brand, Leif; Eggeling, C.; Drexhage, Karl-Heinz; Seidel, Claus A. M.

1997-05-01

Using a mode-locked titanium: sapphire laser at 700 nm for two-photon excitation we studied fluorescence bursts from individual coumarin 120 molecules in water and triacetin. Fluorescence lifetimes and multichannel scaler traces have been measured simultaneously. Due to the fact that scattered excitation light as well as Raman scattered photons can be suppressed by a short-pass filter a very low background level was achieved. To identify the fluorophore by its characteristic fluorescence lifetime the time-resolved fluorescence signals were analyzed by a maximum likelihood estimator. The obtained average fluorescence lifetimes (tau) av equals 4.8 +/- 1.2 ns for coumarin 120 in water and (tau) av equals 3.3 +/- 0.6 for coumarin 120 in triacetin are in good agreement with results obtained from separate measurements at higher concentrations.

The Origin of Fluorescence from Graphene Oxide

PubMed Central

Shang, Jingzhi; Ma, Lin; Li, Jiewei; Ai, Wei; Yu, Ting; Gurzadyan, Gagik G.

2012-01-01

Time-resolved fluorescence measurements of graphene oxide in water show multiexponential decay kinetics ranging from 1 ps to 2 ns. Electron-hole recombination from the bottom of the conduction band and nearby localized states to wide-range valance band is suggested as origin of the fluorescence. Excitation wavelength dependence of the fluorescence was caused by relative intensity changes of few emission species. By introducing the molecular orbital concept, the dominant fluorescence was found to originate from the electronic transitions among/between the non-oxidized carbon regions and the boundary of oxidized carbon atom regions, where all three kinds of functionalized groups C-O, C = O and O = C-OH were participating. In the visible spectral range, the ultrafast fluorescence of graphene oxide was observed for the first time. PMID:23145316

Site-resolved imaging of a bosonic Mott insulator using ytterbium atoms

NASA Astrophysics Data System (ADS)

Miranda, Martin; Inoue, Ryotaro; Tambo, Naoki; Kozuma, Mikio

2017-10-01

We demonstrate site-resolved imaging of a strongly correlated quantum system without relying on laser cooling techniques during fluorescence imaging. We observe the formation of Mott shells in the insulating regime and realize thermometry in an atomic cloud. This work proves the feasibility of the noncooled approach and opens the door to extending the detection technology to new atomic species.

Frequency division multiplexed multi-color fluorescence microscope system

NASA Astrophysics Data System (ADS)

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

2017-10-01

Grayscale camera can only obtain gray scale image of object, while the multicolor imaging technology can obtain the color information to distinguish the sample structures which have the same shapes but in different colors. In fluorescence microscopy, the current method of multicolor imaging are flawed. Problem of these method is affecting the efficiency of fluorescence imaging, reducing the sampling rate of CCD etc. In this paper, we propose a novel multiple color fluorescence microscopy imaging method which based on the Frequency division multiplexing (FDM) technology, by modulating the excitation lights and demodulating the fluorescence signal in frequency domain. This method uses periodic functions with different frequency to modulate amplitude of each excitation lights, and then combine these beams for illumination in a fluorescence microscopy imaging system. The imaging system will detect a multicolor fluorescence image by a grayscale camera. During the data processing, the signal obtained by each pixel of the camera will be processed with discrete Fourier transform, decomposed by color in the frequency domain and then used inverse discrete Fourier transform. After using this process for signals from all of the pixels, monochrome images of each color on the image plane can be obtained and multicolor image is also acquired. Based on this method, this paper has constructed and set up a two-color fluorescence microscope system with two excitation wavelengths of 488 nm and 639 nm. By using this system to observe the linearly movement of two kinds of fluorescent microspheres, after the data processing, we obtain a two-color fluorescence dynamic video which is consistent with the original image. This experiment shows that the dynamic phenomenon of multicolor fluorescent biological samples can be generally observed by this method. Compared with the current methods, this method can obtain the image signals of each color at the same time, and the color video's frame rate is consistent with the frame rate of the camera. The optical system is simpler and does not need extra color separation element. In addition, this method has a good filtering effect on the ambient light or other light signals which are not affected by the modulation process.

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

PubMed Central

Chen, Hongtao

2013-01-01

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

Rapid detection of trace amounts of surfactants using nanoparticles in fluorometric assays

NASA Astrophysics Data System (ADS)

Härmä, Harri; Laakso, Susana; Pihlasalo, Sari; Hänninen, Pekka; Faure, Bertrand; Rana, Subhasis; Bergström, Lennart

2010-01-01

Rapid microtiter assays that utilize the time-resolved fluorescence resonance energy transfer or quenching of dye-labeled proteins adsorbed onto the surfaces of polystyrene or maghemite nanoparticles have been developed for the detection and quantification of trace amounts of surfactants at concentrations down to 10 nM.Rapid microtiter assays that utilize the time-resolved fluorescence resonance energy transfer or quenching of dye-labeled proteins adsorbed onto the surfaces of polystyrene or maghemite nanoparticles have been developed for the detection and quantification of trace amounts of surfactants at concentrations down to 10 nM. Electronic supplementary information (ESI) available: Experimental details and Fig. S1 and S2. See DOI: 10.1039/b9nr00172g

Picosecond-resolved FRET on non-amplified DNA for identifying individuals genetically susceptible to type-1 diabetes

NASA Astrophysics Data System (ADS)

Nardo, Luca; Tosi, Giovanna; Bondani, Maria; Accolla, Roberto; Andreoni, Alessandra

2012-06-01

By tens-of-picosecond resolved fluorescence detection we study Förster resonance energy transfer between a donor and a black-hole-quencher bound at the 5'- and 3'-positions of an oligonucleotide probe matching the highly polymorphic region between codons 51 and 58 of the human leukocyte antigen DQB1 0201 allele, conferring susceptibility to type-1 diabetes. The probe is annealed with non-amplified genomic DNAs carrying either the 0201 sequence or other DQB1 allelic variants. We detect the longest-lived donor fluorescence in the case of hybridization with the 0201 allele and definitely faster and distinct decays for the other allelic variants, some of which are single-nucleotide polymorphic.

High repetition rate laser induced fluorescence applied to Surfatron Induced Plasmas

NASA Astrophysics Data System (ADS)

van der Mullen, J. J. A. M.; Palomares, J. M.; Carbone, E. A. D.; Graef, W.; Hübner, S.

2012-05-01

The reaction kinetics in the excitation space of Ar and the conversion space of Ar-molecule mixtures are explored using a combination of high rep-rate YAG-Dye laser systems with a well defined and easily controllable Surfatron Induced Plasma set-up. Applying the method of Saturation Time Resolved Laser Induced Fluorescence (SaTiRe-LIF), we could trace excitation and conversion channels and determine rates of electron and heavy particle excitation kinetics. The time resolved density disturbances observed in the Ar excitation space, which are initiated by the laser, reveal the excitation channels and corresponding rates; responses of the molecular radiation in Ar-molecule mixtures corresponds to the presence of conversion processes induced by heavy particle excitation kinetics.

Binding and relaxation behavior of Coumarin-153 in lecithin-taurocholate mixed micelles: A time resolved fluorescence spectroscopic study

NASA Astrophysics Data System (ADS)

Chakrabarty, Debdeep; Chakraborty, Anjan; Seth, Debabrata; Hazra, Partha; Sarkar, Nilmoni

2005-09-01

The microenvironment of the bile salt-lecithin mixed aggregates has been investigated using steady state and picosecond time resolved fluorescence spectroscopy. The steady state spectra show that the polarity of the bile salt is higher compared to lecithin vesicles or the mixed aggregates. We have observed slow solvent relaxation in bile salt micelles and lecithin vesicles. The solvation time is gradually slowed down due to gradual addition of the bile salt in lecithin vesicles. Addition of bile salt leads to the tighter head group packing in lecithin. Thus, mobility of the water molecules becomes slower and consequently the solvation time is also retarded. We have observed bimodal slow rotational relaxation time in all these systems.

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

Thermodynamic measurements in a high pressure hydrogen-oxygen flame using Raman scattering from a broadband excimer laser

NASA Technical Reports Server (NTRS)

Hartfield, Roy, Jr.

1996-01-01

Raman scattering is an inelastic molecular scattering process in which incident radiation is reemitted at a fixed change in frequency. Raman spectroscopy can be used to measure the number density and temperature of the irradiated species. The strength of the Raman signal is inversely proportional to the wavelength raised to the fourth power. Consequently, high signal to noise ratios are obtained by using ultraviolet (UV) excitation sources. Using UV sources for Raman Spectroscopy in flames is complicated by the fact that some of the primary constituents in hydrogen-oxygen combustion absorb and reemit light in the UV and these fluorescence processes interfere with the Raman signals. This problem has been handled in atmospheric pressure flames in some instances by using a narrowband tunable excimer laser as a source. This allows for detuning from absorption transitions and the elimination of interfering fluorescence signals at the Raman wavelengths. This approach works well in the atmospheric pressure flame; however, it has two important disadvantages. First, injection-locked narrowband tunable excimer lasers are very expensive. More importantly, however, is the fact that at the high pressures characteristic of rocket engine combustion chambers, the absorption transitions are broadened making it difficult to tune to a spectral location at which substantial absorption would not occur. The approach taken in this work is to separate the Raman signal from the fluorescence background by taking advantage of the fact that Raman signal has nonisotropic polarization characteristics while the fluorescence signals are unpolarized. Specifically, for scattering at right angles to the excitation beam path, the Raman signal is completely polarized. The Raman signal is separated from the fluorescence background by collecting both horizontally and vertically polarized signals separately. One of the polarizations has both the Raman signal and the fluorescence background while the other has only the fluorescence signal. The Raman scatter is the difference between the signals. By choosing an appropriate optical setup, both signals can be obtained simultaneously with the same monochromator; hence, time resolved measurements are possible using this approach.

Study of fluorescence quenching of Barley α-amylase

NASA Astrophysics Data System (ADS)

Bakkialakshmi, S.; Shanthi, B.; Bhuvanapriya, T.

2012-05-01

The fluorescence quenching of Barley α-amylase by acrylamide and succinimide has been studied in water using steady-state and time-resolved fluorescence techniques. The steady-state fluorescence quenching technique has been performed in three different pHs (i.e., 6, 7 and 8) of water. Ground state and excited state binding constants (Kg &Ke) have been calculated. From the calculated binding constants (Kg &Ke) the free energy changes for the ground (ΔGg) and excited (ΔGe) states have been calculated and are presented in tables. UV and FTIR spectra have also been recorded to prove the binding of Barley α-amylase with acrylamide and succinimide.

Mode-resolved frequency comb interferometry for high-accuracy long distance measurement

PubMed Central

van den Berg, Steven. A.; van Eldik, Sjoerd; Bhattacharya, Nandini

2015-01-01

Optical frequency combs have developed into powerful tools for distance metrology. In this paper we demonstrate absolute long distance measurement using a single femtosecond frequency comb laser as a multi-wavelength source. By applying a high-resolution spectrometer based on a virtually imaged phased array, the frequency comb modes are resolved spectrally to the level of an individual mode. Having the frequency comb stabilized against an atomic clock, thousands of accurately known wavelengths are available for interferometry. From the spectrally resolved output of a Michelson interferometer a distance is derived. The presented measurement method combines spectral interferometry, white light interferometry and multi-wavelength interferometry in a single scheme. Comparison with a fringe counting laser interferometer shows an agreement within <10−8 for a distance of 50 m. PMID:26419282

Controlled fluorescence in a beetle's photonic structure and its sensitivity to environmentally induced changes

PubMed Central

Kaczmarek, Anna M.; Vukusic, Peter; Deparis, Olivier; Van Hooijdonk, Eloise

2016-01-01

The scales covering the elytra of the male Hoplia coerulea beetle contain fluorophores embedded within a porous photonic structure. The photonic structure controls both insect colour (reflected light) and fluorescence emission. Herein, the effects of water-induced changes on the fluorescence emission from the beetle were investigated. The fluorescence emission peak wavelength was observed to blue-shift on water immersion of the elytra whereas its reflectance peak wavelength was observed to red-shift. Time-resolved fluorescence measurements, together with optical simulations, confirmed that the radiative emission is controlled by a naturally engineered photonic bandgap while the elytra are in the dry state, whereas non-radiative relaxation pathways dominate the emission response of wet elytra. PMID:28003460

Correction: Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS.

PubMed

Wei, Feng; Xiong, Wei; Li, Wenhui; Lu, Wangting; Allen, Heather C; Zheng, Wanquan

2016-06-14

Correction for 'Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS' by Feng Wei et al., Phys. Chem. Chem. Phys., 2015, 17, 25114-25122.

Room temperature spectrally resolved single-molecule spectroscopy reveals new spectral forms and photophysical versatility of aequorea green fluorescent protein variants.

PubMed

Blum, Christian; Meixner, Alfred J; Subramaniam, Vinod

2004-12-01

It is known from ensemble spectroscopy at cryogenic temperatures that variants of the Aequorea green fluorescent protein (GFP) occur in interconvertible spectroscopically distinct forms which are obscured in ensemble room temperature spectroscopy. By analyzing the fluorescence of the GFP variants EYFP and EGFP by spectrally resolved single-molecule spectroscopy we were able to observe spectroscopically different forms of the proteins and to dynamically monitor transitions between these forms at room temperature. In addition to the predominant EYFP B-form we have observed the blue-shifted I-form thus far only seen at cryogenic temperatures and have followed transitions between these forms. Further we have identified for EYFP and for EGFP three more, so far unknown, forms with red-shifted fluorescence. Transitions between the predominant forms and the red-shifted forms show a dark time which indicates the existence of a nonfluorescent intermediate. The spectral position of the newly-identified red-shifted forms and their formation via a nonfluorescent intermediate hint that these states may account for the possible photoactivation observed in bulk experiments. The comparison of the single-protein spectra of the red-shifted EYFP and EGFP forms with single-molecule fluorescence spectra of DsRed suggest that these new forms possibly originate from an extended chromophoric pi-system analogous to the DsRed chromophore.

Hydrogen-bonding dynamics of free flavins in benzene and FAD in electron-transferring flavoprotein upon excitation.

PubMed

Sato, Kyousuke; Nishina, Yasuzou; Shiga, Kiyoshi; Tanaka, Fumio

2003-01-01

The dynamic natures of two hydrogen-bonding model systems, riboflavin tetrabutylate (RFTB)-trichloroacetic acid (TCA) and RFTB-phenol in benzene, and of electron-transferring flavoprotein (ETF) from pig kidney upon excitation of flavins was investigated by means of steady state and time-resolved fluorescence spectroscopy. In both model systems fluorescence intensities of RFTB decreased as TCA or phenol was added. The spectral characteristics of ETF under steady state excitation were quite similar to those of the RFTB-TCA system, but not to those of the RFTB-phenol system. The observed fluorescence decay curves of ETF fit well with the calculated decay curves with two lifetime components, as in the model systems. Averaged lifetime was 0.9 ns. The time-resolved fluorescence spectrum of ETF shifted toward longer wavelength with time after pulsed excitation, which was also observed in the RFTB-TCA system. In the RFTB-phenol system the emission spectrum did not shift at all with time. These results reveal that the dynamic nature of ETF can be ascribed to aliphatic hydrogen-bonding(s) of the isoalloxazine ring with surrounding amino acid(s). From the fluorescence characteristics of ETF in comparison with the model systems, human ETF and other flavoproteins, it was suggested that ETF from pig kidney does not contain Tyr-16 in the beta subunit, unlike human ETF.

Planar temperature measurement in compressible flows using laser-induced iodine fluorescence

NASA Technical Reports Server (NTRS)

Hartfield, Roy J., Jr.; Hollo, Steven D.; Mcdaniel, James C.

1991-01-01

A laser-induced iodine fluorescence technique that is suitable for the planar measurement of temperature in cold nonreacting compressible air flows is investigated analytically and demonstrated in a known flow field. The technique is based on the temperature dependence of the broadband fluorescence from iodine excited by the 514-nm line of an argon-ion laser. Temperatures ranging from 165 to 245 K were measured in the calibration flow field. This technique makes complete, spatially resolved surveys of temperature practical in highly three-dimensional, low-temperature compressible flows.

Resonance fluorescence in the resolvent-operator formalism

NASA Astrophysics Data System (ADS)

Debierre, V.; Harman, Z.

2017-10-01

The Mollow spectrum for the light scattered by a driven two-level atom is derived in the resolvent operator formalism. The derivation is based on the construction of a master equation from the resolvent operator of the atom-field system. We show that the natural linewidth of the excited atomic level remains essentially unmodified, to a very good level of approximation, even in the strong-field regime, where Rabi flopping becomes relevant inside the self-energy loop that yields the linewidth. This ensures that the obtained master equation and the spectrum derived matches that of Mollow.

Spectral and Temporal Laser Fluorescence Analysis Such as for Natural Aquatic Environments

NASA Technical Reports Server (NTRS)

Chekalyuk, Alexander (Inventor)

2015-01-01

An Advanced Laser Fluorometer (ALF) can combine spectrally and temporally resolved measurements of laser-stimulated emission (LSE) for characterization of dissolved and particulate matter, including fluorescence constituents, in liquids. Spectral deconvolution (SDC) analysis of LSE spectral measurements can accurately retrieve information about individual fluorescent bands, such as can be attributed to chlorophyll-a (Chl-a), phycobiliprotein (PBP) pigments, or chromophoric dissolved organic matter (CDOM), among others. Improved physiological assessments of photosynthesizing organisms can use SDC analysis and temporal LSE measurements to assess variable fluorescence corrected for SDC-retrieved background fluorescence. Fluorescence assessments of Chl-a concentration based on LSE spectral measurements can be improved using photo-physiological information from temporal measurements. Quantitative assessments of PBP pigments, CDOM, and other fluorescent constituents, as well as basic structural characterizations of photosynthesizing populations, can be performed using SDC analysis of LSE spectral measurements.

Complex metabolic oscillations in plants forced by harmonic irradiance.

PubMed Central

Nedbal, Ladislav; Brezina, Vítezslav

2002-01-01

Plants exposed to harmonically modulated irradiance, approximately 1 + cos(omegat), exhibit a complex periodic pattern of chlorophyll fluorescence emission that can be deconvoluted into a steady-state component, a component that is modulated with the frequency of the irradiance (omega), and into at least two upper harmonic components (2omega and 3omega). A model is proposed that accounts for the upper harmonics in fluorescence emission by nonlinear negative feedback regulation of photosynthesis. In contrast to simpler linear models, the model predicts that the steady-state fluorescence component will depend on the frequency of light modulation, and that amplitudes of all fluorescence components will exhibit resonance peak(s) when the irradiance frequency is tuned to an internal frequency of a regulatory component. The experiments confirmed that the upper harmonic components appear and exhibit distinct resonant peaks. The frequency of autonomous oscillations observed earlier upon an abrupt increase in CO(2) concentration corresponds to the sharpest of the resonant peaks of the forced oscillations. We propose that the underlying principles are general for a wide spectrum of negative-feedback regulatory mechanisms. The analysis by forced harmonic oscillations will enable us to examine internal dynamics of regulatory processes that have not been accessible to noninvasive fluorescence monitoring to date. PMID:12324435

Combustion Diagnostics and Flow Visualization of Hypergolic Combustion and Gelled Mixing Behavior

DTIC Science & Technology

1997-12-19

difference. Also, Exciplex Flourescence imaging has been implented to visualize diffusion layers which form at the contact interface of mixing...have been implemented and developed as a result of this effort. Among these techniques the most noteworthy involves a unique application of Exciplex ...fluorescence for visualization of diffusion layers formed between mixing liquids. Time resolved images of Exciplex fluorescence have been obtained

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

Ding, Huanjun; Cho, Hyo-Min; Molloi, Sabee, E-mail: symolloi@uci.edu

Purpose: To investigate the feasibility of characterizing a Si strip photon-counting detector using x-ray fluorescence. Methods: X-ray fluorescence was generated by using a pencil beam from a tungsten anode x-ray tube with 2 mm Al filtration. Spectra were acquired at 90° from the primary beam direction with an energy-resolved photon-counting detector based on an edge illuminated Si strip detector. The distances from the source to target and the target to detector were approximately 19 and 11 cm, respectively. Four different materials, containing silver (Ag), iodine (I), barium (Ba), and gadolinium (Gd), were placed in small plastic containers with a diametermore » of approximately 0.7 cm for x-ray fluorescence measurements. Linear regression analysis was performed to derive the gain and offset values for the correlation between the measured fluorescence peak center and the known fluorescence energies. The energy resolutions and charge-sharing fractions were also obtained from analytical fittings of the recorded fluorescence spectra. An analytical model, which employed four parameters that can be determined from the fluorescence calibration, was used to estimate the detector response function. Results: Strong fluorescence signals of all four target materials were recorded with the investigated geometry for the Si strip detector. The average gain and offset of all pixels for detector energy calibration were determined to be 6.95 mV/keV and −66.33 mV, respectively. The detector’s energy resolution remained at approximately 2.7 keV for low energies, and increased slightly at 45 keV. The average charge-sharing fraction was estimated to be 36% within the investigated energy range of 20–45 keV. The simulated detector output based on the proposed response function agreed well with the experimental measurement. Conclusions: The performance of a spectral imaging system using energy-resolved photon-counting detectors is very dependent on the energy calibration of the detector. The proposed x-ray fluorescence technique offers an accurate and efficient way to calibrate the energy response of a photon-counting detector.« less

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

NASA Astrophysics Data System (ADS)

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

2013-06-01

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

Time-domain laser-induced fluorescence spectroscopy apparatus for clinical diagnostics

NASA Astrophysics Data System (ADS)

Fang, Qiyin; Papaioannou, Thanassis; Jo, Javier A.; Vaitha, Russel; Shastry, Kumar; Marcu, Laura

2004-01-01

We report the design and development of a compact optical fiber-based apparatus for in situ time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) of biological systems. The apparatus is modular, optically robust, and compatible with the clinical environment. It incorporates a dual output imaging spectrograph, a gated multichannel plate photomultiplier (MCP-PMT), an intensified charge-coupled-device (ICCD) camera, and a fast digitizer. It can accommodate various types of light sources and optical fiber probes for selective excitation and remote light delivery/collection as required by different applications. The apparatus allows direct recording of the entire fluorescence decay with high sensitivity (nM range fluorescein dye concentration with signal-to-noise ratio of 46) and with four decades dynamic range. It is capable of resolving a broad range of fluorescence lifetimes from hundreds of picoseconds (as low as 300 ps) using the MCP-PMT coupled to the digitizer to milliseconds using the ICCD. The data acquisition and analysis process is fully automated, enabling fast recording of fluorescence intensity decay across the entire emission spectrum (0.8 s per wavelength or ˜40 s for a 200 nm wavelength range at 5 nm increments). The spectral and temporal responses of the apparatus were calibrated and its performance was validated using fluorescence lifetime standard dyes (Rhodamin B, 9-cyanoanthracene, and rose Bengal) and tissue endogenous fluorophores (elastin, collagen, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide). Fluorescence decay lifetimes and emission spectra of all tested compounds measured with the current tr-LIFS apparatus were found in good agreement with the values reported in the literature. The design and performance of tr-LIFS apparatus have enabled in vivo studies of atherosclerotic plaques and brain tumors.

Direct on-strip analysis of size- and time-resolved aerosol impactor samples using laser induced fluorescence spectra excited at 263 and 351 nm.

PubMed

Wang, Chuji; Pan, Yong-Le; James, Deryck; Wetmore, Alan E; Redding, Brandon

2014-04-11

We report a novel atmospheric aerosol characterization technique, in which dual wavelength UV laser induced fluorescence (LIF) spectrometry marries an eight-stage rotating drum impactor (RDI), namely UV-LIF-RDI, to achieve size- and time-resolved analysis of aerosol particles on-strip. The UV-LIF-RDI technique measured LIF spectra via direct laser beam illumination onto the particles that were impacted on a RDI strip with a spatial resolution of 1.2mm, equivalent to an averaged time resolution in the aerosol sampling of 3.6 h. Excited by a 263 nm or 351 nm laser, more than 2000 LIF spectra within a 3-week aerosol collection time period were obtained from the eight individual RDI strips that collected particles in eight different sizes ranging from 0.09 to 10 μm in Djibouti. Based on the known fluorescence database from atmospheric aerosols in the US, the LIF spectra obtained from the Djibouti aerosol samples were found to be dominated by fluorescence clusters 2, 5, and 8 (peaked at 330, 370, and 475 nm) when excited at 263 nm and by fluorescence clusters 1, 2, 5, and 6 (peaked at 390 and 460 nm) when excited at 351 nm. Size- and time-dependent variations of the fluorescence spectra revealed some size and time evolution behavior of organic and biological aerosols from the atmosphere in Djibouti. Moreover, this analytical technique could locate the possible sources and chemical compositions contributing to these fluorescence clusters. Advantages, limitations, and future developments of this new aerosol analysis technique are also discussed. Published by Elsevier B.V.

Micelle-induced versatile sensing behavior of bispyrene-based fluorescent molecular sensor for picric acid and PYX explosives.

PubMed

Ding, Liping; Bai, Yumei; Cao, Yuan; Ren, Guijia; Blanchard, Gary J; Fang, Yu

2014-07-08

The effect of surfactant micelles on the photophysical properties of a cationic bispyrene fluorophore, Py-diIM-Py, was systemically examined. The results from series of measurements including UV-vis absorption, steady-state fluorescence emission, quantum yield, fluorescence lifetime, and time-resolved emission spectra reveal that the cationic fluorophore is only encapsulated by the anionic sodium dodecyl sulfate (SDS) surfactant micelles and not incorporated in the cationic dodecyltrimethylammonium bromide (DTAB) and neutral Triton X-100 (TX100) surfactant micelles. This different fluorophore location in the micellar solutions significantly influences its sensing behavior to various explosives. Fluorescence quenching studies reveal that the simple variation of micellar systems leads to significant changes in the sensitivity and selectivity of the fluorescent sensor to explosives. The sensor exhibits an on-off response to multiple explosives with the highest sensitivity to picric acid (PA) in the anionic SDS micelles. In the cationic DTAB micelles, it displays the highest on-off responses to PYX. Both the sensitivity and selectivity to PYX in the cationic micelles are enhanced compared with that to PA in the anionic micelles. However, the poor encapsulation in the neutral surfactant TX100 micelles leads to fluorescence instability of the fluorophore and fails to function as a sensor system. Time-resolved fluorescence decays in the presence of explosives reveal that the quenching mechanism of two micellar sensor systems to explosives is static in nature. The present work demonstrates that the electrostatic interaction between the cationic fluorophore and differently charged micelles plays a determinative role in adjusting its distribution in micellar solutions, which further influences the sensing behavior of the obtained micellar sensor systems.

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.

Density relaxation and particle motion characteristics in a non-ionic deep eutectic solvent (acetamide + urea): time-resolved fluorescence measurements and all-atom molecular dynamics simulations.

PubMed

Das, Anuradha; Das, Suman; Biswas, Ranjit

2015-01-21

Temperature dependent relaxation dynamics, particle motion characteristics, and heterogeneity aspects of deep eutectic solvents (DESs) made of acetamide (CH3CONH2) and urea (NH2CONH2) have been investigated by employing time-resolved fluorescence measurements and all-atom molecular dynamics simulations. Three different compositions (f) for the mixture [fCH3CONH2 + (1 - f)NH2CONH2] have been studied in a temperature range of 328-353 K which is ∼120-145 K above the measured glass transition temperatures (∼207 K) of these DESs but much lower than the individual melting temperature of either of the constituents. Steady state fluorescence emission measurements using probe solutes with sharply different lifetimes do not indicate any dependence on excitation wavelength in these metastable molten systems. Time-resolved fluorescence anisotropy measurements reveal near-hydrodynamic coupling between medium viscosity and rotation of a dissolved dipolar solute. Stokes shift dynamics have been found to be too fast to be detected by the time-resolution (∼70 ps) employed, suggesting extremely rapid medium polarization relaxation. All-atom simulations reveal Gaussian distribution for particle displacements and van Hove correlations, and significant overlap between non-Gaussian (α2) and new non-Gaussian (γ) heterogeneity parameters. In addition, no stretched exponential relaxations have been detected in the simulated wavenumber dependent acetamide dynamic structure factors. All these results are in sharp contrast to earlier observations for ionic deep eutectics with acetamide [Guchhait et al., J. Chem. Phys. 140, 104514 (2014)] and suggest a fundamental difference in interaction and dynamics between ionic and non-ionic deep eutectic solvent systems.

Density relaxation and particle motion characteristics in a non-ionic deep eutectic solvent (acetamide + urea): Time-resolved fluorescence measurements and all-atom molecular dynamics simulations

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

Das, Anuradha; Das, Suman; Biswas, Ranjit, E-mail: ranjit@bose.res.in

2015-01-21

Temperature dependent relaxation dynamics, particle motion characteristics, and heterogeneity aspects of deep eutectic solvents (DESs) made of acetamide (CH{sub 3}CONH{sub 2}) and urea (NH{sub 2}CONH{sub 2}) have been investigated by employing time-resolved fluorescence measurements and all-atom molecular dynamics simulations. Three different compositions (f) for the mixture [fCH{sub 3}CONH{sub 2} + (1 − f)NH{sub 2}CONH{sub 2}] have been studied in a temperature range of 328-353 K which is ∼120-145 K above the measured glass transition temperatures (∼207 K) of these DESs but much lower than the individual melting temperature of either of the constituents. Steady state fluorescence emission measurements using probemore » solutes with sharply different lifetimes do not indicate any dependence on excitation wavelength in these metastable molten systems. Time-resolved fluorescence anisotropy measurements reveal near-hydrodynamic coupling between medium viscosity and rotation of a dissolved dipolar solute. Stokes shift dynamics have been found to be too fast to be detected by the time-resolution (∼70 ps) employed, suggesting extremely rapid medium polarization relaxation. All-atom simulations reveal Gaussian distribution for particle displacements and van Hove correlations, and significant overlap between non-Gaussian (α{sub 2}) and new non-Gaussian (γ) heterogeneity parameters. In addition, no stretched exponential relaxations have been detected in the simulated wavenumber dependent acetamide dynamic structure factors. All these results are in sharp contrast to earlier observations for ionic deep eutectics with acetamide [Guchhait et al., J. Chem. Phys. 140, 104514 (2014)] and suggest a fundamental difference in interaction and dynamics between ionic and non-ionic deep eutectic solvent systems.« less

Density relaxation and particle motion characteristics in a non-ionic deep eutectic solvent (acetamide + urea): Time-resolved fluorescence measurements and all-atom molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Das, Anuradha; Das, Suman; Biswas, Ranjit

2015-01-01

Temperature dependent relaxation dynamics, particle motion characteristics, and heterogeneity aspects of deep eutectic solvents (DESs) made of acetamide (CH3CONH2) and urea (NH2CONH2) have been investigated by employing time-resolved fluorescence measurements and all-atom molecular dynamics simulations. Three different compositions (f) for the mixture [fCH3CONH2 + (1 - f)NH2CONH2] have been studied in a temperature range of 328-353 K which is ˜120-145 K above the measured glass transition temperatures (˜207 K) of these DESs but much lower than the individual melting temperature of either of the constituents. Steady state fluorescence emission measurements using probe solutes with sharply different lifetimes do not indicate any dependence on excitation wavelength in these metastable molten systems. Time-resolved fluorescence anisotropy measurements reveal near-hydrodynamic coupling between medium viscosity and rotation of a dissolved dipolar solute. Stokes shift dynamics have been found to be too fast to be detected by the time-resolution (˜70 ps) employed, suggesting extremely rapid medium polarization relaxation. All-atom simulations reveal Gaussian distribution for particle displacements and van Hove correlations, and significant overlap between non-Gaussian (α2) and new non-Gaussian (γ) heterogeneity parameters. In addition, no stretched exponential relaxations have been detected in the simulated wavenumber dependent acetamide dynamic structure factors. All these results are in sharp contrast to earlier observations for ionic deep eutectics with acetamide [Guchhait et al., J. Chem. Phys. 140, 104514 (2014)] and suggest a fundamental difference in interaction and dynamics between ionic and non-ionic deep eutectic solvent systems.

Cellular uptake of modified oligonucleotides: fluorescence approach

NASA Astrophysics Data System (ADS)

Kočišová, Eva; Praus, Petr; Rosenberg, Ivan; Seksek, Olivier; Sureau, Franck; Štěpánek, Josef; Turpin, Pierre-Yves

2005-06-01

Cellular uptake and intracellular distribution of the synthetic antisense analogue of dT 15 oligonucleotide (homogenously containing 3'-O-P-CH 2-O-5' internucleotide linkages and labeled with tetramethylrhodamine dye) was studied on B16 melanoma cell line by fluorescence micro-imaging and time-resolved microspectrofluorimetry. By using amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process, homogenous staining of the cells with rather distinct nucleoli staining was achieved after 4 h of incubation. Two spectral components of 2.7 and 1.3 ns lifetime, respectively, were resolved in the emission collected from the cell nucleus. The way of staining and the long-lived component differed from our previous experiments demonstrating complexity of the intracellular oligonucleotide distribution and in particular of the binding inside the nucleus.

Noninvasive in situ evaluation of osteogenic differentiation by time-resolved laser-induced fluorescence spectroscopy.

PubMed

Ashjian, Peter; Elbarbary, Amir; Zuk, Patricia; DeUgarte, Daniel A; Benhaim, Prosper; Marcu, Laura; Hedrick, Marc H

2004-01-01

The clinical implantation of bioengineered tissues requires an in situ nondestructive evaluation of the quality of tissue constructs developed in vitro before transplantation. Time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) is demonstrated here to noninvasively monitor the formation of osteogenic extracellular matrix (ECM) produced by putative stem cells (PLA cells) derived from human adipose tissue. We show that this optical spectroscopy technique can assess the relative expression of collagens (types I, III, IV, and V) within newly forming osteogenic ECM. The results are consistent with those obtained by conventional histochemical techniques (immunofluorescence and Western blot) and demonstrate that TR-LIFS is a potential tool for monitoring the expression of distinct collagen types and the formation of collagen cross-links in intact tissue constructs.

Conformational States of the Rapana thomasiana Hemocyanin and Its Substructures Studied by Dynamic Light Scattering and Time-Resolved Fluorescence Spectroscopy

PubMed Central

Georgieva, Dessislava; Schwark, Daniel; Nikolov, Peter; Idakieva, Krassimira; Parvanova, Katja; Dierks, Karsten; Genov, Nicolay; Betzel, Christian

2005-01-01

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another. PMID:15533921

Identification of detergents for forensic fiber analysis.

PubMed

Heider, Emily C; Mujumdar, Nirvani; Campiglia, Andres D

2016-11-01

Trace fibers are an important form of trace evidence, and identification of exogenous substances on textile fibers provides valuable information about the origin of the fiber. Laundering textiles can provide a unique fluorescent spectral signature of the whitening agent in the detergent that adsorbs to the fiber. Using fluorescence microscopy, the spectral characteristics of seven detergents adsorbed to single fibers drawn from laundered textiles were investigated, and principal component analysis of clusters was used to characterize the type of detergent on the fiber. On dyed nylon fibers, spectra from eight different detergent pairs could be resolved and washed validation fibers correctly classified. On dyed acrylic fibers, five different detergent pairs could be resolved and identified. Identification of the detergent type may prove useful in matching a trace fiber to its bulk specimen of origin.

A CMOS Time-Resolved Fluorescence Lifetime Analysis Micro-System

PubMed Central

Rae, Bruce R.; Muir, Keith R.; Gong, Zheng; McKendry, Jonathan; Girkin, John M.; Gu, Erdan; Renshaw, David; Dawson, Martin D.; Henderson, Robert K.

2009-01-01

We describe a CMOS-based micro-system for time-resolved fluorescence lifetime analysis. It comprises a 16 × 4 array of single-photon avalanche diodes (SPADs) fabricated in 0.35 μm high-voltage CMOS technology with in-pixel time-gated photon counting circuitry and a second device incorporating an 8 × 8 AlInGaN blue micro-pixellated light-emitting diode (micro-LED) array bump-bonded to an equivalent array of LED drivers realized in a standard low-voltage 0.35 μm CMOS technology, capable of producing excitation pulses with a width of 777 ps (FWHM). This system replaces instrumentation based on lasers, photomultiplier tubes, bulk optics and discrete electronics with a PC-based micro-system. Demonstrator lifetime measurements of colloidal quantum dot and Rhodamine samples are presented. PMID:22291564

Simultaneous Spectral Temporal Adaptive Raman Spectrometer - SSTARS

NASA Technical Reports Server (NTRS)

Blacksberg, Jordana

2010-01-01

Raman spectroscopy is a prime candidate for the next generation of planetary instruments, as it addresses the primary goal of mineralogical analysis, which is structure and composition. However, large fluorescence return from many mineral samples under visible light excitation can render Raman spectra unattainable. Using the described approach, Raman and fluorescence, which occur on different time scales, can be simultaneously obtained from mineral samples using a compact instrument in a planetary environment. This new approach is taken based on the use of time-resolved spectroscopy for removing the fluorescence background from Raman spectra in the laboratory. In the SSTARS instrument, a visible excitation source (a green, pulsed laser) is used to generate Raman and fluorescence signals in a mineral sample. A spectral notch filter eliminates the directly reflected beam. A grating then disperses the signal spectrally, and a streak camera provides temporal resolution. The output of the streak camera is imaged on the CCD (charge-coupled device), and the data are read out electronically. By adjusting the sweep speed of the streak camera, anywhere from picoseconds to milliseconds, it is possible to resolve Raman spectra from numerous fluorescence spectra in the same sample. The key features of SSTARS include a compact streak tube capable of picosecond time resolution for collection of simultaneous spectral and temporal information, adaptive streak tube electronics that can rapidly change from one sweep rate to another over ranges of picoseconds to milliseconds, enabling collection of both Raman and fluorescence signatures versus time and wavelength, and Synchroscan integration that allows for a compact, low-power laser without compromising ultimate sensitivity.

Fluorescence lifetime of normal, benign, and malignant thyroid tissues

NASA Astrophysics Data System (ADS)

Brandao, Mariana; Iwakura, Ricardo; Basilio, Fagne; Haleplian, Kaique; Ito, Amando; de Freitas, Luiz Carlos Conti; Bachmann, Luciano

2015-06-01

Fine-needle aspiration cytology is the standard technique to diagnose thyroid pathologies. However, this method results in a high percentage of inconclusive and false negatives. The use of time-resolved fluorescence techniques to detect biochemical composition and tissue structure alterations could help to develop a portable, minimally invasive, and nondestructive method to assist during surgical procedures. This study aimed to use fluorescence lifetimes to differentiate healthy and benign tissues from malignant thyroid tissue. The thyroid tissue was excited at 298-300 nm and the fluorescence decay registered at 340 and 450 nm. We observed fluorescence lifetimes at 340 nm emission of 0.80±0.26 and 3.94±0.47 ns for healthy tissue; 0.90±0.24 and 4.05±0.46 ns for benign lesions; and 1.21±0.14 and 4.63±0.25 ns for malignant lesions. For 450 nm emissions, we obtain lifetimes of 0.25±0.18 and 3.99±0.39 ns for healthy tissue, 0.24±0.17 and 4.20±0.48 ns for benign lesions, 0.33±0.32 and 4.55±0.55 ns for malignant lesions. Employing analysis of variance, we differentiate malignant lesions from benign and healthy tissues. In addition, we use quadratic discriminant analysis to distinguish malignant from benign and healthy tissues with an accuracy of 76.1%, sensitivity of 74.7%, and specificity of 83.3%. These results indicate that time-resolved fluorescence can assist medical evaluation of thyroid pathologies during surgeries.

A MEMS-based high frequency x-ray chopper.

PubMed

Siria, A; Dhez, O; Schwartz, W; Torricelli, G; Comin, F; Chevrier, J

2009-04-29

Time-resolved x-ray experiments require intensity modulation at high frequencies (advanced rotating choppers have nowadays reached the kHz range). We here demonstrate that a silicon microlever oscillating at 13 kHz with nanometric amplitude can be used as a high frequency x-ray chopper. We claim that using micro-and nanoelectromechanical systems (MEMS and NEMS), it will be possible to achieve higher frequencies in excess of hundreds of megahertz. Working at such a frequency can open a wealth of possibilities in chemistry, biology and physics time-resolved experiments.

Demonstration of x-ray fluorescence imaging of a high-energy-density plasma.

PubMed

MacDonald, M J; Keiter, P A; Montgomery, D S; Biener, M M; Fein, J R; Fournier, K B; Gamboa, E J; Klein, S R; Kuranz, C C; LeFevre, H J; Manuel, M J-E; Streit, J; Wan, W C; Drake, R P

2014-11-01

Experiments at the Trident Laser Facility have successfully demonstrated the use of x-ray fluorescence imaging (XRFI) to diagnose shocked carbonized resorcinol formaldehyde (CRF) foams doped with Ti. One laser beam created a shock wave in the doped foam. A second laser beam produced a flux of vanadium He-α x-rays, which in turn induced Ti K-shell fluorescence within the foam. Spectrally resolved 1D imaging of the x-ray fluorescence provided shock location and compression measurements. Additionally, experiments using a collimator demonstrated that one can probe specific regions within a target. These results show that XRFI is a capable alternative to path-integrated measurements for diagnosing hydrodynamic experiments at high energy density.

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

NASA Astrophysics Data System (ADS)

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

2018-04-01

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

Studies of bio-mimetic medium of ionic and non-ionic micelles by a simple charge transfer fluorescence probe N,N-dimethylaminonapthyl-(acrylo)-nitrile

NASA Astrophysics Data System (ADS)

Samanta, Anuva; Paul, Bijan Kumar; Guchhait, N.

2011-05-01

In this report we have studied micellization process of anionic, cationic and non-ionic surfactants using N,N-dimethylaminonapthyl-(acrylo)-nitrile (DMANAN) as an external fluorescence probe. Micropolarity, microviscosity, critical micellar concentration of these micelles based on steady state absorption and fluorescence and time resolved emission spectroscopy of the probe DMANAN show that the molecule resides in the micelle-water interface for ionic micelles and in the core for the non-ionic micelle. The effect of variation of pH of the micellar solution as well as fluorescence quenching measurements of DMANAN provide further support for the location of the probe in the micelles.

Spectroscopic characterization of Venus at the single molecule level.

PubMed

David, Charlotte C; Dedecker, Peter; De Cremer, Gert; Verstraeten, Natalie; Kint, Cyrielle; Michiels, Jan; Hofkens, Johan

2012-02-01

Venus is a recently developed, fast maturating, yellow fluorescent protein that has been used as a probe for in vivo applications. In the present work the photophysical characteristics of Venus were analyzed spectroscopically at the bulk and single molecule level. Through time-resolved single molecule measurements we found that single molecules of Venus display pronounced fluctuations in fluorescence emission, with clear fluorescence on- and off-times. These fluorescence intermittencies were found to occupy a broad range of time scales, ranging from milliseconds to several seconds. Such long off-times can complicate the analysis of single molecule counting experiments or single-molecule FRET experiments. This journal is © The Royal Society of Chemistry and Owner Societies 2012

Effect of ion pairing on the fluorescence of berberine, a natural isoquinoline alkaloid

NASA Astrophysics Data System (ADS)

Megyesi, Mónika; Biczók, László

2007-10-01

Effect of association with chloride or perchlorate anions on the fluorescence properties of berberine, a cationic isoquinoline alkaloid, has been studied. Interaction with Cl - caused more efficient fluorescence quenching; it significantly accelerated the radiationless deactivation and slowed down the radiative transition. Combined analysis of spectrophotometric, steady-state and time-resolved fluorescence results provided 1.5 × 10 5 M -1 for the equilibrium constant of ion pairing with Cl - in CH 2Cl 2. Both ion pairing and enrichment of the microenvironment of berberine in ions led to excited state quenching in solvents of medium polarity, but only the latter effect was observed in the presence of perchlorates in butyronitrile.

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

Cortical pitch regions in humans respond primarily to resolved harmonics and are located in specific tonotopic regions of anterior auditory cortex.

PubMed

Norman-Haignere, Sam; Kanwisher, Nancy; McDermott, Josh H

2013-12-11

Pitch is a defining perceptual property of many real-world sounds, including music and speech. Classically, theories of pitch perception have differentiated between temporal and spectral cues. These cues are rendered distinct by the frequency resolution of the ear, such that some frequencies produce "resolved" peaks of excitation in the cochlea, whereas others are "unresolved," providing a pitch cue only via their temporal fluctuations. Despite longstanding interest, the neural structures that process pitch, and their relationship to these cues, have remained controversial. Here, using fMRI in humans, we report the following: (1) consistent with previous reports, all subjects exhibited pitch-sensitive cortical regions that responded substantially more to harmonic tones than frequency-matched noise; (2) the response of these regions was mainly driven by spectrally resolved harmonics, although they also exhibited a weak but consistent response to unresolved harmonics relative to noise; (3) the response of pitch-sensitive regions to a parametric manipulation of resolvability tracked psychophysical discrimination thresholds for the same stimuli; and (4) pitch-sensitive regions were localized to specific tonotopic regions of anterior auditory cortex, extending from a low-frequency region of primary auditory cortex into a more anterior and less frequency-selective region of nonprimary auditory cortex. These results demonstrate that cortical pitch responses are located in a stereotyped region of anterior auditory cortex and are predominantly driven by resolved frequency components in a way that mirrors behavior.

An analog filter approach to frequency domain fluorescence spectroscopy

DOE PAGES

Trainham, Clifford P.; O'Neill, Mary D.; McKenna, Ian J.

2015-10-01

The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modeled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as Spicea can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modeling of the entiremore » system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. Furthermore, the techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response.« less

Frequency-domain phase fluorometry in the presence of dark states: A numerical study

NASA Astrophysics Data System (ADS)

Zhu, Xinxin; Min, Wei

2011-11-01

Fluorescence anomalous phase advance (FAPA) is a newly discovered spectroscopy phenomenon: instead of lagging behind the modulated light, fluorescence signal can exhibit FAPA as if it precedes the excitation source in time. While FAPA offers a promising technique for probing dark state lifetime, the underlying mechanism is not fully elucidated. Herein we investigate frequency-domain phase fluorometry as a result of intricate interplay between a short-lived fluorescent state and a long-lived dark state. In particular, the quantitative dependence on modulation frequency, excitation intensity, nonradiative decay, intersystem crossing and dark-state lifetime are explored respectively. A comprehensive view of phase fluorometry emerges consequently.

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

PubMed Central

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

2013-01-01

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

Velocity landscape correlation resolves multiple flowing protein populations from fluorescence image time series.

PubMed

Pandžić, Elvis; Abu-Arish, Asmahan; Whan, Renee M; Hanrahan, John W; Wiseman, Paul W

2018-02-16

Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells. Copyright © 2018 Elsevier Inc. All rights reserved.

A Monte Carlo study of fluorescence generation probability in a two-layered tissue model

NASA Astrophysics Data System (ADS)

Milej, Daniel; Gerega, Anna; Wabnitz, Heidrun; Liebert, Adam

2014-03-01

It was recently reported that the time-resolved measurement of diffuse reflectance and/or fluorescence during injection of an optical contrast agent may constitute a basis for a technique to assess cerebral perfusion. In this paper, we present results of Monte Carlo simulations of the propagation of excitation photons and tracking of fluorescence photons in a two-layered tissue model mimicking intra- and extracerebral tissue compartments. Spatial 3D distributions of the probability that the photons were converted from excitation to emission wavelength in a defined voxel of the medium (generation probability) during their travel between source and detector were obtained for different optical properties in intra- and extracerebral tissue compartments. It was noted that the spatial distribution of the generation probability depends on the distribution of the fluorophore in the medium and is influenced by the absorption of the medium and of the fluorophore at excitation and emission wavelengths. Simulations were also carried out for realistic time courses of the dye concentration in both layers. The results of the study show that the knowledge of the absorption properties of the medium at excitation and emission wavelengths is essential for the interpretation of the time-resolved fluorescence signals measured on the surface of the head.

Diode laser-induced infrared fluorescence of water vapour

NASA Astrophysics Data System (ADS)

Li, Hejie; Hanson, Ronald K.; Jeffries, Jay B.

2004-07-01

Infrared laser-induced fluorescence (LIF) of water vapour was investigated for its potential as a spatially resolved gasdynamic diagnostic. A cw diode laser operating near 1392 nm was scanned across a single absorption transition in the ngr1 + ngr3 band of H2O in a static cell, and the resulting fluorescence signal was collected near 2.7 µm (both ngr1 and ngr3 bands). Experiments were conducted at low pressure in pure water vapour and mixtures of water vapour and N2 using a 20 mW laser in a double-pass arrangement. A simple analytical model was developed to relate LIF intensity to gas properties as a function of laser power. The spectrally resolved, single-line excitation spectrum was fitted with a Voigt profile, allowing inference of the water vapour temperature from the Doppler-broadened component of the measured fluorescence lineshape. A two-line excitation scheme was also investigated as a means of measuring temperature with reduced measurement time. From these initial measurements, we estimate that a practical sensor for atmospheric pressure applications would require a minimum of 1-2 W of laser power for two-line, fixed-wavelength temperature measurements and a minimum of about 70 W of power for scanned-wavelength measurements.

Ultrafast fluorescence spectroscopy of biologically relevant chromophores using type II difference frequency generation

NASA Astrophysics Data System (ADS)

Léonard, J.; Gelot, T.; Torgasin, K.; Haacke, S.

2011-01-01

A novel femtosecond fluorescence experiment based on type II difference frequency mixing is demonstrated. This approach is particularly interesting for near-UV emitting biological chromophores like amino acids and nucleotides, as the fluorescence is converted into the spectral range where CCD have their highest quantum efficiencies. The method is implemented with a 5-kHz amplified Ti:Sapphire laser system and first results obtained with 2,5-diphenyloxazole (PPO) in ethanol are reported.

Multimodal hyperspectral optical microscopy

DOE PAGES

Novikova, Irina V.; Smallwood, Chuck R.; Gong, Yu; ...

2017-09-02

We describe a unique and convenient approach to multimodal hyperspectral optical microscopy, herein achieved by coupling a portable and transferable hyperspectral imager to various optical microscopes. The experimental and data analysis schemes involved in recording spectrally and spatially resolved fluorescence, dark field, and optical absorption micrographs are illustrated through prototypical measurements targeting selected model systems. Namely, hyperspectral fluorescence micrographs of isolated fluorescent beads are employed to ensure spectral calibration of our detector and to gauge the attainable spatial resolution of our measurements; the recorded images are diffraction-limited. Moreover, spatially over-sampled absorption spectroscopy of a single lipid (18:1 Liss Rhod PE)more » layer reveals that optical densities on the order of 10-3 may be resolved by spatially averaging the recorded optical signatures. We also briefly illustrate two applications of our setup in the general areas of plasmonics and cell biology. Most notably, we deploy hyperspectral optical absorption microscopy to identify and image algal pigments within a single live Tisochrysis lutea cell. Overall, this work paves the way for multimodal multidimensional spectral imaging measurements spanning the realms of several scientific disciples.« less

Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

PubMed

Chowdhury, Rajdeep; Amin, Md Asif; Bhattacharyya, Kankan

2015-08-27

Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

Energy transfer in Anabaena variabilis filaments adapted to nitrogen-depleted and nitrogen-enriched conditions studied by time-resolved fluorescence.

PubMed

Onishi, Aya; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

2017-09-01

Nitrogen is among the most important nutritious elements for photosynthetic organisms such as plants, algae, and cyanobacteria. Therefore, nitrogen depletion severely compromises the growth, development, and photosynthesis of these organisms. To preserve their integrity under nitrogen-depleted conditions, filamentous nitrogen-fixing cyanobacteria reduce atmospheric nitrogen to ammonia, and self-adapt by regulating their light-harvesting and excitation energy-transfer processes. To investigate the changes in the primary processes of photosynthesis, we measured the steady-state absorption and fluorescence spectra and time-resolved fluorescence spectra (TRFS) of whole filaments of the nitrogen-fixing cyanobacterium Anabaena variabilis at 77 K. The filaments were grown in standard and nitrogen-free media for 6 months. The TRFS were measured with a picosecond time-correlated single photon counting system. Despite the phycobilisome degradation, the energy-transfer paths within phycobilisome and from phycobilisome to both photosystems were maintained. However, the energy transfer from photosystem II to photosystem I was suppressed and a specific red chlorophyll band appeared under the nitrogen-depleted condition.

Nanowires formed by the co-assembly of a negatively charged low-molecular weight gelator and a zwitterionic polythiophene.

PubMed

Li, Feng; Palaniswamy, Ganesan; de Jong, Menno R; Aslund, Andreas; Konradsson, Peter; Marcelis, Antonius T M; Sudhölter, Ernst J R; Stuart, Martien A Cohen; Leermakers, Frans A M

2010-06-21

Conjugated organic nanowires have been prepared by co-assembling a carboxylate containing low-molecular weight gelator (LMWG) and an amino acid substituted polythiophene derivative (PTT). Upon introducing the zwitterionic polyelectrolyte PTT to a basic molecular solution of the organogelator, the negative charges on the LMWG are compensated by the positive charges of the PTT. As a result, nanowires form through co-assembly. These nanowires are visualized by both transmission electron microscopy (TEM) and atomic force microscopy (AFM). Depending on the concentration and ratio of the components these nanowires can be micrometers long. These measurements further suggest that the aggregates adopt a helical conformation. The morphology of these nanowires are studied with fluorescent confocal laser scanning microscopy (CLSM). The interactions between LMWG and PTT are characterized by steady-state and time-resolved fluorescence spectroscopy studies. The steady-state spectra indicate that the backbone of the PTT adopts a more planar and more aggregated conformation when interacting with LMWG. The time- resolved fluorescence decay studies confirm this interpretation.

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675.

PubMed

Konold, Patrick E; Yoon, Eunjin; Lee, Junghwa; Allen, Samantha L; Chapagain, Prem P; Gerstman, Bernard S; Regmi, Chola K; Piatkevich, Kiryl D; Verkhusha, Vladislav V; Joo, Taiha; Jimenez, Ralph

2016-08-04

Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.

Multimodal hyperspectral optical microscopy

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

Novikova, Irina V.; Smallwood, Chuck R.; Gong, Yu

We describe a unique and convenient approach to multimodal hyperspectral optical microscopy, herein achieved by coupling a portable and transferable hyperspectral imager to various optical microscopes. The experimental and data analysis schemes involved in recording spectrally and spatially resolved fluorescence, dark field, and optical absorption micrographs are illustrated through prototypical measurements targeting selected model systems. Namely, hyperspectral fluorescence micrographs of isolated fluorescent beads are employed to ensure spectral calibration of our detector and to gauge the attainable spatial resolution of our measurements; the recorded images are diffraction-limited. Moreover, spatially over-sampled absorption spectroscopy of a single lipid (18:1 Liss Rhod PE)more » layer reveals that optical densities on the order of 10-3 may be resolved by spatially averaging the recorded optical signatures. We also briefly illustrate two applications of our setup in the general areas of plasmonics and cell biology. Most notably, we deploy hyperspectral optical absorption microscopy to identify and image algal pigments within a single live Tisochrysis lutea cell. Overall, this work paves the way for multimodal multidimensional spectral imaging measurements spanning the realms of several scientific disciples.« less

Spirally-patterned pinhole arrays for long-term fluorescence cell imaging.

PubMed

Koo, Bon Ung; Kang, YooNa; Moon, SangJun; Lee, Won Gu

2015-11-07

Fluorescence cell imaging using a fluorescence microscope is an extensively used technique to examine the cell nucleus, internal structures, and other cellular molecules with fluorescence response time and intensity. However, it is difficult to perform high resolution cell imaging for a long period of time with this technique due to necrosis and apoptosis depending on the type and subcellular location of the damage caused by phototoxicity. A large number of studies have been performed to resolve this problem, but researchers have struggled to meet the challenge between cellular viability and image resolution. In this study, we employ a specially designed disc to reduce cell damage by controlling total fluorescence exposure time without deterioration of the image resolution. This approach has many advantages such as, the apparatus is simple, cost-effective, and easily integrated into the optical pathway through a conventional fluorescence microscope.

Laguerre-based method for analysis of time-resolved fluorescence data: application to in-vivo characterization and diagnosis of atherosclerotic lesions.

PubMed

Jo, Javier A; Fang, Qiyin; Papaioannou, Thanassis; Baker, J Dennis; Dorafshar, Amir H; Reil, Todd; Qiao, Jian-Hua; Fishbein, Michael C; Freischlag, Julie A; Marcu, Laura

2006-01-01

We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability.

Diagnosis of vulnerable atherosclerotic plaques by time-resolved fluorescence spectroscopy and ultrasound imaging.

PubMed

Jo, J A; Fang, Q; Papaioannou, T; Qiao, J H; Fishbein, M C; Beseth, B; Dorafshar, A H; Reil, T; Baker, D; Freischlag, J; Shung, K K; Sun, L; Marcu, L

2006-01-01

In this study, time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonography were applied to detect vulnerable (high-risk) atherosclerotic plaque. A total of 813 TR-LIFS measurements were taken from carotid plaques of 65 patients, and subsequently analyzed using the Laguerre deconvolution technique. The investigated spots were classified by histopathology as thin, fibrotic, calcified, low-inflamed, inflamed and necrotic lesions. Spectral and time-resolved parameters (normalized intensity values and Laguerre expansion coefficients) were extracted from the TR-LIFS data. Feature selection for classification was performed by either analysis of variance (ANOVA) or principal component analysis (PCA). A stepwise linear discriminant analysis algorithm was developed for detecting inflamed and necrotic lesion, representing the most vulnerable plaques. These vulnerable plaques were detected with high sensitivity (>80%) and specificity (>90%). Ultrasound (US) imaging was obtained in 4 carotid plaques in addition to TR-LIFS examination. Preliminary results indicate that US provides important structural information of the plaques that could be combined with the compositional information obtained by TR-LIFS, to obtain a more accurate diagnosis of vulnerable atherosclerotic plaque.

Laguerre-based method for analysis of time-resolved fluorescence data: application to in-vivo characterization and diagnosis of atherosclerotic lesions

NASA Astrophysics Data System (ADS)

Jo, Javier A.; Fang, Qiyin; Papaioannou, Thanassis; Baker, J. Dennis; Dorafshar, Amir; Reil, Todd; Qiao, Jianhua; Fishbein, Michael C.; Freischlag, Julie A.; Marcu, Laura

2006-03-01

We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability.

Laguerre-based method for analysis of time-resolved fluorescence data: application to in-vivo characterization and diagnosis of atherosclerotic lesions

PubMed Central

Jo, Javier A.; Fang, Qiyin; Papaioannou, Thanassis; Baker, J. Dennis; Dorafshar, Amir H.; Reil, Todd; Qiao, Jian-Hua; Fishbein, Michael C.; Freischlag, Julie A.; Marcu, Laura

2007-01-01

We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability. PMID:16674179

Super-resolution optical microscopy resolves network morphology of smart colloidal microgels.

PubMed

Bergmann, Stephan; Wrede, Oliver; Huser, Thomas; Hellweg, Thomas

2018-02-14

We present a new method to resolve the network morphology of colloidal particles in an aqueous environment via super-resolution microscopy. By localization of freely diffusing fluorophores inside the particle network we can resolve the three dimensional structure of one species of colloidal particles (thermoresponsive microgels) without altering their chemical composition through copolymerization with fluorescent monomers. Our approach utilizes the interaction of the fluorescent dye rhodamine 6G with the polymer network to achieve an indirect labeling. We calculate the 3D structure from the 2D images and compare the structure to previously published models for the microgel morphology, e.g. the fuzzy sphere model. To describe the differences in the data an extension of this model is suggested. Our method enables the tailor-made fabrication of colloidal particles which are used in various applications, such as paints or cosmetics, and are promising candidates for drug delivery, smart surface coatings, and nanocatalysis. With the precise knowledge of the particle morphology an understanding of the underlying structure-property relationships for various colloidal systems is possible.

An angle-resolved, wavelength-dispersive x-ray fluorescence spectrometer for depth profile analysis of ion-implanted semiconductors using synchrotron radiation

NASA Astrophysics Data System (ADS)

Schmitt, W.; Hormes, J.; Kuetgens, U.; Gries, W. H.

1992-01-01

An apparatus for angle-resolved, wavelength-dispersive x-ray fluorescence spectroscopy with synchrotron radiation has been built and tested at the beam line BN2 of the Bonn electron stretcher and accelerator (ELSA). The apparatus is to be used for nondestructive depth profile analysis of ion-implanted semiconductors as part of the multinational Versailles Project of Advanced Materials and Standards (VAMAS) project on ion-implanted reference materials. In particular, the centroid depths of depth profiles of various implants is to be determined by use of the angle-resolved signal ratio technique. First results of measurements on implants of phosphorus (100 keV, 1016 cm-2) and sulfur (200 keV, 1014 cm-2) in silicon wafers using ``white'' synchrotron radiation are presented and suggest that it should be generally possible to measure the centroid depth of an implant at dose densities as low as 1014 cm-2. Some of the apparative and technical requirements are discussed which are peculiar to the use of synchrotron radiation in general and to the use of nonmonochromatized radiation in particular.

Ultrafast dynamics of photoactive yellow protein via the photoexcitation and emission processes.

PubMed

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

2007-01-01

Pump-dump fluorescence spectroscopy was performed for photoactive yellow protein (PYP) at room temperature. The effect of the dump pulse on the population of the potential energy surface of the electronic excited state was examined as depletion in the stationary fluorescence intensity. The dynamic behavior of the population in the electronic excited state was successfully probed in the various combinations of the pump-dump delay, the dump-pulse wavelength, the dump-pulse energy and the observation wavelength. The experimental results were compared with the results obtained by the femtosecond time-resolved fluorescence spectroscopy.

Heat release and flame structure measurements of self-excited acoustically-driven premixed methane flames

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

Kopp-Vaughan, Kristin M.; Tuttle, Steven G.; Renfro, Michael W.

An open-open organ pipe burner (Rijke tube) with a bluff-body ring was used to create a self-excited, acoustically-driven, premixed methane-air conical flame, with equivalence ratios ranging from 0.85 to 1.05. The feed tube velocities corresponded to Re = 1780-4450. Coupled oscillations in pressure, velocity, and heat release from the flame are naturally encouraged at resonant frequencies in the Rijke tube combustor. This coupling creates sustainable self-excited oscillations in flame front area and shape. The period of the oscillations occur at the resonant frequency of the combustion chamber when the flame is placed {proportional_to}1/4 of the distance from the bottom ofmore » the tube. In this investigation, the shape of these acoustically-driven flames is measured by employing both OH planar laser-induced fluorescence (PLIF) and chemiluminescence imaging and the images are correlated to simultaneously measured pressure in the combustor. Past research on acoustically perturbed flames has focused on qualitative flame area and heat release relationships under imposed velocity perturbations at imposed frequencies. This study reports quantitative empirical fits with respect to pressure or phase angle in a self-generated pressure oscillation. The OH-PLIF images were single temporal shots and the chemiluminescence images were phase averaged on chip, such that 15 exposures were used to create one image. Thus, both measurements were time resolved during the flame oscillation. Phase-resolved area and heat release variations throughout the pressure oscillation were computed. A relation between flame area and the phase angle before the pressure maximum was derived for all flames in order to quantitatively show that the Rayleigh criterion was satisfied in the combustor. Qualitative trends in oscillating flame area were found with respect to feed tube flow rates. A logarithmic relation was found between the RMS pressure and both the normalized average area and heat release rate for all flames. (author)« less

Magnetic resonance-coupled fluorescence tomography scanner for molecular imaging of tissue

NASA Astrophysics Data System (ADS)

Davis, Scott C.; Pogue, Brian W.; Springett, Roger; Leussler, Christoph; Mazurkewitz, Peter; Tuttle, Stephen B.; Gibbs-Strauss, Summer L.; Jiang, Shudong S.; Dehghani, Hamid; Paulsen, Keith D.

2008-06-01

A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1nM in a 70mm diameter homogeneous phantom, and detection is feasible to near 10pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.

Internalization of aggregated photosensitizers by tumor cells: subcellular time-resolved fluorescence spectroscopy on derivatives of pyropheophorbide-a ethers and chlorin e6 under femtosecond one- and two-photon excitations.

PubMed

Kelbauskas, L; Dietel, W

2002-12-01

Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.

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

PubMed

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

2016-09-19

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

Correlation of conformational heterogeneity of the tryptophyl side chain and time-resolved fluorescence intensity decay kinetics

NASA Astrophysics Data System (ADS)

Laws, William R.; Ross, J. B. Alexander

1992-04-01

The time-resolved fluorescence properties of a tryptophan residue should be useful for probing protein structure, function, and dynamics. To date, however, the non-single exponential fluorescence intensity decay kinetics for numerous peptides and proteins having a single tryptophan residue have not been adequately explained. Many possibilities have been considered and include: (1) contributions from the 1La and 1Lb states of indole; (2) excited-state hydrogen exchange; and (3) environmental heterogeneity from (chi) 1 and (chi) 2 rotamers. In addition, it has been suggested that generally many factors contribute to the decay and a distribution of probabilities may be more appropriate. Two recent results support multiple species due to conformational heterogeneity as the major contributor to complex kinetics. First, a rotationally constrained tryptophan analogue has fluorescence intensity decay kinetics that can be described by the sum of two exponentials with amplitudes comparable to the relative populations of the two rotational isomers. Second, the multiple exponentials observed for tyrosine-containing model compounds and peptides correlate with the (chi) 1 rotamer populations independently determined by 1H NMR. We now report similar correlations between rotamer populations and fluorescence intensity decay kinetics for a tryptophan analogue of oxytocin. It appears for this compound that either (chi) 2 rotations do not appreciably alter the indole environment, (chi) 2 rotations are rapid enough to average the observed dependence, or only one of two possible (chi) 2 populations is associated with each (chi) 1 rotamer.

Applications of time-resolved laser fluorescence spectroscopy to the environmental biogeochemistry of actinides.

PubMed

Collins, Richard N; Saito, Takumi; Aoyagi, Noboru; Payne, Timothy E; Kimura, Takaumi; Waite, T David

2011-01-01

Time-resolved laser fluorescence spectroscopy (TRLFS) is a useful means of identifying certain actinide species resulting from various biogeochemical processes. In general, TRLFS differentiates chemical species of a fluorescent metal ion through analysis of different excitation and emission spectra and decay lifetimes. Although this spectroscopic technique has largely been applied to the analysis of actinide and lanthanide ions having fluorescence decay lifetimes on the order of microseconds, such as UO , Cm, and Eu, continuing development of ultra-fast and cryogenic TRLFS systems offers the possibility to obtain speciation information on metal ions having room-temperature fluorescence decay lifetimes on the order of nanoseconds to picoseconds. The main advantage of TRLFS over other advanced spectroscopic techniques is the ability to determine in situ metal speciation at environmentally relevant micromolar to picomolar concentrations. In the context of environmental biogeochemistry, TRLFS has principally been applied to studies of (i) metal speciation in aqueous and solid phases and (ii) the coordination environment of metal ions sorbed to mineral and bacterial surfaces. In this review, the principles of TRLFS are described, and the literature reporting the application of this methodology to the speciation of actinides in systems of biogeochemical interest is assessed. Significant developments in TRLFS methodology and advanced data analysis are highlighted, and we outline how these developments have the potential to further our mechanistic understanding of actinide biogeochemistry. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

PubMed Central

Warren, Sean C.; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J.; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda

2013-01-01

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment. PMID:23940626

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

Ding, H; Cho, H; Molloi, S

Purpose: To investigate the feasibility of energy response calibration of a Si strip photon-counting detector by using the x-ray fluorescence technique. Methods: X-ray fluorescence was generated by using a pencil beam from a tungsten anode x-ray tube with 2 mm Al filtration. Spectra were acquired at 90° from the primary beam direction with an energy-resolved photon-counting detector based on Si strips. The distances from the source to target and the target to detector were approximately 19 and 11 cm, respectively. Four different materials, containing Ag, I, Ba, and Gd, were placed in small plastic aliquots with a diameter of approximatelymore » 0.7 cm for x-ray fluorescence measurements. Linear regression analysis was performed to derive the gain and offset values for the correlation between the measured fluorescence peak center and the known energies for materials. The energy resolution was derived from the full width at half maximum (FWHM) of the fluorescence peaks. In addition, the angular dependence of the recorded fluorescence spectra was studied at 30°, 60°, and 120°. Results: Strong fluorescence signals of all four target materials were recorded with the investigated geometry for the Si strip detector. The recorded pulse height was calibrated with respect to photon energy and the gain and offset values were calculated to be 7.0 mV/keV and −69.3 mV, respectively. Negligible variation in energy calibration was observed among the four energy thresholds. The variation among different pixels was estimated to be approximately 1 keV. The energy resolution of the detector was estimated to be 7.9% within the investigated energy range. Conclusion: The performance of a spectral imaging system using energy-resolved photon-counting detectors is very dependent on the energy calibration of the detector. The proposed x-ray fluorescence technique provides an accurate and efficient way to calibrate the energy response of a photon-counting detector.« less

Characterization of energy response for photon-counting detectors using x-ray fluorescence

PubMed Central

Ding, Huanjun; Cho, Hyo-Min; Barber, William C.; Iwanczyk, Jan S.; Molloi, Sabee

2014-01-01

Purpose: To investigate the feasibility of characterizing a Si strip photon-counting detector using x-ray fluorescence. Methods: X-ray fluorescence was generated by using a pencil beam from a tungsten anode x-ray tube with 2 mm Al filtration. Spectra were acquired at 90° from the primary beam direction with an energy-resolved photon-counting detector based on an edge illuminated Si strip detector. The distances from the source to target and the target to detector were approximately 19 and 11 cm, respectively. Four different materials, containing silver (Ag), iodine (I), barium (Ba), and gadolinium (Gd), were placed in small plastic containers with a diameter of approximately 0.7 cm for x-ray fluorescence measurements. Linear regression analysis was performed to derive the gain and offset values for the correlation between the measured fluorescence peak center and the known fluorescence energies. The energy resolutions and charge-sharing fractions were also obtained from analytical fittings of the recorded fluorescence spectra. An analytical model, which employed four parameters that can be determined from the fluorescence calibration, was used to estimate the detector response function. Results: Strong fluorescence signals of all four target materials were recorded with the investigated geometry for the Si strip detector. The average gain and offset of all pixels for detector energy calibration were determined to be 6.95 mV/keV and −66.33 mV, respectively. The detector’s energy resolution remained at approximately 2.7 keV for low energies, and increased slightly at 45 keV. The average charge-sharing fraction was estimated to be 36% within the investigated energy range of 20–45 keV. The simulated detector output based on the proposed response function agreed well with the experimental measurement. Conclusions: The performance of a spectral imaging system using energy-resolved photon-counting detectors is very dependent on the energy calibration of the detector. The proposed x-ray fluorescence technique offers an accurate and efficient way to calibrate the energy response of a photon-counting detector. PMID:25471962

Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

NASA Technical Reports Server (NTRS)

Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

Comb-Resolved Dual-Comb Spectroscopy Stabilized by Free-Running Continuous-Wave Lasers

NASA Astrophysics Data System (ADS)

Kuse, Naoya; Ozawa, Akira; Kobayashi, Yohei

2012-11-01

We demonstrate dual-comb spectroscopy with relatively phase-locked two frequency combs, instead of frequency combs firmly fixed to the absolute frequency references. By stabilizing two beat frequencies between two mode-locked lasers at different wavelengths observed via free-running continuous-wave (CW) lasers, two combs are tightly phase locked to each other. The frequency noise of the CW lasers barely affects the performance of dual-comb spectroscopy because of the extremely fast common-mode noise rejection. Transform-limited comb-resolved dual-comb spectroscopy with a 6 Hz radio frequency linewidth is demonstrated by the use of Yb-fiber oscillators.

Quantitative spatial frequency fluorescence imaging in the sub-diffusive domain for image-guided glioma resection

PubMed Central

Sibai, Mira; Veilleux, Israel; Elliott, Jonathan T.; Leblond, Frederic; Wilson, Brian C.

2015-01-01

Intraoperative 5- aminolevulinic acid induced-Protoporphyrin IX (PpIX) fluorescence guidance enables maximum safe resection of glioblastomas by providing surgeons with real-time tumor optical contrast. However, visual assessment of PpIX fluorescence is subjective and limited by the distorting effects of light attenuation and tissue autofluorescence. We have previously shown that non-invasive point measurements of absolute PpIX concentration identifies residual tumor that is otherwise non-detectable. Here, we extend this approach to wide-field quantitative fluorescence imaging by implementing spatial frequency domain imaging to recover tissue optical properties across the field-of-view in phantoms and ex vivo tissue. PMID:26713206

SHERLOC: An investigation for Mars 2020

NASA Astrophysics Data System (ADS)

Beegle, Luther; Bhartia, Rohit

2016-04-01

SHERLOC is a Deep UV (DUV) native fluorescence and resonance Raman spectrometer that was selected as part of the Mars 2020 payload. It is a robotic arm mounted instrument that utilizes a DUV laser to generate characteristic Raman and fluorescence photons from a targeted spot. The DUV laser is co-boresighted to a context imager and integrated into an autofocusing/scanning optical system that allows us to correlate spectral signatures to surface textures, morphology and visible features. Additionally, it has recently been augmented with an imaging system that is a built-to-print version of the MArs Hand Lens Imager (MAHLI) instrument on the Mars Science Laboratory (MSL). Through the use of an internal scanning mirror, autofocusing lens, and a depth of focus of ±500 μm, the 100 μm laser spot can be systematically scanned over a 7x7 mm area with a fine-scale spatial resolution on natural or abraded surfaces and boreholes to a depth of at least 13 mm, without further arm movement. Through the use of the context imager, SHERLOC's data products can be combined with observations made by other instruments on the Mars 2020 payload. By bringing to bear multiple scientific instruments on a single sample, our ability to assess the habitability of ancient environments and search for potential biosignatures preserved within the geologic record will be greatly enhanced, making possible the selection of high-priority samples for caching. The SHERLOC investigation combines two spectral phenomena, fluorescence and pre-resonance/resonance DUV Raman scattering. These spectral features are resolvable when a high-radiance, narrow line-width, laser source illuminates a sample. In fluorescence, the incident photons are absorbed and re-emitted at a longer wavelength. The difference between the excitation and emission wavelength is the difference between the excitation frequency and the lowest electronic state frequency that increases with increasing aromatic structure (i.e., number of aromatic rings). Typical fluorescence cross-sections are 104 greater than traditional Raman, enabling the detection of sub-picograms levels of aromatic organic compounds. SHERLOC's narrow-linewidth (3 GHz) DUV laser (248.6 nm) enables fluorescence-free Raman scattering for additional classification of aromatics and aliphatic organics and minerals. Raman scattering is inelastic scattering where the loss of energy from the excitation energy (40225 cm-1) and defines the vibrational energy of a bond with which it interacted. This enables classification of bonds such as C-H, CN, C=O, C=C, NHx, NOx, SOx, POx, ClOx, and OH. It should be noted that Raman shifts (cm-1) (i.e., peak position as energy loss from the excitation energy) are invariant to excitation wavelength. Thus peak positions in Raman databases (at 229, 248, 488, 532 and 785 nm) can be compared (c.f. http://rruff.info/). Fluorescence emission of organics extends from ~270 nm into visible wavelengths. On the other hand, mineral fluorescence emission stemming from crystalline defects and impurities is weak in the deep UV, and typically begins longward of 360 nm continuing into the NIR. Mineral fluorescence is very unlikely to be seen in samples found on Mars. The DUV fluorescence technique employed by SHERLOC is well-suited to the detection of organics on mineral surfaces.

Thermally activated delayed fluorescence organic dots for two-photon fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

He, Tingchao; Ren, Can; Li, Zhuohua; Xiao, Shuyu; Li, Junzi; Lin, Xiaodong; Ye, Chuanxiang; Zhang, Junmin; Guo, Lihong; Hu, Wenbo; Chen, Rui

2018-05-01

Autofluorescence is a major challenge in complex tissue imaging when molecules present in the biological tissue compete with the fluorophore. This issue may be resolved by designing organic molecules with long fluorescence lifetimes. The present work reports the two-photon absorption (TPA) properties of a thermally activated delayed fluorescence (TADF) molecule with carbazole as the electron donor and dicyanobenzene as the electron acceptor (i.e., 4CzIPN). The results indicate that 4CzIPN exhibits a moderate TPA cross-section (˜9 × 10-50 cm4 s photon-1), high fluorescence quantum yield, and a long fluorescence lifetime (˜1.47 μs). 4CzIPN was compactly encapsulated into an amphiphilic copolymer via nanoprecipitation to achieve water-soluble organic dots. Interestingly, 4CzIPN organic dots have been utilized in applications involving two-photon fluorescence lifetime imaging (FLIM). Our work aptly demonstrates that TADF molecules are promising candidates of nonlinear optical probes for developing next-generation multiphoton FLIM applications.

A detailed spectroscopic study on the interaction of Rhodamine 6G with human hemoglobin.

PubMed

Mandal, Paulami; Bardhan, Munmun; Ganguly, Tapan

2010-05-03

UV-vis, time-resolved fluorescence and circular dichroism spectroscopic investigations have been made to reveal the nature of the interactions between xanthene dye Rhodamine 6G and the well known protein hemoglobin. From the analysis of the steady-state and time-resolved fluorescence quenching of Rhodamine 6G in aqueous solutions in presence of hemoglobin, it is revealed that the quenching is static in nature. The primary binding pattern between Rhodamine and hemoglobin has been interpreted as combined effect of hydrophobic association and electrostatic interaction. The binding constants, number of binding sites and thermodynamic parameters at various pH of the environment have been computed. The binding average distance between the energy donor Rhodamine and acceptor hemoglobin has been determined from the Forster's theory. Copyright 2010 Elsevier B.V. All rights reserved.

Nonradiative inter- and intramolecular energy transfer from the aromatic donor anisole to a synthesized photoswitchable acceptor system.

PubMed

Bardhan, Munmun; Bhattacharya, Sudeshna; Misra, Tapas; Mukhopadhyay, Rupa; De, Asish; Chowdhury, Joydeep; Ganguly, Tapan

2010-02-01

We report steady state and time resolved fluorescence measurements on acetonitrile (ACN) solutions of the model compounds, energy donor anisole (A) and a photoswitchable acceptor N,N'-1,2-phenylene di-p-tosylamide (B) and the multichromophore (M) where A and B are connected by a spacer containing both rigid triple (acetylenic) and flexible methylene bonds. Both steady state and time correlated single photon counting measurements demonstrate that though intermolecular energy transfer, of Forster type, between the donor and acceptor moieties occurs with rate 10(8)s(-1) but when these two reacting components are linked by a spacer (multichromophore, M) the observed transfer rate ( approximately 10(11)s(-1)) enhances. This seemingly indicates that the imposition of the spacer by inserting a triple bond may facilitate in the propagation of electronic excitation energy through bond. The time resolved fluorescence measurements along with the theoretical predictions using Configuration interaction singles (CIS) method by using 6-31G (d,p) basis set, implemented in the Gaussian package indicate the formations of the two excited conformers of B. The experimental findings made from the steady state and time resolved fluorescence measurements demonstrate that, though two different isomeric species of the acceptor B are formed in the excited singlet states, the prevailing singlet-singlet nonradiative energy transfer route was found from the donor A to the relatively longer-lived isomeric species of B. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Imaging a photodynamic therapy photosensitizer in vivo with a time-gated fluorescence tomography system

NASA Astrophysics Data System (ADS)

Mo, Weirong; Rohrbach, Daniel; Sunar, Ulas

2012-07-01

We report the tomographic imaging of a photodynamic therapy (PDT) photosensitizer, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) in vivo with time-domain fluorescence diffuse optical tomography (TD-FDOT). Simultaneous reconstruction of fluorescence yield and lifetime of HPPH was performed before and after PDT. The methodology was validated in phantom experiments, and depth-resolved in vivo imaging was achieved through simultaneous three-dimensional (3-D) mappings of fluorescence yield and lifetime contrasts. The tomographic images of a human head-and-neck xenograft in a mouse confirmed the preferential uptake and retention of HPPH by the tumor 24-h post-injection. HPPH-mediated PDT induced significant changes in fluorescence yield and lifetime. This pilot study demonstrates that TD-FDOT may be a good imaging modality for assessing photosensitizer distributions in deep tissue during PDT monitoring.

Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation

NASA Astrophysics Data System (ADS)

Luchowski, R.; Kapusta, P.; Szabelski, M.; Sarkar, P.; Borejdo, J.; Gryczynski, Z.; Gryczynski, I.

2009-09-01

Förster resonance energy transfer (FRET) can be utilized to achieve ultrashort fluorescence responses in time-domain fluorometry. In a poly(vinyl) alcohol matrix, the presence of 60 mM Rhodamine 800 acceptor shortens the fluorescence lifetime of a pyridine 1 donor to about 20 ps. Such a fast fluorescence response is very similar to the instrument response function (IRF) obtained using scattered excitation light. A solid fluorescent sample (e.g a film) with picosecond lifetime is ideal for IRF measurements and particularly useful for time-resolved microscopy. Avalanche photodiode detectors, commonly used in this field, feature color- dependent-timing responses. We demonstrate that recording the fluorescence decay of the proposed FRET-based reference sample yields a better IRF approximation than the conventional light-scattering method and therefore avoids systematic errors in decay curve analysis.

Demonstration of FRET in solutions

NASA Astrophysics Data System (ADS)

Shah, Sunil; Gryczynski, Zygmunt; Chib, Rahul; Fudala, Rafal; Baxi, Aatmun; Borejdo, Julian; Synak, Anna; Gryczynski, Ignacy

2016-03-01

We measured the Förster resonance energy transfer (FRET) from Uranin (U) donor to Rhodamine 101 (R101) acceptor in propylene glycol. Steady-state fluorescence measurements show a significant difference between mixed and unmixed fluorophore solutions. In the solution with mixed fluorophores, fluorescence intensity of the U donor decreases and intensity of R101 fluorescence increases. This is visualized as a color change from green to orange. Fluorescence anisotropy of the mixture solution increases in the donor emission wavelength region and decreases in the acceptor emission wavelengths; which is consistent with FRET occurrence. Time-resolved (lifetime) measurements show a decrease of the U lifetime in the presence of R101 acceptor. In the intensity decay of R101 acceptor appears a negative component indicating excited state process. All these measurements prove the presence of FRET in U/R101 mixture fluorescence.

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

Yu, Chongqi; Harbich, Wolfgang; Sementa, Luca

Ligand-protected Au clusters are non-bleaching fluorescence markers in bio- and medical applications. We show that their fluorescence is an intrinsic property of the Au cluster itself. We find a very intense and sharp fluorescence peak located at λ =739.2 nm (1.68 eV) for Au20 clusters in a Ne matrix held at 6 K. The fluorescence reflects the HOMO-LUMO diabatic bandgap of the cluster. The cluster shows a very rich absorption fine structure reminiscent of well defined molecule-like quantum levels. These levels are resolved since Au20 has only one stable isomer (tetrahedral), therefore our sample is mono-disperse in cluster size andmore » conformation. Density-functional theory (DFT) and time-dependent DFT calculations clarify the nature of optical absorptionand predict both main absorption peaks and intrinsic fluorescence in good agreement with experiment.« less

Quantitative fluorescence and elastic scattering tissue polarimetry using an Eigenvalue calibrated spectroscopic Mueller matrix system.

PubMed

Soni, Jalpa; Purwar, Harsh; Lakhotia, Harshit; Chandel, Shubham; Banerjee, Chitram; Kumar, Uday; Ghosh, Nirmalya

2013-07-01

A novel spectroscopic Mueller matrix system has been developed and explored for both fluorescence and elastic scattering polarimetric measurements from biological tissues. The 4 × 4 Mueller matrix measurement strategy is based on sixteen spectrally resolved (λ = 400 - 800 nm) measurements performed by sequentially generating and analyzing four elliptical polarization states. Eigenvalue calibration of the system ensured high accuracy of Mueller matrix measurement over a broad wavelength range, either for forward or backscattering geometry. The system was explored for quantitative fluorescence and elastic scattering spectroscopic polarimetric studies on normal and precancerous tissue sections from human uterine cervix. The fluorescence spectroscopic Mueller matrices yielded an interesting diattenuation parameter, exhibiting differences between normal and precancerous tissues.

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).

Airborne detection of oceanic turbidity cell structure using depth-resolved laser-induced water Raman backscatter

NASA Technical Reports Server (NTRS)

Hoge, F. E.; Swift, R. N.

1983-01-01

Airborne laser-induced, depth-resolved water Raman backscatter is useful in the detection and mapping of water optical transmission variations. This test, together with other field experiments, has identified the need for additional field experiments to resolve the degree of the contribution to the depth-resolved, Raman-backscattered signal waveform that is due to (1) sea surface height or elevation probability density; (2) off-nadir laser beam angle relative to the mean sea surface; and (3) the Gelbstoff fluorescence background, and the analytical techniques required to remove it. When converted to along-track profiles, the waveforms obtained reveal cells of a decreased Raman backscatter superimposed on an overall trend of monotonically decreasing water column optical transmission.

Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

NASA Astrophysics Data System (ADS)

Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

2015-05-01

Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1 immunolabeling. See DOI: 10.1039/c5nr01539a

Study of the Many Fluorescent Lines and the Absorption Variability in GX 301-2 with XMM-Newton

NASA Technical Reports Server (NTRS)

Fuerst, F.; Suchy, S.; Kreykenbohm, I.; Barragan, L.; Wilms, J.; Pottschmidt, K.; Caballero, I.; Kretschmar, P.; Ferrigno, C.; Rothschild, R. E.

2011-01-01

We present an in-depth study of the High Mass X-ray Binary (HMXB) GX 301-2 during its pre-periastron flare using data from the XMM-Newton satellite. The energy spectrum shows a power law continuum absorbed by a large equivalent hydrogen column on the order of 10(exp 24)/ sq cm and a prominent Fe K-alpha fluorescent emission line. Besides the Fe K-alpha line, evidence for Fe K-Beta, Ni K-alpha, Ni K-Beta, S K-alpha, Ar K-alpha, Ca K-alpha, and Cr K-alpha fluorescent lines is found. The observed line strengths are consistent with fluorescence in a cold absorber. This is the first time that Cr K-alpha is seen in emission in the X-ray spectrum of a HMXB. In addition to the modulation by the strong pulse period of approx 685 sec the source is highly variable and shows different states of activity. We perform time-resolved as well as pulse-to-pulse resolved spectroscopy to investigate differences between these states of activity. We find that fluorescent line fluxes are strongly variable and generally follow the overall flux. The N-H value is variable by a factor of 2, but not correlated to continuum normalization. We find an interval of low flux in the light curve in which the pulsations cease almost completely, without any indication of an increasing absorption column. We investigate this dip in detail and argue that it is most likely that during the dip the accretion ceased and the afterglow of the fluorescent iron accounted for the main portion of the X-ray flux. A similar dip was found earlier in RXTE data, and we compare our findings to these results.

Characterization of the lipid and protein organization in HBsAg viral particles by steady-state and time-resolved fluorescence spectroscopy.

PubMed

Greiner, Vanille J; Egelé, Caroline; Oncul, Sule; Ronzon, Frédéric; Manin, Catherine; Klymchenko, Andrey; Mély, Yves

2010-08-01

Hepatitis B surface antigen (HBsAg) particles, produced in the yeast Hansenula polymorpha, are 20 nm particles, composed of S surface viral proteins and host-derived lipids. Since the detailed structure of these particles is still missing, we further characterized them by fluorescence techniques. Fluorescence correlation spectroscopy indicated that the particles are mainly monomeric, with about 70 S proteins per particle. The S proteins were characterized through the intrinsic fluorescence of their thirteen Trp residues. Fluorescence quenching and time-resolved fluorescence experiments suggest the presence of both low emissive embedded Trp residues and more emissive Trp residues at the surface of the HBsAg particles. The low emission of the embedded Trp residues is consistent with their close proximity in alpha-helices. Furthermore, S proteins exhibit restricted movement, as expected from their tight association with lipids. The lipid organization of the particles was studied using viscosity-sensitive DPH-based probes and environment sensitive 3-hydroxyflavone probes, and compared to lipid vesicles and low density lipoproteins (LDLs), taken as models. Like LDLs, the HBsAg particles were found to be composed of an ordered rigid lipid interface, probably organized as a phospholipid monolayer, and a more hydrophobic and fluid inner core, likely composed of triglycerides and free fatty acids. However, the lipid core of HBsAg particles was substantially more polar than the LDL one, probably due to its larger content in proteins and its lower content in sterols. Based on our data, we propose a structural model for HBsAg particles where the S proteins deeply penetrate into the lipid core. Copyright 2010 Elsevier Masson SAS. All rights reserved.

Improved two-photon imaging of living neurons in brain tissue through temporal gating

PubMed Central

Gautam, Vini; Drury, Jack; Choy, Julian M. C.; Stricker, Christian; Bachor, Hans-A.; Daria, Vincent R.

2015-01-01

We optimize two-photon imaging of living neurons in brain tissue by temporally gating an incident laser to reduce the photon flux while optimizing the maximum fluorescence signal from the acquired images. Temporal gating produces a bunch of ~10 femtosecond pulses and the fluorescence signal is improved by increasing the bunch-pulse energy. Gating is achieved using an acousto-optic modulator with a variable gating frequency determined as integral multiples of the imaging sampling frequency. We hypothesize that reducing the photon flux minimizes the photo-damage to the cells. Our results, however, show that despite producing a high fluorescence signal, cell viability is compromised when the gating and sampling frequencies are equal (or effectively one bunch-pulse per pixel). We found an optimum gating frequency range that maintains the viability of the cells while preserving a pre-set fluorescence signal of the acquired two-photon images. The neurons are imaged while under whole-cell patch, and the cell viability is monitored as a change in the membrane’s input resistance. PMID:26504651

Velocity measurement using frequency domain interferometer and chirped pulse laser

NASA Astrophysics Data System (ADS)

Ishii, K.; Nishimura, Y.; Mori, Y.; Hanayama, R.; Kitagawa, Y.; Sekine, T.; Sato, N.; Kurita, T.; Kawashima, T.; Sunahara, A.; Sentoku, Y.; Miura, E.; Iwamoto, A.; Sakagami, H.

2017-02-01

An ultra-intense short pulse laser induces a shock wave in material. The pressure of shock compression is stronger than a few tens GPa. To characterize shock waves, time-resolved velocity measurement in nano- or pico-second time scale is needed. Frequency domain interferometer and chirped pulse laser provide single-shot time-resolved measurement. We have developed a laser-driven shock compression system and frequency domain interferometer with CPA laser. In this paper, we show the principle of velocity measurement using a frequency domain interferometer and a chirped pulse laser. Next, we numerically calculated spectral interferograms and show the time-resolved velocity measurement can be done from the phase analysis of spectral interferograms. Moreover we conduct the laser driven shock generation and shock velocity measurement. From the spectral fringes, we analyze the velocities of the sample and shockwaves.

Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging.

PubMed

Zhao, Qiaole; Schelen, Ben; Schouten, Raymond; van den Oever, Rein; Leenen, René; van Kuijk, Harry; Peters, Inge; Polderdijk, Frank; Bosiers, Jan; Raspe, Marcel; Jalink, Kees; Geert Sander de Jong, Jan; van Geest, Bert; Stoop, Karel; Young, Ian Ted

2012-12-01

We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

Systematic characterization and fluorescence threshold strategies for the wideband integrated bioaerosol sensor (WIBS) using size-resolved biological and interfering particles

NASA Astrophysics Data System (ADS)

Savage, Nicole J.; Krentz, Christine E.; Könemann, Tobias; Han, Taewon T.; Mainelis, Gediminas; Pöhlker, Christopher; Huffman, J. Alex

2017-11-01

Atmospheric particles of biological origin, also referred to as bioaerosols or primary biological aerosol particles (PBAP), are important to various human health and environmental systems. There has been a recent steep increase in the frequency of published studies utilizing commercial instrumentation based on ultraviolet laser/light-induced fluorescence (UV-LIF), such as the WIBS (wideband integrated bioaerosol sensor) or UV-APS (ultraviolet aerodynamic particle sizer), for bioaerosol detection both outdoors and in the built environment. Significant work over several decades supported the development of the general technologies, but efforts to systematically characterize the operation of new commercial sensors have remained lacking. Specifically, there have been gaps in the understanding of how different classes of biological and non-biological particles can influence the detection ability of LIF instrumentation. Here we present a systematic characterization of the WIBS-4A instrument using 69 types of aerosol materials, including a representative list of pollen, fungal spores, and bacteria as well as the most important groups of non-biological materials reported to exhibit interfering fluorescent properties. Broad separation can be seen between the biological and non-biological particles directly using the five WIBS output parameters and by taking advantage of the particle classification analysis introduced by Perring et al. (2015). We highlight the importance that particle size plays on observed fluorescence properties and thus in the Perring-style particle classification. We also discuss several particle analysis strategies, including the commonly used fluorescence threshold defined as the mean instrument background (forced trigger; FT) plus 3 standard deviations (σ) of the measurement. Changing the particle fluorescence threshold was shown to have a significant impact on fluorescence fraction and particle type classification. We conclude that raising the fluorescence threshold from FT + 3σ to FT + 9σ does little to reduce the relative fraction of biological material considered fluorescent but can significantly reduce the interference from mineral dust and other non-biological aerosols. We discuss examples of highly fluorescent interfering particles, such as brown carbon, diesel soot, and cotton fibers, and how these may impact WIBS analysis and data interpretation in various indoor and outdoor environments. The performance of the particle asymmetry factor (AF) reported by the instrument was assessed across particle types as a function of particle size, and comments on the reliability of this parameter are given. A comprehensive online supplement is provided, which includes size distributions broken down by fluorescent particle type for all 69 aerosol materials and comparing threshold strategies. Lastly, the study was designed to propose analysis strategies that may be useful to the broader community of UV-LIF instrumentation users in order to promote deeper discussions about how best to continue improving UV-LIF instrumentation and results.

Determination of the orientation of fluorescent labels relative to myosin S1 in solution from time-resolved fluorescence anisotropy experiments

NASA Astrophysics Data System (ADS)

van der Heide, Uulke A.; Gerritsen, Hans C.; Trayer, Ian P.; Levine, Yehudi K.

1992-04-01

The time-resolved fluorescence anisotropy of myosin S1 covalently labeled with Eosin-5- maleimide and 1,5-I-AEDANS was measured in solution. Each probe was specifically attached at one SH-group on the S1. The two most reactive SH sites on the heavy chain of the myosin S1 were used. The fluorescence anisotropy was measured at different excitation wavelengths. In this way, several absorption moments were utilized, each having a distinct orientation in the frame of the dye. The orientations of the transition moments in the dyes were determined in a separate experiment using an angle resolved fluorescence depolarization experiment on dyes embedded in stretched matrices of PVA polymers. The anisotropy decay curves exhibit fast (<3 ns) and slow (> 100 ns) components. The slow decay components reflect the motion of the large protein molecules. The fast anisotropy decay are attributed to a fast, but restricted, motion of the bound dye relative to the protein as experiments on free dyes in solution reveal subnanosecond anisotropy decays. The anisotropy decays have been analyzed in terms of a model which describes the restricted motion of the dye molecule relative to the protein and the overall rotation of the dye-protein complex in solution. An important element in the model is the incorporation of the orientational distribution of the dye relative to the protein. The observed anisotropy decays were analyzed using a global target approach in which the experimental data obtained at different excitation wavelengths are fitted simultaneously to the theoretical model. It is important to note that the orientational distribution of the dye relative to the protein, as well as the rotational correlation times of the motions for a dye attached to a given binding site, are independent of the excitation wavelength used. This leads to a reduction in the number of independent parameters optimized by the nonlinear least squares procedure. The orientational distribution of the dye relative to the protein obtained in this way is particularly useful for the interpretation of fluorescence depolarization data obtained from labeled muscle fibers. Indeed, knowledge of the distribution function of a dye attached to a binding site of the S1 protein is a prerequisite for a probe-independent determination of the orientational distribution of the S1 proteins themselves in the muscle fiber.

In situ optical measurements for characterization of flame species and remote sensing

NASA Astrophysics Data System (ADS)

Cullum, Brian Michael

1998-12-01

The following dissertation describes the use of spectroscopic techniques for both characterization of combustion intermediates and remote chemical sensing. The primary techniques that have been used for these measurements include, laser-induced fluorescence (LIF), time resolved LIF, resonance enhanced multiphoton ionization (REMPI) and Raman spectroscopy. A simple and quantitative means of measuring the efficiency of halogenated flame retardants is described, using laser-induced fluorescence (LIF). Intensity based LIF measurements of OH radical have been used to quantitatively measure the efficacy of halogenated flame retardant/polymer plaques. Temporally resolved LIF has been used to determine the extent to which the chemical kinetic theory of flame retardation applies to the effect of these compounds on combustion. We have shown that LIF of OH radicals is a very sensitive means of measuring the efficiency of these flame retardants as well as the giving information about the nature of flame retardation. In addition, we have developed a technique for the introduction of insoluble polymer plaques into a flame for fluorescence analysis. A high power pulsed Nd:YAG laser is used to ablate the sample into the flame while a second pulse from a dye laser is used to measure the LIF of OH radicals. Spectroscopic techniques are also very useful for trace remote analysis of environmental pollutants via optical fibers. A simple fiber-optic probe suitable for remote analysis using resonance enhanced multiphoton ionization (REMPI) has been developed for this purpose and is used to determine the toluene/gasoline concentration in water samples via a headspace measurement. The limit of detection for toluene in water using this probe is 0.54 ppb (wt/wt) with a sample standard deviation of 0.02 ppb (wt/wt). Another technique that has great potential for optical sensing is fluorescence lifetime imaging. A new method for measuring fluorescence lifetime images of quickly decaying species has been developed. This method employs a high powered pulsed laser that excites the fluorescent species in a dual pulse manner, and a non-gated charge coupled device (CCD) for detection of the fluorescence. Unlike other fluorescence lifetime imaging methods, this technique has the potential of monitoring fluorescent species with picosecond lifetimes.

NADPH as a potential intrinsic probe for tumour margin estimation

NASA Astrophysics Data System (ADS)

Stewart, Hazel; Hupp, Ted R.; Birch, David J. S.

2018-03-01

The fluorescent properties of the reduced coenzyme NADH and its phosphorylated derivative (NADPH) have been explored in order to assess their potential as an intrinsic probe for cancer surgery. NADPH production is increased in cancer cells to quench reactive oxygen species and meet higher demands for biosynthesis, and has attractive fluorescent properties such as emission towards the visible part of the spectrum and a relatively long fluorescence lifetime upon binding to enzymes ( 1 - 6.5 ns) that helps discriminate against other endogenous species. Different environmental effects on NAD(P)H fluorescence are reported here, including an increase in lifetime upon oxygen removal, an ability to retain its fluorescent properties in a complex medium (a silica phantom) and its fluorescence lifetime also being distinguishable in a cell environment. In addition, the development of a miniaturized liquid light guide filter-based timecorrelated single photon counting fluorescence lifetime system is reported as a step towards time-resolved visual imaging in cancer surgery. This system has been demonstrated as being capable of accurately measuring NAD(P)H fluorescence lifetimes in both simple solvent and cellular environments.

Resonance Raman and surface-enhanced resonance Raman spectra of LH2 antenna complex from Rhodobacter sphaeroides and Ectothiorhodospira sp. excited in the Qx and Qy transitions.

PubMed

Chumanov, G; Picorel, R; Ortiz de Zarate, I; Cotton, T M; Seibert, M

2000-05-01

Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment-pigment and pigment-protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment-protein complexes from different sources or in complexes altered chemically or genetically.

Simultaneous X-ray fluorescence and scanning X-ray diffraction microscopy at the Australian Synchrotron XFM beamline

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

Jones, Michael W. M.; Phillips, Nicholas W.; van Riessen, Grant A.

2016-08-11

Owing to its extreme sensitivity, quantitative mapping of elemental distributionsviaX-ray fluorescence microscopy (XFM) has become a key microanalytical technique. The recent realisation of scanning X-ray diffraction microscopy (SXDM) meanwhile provides an avenue for quantitative super-resolved ultra-structural visualization. The similarity of their experimental geometries indicates excellent prospects for simultaneous acquisition. Here, in both step- and fly-scanning modes, robust, simultaneous XFM-SXDM is demonstrated.

Spectrally And Temporally Resolved Low-Light Level Video Microscopy

NASA Astrophysics Data System (ADS)

Wampler, John E.; Furukawa, Ruth; Fechheimer, Marcus

1989-12-01

The IDG law-light video microscope system was designed to aid studies of localization of subcellular luminescence sources and stimulus/response coupling in single living cells using luminescent probes. Much of the motivation for design of this instrument system came from the pioneering efforts of Dr. Reynolds (Reynolds, Q. Rev. Biophys. 5, 295-347; Reynolds and Taylor, Bioscience 30, 586-592) who showed the value of intensified video camera systems for detection and localizion of fluorescence and bioluminescence signals from biological tissues. Our instrument system has essentially two roles, 1) localization and quantitation of very weak bioluminescence signals and 2) quantitation of intracellular environmental characteristics such as pH and calcium ion concentrations using fluorescent and bioluminescent probes. The instrument system exhibits over one million fold operating range allowing visualization and enhancement of quantum limited images with quantum limited response, spectral analysis of fluorescence signals, and transmitted light imaging. The computer control of the system implements rapid switching between light regimes, spatially resolved spectral scanning, and digital data processing for spectral shape analysis and for detailed analysis of the statistical distribution of single cell measurements. The system design and software algorithms used by the system are summarized. These design criteria are illustrated with examples taken from studies of bioluminescence, applications of bioluminescence to study developmental processes and gene expression in single living cells, and applications of fluorescent probes to study stimulus/response coupling in living cells.

Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy.

PubMed

Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N; Wientjes, Emilie; van Amerongen, Herbert

2016-09-01

Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was achieved by separating the time-resolved fluorescence of PSI and PSII in the leaf. It is found that the PSII antenna size is larger on the abaxial side of A. thaliana leaves, presumably because chloroplasts in the spongy mesophyll are "shaded" by the palisade cells. The number of chlorophylls in PSI on the adaxial side of the A. thaliana leaf is slightly higher. The C4 plant M. x giganteus contains both mesophyll and bundle sheath cells, which have a different PSI/PSII ratio. It is shown that the time-resolved fluorescence of bundle sheath and mesophyll cells can be analysed separately. The relative number of chlorophylls, which belong to PSI (as compared to PSII) in the bundle sheath cells is at least 2.5 times higher than in mesophyll cells. FLIM is thus demonstrated to be a useful technique to study the PSI/PSII ratio and PSII antenna size in well-defined regions of plant leaves without having to isolate pigment-protein complexes. Copyright © 2016 Elsevier B.V. All rights reserved.

Single-photon counting multicolor multiphoton fluorescence microscope.

PubMed

Buehler, Christof; Kim, Ki H; Greuter, Urs; Schlumpf, Nick; So, Peter T C

2005-01-01

We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV-Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 10(8) counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte lambda-stacks composed of 16 spectral channels, 256 x 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.

Eddy current imaging with an atomic radio-frequency magnetometer

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

Wickenbrock, Arne, E-mail: wickenbr@uni-mainz.de; Leefer, Nathan; Blanchard, John W.

2016-05-02

We use a radio-frequency {sup 85}Rb alkali-vapor cell magnetometer based on a paraffin-coated cell with long spin-coherence time and a small, low-inductance driving coil to create highly resolved conductivity maps of different objects. We resolve sub-mm features in conductive objects, we characterize the frequency response of our technique, and by operating at frequencies up to 250 kHz we are able to discriminate between differently conductive materials based on the induced response. The method is suited to cover a wide range of driving frequencies and can potentially be used for detecting non-metallic objects with low DC conductivity.

Design and synthesis of the BODIPY-BSA complex for biological applications.

PubMed

Vedamalai, Mani; Gupta, Iti

2018-02-01

A quinoxaline-functionalized styryl-BODIPY derivative (S1) was synthesized by microwave-assisted Knoevenagel condensation. It exhibited fluorescence enhancement upon micro-encapsulation into the hydrophobic cavity of bovine serum albumin (BSA). The S1-BSA complex was characterized systematically using ultraviolet (UV)-visible absorption, fluorescence emission, kinetics, circular dichroism and time-resolved lifetime measurements. The binding nature of BSA towards S1 was strong, and was found to be stable over a period of days. The studies showed that the S1-BSA complex could be used as a new biomaterial for fluorescence-based high-throughput assay for kinase enzymes. Copyright © 2017 John Wiley & Sons, Ltd.

Time-resolved fluorescence spectroscopy for chemical sensors

NASA Astrophysics Data System (ADS)

Draxler, Sonja; Lippitsch, Max E.

1996-07-01

A family of sensors is presented with fluorescence decay-time measurements used as the sensing technique. The concept is to take a single fluorophore with a suitably long fluorescence decay time as the basic building block for numerous different sensors. Analyte recognition can be performed by different functional groups that are necessary for selective interaction with the analyte. To achieve this, the principle of excited-state electron transfer is applied with pyrene as the fluorophore. Therefore the same instrumentation based on a small, ambient air-nitrogen laser and solid-state electronics can be used to measure different analytes, for example, oxygen, pH, carbon dioxide, potassium, ammonium, lead, cadmium, zinc, and phosphate.

A lanthanide-based chemosensor for bioavailable Fe3+ using a fluorescent siderophore: an assay displacement approach.

PubMed

Orcutt, Karen M; Jones, W Scott; McDonald, Andrea; Schrock, David; Wallace, Karl J

2010-01-01

The measurement of trace analytes in aqueous systems has become increasingly important for understanding ocean primary productivity. In oceanography, iron (Fe) is a key element in regulating ocean productivity, microplankton assemblages and has been identified as a causative element in the development of some harmful algal blooms. The chemosenor developed in this study is based on an indicator displacement approach that utilizes time-resolved fluorescence and fluorescence resonance energy transfer as the sensing mechanism to achieve detection of Fe3+ ions as low as 5 nM. This novel approach holds promise for the development of photoactive chemosensors for ocean deployment.

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.

Which model based on fluorescence quenching is suitable to study the interaction between trans-resveratrol and BSA?

NASA Astrophysics Data System (ADS)

Wei, Xin Lin; Xiao, Jian Bo; Wang, Yuanfeng; Bai, Yalong

2010-01-01

There are several models by means of quenching fluorescence of BSA to determine the binding parameters. The binding parameters obtained from different models are quite different from each other. Which model is suitable to study the interaction between trans-resveratrol and BSA? Herein, twelve models based fluorescence quenching of BSA were compared. The number of binding sites increasing with increased binding constant for similar compounds binding to BSA maybe one approach to resolve this question. For example, here eleven flavonoids were tested to illustrate that the double logarithm regression curve is suitable to study binding polyphenols to BSA.

Multipixel frequency-domain imaging of spontaneous canine breast disease using fluorescent contrast agents

NASA Astrophysics Data System (ADS)

Reynolds, Jeffery S.; Thompson, Alan B.; Troy, Tamara L.; Mayer, Ralf H.; Waters, David J.; Sevick-Muraca, Eva M.

1999-07-01

In this paper we demonstrate the ability to detect the frequency-domain fluorescent signal from the contrast agent indocyanine green within the mammary chain of dogs with spontaneous mammary tumors. We use a gain-modulated image intensifier to rapidly capture multi-pixel images of the fluorescent modulation amplitude, modulation phase, and average intensity signals. Excitation is provided by a 100 MHz amplitude-modulated, 780 nm laser diode. Time series images of the uptake and clearance of the contrast agent in the diseased tissue are also presented.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Application of time-resolved fluorescence for direct and continuous probing of release from polymeric delivery vehicles.

PubMed

Viger, Mathieu L; Sheng, Wangzhong; McFearin, Cathryn L; Berezin, Mikhail Y; Almutairi, Adah

2013-11-10

Though accurately evaluating the kinetics of release is critical for validating newly designed therapeutic carriers for in vivo applications, few methods yet exist for release measurement in real time and without the need for any sample preparation. Many of the current approaches (e.g. chromatographic methods, absorption spectroscopy, or NMR spectroscopy) rely on isolation of the released material from the loaded vehicles, which require additional sample purification and can lead to loss of accuracy when probing fast kinetics of release. In this study we describe the use of time-resolved fluorescence for in situ monitoring of small molecule release kinetics from biodegradable polymeric drug delivery systems. This method relies on the observation that fluorescent reporters being released from polymeric drug delivery systems possess distinct excited-state lifetime components, reflecting their different environments in the particle suspensions, i.e., confined in the polymer matrices or free in the aqueous environment. These distinct lifetimes enable real-time quantitative mapping of the relative concentrations of dye in each population to obtain precise and accurate temporal information on the release profile of particular carrier/payload combinations. We found that fluorescence lifetime better distinguishes subtle differences in release profiles (e.g. differences associated with dye loading) than conventional steady-state fluorescence measurements, which represent the averaged dye behavior over the entire scan. Given the method's applicability to both hydrophobic and hydrophilic cargo, it could be employed to model the release of any drug-carrier combination. Copyright © 2013 Elsevier B.V. All rights reserved.

Anomalous fluorescence enhancement and fluorescence quenching of graphene quantum dots by single walled carbon nanotubes.

PubMed

Das, Ruma; Rajender, Gone; Giri, P K

2018-02-07

We explore the mechanism of the fluorescence enhancement and fluorescence quenching effect of single walled carbon nanotubes (SWCNTs) on highly fluorescent graphene quantum dots (GQDs) over a wide range of concentrations of SWCNTs. At very low concentrations of SWCNTs, the fluorescence intensity of the GQDs is enhanced, while at higher concentrations, systematic quenching of fluorescence is observed. The nature of the Stern-Volmer plot for the latter case was found to be non-linear indicating a combined effect of dynamic and static quenching. The contribution of the dynamic quenching component was assessed through the fluorescence lifetime measurements. The contribution of static quenching is confirmed from the red shift of the fluorescence spectra of the GQDs after addition of SWCNTs. The fluorescence intensity is first enhanced at very low concentration due to improved dispersion and higher absorption by GQDs, while at higher concentration, the fluorescence of GQDs is quenched due to the complex formation and associated reduction of the radiative sites of the GQDs, which is confirmed from time-resolved fluorescence measurements. Laser confocal microscopy imaging provides direct evidence of the enhancement and quenching of fluorescence at low and high concentrations of SWCNTs, respectively. This study provides an important insight into tuning the fluorescence of GQDs and understanding the interaction between GQDs and different CNTs, which is important for bio-imaging and drug delivery applications.

Comparative studies on the interaction of caffeic acid, chlorogenic acid and ferulic acid with bovine serum albumin

NASA Astrophysics Data System (ADS)

Li, Shuang; Huang, Kelong; Zhong, Ming; Guo, Jun; Wang, Wei-zheng; Zhu, Ronghua

2010-10-01

The substitution of the hydrogen on aromatic and esterification of carboxyl group of the phenol compounds plays an important role in their bio-activities. In this paper, caffeic acid (CaA), chlorogenic acid (ChA) and ferulic acid (FA) were selected to investigate the binding to bovine serum albumin (BSA) using UV absorption spectroscopy, fluorescence spectroscopy and synchronous fluorescence spectroscopy. It was found that the methoxyl group substituting for the 3-hydroxyl group of CaA decreased the affinity for BSA and the esterification of carboxyl group of CaA with quinic acid increased the affinities. The affinities of ChA and FA with BSA were more sensitive to the temperature than that of CaA with BSA. Synchronous fluorescence spectroscopy and time-resolved fluorescence indicated that the Stern-Volmer plots largely deviated from linearity at high concentrations and were caused by complete quenching of the tyrosine fluorescence of BSA.

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

PubMed Central

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

2012-01-01

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

Resolution Limits of Nanoimprinted Patterns by Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Kubo, Shoichi; Tomioka, Tatsuya; Nakagawa, Masaru

2013-06-01

The authors investigated optical resolution limits to identify minimum distances between convex lines of fluorescent dye-doped nanoimprinted resist patterns by fluorescence microscopy. Fluorescent ultraviolet (UV)-curable resin and thermoplastic resin films were transformed into line-and-space patterns by UV nanoimprinting and thermal nanoimprinting, respectively. Fluorescence immersion observation needed an immersion medium immiscible to the resist films, and an ionic liquid of triisobutyl methylphosphonium tosylate was appropriate for soluble thermoplastic polystyrene patterns. Observation with various numerical aperture (NA) values and two detection wavelength ranges showed that the resolution limits were smaller than the values estimated by the Sparrow criterion. The space width to identify line patterns became narrower as the line width increased. The space width of 100 nm was demonstrated to be sufficient to resolve 300-nm-wide lines in the detection wavelength range of 575-625 nm using an objective lens of NA= 1.40.

Light on fluorescent lipids in rafts: a lesson from model membranes.

PubMed

Kahya, Nicoletta

2010-09-15

Tracking fluorescent lipids in cellular membranes has been applied for decades to shed light on membrane trafficking, sorting, endocytosis and exocytosis, viral entry, and to understand the functional relevance of membrane heterogeneity, phase separation and lipid rafts. However, fluorescent probes may display different organizing behaviour from their corresponding endogenous lipids. A full characterization of these probes is therefore required for proper interpretation of fluorescence microscopy data in complex membrane systems. Model membrane studies provide essential clues that guide us to design and interpret our experiments, help us to avoid pitfalls and resolve artefacts in complex cellular environments. In the present issue of the Biochemical Journal, Juhasz, Davis and Sharom demonstrate the importance of testing lipid probes systematically in heterogeneous model membranes of specific composition and well-defined thermodynamic properties. The phase-partitioning behaviour of fluorescent probes, alone and/or in combination, cannot simply be assumed, but has to be fully characterized.

Implementation of laser induced fluorescence in a pulse radiolysis experiment--a new way to analyze resazurin-like reduction mechanisms.

PubMed

Balcerzyk, A; Baldacchino, G

2014-04-07

Resazurin (RNO) reduction by hydrated electrons produces a fluorescent molecule: resorufin (RN). To take advantage of RN fluorescence, a novel setup is designed by implementing fluorescence detection induced by laser in a pulse radiolysis experiment. Time resolved fluorescence spectra were recorded with a fast gated intensified CCD camera during the reduction of RNO from μs to ms. Two 532 nm laser types have been used to describe the short μs range by a 5 ns Q-switch laser and the μs-ms range by a CW DPSS laser. By fitting the simulated model to the experimental data a second order rate constant of 10(9) M(-1) s(-1) was re-evaluated. This method should be considered in the near future in many in situ and real time measurements for evaluating radical production.

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

NASA Astrophysics Data System (ADS)

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

2011-01-01

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

Plasmonic platforms of self-assembled silver nanostructures in application to fluorescence

PubMed Central

Luchowski, Rafal; Calander, Nils; Shtoyko, Tanya; Apicella, Elisa; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

2011-01-01

Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2–3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from localized areas of silver nanostructures. PMID:21403765

Characterization of time-resolved fluorescence response measurements for distributed optical-fiber sensing.

PubMed

Sinchenko, Elena; Gibbs, W E Keith; Davis, Claire E; Stoddart, Paul R

2010-11-20

A distributed optical-fiber sensing system based on pulsed excitation and time-gated photon counting has been used to locate a fluorescent region along the fiber. The complex Alq3 and the infrared dye IR-125 were examined with 405 and 780 nm excitation, respectively. A model to characterize the response of the distributed fluorescence sensor to a Gaussian input pulse was developed and tested. Analysis of the Alq3 fluorescent response confirmed the validity of the model and enabled the fluorescence lifetime to be determined. The intrinsic lifetime obtained (18.2±0.9 ns) is in good agreement with published data. The decay rate was found to be proportional to concentration, which is indicative of collisional deactivation. The model allows the spatial resolution of a distributed sensing system to be improved for fluorophores with lifetimes that are longer than the resolution of the sensing system.

Complexation induced fluorescence and acid-base properties of dapoxyl dye with γ-cyclodextrin: a drug-binding application using displacement assays.

PubMed

Pal, Kaushik; Mallick, Suman; Koner, Apurba L

2015-06-28

Host-guest complexation of dapoxyl sodium sulphonate (DSS), an intramolecular charge transfer dye with water-soluble and non-toxic macrocycle γ-cyclodextrin (γ-CD), has been investigated in a wide pH range. Steady-state absorption, fluorescence and time-resolved fluorescence measurements confirm the positioning of DSS into the hydrophobic cavity of γ-CD. A large fluorescence enhancement ca. 30 times, due to 1 : 2 complex formation and host-assisted guest-protonation have been utilised for developing a method for the utilisation of CD based drug-delivery applications. A simple fluorescence-displacement based approach is implemented at physiological pH for the assessment of binding strength of pharmaceutically useful small drug molecules (ibuprofen, paracetamol, methyl salicylate, salicylic acid, aspirin, and piroxicam) and six important antibiotic drugs (resazurin, thiamphenicol, chloramphenicol, ampicillin, kanamycin, and sorbic acid) with γ-CD.

Interaction of the iron(II) cage complexes with proteins: protein fluorescence quenching study.

PubMed

Losytskyy, Mykhaylo Y; Kovalska, Vladyslava B; Varzatskii, Oleg A; Sergeev, Alexander M; Yarmoluk, Sergiy M; Voloshin, Yan Z

2013-09-01

Interaction of the iron(II) mono- and bis-clathrochelates with bovine serum albumin (BSA), β-lactoglobulin, lysozyme and insulin was studied by the steady-state and time-resolved fluorescent spectroscopies. These cage complexes do not make significant impact on fluorescent properties of β-lactoglobulin, lysozyme and insulin. At the same time, the monoclathrochelates strongly quench a fluorescence intensity of BSA and substantially decrease its excited state lifetime due to their binding to this protein. This occurs due to the excitation energy transfer from a tryptophan residue to a cage molecule or/and to the change of the tryptophan nearest environment caused by either clathrochelate binding or an alteration of the BSA conformation. The effect of the iron(II) bis-clathrochelate on BSA fluorescence is much weaker as compared to its monomacrobicyclic analogs as a result of an increase in its size.

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

PubMed Central

Vallaghe, Julie; Gregor, Nathalie; Donthamsetti, Prashant; Harris, Paul E.; Pierre, Nicolas; Freyberg, Robin; Charrier-Savournin, Fabienne; Javitch, Jonathan A.; Freyberg, Zachary

2016-01-01

Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment. PMID:26849707

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera

NASA Astrophysics Data System (ADS)

Cruz Perez, Carlos; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera.

PubMed

Perez, Carlos Cruz; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Amine-functionalized lanthanide-doped zirconia nanoparticles: optical spectroscopy, time-resolved fluorescence resonance energy transfer biodetection, and targeted imaging.

PubMed

Liu, Yongsheng; Zhou, Shanyong; Tu, Datao; Chen, Zhuo; Huang, Mingdong; Zhu, Haomiao; Ma, En; Chen, Xueyuan

2012-09-12

Ultrasmall inorganic oxide nanoparticles doped with trivalent lanthanide ions (Ln(3+)), a new and huge family of luminescent bioprobes, remain nearly untouched. Currently it is a challenge to synthesize biocompatible ultrasmall oxide bioprobes. Herein, we report a new inorganic oxide bioprobe based on sub-5 nm amine-functionalized tetragonal ZrO(2)-Ln(3+) nanoparticles synthesized via a facile solvothermal method and ligand exchange. By utilizing the long-lived luminescence of Ln(3+), we demonstrate its application as a sensitive time-resolved fluorescence resonance energy transfer (FRET) bioprobe to detect avidin with a record-low detection limit of 3.0 nM. The oxide nanoparticles also exhibit specific recognition of cancer cells overexpressed with urokinase plasminogen activator receptor (uPAR, an important marker of tumor biology and metastasis) and thus may have great potentials in targeted bioimaging.

Perspectives in Super-resolved Fluorescence Microscopy: What comes next?

NASA Astrophysics Data System (ADS)

Cremer, Christoph; Birk, Udo

2016-04-01

The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM) methods. They have proven that it is possible to overcome the hundred year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light), which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.

Resolving fluorophores by unmixing multispectral fluorescence tomography with independent component analysis

NASA Astrophysics Data System (ADS)

Pu, Huangsheng; Zhang, Guanglei; He, Wei; Liu, Fei; Guang, Huizhi; Zhang, Yue; Bai, Jing; Luo, Jianwen

2014-09-01

It is a challenging problem to resolve and identify drug (or non-specific fluorophore) distribution throughout the whole body of small animals in vivo. In this article, an algorithm of unmixing multispectral fluorescence tomography (MFT) images based on independent component analysis (ICA) is proposed to solve this problem. ICA is used to unmix the data matrix assembled by the reconstruction results from MFT. Then the independent components (ICs) that represent spatial structures and the corresponding spectrum courses (SCs) which are associated with spectral variations can be obtained. By combining the ICs with SCs, the recovered MFT images can be generated and fluorophore concentration can be calculated. Simulation studies, phantom experiments and animal experiments with different concentration contrasts and spectrum combinations are performed to test the performance of the proposed algorithm. Results demonstrate that the proposed algorithm can not only provide the spatial information of fluorophores, but also recover the actual reconstruction of MFT images.

Time-resolved fluorescence sensing of pesticides chlorpyrifos, crotoxyphos and endosulfan by the luminescent Eu(III)-8-allyl-3-carboxycoumarin probe

NASA Astrophysics Data System (ADS)

Azab, Hassan A.; Khairy, Gasser M.; Kamel, Rasha M.

2015-09-01

This work describes the application of time resolved fluorescence in microtiter plates for investigating the interactions of europium-allyl-3-carboxycoumarin with pesticides chlorpyrifos, endosulfan and crotoxyphos. Stern-Volmer studies at different temperatures for chlorpyrifos and crotoxyphos shows dynamic and static quenching mechanisms respectively. Direct methods for the determination of the pesticides under investigation have been developed using the luminescence variations of the probe in solution. The detection limits are 6.53, 0.004, 3.72 μmol/L for chlorpyrifos, endosulfan, and crotoxyphos, respectively. The binding constants and thermodynamic parameters of the pesticides with probe were evaluated. A thermodynamic analysis showed that the reaction is spontaneous with negative ΔG. Effect of some relevant interferents on the detection of pesticides has been investigated. The new method was applied to the determination of the pesticides in different types of water samples (tap, mineral, and waste water).

Extracting Optical Fiber Background from Surface-Enhanced Raman Spectroscopy Spectra Based on Bi-Objective Optimization Modeling.

PubMed

Huang, Jie; Shi, Tielin; Tang, Zirong; Zhu, Wei; Liao, Guanglan; Li, Xiaoping; Gong, Bo; Zhou, Tengyuan

2017-08-01

We propose a bi-objective optimization model for extracting optical fiber background from the measured surface-enhanced Raman spectroscopy (SERS) spectrum of the target sample in the application of fiber optic SERS. The model is built using curve fitting to resolve the SERS spectrum into several individual bands, and simultaneously matching some resolved bands with the measured background spectrum. The Pearson correlation coefficient is selected as the similarity index and its maximum value is pursued during the spectral matching process. An algorithm is proposed, programmed, and demonstrated successfully in extracting optical fiber background or fluorescence background from the measured SERS spectra of rhodamine 6G (R6G) and crystal violet (CV). The proposed model not only can be applied to remove optical fiber background or fluorescence background for SERS spectra, but also can be transferred to conventional Raman spectra recorded using fiber optic instrumentation.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

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

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

Experimental feasibility of the airborne measurement of absolute oil fluorescence spectral conversion efficiency

NASA Technical Reports Server (NTRS)

Hoge, F. E.; Swift, R. N.

1983-01-01

Airborne lidar oil spill experiments carried out to determine the practicability of the AOFSCE (absolute oil fluorescence spectral conversion efficiency) computational model are described. The results reveal that the model is suitable over a considerable range of oil film thicknesses provided the fluorescence efficiency of the oil does not approach the minimum detection sensitivity limitations of the lidar system. Separate airborne lidar experiments to demonstrate measurement of the water column Raman conversion efficiency are also conducted to ascertain the ultimate feasibility of converting such relative oil fluorescence to absolute values. Whereas the AOFSCE model is seen as highly promising, further airborne water column Raman conversion efficiency experiments with improved temporal or depth-resolved waveform calibration and software deconvolution techniques are thought necessary for a final determination of suitability.

Mechanism of the reaction, CH4+O(1D2)→CH3+OH, studied by ultrafast and state-resolved photolysis/probe spectroscopy of the CH4ṡO3 van der Waals complex

NASA Astrophysics Data System (ADS)

Miller, C. Cameron; van Zee, Roger D.; Stephenson, John C.

2001-01-01

The mechanism of the reaction CH4+O(1D2)→CH3+OH was investigated by ultrafast, time-resolved and state-resolved experiments. In the ultrafast experiments, short ultraviolet pulses photolyzed ozone in the CH4ṡO3 van der Waals complex to produce O(1D2). The ensuing reaction with CH4 was monitored by measuring the appearance rate of OH(v=0,1;J,Ω,Λ) by laser-induced fluorescence, through the OH A←X transition, using short probe pulses. These spectrally broad pulses, centered between 307 and 316 nm, probe many different OH rovibrational states simultaneously. At each probe wavelength, both a fast and a slow rise time were evident in the fluorescence signal, and the ratio of the fast-to-slow signal varied with probe wavelength. The distribution of OH(v,J,Ω,Λ) states, Pobs(v,J,Ω,Λ), was determined by laser-induced fluorescence using a high-resolution, tunable dye laser. The Pobs(v,J,Ω,Λ) data and the time-resolved data were analyzed under the assumption that different formation times represent different reaction mechanisms and that each mechanism produces a characteristic rovibrational distribution. The state-resolved and the time-resolved data can be fit independently using a two-mechanism model: Pobs(v,J,Ω,Λ) can be decomposed into two components, and the appearance of OH can be fit by two exponential rise times. However, these independent analyses are not mutually consistent. The time-resolved and state-resolved data can be consistently fit using a three-mechanism model. The OH appearance signals, at all probe wavelengths, were fit with times τfast≈0.2 ps, τinter≈0.5 ps and τslow≈5.4 ps. The slowest of these three is the rate for dissociation of a vibrationally excited methanol intermediate (CH3OH*) predicted by statistical theory after complete intramolecular energy redistribution following insertion of O(1D2) into CH4. The Pobs(v,J,Ω,Λ) was decomposed into three components, each with a linear surprisal, under the assumption that the mechanism producing OH at a statistical rate would be characterized by a statistical prior. Dissociation of a CH4O* intermediate before complete energy randomization was identified as producing OH at the intermediate rate and was associated with a population distribution with more rovibrational energy than the slow mechanism. The third mechanism produces OH promptly with a cold rovibrational distribution, indicative of a collinear abstraction mechanism. After these identifications were made, it was possible to predict the fraction of signal associated with each mechanism at different probe wavelengths in the ultrafast experiment, and the predictions proved consistent with measured appearance signals. This model also reconciles data from a variety of previous experiments. While this model is the simplest that is consistent with the data, it is not definitive for several reasons. First, the appearance signals measured in these experiments probe simultaneously many OH(v,J,Ω,Λ) states, which would tend to obfuscate differences in the appearance rate of specific rovibrational states. Second, only about half of the OH(v,J,Ω,Λ) states populated by this reaction could be probed by laser-induced fluorescence through the OH A←X band with our apparatus. Third, the cluster environment might influence the dynamics compared to the free bimolecular reaction.

Characterizing chlorine oxidation of dissolved organic matter and disinfection by-product formation with fluorescence spectroscopy and parallel factor analysis

NASA Astrophysics Data System (ADS)

Beggs, Katherine M. H.; Summers, R. Scott; McKnight, Diane M.

2009-12-01

Relationships between chlorine demand and disinfection by-product (DBP) formation during chlorination and fluorescence of dissolved organic matter (DOM) were developed. Fluorescence excitation and emission (EEM) spectroscopy was employed, and parameters including fluorescence index, redox index, and overall fluorescence intensity (OFI) were correlated to chlorine demand and DBP formation. The EEMs were also analyzed using a well established global parallel factor analysis (PARAFAC) model which resolves the fluorescence signal into 13 components, including quinone-like and protein-like components. Over an 8-day chlorination period the OFI and sum of the 13 PARAFAC loadings decreased by more than 70%. The remaining identified quinone-like compounds within the DOM were shifted to a more oxidized state. Quinone fluorescence was strongly correlated to both reduced fluorescence intensity and to chlorine demand which indicates that fluorescence may be used to track the chlorine oxidation of DOM. Quinone fluorescence was also correlated strongly with both classes of regulated DBPs: total trihalomethanes and haloacetic acids. Quinone-like components were found to be strongly correlated to overall, short-term, and long-term specific DBP formation. The results of this study show that fluorescence is a useful tool in tracking both DOM oxidation and DBP formation during chlorination.

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.

Multilinear analysis of Time-Resolved Laser-Induced Fluorescence Spectra of U(VI) containing natural water samples

NASA Astrophysics Data System (ADS)

Višňák, Jakub; Steudtner, Robin; Kassahun, Andrea; Hoth, Nils

2017-09-01

Natural waters' uranium level monitoring is of great importance for health and environmental protection. One possible detection method is the Time-Resolved Laser-Induced Fluorescence Spectroscopy (TRLFS), which offers the possibility to distinguish different uranium species. The analytical identification of aqueous uranium species in natural water samples is of distinct importance since individual species differ significantly in sorption properties and mobility in the environment. Samples originate from former uranium mine sites and have been provided by Wismut GmbH, Germany. They have been characterized by total elemental concentrations and TRLFS spectra. Uranium in the samples is supposed to be in form of uranyl(VI) complexes mostly with carbonate (CO32- ) and bicarbonate (HCO3- ) and to lesser extend with sulphate (SO42- ), arsenate (AsO43- ), hydroxo (OH- ), nitrate (NO3- ) and other ligands. Presence of alkaline earth metal dications (M = Ca2+ , Mg2+ , Sr2+ ) will cause most of uranyl to prefer ternary complex species, e.g. Mn(UO2)(CO3)32n-4 (n ɛ {1; 2}). From species quenching the luminescence, Cl- and Fe2+ should be mentioned. Measurement has been done under cryogenic conditions to increase the luminescence signal. Data analysis has been based on Singular Value Decomposition and monoexponential fit of corresponding loadings (for separate TRLFS spectra, the "Factor analysis of Time Series" (FATS) method) and Parallel Factor Analysis (PARAFAC, all data analysed simultaneously). From individual component spectra, excitation energies T00, uranyl symmetric mode vibrational frequencies ωgs and excitation driven U-Oyl bond elongation ΔR have been determined and compared with quasirelativistic (TD)DFT/B3LYP theoretical predictions to cross -check experimental data interpretation. Note to the reader: Several errors have been produced in the initial version of this article. This new version published on 23 October 2017 contains all the corrections.

Light adaptation of the unicellular red alga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.

PubMed

Ueno, Yoshifumi; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

2015-08-01

Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed.

340nm UV LED excitation in time-resolved fluorescence system for europium-based immunoassays detection

NASA Astrophysics Data System (ADS)

Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Pedersen, Christian

2017-02-01

In immunoassay analyzers for in-vitro diagnostics, Xenon flash lamps have been widely used as excitation light sources. Recent advancements in UV LED technology and its advantages over the flash lamps such as smaller footprint, better wall-plug efficiency, narrow emission spectrum, and no significant afterglow, have made them attractive light sources for gated detection systems. In this paper, we report on the implementation of a 340 nm UV LED based time-resolved fluorescence system based on europium chelate as a fluorescent marker. The system performance was tested with the immunoassay based on the cardiac marker, TnI. The same signal-to-noise ratio as for the flash lamp based system was obtained, operating the LED below specified maximum current. The background counts of the system and its main contributors were measured and analyzed. The background of the system of the LED based unit was improved by 39% compared to that of the Xenon flash lamp based unit, due to the LEDs narrower emission spectrum and longer pulse width. Key parameters of the LED system are discussed to further optimize the signal-to-noise ratio and signal-to-background, and hence the sensitivity of the instrument.

Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography

NASA Astrophysics Data System (ADS)

Coquelle, Nicolas; Sliwa, Michel; Woodhouse, Joyce; Schirò, Giorgio; Adam, Virgile; Aquila, Andrew; Barends, Thomas R. M.; Boutet, Sébastien; Byrdin, Martin; Carbajo, Sergio; de La Mora, Eugenio; Doak, R. Bruce; Feliks, Mikolaj; Fieschi, Franck; Foucar, Lutz; Guillon, Virginia; Hilpert, Mario; Hunter, Mark S.; Jakobs, Stefan; Koglin, Jason E.; Kovacsova, Gabriela; Lane, Thomas J.; Lévy, Bernard; Liang, Mengning; Nass, Karol; Ridard, Jacqueline; Robinson, Joseph S.; Roome, Christopher M.; Ruckebusch, Cyril; Seaberg, Matthew; Thepaut, Michel; Cammarata, Marco; Demachy, Isabelle; Field, Martin; Shoeman, Robert L.; Bourgeois, Dominique; Colletier, Jacques-Philippe; Schlichting, Ilme; Weik, Martin

2018-01-01

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.

Potential toxicity and affinity of triphenylmethane dye malachite green to lysozyme.

PubMed

Ding, Fei; Li, Xiu-Nan; Diao, Jian-Xiong; Sun, Ye; Zhang, Li; Ma, Lin; Yang, Xin-Ling; Zhang, Li; Sun, Ying

2012-04-01

Malachite green is a triphenylmethane dye that is used extensively in many industrial and aquacultural processes, generating environmental concerns and health problems to human being. In this contribution, the complexation between lysozyme and malachite green was verified by means of computer-aided molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) approaches. The precise binding patch of malachite green in lysozyme has been identified from molecular modeling and ANS displacement, Trp-62, Trp-63, and Trp-108 residues of lysozyme were earmarked to possess high-affinity for this dye, the principal forces in the lysozyme-malachite green adduct are hydrophobic and π-π interactions. Steady state fluorescence proclaimed the complex of malachite green with lysozyme yields quenching through static type, which substantiates time-resolved fluorescence measurements that lysozyme-malachite green conjugation formation has an affinity of 10(3)M(-1). Moreover, via molecular modeling and also CD data, we can safely arrive at a conclusion that the polypeptide chain of lysozyme partially destabilized upon complexation with malachite green. The data emerged here will help to further understand the toxicological action of malachite green in human body. Copyright © 2012 Elsevier Inc. All rights reserved.

Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography

DOE PAGES

Coquelle, Nicolas; Sliwa, Michel; Woodhouse, Joyce; ...

2017-09-11

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here in this paper we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecondmore » timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.« less

Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography

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

Coquelle, Nicolas; Sliwa, Michel; Woodhouse, Joyce

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here in this paper we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecondmore » timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.« less

Wavelength-Resolved Photon Fluxes of Indoor Light Sources: Implications for HOx Production

NASA Astrophysics Data System (ADS)

Kowal, S.; Kahan, T.

2017-12-01

Only a handful of studies have considered photolytic reactions indoors because photon fluxes at short wavelengths are generally considered to be negligible. We have measured wavelength resolved photon fluxes from indoor light sources including incandescent, halogen, compact fluorescent (CFL), and light emitting diodes (LED). In addition, fluorescent tubes, used in many offices and industrial buildings, and sunlight through windows were measured. The measured photon fluxes were used to calculate photolysis rate constants for potential indoor hydroxyl and peroxy radical (OH and HO2, "HOx") precursors: acetaldehyde (CH3CHO), formaldehyde (HCHO), hydrogen peroxide (H2O2), nitrous acid (HONO) and ozone (O3). Rate constants in conjunction with typical indoor concentrations were used to predict HOx production rates under various lighting conditions. Our results illustrate that all light sources except LEDs emit light at high enough energy to photolyze HOx precursors. Under typical lighting conditions only fluorescent tubes and sunlight will initiate significant photochemical HOx formation, and HONO and HCHO will be the only molecules that will have a strong influence on HOx levels indoors. Data from our experiments can be used in indoor air models to better predict HOx levels indoors.

Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy.

PubMed

Gröner, Nadine; Capoulade, Jérémie; Cremer, Christoph; Wachsmuth, Malte

2010-09-27

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

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.

Understanding Unimer Exchange Processes in Block Copolymer Micelles using NMR Diffusometry, Time-Resolved NMR, and SANS

NASA Astrophysics Data System (ADS)

Madsen, Louis; Kidd, Bryce; Li, Xiuli; Miller, Katherine; Cooksey, Tyler; Robertson, Megan

Our team seeks to understand dynamic behaviors of block copolymer micelles and their interplay with encapsulated cargo molecules. Quantifying unimer and cargo exchange rates micelles can provide critical information for determining mechanisms of unimer exchange as well as designing systems for specific cargo release dynamics. We are exploring the utility of NMR spectroscopy and diffusometry techniques as complements to existing SANS and fluorescence methods. One promising new method involves time-resolved NMR spin relaxation measurements, wherein mixing of fully protonated and 2H-labeled PEO-b-PCL micelles solutions shows an increase in spin-lattice relaxation time (T1) with time after mixing. This is due to a weakening in magnetic environment surrounding 1H spins as 2H-bearing unimers join fully protonated micelles. We are measuring time constants for unimer exchange of minutes to hours, and we expect to resolve times of <1 min. This method can work on any solution NMR spectrometer and with minimal perturbation to chemical structure (as in dye-labelled fluorescence methods). Multimodal NMR can complement existing characterization tools, expanding and accelerating dynamics measurements for polymer micelle, nanogel, and nanoparticle developers.

Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

PubMed

Schmid, Volker J; Cremer, Marion; Cremer, Thomas

2017-07-01

Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data. Copyright © 2017 Elsevier Inc. All rights reserved.

Connecting active to passive fluorescence with photosynthesis: a method for evaluating remote sensing measurements of Chl fluorescence.

PubMed

Magney, Troy S; Frankenberg, Christian; Fisher, Joshua B; Sun, Ying; North, Gretchen B; Davis, Thomas S; Kornfeld, Ari; Siebke, Katharina

2017-09-01

Recent advances in the retrieval of Chl fluorescence from space using passive methods (solar-induced Chl fluorescence, SIF) promise improved mapping of plant photosynthesis globally. However, unresolved issues related to the spatial, spectral, and temporal dynamics of vegetation fluorescence complicate our ability to interpret SIF measurements. We developed an instrument to measure leaf-level gas exchange simultaneously with pulse-amplitude modulation (PAM) and spectrally resolved fluorescence over the same field of view - allowing us to investigate the relationships between active and passive fluorescence with photosynthesis. Strongly correlated, slope-dependent relationships were observed between measured spectra across all wavelengths (F λ , 670-850 nm) and PAM fluorescence parameters under a range of actinic light intensities (steady-state fluorescence yields, F t ) and saturation pulses (maximal fluorescence yields, F m ). Our results suggest that this method can accurately reproduce the full Chl emission spectra - capturing the spectral dynamics associated with changes in the yields of fluorescence, photochemical (ΦPSII), and nonphotochemical quenching (NPQ). We discuss how this method may establish a link between photosynthetic capacity and the mechanistic drivers of wavelength-specific fluorescence emission during changes in environmental conditions (light, temperature, humidity). Our emphasis is on future research directions linking spectral fluorescence to photosynthesis, ΦPSII, and NPQ. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Submicrosecond phospholipid dynamics using a long-lived fluorescence emission anisotropy probe.

PubMed Central

Davenport, L; Targowski, P

1996-01-01

The use of the long-lived fluorescence probe coronene (mean value of tau(FL) approximately 200 ns) is described for investigating submicrosecond lipid dynamics in DPPC model bilayer systems occurring below the lipid phase transition. Time-resolved fluorescence emission anisotropy decay profiles, measures as a function of increasing temperature toward the lipid-phase transition temperature (T(C)), for coronene-labeled DPPC small unilamellar vesicles (SUVs), are best described in most cases by three rotational decay components (phi(i = 3)). We have interpreted these data using two dynamic lipid bilayer models. In the first, a compartmental model, the long correlation time (phi(N)) is assigned to immobilized coronene molecules located in "gel-like" or highly ordered lipid phases (S-->1) of the bilayer, whereas a second fast rotational time (phi(F) approximately 2-5 ns) is associated with probes residing in more "fluid-like" regions (with corresponding lower ordering, S-->0). Interests here have focused on the origins of an intermediate correlation time (50-100 ns), the associated amplitude (beta(G)) of which increases with increasing temperature. Such behavior suggests a changing rotational environment surrounding the coronene molecules, arising from fluidization of gel lipid. The observed effective correlation time (phi(EFF)) thus reflects a discrete gel-fluid lipid exchange rate (k(FG)). A refinement of the compartmental model invokes a distribution of gel-fluid exchange rates (d(S,T)) corresponding to a distribution of lipid order parameters and is based on an adapted Landau expression for describing "gated" packing fluctuations. A total of seven parameters (five thermodynamic quantities, defined by the free energy versus temperature expansion; one gating parameter (gamma) defining a cooperative "melting" requirement; one limiting diffusion rate (or frequency factor: d(infinity))) suffice to predict complete anisotropy decay curves measured for coronene at several temperatures below the phospholipid T(C). The thermodynamic quantities are associated with the particular lipid of interest (in this case DPPC) and have been determined previously from ultrasound studies, thus representing fixed constants. Hence resolved variables are r(O), temperature-dependent gate parameters (gamma), and limiting diffusion rates (d(infinity)). This alternative distribution model is attractive because it provides a general probe-independent expression for distributed lipid fluctuation-induced probe rotational rates occurring within bilayer membranes below the phospholipid phase transition on the submicrosecond time scale. PMID:8889160

Reconstruction for limited-projection fluorescence molecular tomography based on projected restarted conjugate gradient normal residual.

PubMed

Cao, Xu; Zhang, Bin; Liu, Fei; Wang, Xin; Bai, Jing

2011-12-01

Limited-projection fluorescence molecular tomography (FMT) can greatly reduce the acquisition time, which is suitable for resolving fast biology processes in vivo but suffers from severe ill-posedness because of the reconstruction using only limited projections. To overcome the severe ill-posedness, we report a reconstruction method based on the projected restarted conjugate gradient normal residual. The reconstruction results of two phantom experiments demonstrate that the proposed method is feasible for limited-projection FMT. © 2011 Optical Society of America

Radiative lifetimes in B I using ultraviolet and vacuum-ultraviolet laser-induced fluorescence

NASA Technical Reports Server (NTRS)

O'Brian, T. R.; Lawler, J. E.

1992-01-01

Radiative lifetimes of the eight lowest even parity levels in the doublet system of B I are measured using time-resolved laser-induced fluorescence in the UV and VUV on an atomic beam of boron. The accurate lifetimes provide a base for improved determination of absolute transition probabilities in B I. The techniques described are broadly applicable to measurement of lifetimes of levels with transitions in the visible, UV, and VUV in almost any element.

Application of Time-Resolved Spectroscopies to the Study of Energetic Materials - 1982

DTIC Science & Technology

1983-05-24

fluores- cence intensity as a function of UV pulse energy, for individual laser shots. The lower curve shows the UV + probe induced fluorescence... intensity as a function of UV pulse energy, for individual laser shots. The lower curve shows the UV + probe Induced fluorescence, at 1 ns delay...locked Nd:YAG Laser Pulse ", Appl. Phys. Lett 26, 501-503 (1975). 97 43. A. J. Campillo, V. H. Kollman and S. L. Shapiro, " Intensity Dependence of

Experimental and Theoretical Aspects of Excited State Electron Transfer and Related Phenomena: Conference Held in Honour of Zbigniew R. Grabowski in Pultusk, Poland on September 27-October 2, 1992

DTIC Science & Technology

1992-10-01

DBMBF2 ) undergoes photoreaction with olefins through a partial electron transfer that leads to cycloaddition or sensitized Diels - Alder reactions. We...8217 Fluorescence. 10:00 J.M. WARMAN: Photon-induced Intramolecular Charge Sepaiation Studied byTime-Resolved Microwave Conductivity. 10:30 Coffee 11:)) W...26 Photon-Induced Intramolecular Charge Separation Studied by Time-Resolved Microwave Conductivity John M. Warman IRI, Delft University of Technology

Serum albumin binding sites properties in donors and in schizophrenia patients: the study of fluorescence decay of the probe K-35 using S-60 synchrotron pulse excitation

NASA Astrophysics Data System (ADS)

Gryzunov, Yu. A.; Syrejshchikova, T. I.; Komarova, M. N.; Misionzhnik, E. Yu; Uzbekov, M. G.; Molodetskich, A. V.; Dobretsov, G. E.; Yakimenko, M. N.

2000-06-01

The properties of serum albumin obtained from donors and from paranoid schizophrenia patients were studied with the fluorescent probe K-35 (N-carboxyphenylimide of dimethylaminonaphthalic acid) and time-resolved fluorescence spectroscopy on the SR beam station of the S-60 synchrotron of the Lebedev Physical Institute. The mean fluorescence quantum yield of K-35 in patients serum was decreased significantly by 25-60% comparing with donors. The analysis of pre-exponential factors of fluorescence decay using "amplitude standard" method has shown that in patient sera the fraction of K-35 molecules bound with albumin and inaccessible to fluorescence quenchers ("bright" K-35 molecules with τ1=8.0±0.4 ns) is 1.2-3 times less than in the donor sera. The fraction of K-35 molecules with partly quenched fluorescence ( τ2=1.44±0.22 ns) was significantly increased in schizophrenia patients. The results obtained suggest that the properties of binding region in serum albumin molecules of acute paranoid schizophrenia patients change significantly.

Using time-frequency analysis to determine time-resolved detonation velocity with microwave interferometry.

PubMed

Kittell, David E; Mares, Jesus O; Son, Steven F

2015-04-01

Two time-frequency analysis methods based on the short-time Fourier transform (STFT) and continuous wavelet transform (CWT) were used to determine time-resolved detonation velocities with microwave interferometry (MI). The results were directly compared to well-established analysis techniques consisting of a peak-picking routine as well as a phase unwrapping method (i.e., quadrature analysis). The comparison is conducted on experimental data consisting of transient detonation phenomena observed in triaminotrinitrobenzene and ammonium nitrate-urea explosives, representing high and low quality MI signals, respectively. Time-frequency analysis proved much more capable of extracting useful and highly resolved velocity information from low quality signals than the phase unwrapping and peak-picking methods. Additionally, control of the time-frequency methods is mainly constrained to a single parameter which allows for a highly unbiased analysis method to extract velocity information. In contrast, the phase unwrapping technique introduces user based variability while the peak-picking technique does not achieve a highly resolved velocity result. Both STFT and CWT methods are proposed as improved additions to the analysis methods applied to MI detonation experiments, and may be useful in similar applications.

New developments in electronic reference controls for frequency domain optical sensing

NASA Astrophysics Data System (ADS)

Chatni, M. R.; Li, G.; Porterfield, D. M.

2009-05-01

The reference optical path is essential for optical systems which function on the basis of light interference. In the case of frequency domain (FD) fluorescence life-time optrodes, a reference LED is used as a standard for calculating the phase angle. The reference LED is configured so that radiation travels the same length to the detector as that of the fluorescence signal being analyzed. The phase shift, which provides details of fluorescence lifetime, is measured between these two signals - the fluorescence signal and reference LED signal, using a photodetector. We have designed, developed and implemented a FD optrode system without a reference LED. The key requirement of such a system is that phase shifts due to optics at wavelength of fluorescence and electronics have to be calibrated. In the reference-free system, the reference signal comes from the lock-in-amplifier which also drives the excitation LED. The lock-in-amplifier measures the phase shift between the excitation signal and the fluorescence emission signal from the photodetector and is locked at the frequency of modulation of the excitation signal. This insures higher signal to noise ratio and low-noise measurements. The reference-free optrode system removes some constraints on the coupling optics, which help improve the overall performance of the system. After development of electronics, and optimization of coupling optics, the system was calibrated in different oxygen concentration solutions to measure fluorescence intensity and lifetime of the oxygen sensitive dye platinum tetrakis (pentafluorophenyl) porphine (PtTFPP).

Cortical Pitch Regions in Humans Respond Primarily to Resolved Harmonics and Are Located in Specific Tonotopic Regions of Anterior Auditory Cortex

PubMed Central

Kanwisher, Nancy; McDermott, Josh H.

2013-01-01

Pitch is a defining perceptual property of many real-world sounds, including music and speech. Classically, theories of pitch perception have differentiated between temporal and spectral cues. These cues are rendered distinct by the frequency resolution of the ear, such that some frequencies produce “resolved” peaks of excitation in the cochlea, whereas others are “unresolved,” providing a pitch cue only via their temporal fluctuations. Despite longstanding interest, the neural structures that process pitch, and their relationship to these cues, have remained controversial. Here, using fMRI in humans, we report the following: (1) consistent with previous reports, all subjects exhibited pitch-sensitive cortical regions that responded substantially more to harmonic tones than frequency-matched noise; (2) the response of these regions was mainly driven by spectrally resolved harmonics, although they also exhibited a weak but consistent response to unresolved harmonics relative to noise; (3) the response of pitch-sensitive regions to a parametric manipulation of resolvability tracked psychophysical discrimination thresholds for the same stimuli; and (4) pitch-sensitive regions were localized to specific tonotopic regions of anterior auditory cortex, extending from a low-frequency region of primary auditory cortex into a more anterior and less frequency-selective region of nonprimary auditory cortex. These results demonstrate that cortical pitch responses are located in a stereotyped region of anterior auditory cortex and are predominantly driven by resolved frequency components in a way that mirrors behavior. PMID:24336712

Fluorescence quantum yield of carbon dioxide for quantitative UV laser-induced fluorescence in high-pressure flames

NASA Astrophysics Data System (ADS)

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

2008-11-01

The fluorescence quantum yield for ultraviolet laser-induced fluorescence of CO2 is determined for selected excitation wavelengths in the range 215-250 nm. Wavelength-resolved laser-induced fluorescence (LIF) spectra of CO2, NO, and O2 are measured in the burned gases of a laminar CH4/air flame ( φ=0.9 and 1.1) at 20 bar with additional NO seeded into the flow. The fluorescence spectra are fit to determine the relative contribution of the three species to infer an estimate of fluorescence quantum yield for CO2 that ranges from 2-8×10-6 depending on temperature and excitation wavelength with an estimated uncertainty of ±0.5×10-6. The CO2 fluorescence signal increases linearly with gas pressure for flames with constant CO2 mole fraction for the 10 to 60 bar range, indicating that collisional quenching is not an important contributor to the CO2 fluorescence quantum yield. Spectral simulation calculations are used to choose two wavelengths for excitation of CO2, 239.34 and 242.14 nm, which minimize interference from LIF of NO and O2. Quantitative LIF images of CO2 are demonstrated using these two excitation wavelengths and the measured fluorescence quantum yield.

Three-photon excitation of 2,5-bis(4-biphenyl)oxazole: steady-state and time-resolved intensities and anisotropies

NASA Astrophysics Data System (ADS)

Gryczynski, Ignacy; Malak, Henryk; Hell, Stefan W.; Lakowicz, Joseph R.

1996-10-01

Three-photon excitation of 2,5-bis(4-biphenyl) oxazole (BBO) was observed when it was excited with the fundamental output of a femtosecond Ti:sapphire laser above 820 nm. The emission spectrum of BBO was identical for one-, two-, and three-photon excitation at 320, 640, and 960 nm, respectively. In toluene and triacetin, the emission intensity of BBO depended on the square of the laser power for wavelengths below 820 nm and displayed a sharp transition to a cubic dependence at longer wavelengths. The spatial distribution of the emission of BBO with three- photon excitation was more strongly localized than for two- photon excitation of a coumarin fluorophore at the same wavelength. The same single exponential intensity decay was observed for one-, two-, and three-photon excitation. However, the frequency domain anisotropy decay with three- photon excitation at 960 nm revealed a larger time-zero anisotropy, larger differential polarized phase angle, and larger modulated anisotropy than is possible for two-photon excitation with colinear oscillators. In triacetin, the anisotropy is not constant for three-photon excitation at different wavelengths. Surprisingly, the fluorescence intensities for three-photon excitation were only about 100- fold less than for two-photon excitation. The increasing availability of Ti:sapphire lasers suggests that multiphoton excitation can become a common tool in fluorescence spectroscopy.

Super-resolution from single photon emission: toward biological application

NASA Astrophysics Data System (ADS)

Moreva, E.; Traina, P.; Forneris, J.; Ditalia Tchernij, S.; Guarina, L.; Franchino, C.; Picollo, F.; Ruo Berchera, I.; Brida, G.; Degiovanni, I. P.; Carabelli, V.; Olivero, P.; Genovese, M.

2017-08-01

Properties of quantum light represent a tool for overcoming limits of classical optics. Several experiments have demonstrated this advantage ranging from quantum enhanced imaging to quantum illumination. In this work, experimental demonstration of quantum-enhanced resolution in confocal fluorescence microscopy will be presented. This is achieved by exploiting the non-classical photon statistics of fluorescence emission of single nitrogen-vacancy (NV) color centers in diamond. By developing a general model of super-resolution based on the direct sampling of the kth-order autocorrelation function of the photoluminescence signal, we show the possibility to resolve, in principle, arbitrarily close emitting centers. Finally, possible applications of NV-based fluorescent nanodiamonds in biosensing and future developments will be presented.

A Single-Photon Avalanche Diode Array for Fluorescence Lifetime Imaging Microscopy.

PubMed

Schwartz, David Eric; Charbon, Edoardo; Shepard, Kenneth L

2008-11-21

We describe the design, characterization, and demonstration of a fully integrated single-photon avalanche diode (SPAD) imager for use in time-resolved fluorescence imaging. The imager consists of a 64-by-64 array of active SPAD pixels and an on-chip time-to-digital converter (TDC) based on a delay-locked loop (DLL) and calibrated interpolators. The imager can perform both standard time-correlated single-photon counting (TCSPC) and an alternative gated-window detection useful for avoiding pulse pile-up when measuring bright signal levels. To illustrate the use of the imager, we present measurements of the decay lifetimes of fluorescent dyes of several types with a timing resolution of 350 ps.

Design of mitochondria-targeted colorimetric and ratiometric fluorescent probes for rapid detection of SO2 derivatives in living cells

NASA Astrophysics Data System (ADS)

Yang, Yutao; Zhou, Tingting; Bai, Bozan; Yin, Caixia; Xu, Wenzhi; Li, Wei

2018-05-01

Two mitochondria-targeted colorimetric and ratiometric fluorescent probes for SO2 derivatives were constructed based on the SO2 derivatives-triggered Michael addition reaction. The probes exhibit high specificity toward HSO3-/SO32- by interrupting their conjugation system resulting in a large ratiometric blue shift of 46-121 nm in their emission spectrum. The two well-resolved emission bands can ensure accurate detection of HSO3-. The detection limits were calculated to be 1.09 and 1.35 μM. Importantly, probe 1 and probe 2 were successfully used to fluorescence ratiometric imaging of endogenous HSO3- in BT-474 cells.

Fluorescence lifetime microscopy with a time- and space-resolved single-photon counting detector

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Pinaud, F. F.; Millaud, J.E.; Weiss, S.

2017-01-01

We have recently developed a wide-field photon-counting detector (the H33D detector) having high-temporal and high-spatial resolutions and capable of recording up to 500,000 photons per sec. Its temporal performance has been previously characterized using solutions of fluorescent materials with different lifetimes, and its spatial resolution using sub-diffraction objects (beads and quantum dots). Here we show its application to fluorescence lifetime imaging of live cells and compare its performance to a scanning confocal TCSPC approach. With the expected improvements in photocathode sensitivity and increase in detector throughput, this technology appears as a promising alternative to the current lifetime imaging solutions. PMID:29449756

Two-photon excitation of nitric oxide fluorescence as a temperature indicator in unsteady gas-dynamic processes

NASA Technical Reports Server (NTRS)

Mckenzie, R. L.; Gross, K. P.

1980-01-01

A laser induced fluorescence technique, suitable for measuring fluctuating temperatures in cold turbulent flows containing very low concentrations of nitric oxide is described. Temperatures below 300 K may be resolved with signal to noise ratios greater than 50 to 1 using high peak power, tunable dye lasers. The method relies on the two photon excitation of selected ro-vibronic transitions. The analysis includes the effects of fluorescence quenching and shows the technique to be effective at all densities below ambient. Signal to noise ratio estimates are based on a preliminary measurement of the two photon absorptivity for a selected rotational transition in the NO gamma (0,0) band.

Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells.

PubMed

Grecco, H E; Lidke, K A; Heintzmann, R; Lidke, D S; Spagnuolo, C; Martinez, O E; Jares-Erijman, E A; Jovin, T M

2004-11-01

In this work, we characterized streptavidin-conjugated quantum dots (QDs) manufactured by Quantum Dot Corporation. We present data on: (1) two-photon excitation; (2) fluorescence lifetimes; (3) ensemble and single QD emission anisotropy; (4) QDs as donors for Forster resonance energy transfer (FRET); and (5) spectral conversion of QDs exposed to high-intensity illumination. We also demonstrate the utility of QDs for (1) imaging the binding and uptake of biotinylated transferrin on living cells, and (2) resolving by fluorescence lifetime imaging microscopy (FLIM) signals originating from QDs from those of spatially and spectrally overlapping visible fluorescent proteins (VFPs). (c) 2005 Wiley-Liss, Inc.

A Single-Photon Avalanche Diode Array for Fluorescence Lifetime Imaging Microscopy

PubMed Central

Schwartz, David Eric; Charbon, Edoardo; Shepard, Kenneth L.

2013-01-01

We describe the design, characterization, and demonstration of a fully integrated single-photon avalanche diode (SPAD) imager for use in time-resolved fluorescence imaging. The imager consists of a 64-by-64 array of active SPAD pixels and an on-chip time-to-digital converter (TDC) based on a delay-locked loop (DLL) and calibrated interpolators. The imager can perform both standard time-correlated single-photon counting (TCSPC) and an alternative gated-window detection useful for avoiding pulse pile-up when measuring bright signal levels. To illustrate the use of the imager, we present measurements of the decay lifetimes of fluorescent dyes of several types with a timing resolution of 350 ps. PMID:23976789

Green Synthesis of Red-Emitting Carbon Nanodots as a Novel "Turn-on" Nanothermometer in Living Cells.

PubMed

Wang, Chuanxi; Jiang, Kaili; Wu, Qian; Wu, Jiapeng; Zhang, Chi

2016-10-04

Temperature measurements in biology and medical diagnostics, along with sensitive temperature probing of living cells, is of great importance; however, it still faces significant challenges. Herein, a novel "turn-on" carbon-dot-based fluorescent nanothermometry device for spatially resolved temperature measurements in living cells is presented. The carbon nanodots (CNDs) are prepared by a green microwave-assisted method and exhibit red fluorescence (λem =615 nm) with high quantum yields (15 %). Then, an on-off fluorescent probe is prepared for detecting glutathione (GSH) based on aggregation-induced fluorescence quenching. Interestingly, the quenched fluorescence could be recovered by increasing temperature and the CNDs-GSH mixture could behave as an off-on fluorescent probe for temperature. Thus, red-emitting CNDs can be utilized for "turn-on" fluorescent nanothermometry through the fluorescence quenching and recovery processes, respectively. We employ MC3T3-E1 cells as an example model to demonstrate the red-emitting CNDs can function as "non-contact" tools for the accurate measurement of temperature and its gradient inside a living cell. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescence probe techniques to monitor protein adsorption-induced conformation changes on biodegradable polymers.

PubMed

Benesch, Johan; Hungerford, Graham; Suhling, Klaus; Tregidgo, Carolyn; Mano, João F; Reis, Rui L

2007-08-15

The study of protein adsorption and any associated conformational changes on interaction with biomaterials is of great importance in the area of implants and tissue constructs. This study aimed to evaluate some fluorescent techniques to probe protein conformation on a selection of biodegradable polymers currently under investigation for biomedical applications. Because of the fluorescence emanating from the polymers, the use of monitoring intrinsic protein fluorescence was precluded. A highly solvatochromic fluorescent dye, Nile red, and a well-known protein label, fluorescein isothiocyanate, were employed to study the adsorption of serum albumin to polycaprolactone and to some extent also to two starch-containing polymer blends (SPCL and SEVA-C). A variety of fluorescence techniques, steady state, time resolved, and imaging were employed. Nile red was found to leach from the protein, while fluorescein isothiocyanate proved useful in elucidating a conformational change in the protein and the observation of protein aggregates adsorbed to the polymer surface. These effects were seen by making use of the phenomenon of energy migration between the fluorescent tags to monitor interprobe distance and the use of fluorescence lifetime imaging to ascertain the surface packing of the protein on polymer.

Multimodal quantitative phase and fluorescence imaging of cell apoptosis

NASA Astrophysics Data System (ADS)

Fu, Xinye; Zuo, Chao; Yan, Hao

2017-06-01

Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

Gas response behaviour and photochemistry of borondiketonate in acrylic polymer matrices for sensing applications.

PubMed

Arias Espinoza, Juan Diego; Sazhnikov, Viacheslav; Smits, Edsger C P; Ionov, Dmirity; Kononevich, Yuriy; Yakimets, Iryna; Alfimov, Mikael; Schoo, Herman F M

2014-11-01

The fluorescent spectra in combination with gas response behavior of acrylic polymers doped with dibenzoyl(methanato)boron difluoride (DBMBF2) were studied by fluorescence spectroscopy and time-resolved fluorescence lifetime. The role of acrylic matrix polarity upon the fluorescence spectra and fluorescence lifetime was analyzed. Changes in emission of the dye doped polymers under exposure to toluene, n-hexane and ethanol were monitored. The fluorescence lifetimes were measured for the singlet excited state as well as the exciplex formed between DBMBF2 and toluene. A reduction of the transition energy to the first singlet-excited state in the four polymers was observed, compared to solution. Reversible exciplex formation, viz. a red shifted fluorescence emission was perceived when exposing the polymers to toluene, while for hexane and ethanol only reversible reduction of the fluorescence occurred. Longer singlet and shorter exciplex lifetimes were observed for non-polar matrixes. The latter mechanism is explained in function of the lower charge transfer character of the exciplex in non-polar matrixes. Additionally, the quantum yield of the dye in the polymer matrix increased almost seventh-fold compared to values for solution.

Singlet and triplet energy transfer in a benzil-doped, light emitting, solid-state conjugated polymer

NASA Astrophysics Data System (ADS)

Rothe, C.; Pålsson, L. O.; Monkman, A. P.

2002-12-01

The luminescence emitted from pure and benzil-doped thin films of the conjugated polymer polyfluorene [PF2/6] are compared. The prompt fluorescence from the first singlet-excited state of the polymer is quenched by 90% in the presence of 10% per weight benzil. In addition to the prompt fluorescence, time-resolved spectroscopy at low temperature also allows the detection of phosphorescence and delayed fluorescence from the host polymer. Again the delayed fluorescence is strongly quenched but the phosphorescence is enhanced in doped samples. An explanation of the results is given in terms of singlet energy transfer from the host to benzil and triplet energy transfer from the dopant back to PF2/6. We have applied this to enable better understanding of the photophysics in PF2/6 doped with a platinum porphyrin complex.

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy.

PubMed

Sun, Yang; Park, Jesung; Stephens, Douglas N; Jo, Javier A; Sun, Lei; Cannata, Jonathan M; Saroufeem, Ramez M G; Shung, K Kirk; Marcu, Laura

2009-06-01

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 microm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque.

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy

PubMed Central

Sun, Yang; Park, Jesung; Stephens, Douglas N.; Jo, Javier A.; Sun, Lei; Cannata, Jonathan M.; Saroufeem, Ramez M. G.; Shung, K. Kirk; Marcu, Laura

2009-01-01

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 μm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque. PMID:19566223

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy

NASA Astrophysics Data System (ADS)

Sun, Yang; Park, Jesung; Stephens, Douglas N.; Jo, Javier A.; Sun, Lei; Cannata, Jonathan M.; Saroufeem, Ramez M. G.; Shung, K. Kirk; Marcu, Laura

2009-06-01

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 μm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque.

Kinetics model for the wavelength-dependence of excited-state dynamics of hetero-FRET sensors

NASA Astrophysics Data System (ADS)

Schwarz, Jacob; Leighton, Ryan; Leopold, Hannah J.; Currie, Megan; Boersma, Arnold J.; Sheets, Erin D.; Heikal, Ahmed A.

2017-08-01

Foerster (or fluorescence) resonance energy transfer (FRET) is a powerful tool for investigating protein-protein interactions, in both living cells and in controlled environments. A typical hetero-FRET pair consists of a donor and acceptor tethered together with a linker. The corresponding energy transfer efficiency of a hetero-FRET pair probe depends upon the donor-acceptor distance, relative dipole orientation, and spectral overlap. Because of the sensitivity of the energy transfer efficiency on the donor-acceptor distance, FRET is often referred to as a "molecular ruler". Time-resolved fluorescence approach for measuring the excited-state lifetime of the donor and acceptor emissions is one of the most reliable approaches for quantitative assessment of the energy transfer efficiency in hetero-FRET pairs. In this contribution, we provide an analytical kinetics model that describes the excited-state depopulation of a FRET probe as a means to predicts the time-resolved fluorescence profile as a function of excitation and detection wavelengths. In addition, we used this developed kinetics model to simulate the time-dependence of the excited-state population of both the donor and acceptor. These results should serve as a guide for our ongoing studies of newly developed hetero-FRET sensors (mCerulean3-linker-mCitrine) that are designed specifically for in vivo studies of macromolecular crowding. The same model is applicable to other FRET pairs with the careful consideration of their steady-state spectroscopy and the experimental design for wavelength- dependence of the fluorescence lifetime measurements.

ESIPT and photodissociation of 3-hydroxychromone in solution: photoinduced processes studied by static and time-resolved UV/Vis, fluorescence, and IR spectroscopy.

PubMed

Chevalier, Katharina; Grün, Anneken; Stamm, Anke; Schmitt, Yvonne; Gerhards, Markus; Diller, Rolf

2013-11-07

The spectral properties of fluorescence sensors such as 3-hydroxychromone (3-HC) and its derivatives are sensitive to interaction with the surrounding medium as well as to substitution. 3-HC is a prototype system for other derivatives because it is the basic unit of all flavonoides undergoing ESIPT and is not perturbed by a substituent. In this study, the elementary processes and intermediate states in the photocycle of 3-HC as well as its anion were identified and characterized by the use of static and femtosecond time-resolved spectroscopy in different solvents (methylcyclohexane, acetonitrile, ethanol, and water at different pH). Electronic absorption and fluorescence spectra and lifetimes of the intermediate states were obtained for the normal, tautomer and anionic excited state, while mid-IR vibrational spectra yielded structural information on ground and excited states of 3-HC. A high sensitivity on hydrogen-bonding perturbations was observed, leading to photoinduced anion formation in water, while in organic solvents, different processes are suggested, including slow picosecond ESIPT and contribution of the trans-structure excited state or a different stable solvation state with different direction of OH. The formation of the latter could be favored by the lack of a substituent increasing contact points for specific solute-solvent interactions at the hydroxyl group compared to substituted derivatives. The effect of substituents has to be considered for the design of future fluorescence sensors based on 3-HC.

Alterations of pigment composition and their interactions in response to different light conditions in the diatom Chaetoceros gracilis probed by time-resolved fluorescence spectroscopy.

PubMed

Nagao, Ryo; Ueno, Yoshifumi; Yokono, Makio; Shen, Jian-Ren; Akimoto, Seiji

2018-07-01

Maintenance of energy balance under changeable light conditions is an essential function of photosynthetic organisms to achieve efficient photochemical reactions. Among the photosynthetic organisms, diatoms possess light-harvesting fucoxanthin chlorophyll (Chl) a/c-binding protein (FCP) as peripheral antennas. However, how diatoms regulate excitation-energy distribution between FCP and the two photosystem cores during light adaptation is poorly understood. In this study, we examined spectroscopic properties of a marine diatom Chaetoceros gracilis adapted in the dark and at photosynthetic photon flux density at 30 and 300 μmol photons m -2  s -1 . Absorption spectra at 77 K showed significant changes in the Soret region, and 77-K steady-state fluorescence spectra showed significant differences in the spectral shape and relative fluorescence intensity originating from both PSII and PSI, among the cells grown under different light conditions. These results suggest alterations of pigment composition and their interactions under the different light conditions. These alterations affected the excitation-energy dynamics monitored by picosecond time-resolved fluorescence analyses at 77 K significantly. The contributions of Chls having lower energy levels than the reaction center Chls in the two photosystems to the energy dynamics were clearly identified in the three cells but with presumably different roles. These findings provide insights into the regulatory mechanism of excitation-energy balance in diatoms under various light conditions. Copyright © 2018 Elsevier B.V. All rights reserved.

Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy

PubMed Central

Butte, Pramod V.; Fang, Qiyin; Jo, Javier A.; Yong, William H.; Pikul, Brian K.; Black, Keith L.; Marcu, Laura

2010-01-01

The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337nm, 700ps), and the intensity decay profiles were recorded in the 360-to550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390nm(lifetime=1.8±0.3ns) and 460nm(lifetime=0.8±0.1ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1ns) and reduced in high-grade glioma (N=9; lifetime=1.7±0.4ns). The emission characteristics at 460nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440to460nm; lifetime: 0.8to1.0ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens. PMID:20459282

Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy.

PubMed

Butte, Pramod V; Fang, Qiyin; Jo, Javier A; Yong, William H; Pikul, Brian K; Black, Keith L; Marcu, Laura

2010-01-01

The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8+/-0.3 ns) and 460 nm (lifetime=0.8+/-0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7+/-0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm; lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens.

Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Butte, Pramod V.; Fang, Qiyin; Jo, Javier A.; Yong, William H.; Pikul, Brian K.; Black, Keith L.; Marcu, Laura

2010-03-01

The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8+/-0.3 ns) and 460 nm (lifetime=0.8+/-0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7+/-0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens.

Interaction of Merocyanine 540 with serum albumins: photophysical and binding studies.

PubMed

Banerjee, Mousumi; Pal, Uttam; Subudhhi, Arijita; Chakrabarti, Abhijit; Basu, Samita

2012-03-01

Photophysical studies on binding interactions of a negatively charged anti-tumor photosensitizer, Merocyanine 540 (MC 540), with serum proteins, bovine serum albumin (BSA) and human serum albumin (HSA), have been performed using absorption and steady-state as well as time-resolved fluorescence techniques. Formation of ground state complex has been confirmed from the detailed studies of absorption spectra of MC 540 in presence of SAs producing isosbestic points. Binding between the proteins and MC 540, which perturbs the existing equilibrium between the fluorescent monomer and its non-fluorescent dimer, induces a remarkable enhancement in fluorescence anisotropy and intensity of MC 540 along with a red shift of its maximum. The binding stoichiometry of MC 540 and SAs are more than 1.0 which depicts that two types of complexes, i.e., 1:1 and 2:1 are formed with addition of varied concentration of protein. Both the steady-state and time-resolved fluorescence results show that in 2:1 complex one of the MC 540 molecules is exposed towards aqueous environment with a greater extent when bound with HSA compared to BSA due to the structural flexibility of that protein. Thermodynamic analyses using van't Hoff plot indicate that the binding between MC 540 and individual SA is an entropy-driven phenomenon. The probable hydrophobic binding site has been located by denaturation of proteins, micropolarity measurement and Förster resonance energy transfer and that is further supported by molecular docking studies. Changes in circular dichroism spectra of BSA in presence of MC 540 depict secondary structural changes of the protein. The induced-CD shows that BSA due to its rigid structure generates chirality in MC 540 much more efficiently compared to HSA. Copyright © 2011 Elsevier B.V. All rights reserved.

Combinatorial mutagenesis of the voltage-sensing domain enables the optical resolution of action potentials firing at 60 Hz by a genetically encoded fluorescent sensor of membrane potential.

PubMed

Piao, Hong Hua; Rajakumar, Dhanarajan; Kang, Bok Eum; Kim, Eun Ha; Baker, Bradley J

2015-01-07

ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase from Ciona intestinalis that gives a large but slow-responding optical signal in response to changes in membrane potential (Jin et al., 2012). Fluorescent voltage sensors using the voltage-sensing domain from other species give faster yet weaker optical signals (Baker et al., 2012; Han et al., 2013). Sequence alignment of voltage-sensing phosphatases from different species revealed conserved polar and charged residues at 7 aa intervals in the S1-S3 transmembrane segments of the voltage-sensing domain, suggesting potential coil-coil interactions. The contribution of these residues to the voltage-induced optical signal was tested using a cassette mutagenesis screen by flanking each transmembrane segment with unique restriction sites to allow for the testing of individual mutations in each transmembrane segment, as well as combinations in all four transmembrane segments. Addition of a counter charge in S2 improved the kinetics of the optical response. A double mutation in the S4 domain dramatically reduced the slow component of the optical signal seen in ArcLight. Combining that double S4 mutant with the mutation in the S2 domain yielded a probe with kinetics <10 ms. Optimization of the linker sequence between S4 and the fluorescent protein resulted in a new ArcLight-derived probe, Bongwoori, capable of resolving action potentials in a hippocampal neuron firing at 60 Hz. Additional manipulation of the voltage-sensing domain could potentially lead to fluorescent sensors capable of optically resolving neuronal inhibition and subthreshold synaptic activity. Copyright © 2015 the authors 0270-6474/15/350372-15$15.00/0.

Detection of colorectal cancer using time-resolved autofluorescence spectrometer

NASA Astrophysics Data System (ADS)

Fu, Sheng; Kwek, Leong-Chuan; Chia, Teck-Chee; Lim, Chu-Sing; Tang, Choong-Leong; Ang, Wuan-Suan; Zhou, Miao-Chang; Loke, Po-Ling

2006-04-01

As we know Quantum mechanics is a mathematical theory that can describe the behavior of objects that are at microscopic level. Time-resolved autofluorescence spectrometer monitors events that occur during the lifetime of the excited state. This time ranges from a few picoseconds to hundreds of nanoseconds. That is an extremely important advance as it allows environmental parameters to be monitored in a spatially defined manner in the specimen under study. This technique is based on the application of Quantum Mechanics. This principle is applied in our project as we are trying to use different fluorescence spectra to detect biological molecules commonly found in cancerous colorectal tissue and thereby differentiate the cancerous and non-cancerous colorectal polyps more accurately and specifically. In this paper, we use Fluorescence Lifetime Spectrometer (Edinburgh Instruments FL920) to measure decay time of autofluorescence of colorectal cancerous and normal tissue sample. All specimens are from Department of Colorectal Surgery, Singapore General Hospital. The tissues are placed in the time-resolved autofluorescence instrument, which records and calculates the decay time of the autofluorescence in the tissue sample at the excitation and emission wavelengths pre-determined from a conventional spectrometer. By studying the decay time,τ, etc. for cancerous and normal tissue, we aim to present time-resolved autofluorescence as a feasible technique for earlier detection of malignant colorectal tissues. By using this concept, we try to contribute an algorithm even an application tool for real time early diagnosis of colorectal cancer for clinical services.

Hyperfine-resolved transition frequency list of fundamental vibration bands of H35Cl and H37Cl

NASA Astrophysics Data System (ADS)

Iwakuni, Kana; Sera, Hideyuki; Abe, Masashi; Sasada, Hiroyuki

2014-12-01

Sub-Doppler resolution spectroscopy of the fundamental vibration bands of H35Cl and H37Cl has been carried out from 87.1 to 89.9 THz. We have determined the absolute transition frequencies of the hyperfine-resolved R(0) to R(4) transitions with a typical uncertainty of 10 kHz. We have also yielded six molecular constants for each isotopomer in the vibrational excited state, which reproduce the determined frequencies with a standard deviation of about 10 kHz.

Polarization-insensitive ultralow-power second-harmonic generation frequency-resolved optical gating.

PubMed

Miao, Houxun; Weiner, Andrew M; Langrock, Carsten; Roussev, Rostislav V; Fejer, Martin M

2007-04-01

We demonstrate polarization-insensitive ultralow-power second-harmonic generation frequency-resolved optical gating (FROG) measurements with a fiber-pigtailed, aperiodically poled lithium niobate waveguide. By scrambling the polarization much faster than the measurement integration time, we eliminate the impairment that frequency-independent random polarization fluctuations induce in FROG measurements. As a result we are able to retrieve intensity and phase profiles of few hundred femtosecond optical pulses with 50 MHz repetition rates at 5.2 nW coupled average power without control of the input polarization.

Near infrared spatial frequency domain fluorescence imaging of tumor phantoms containing erythrocyte-derived optical nanoplatforms

NASA Astrophysics Data System (ADS)

Burns, Joshua M.; Schaefer, Elise; Anvari, Bahman

2018-02-01

Light-activated theranostic constructs provide a multi-functional platform for optical imaging and phototherapeutic applications. Our group has engineered nano-sized vesicles derived from erythrocytes that encapsulate the FDAapproved near infrared (NIR) absorber indocyanine green (ICG). We refer to these constructs as NIR erythrocytemimicking transducers (NETs). Once photo-excited by NIR light these constructs can transduce the photons energy to emit fluorescence, generate heat, or induce chemical reactions. In this study, we investigated fluorescence imaging of NETs embedded within tumor phantoms using spatial frequency domain imaging (SFDI). Using SFDI, we were able to fluorescently image simulated tumors doped with different concentration of NETs. These preliminary results suggest that NETs can be used in conjunction with SFDI for potential tumor imaging applications.

Picosecond time-resolved fluorescence spectroscopy of K-590 in the bacteriorhodopsin photocycle.

PubMed Central

Atkinson, G H; Blanchard, D; Lemaire, H; Brack, T L; Hayashi, H

1989-01-01

The fluorescence spectrum of a distinct isometric and conformational intermediate formed on the 10(-11) s time scale during the bacteriorhodopsin (BR) photocycle is observed at room temperature using a two laser, pump-probe technique with picosecond time resolution. The BR photocycle is initiated by pulsed (8 ps) excitation at 565 nm, whereas the fluorescence is generated by 4-ps laser pulses at 590 nm. The unstructured fluorescence extends from 650 to 880 nm and appears in the same general spectral region as the fluorescence spectrum assigned to BR-570. The transient fluorescence spectrum can be distinguished from that assigned to BR-570 by a larger emission quantum yield (approximately twice that of BR-570) and by a maximum intensity near 731 nm (shifted 17 nm to higher energy from the maximum of the BR-570 fluorescence spectrum). The fluorescence spectrum of BR-570 only is measured with low energy, picosecond pulsed excitation at 590 nm and is in good agreement with recent data in the literature. The assignment of the transient fluorescence spectrum to the K-590 intermediate is based on its appearance at time delays longer than 40 ps. The K-590 fluorescence spectrum remains unchanged over the entire 40-100-ps interval. The relevance of these fluorescence data with respect to the molecular mechanism used to model the primary processes in the BR photocycle also is discussed. PMID:2713439

Chlorophyll fluorescence emission as a reporter on cold tolerance in Arabidopsis thaliana accessions

PubMed Central

Mishra, Anamika; Höermiller, Imke I; Heyer, Arnd G; Nedbal, Ladislav

2011-01-01

Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around −15°C. This, together with the method's easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method. PMID:21427532

The complex nature of calcium cation interactions with phospholipid bilayers

PubMed Central

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-01-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association. PMID:27905555

Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices

PubMed Central

Sriram, K. K.; Yeh, Jia-Wei; Lin, Yii-Lih; Chang, Yi-Ren; Chou, Chia-Fu

2014-01-01

Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein–DNA interaction sites. PMID:24753422

Raman scattering measurements in flames using a tunable KrF excimer laser

NASA Technical Reports Server (NTRS)

Wehrmeyer, Joseph A.; Cheng, Tsarng-Sheng; Pitz, Robert W.

1992-01-01

A narrow-band tunable KrF excimer laser is used as a spontaneous vibrational Raman scattering source to demonstrate that single-pulse concentration and temperature measurements, with only minimal fluorescence interference, are possible for all major species (O2, N2, H2O, and H2) at all stoichiometries (fuel-lean to fuel rich) of H2-air flames. Photon-statistics-limited precisions in these instantaneous and spatially resolved single-pulse measurements are typically 5 percent, which are based on the relative standard deviations of single-pulse probability distributions. In addition to the single-pulse N2 Stokes/anti-Stokes ratio temperature measurement technique, a time-averaged temperature measurement technique is presented that matches the N2 Stokes Raman spectrum to theoretical spectra by using a single intermediate state frequency to account for near-resonance enhancement. Raman flame spectra in CH4-air flames are presented that have good signal-to-noise characteristics and show promise for single-pulse UV Raman measurements in hydrocarbon flames.

Laser characterization of electric field oscillations in the Hall thruster breathing mode

NASA Astrophysics Data System (ADS)

Young, Christopher; Lucca Fabris, Andrea; MacDonald-Tenenbaum, Natalia; Hargus, William, Jr.; Cappelli, Mark

2016-10-01

Hall thrusters are a mature technology for space propulsion applications that exhibit a wide array of dynamic behavior, including plasma waves, instabilities and turbulence. One common low frequency (10-50 kHz) discharge current oscillation is the breathing mode, a cycle of neutral propellant injection, strong ionization, and ion acceleration by a steep potential gradient. A time-resolved laser-induced fluorescence diagnostic non-intrusively captures this propagating ionization front in the channel of a commercial BHT-600 Hall thruster manufactured by Busek Co. Measurements of ion velocity and relative ion density (using the 5 d[ 4 ] 7 / 2 - 6 p[ 3 ] 5 / 2 Xe II transition at 834.95 nm, vacuum) reveal a dynamic electric field structure traversing the channel throughout the breathing mode cycle. This work is sponsored by the U.S. Air Force Office of Scientific Research, with Dr. M. Birkan as program manager. C.Y. acknowledges support from the DOE NSSA Stewardship Science Graduate Fellowship under contract DE-FC52-08NA28752.

The complex nature of calcium cation interactions with phospholipid bilayers

NASA Astrophysics Data System (ADS)

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-12-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Observations of a field-aligned ion/ion-beam instability in a magnetized laboratory plasma

NASA Astrophysics Data System (ADS)

Heuer, P. V.; Weidl, M. S.; Dorst, R. S.; Schaeffer, D. B.; Bondarenko, A. S.; Tripathi, S. K. P.; Van Compernolle, B.; Vincena, S.; Constantin, C. G.; Niemann, C.; Winske, D.

2018-03-01

Collisionless coupling between super Alfvénic ions and an ambient plasma parallel to a background magnetic field is mediated by a set of electromagnetic ion/ion-beam instabilities including the resonant right hand instability (RHI). To study this coupling and its role in parallel shock formation, a new experimental configuration at the University of California, Los Angeles utilizes high-energy and high-repetition-rate lasers to create a super-Alfvénic field-aligned debris plasma within an ambient plasma in the Large Plasma Device. We used a time-resolved fluorescence monochromator and an array of Langmuir probes to characterize the laser plasma velocity distribution and density. The debris ions were observed to be sufficiently super-Alfvénic and dense to excite the RHI. Measurements with magnetic flux probes exhibited a right-hand circularly polarized frequency chirp consistent with the excitation of the RHI near the laser target. We compared measurements to 2D hybrid simulations of the experiment.

Observations of a field-aligned ion/ion-beam instability in a magnetized laboratory plasma

DOE PAGES

Heuer, P. V.; Weidl, M. S.; Dorst, R. S.; ...

2018-03-01

Collisionless coupling between super Alfvénic ions and an ambient plasma parallel to a background magnetic field is mediated by a set of electromagnetic ion/ion-beam instabilities including the resonant right hand instability (RHI). To study this coupling and its role in parallel shock formation, a new experimental configuration at the University of California, Los Angeles utilizes high-energy and high-repetition-rate lasers to create a super-Alfvénic field-aligned debris plasma within an ambient plasma in the Large Plasma Device. We used a time-resolved fluorescence monochromator and an array of Langmuir probes to characterize the laser plasma velocity distribution and density. The debris ions weremore » observed to be sufficiently super-Alfvénic and dense to excite the RHI. Measurements with magnetic flux probes exhibited a right-hand circularly polarized frequency chirp consistent with the excitation of the RHI near the laser target. To conclude, we compared measurements to 2D hybrid simulations of the experiment.« less