Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

PubMed

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

PubMed

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer.

PubMed

Karasawa, Satoshi; Araki, Toshio; Nagai, Takeharu; Mizuno, Hideaki; Miyawaki, Atsushi

2004-07-01

GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) technology has facilitated the exploration of the spatio-temporal patterns of cellular signalling. While most studies have used cyan- and yellow-emitting FPs (fluorescent proteins) as FRET donors and acceptors respectively, this pair of proteins suffers from problems of pH-sensitivity and bleeding between channels. In the present paper, we demonstrate the use of an alternative additional donor/acceptor pair. We have cloned two genes encoding FPs from stony corals. We isolated a cyan-emitting FP from Acropara sp., whose tentacles exhibit cyan coloration. Similar to GFP from Renilla reniformis, the cyan FP forms a tight dimeric complex. We also discovered an orange-emitting FP from Fungia concinna. As the orange FP exists in a complex oligomeric structure, we converted this protein into a monomeric form through the introduction of three amino acid substitutions, recently reported to be effective for converting DsRed into a monomer (Clontech). We used the cyan FP and monomeric orange FP as a donor/acceptor pair to monitor the activity of caspase 3 during apoptosis. Due to the close spectral overlap of the donor emission and acceptor absorption (a large Förster distance), substantial pH-resistance of the donor fluorescence quantum yield and the acceptor absorbance, as well as good separation of the donor and acceptor signals, the new pair can be used for more effective quantitative FRET imaging.

Emission wavelength tuning of fluorescence by fine structural control of optical metamaterials with Fano resonance

PubMed Central

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

2016-01-01

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

Spectral dependence of fluorescence near plasmon resonant metal nanoparticles

NASA Astrophysics Data System (ADS)

Chen, Yeechi

The optical properties of fluorophores are significantly modified when placed within the near field (0--100 nm) of plasmon resonant metal nanostructures, due to the competition between increased decay rates and "hotspots" of concentrated electric fields. The decay rates and effective electric field intensities are highly dependent on the relative position of dye and metal and the overlap between plasmon resonance and dye absorption and emission. Understanding these dependencies can greatly improve the performance of biosensing and nanophotonic devices. In this dissertation, the fluorescence intensity of organic dyes and CdSe quantum dots near single metal nanoparticles is studied as a function of the local surface plasmon resonance (LSPR) of the nanoparticle. Single metal nanoparticles have narrow, well-defined, intense local surface plasmon resonances that are tunable across the visible spectrum by changes in size and shape. First, we show that organic dyes can be self-assembled on single silver nanoprisms into known configurations by the hybridization of thiolated DNA oligomers. We correlate the fluorescence intensity of the dyes to the LSPR of the individual nanoprism to which they are attached. For each of three different organic dyes, we observe a strong correlation between the fluorescence intensity of the dye and the degree of spectral overlap with the plasmon resonance of the nanoparticle. On average, we observe the brightest fluorescence from dyes attached to metal nanoparticles that have a LSPR scattering peak 40--120 meV higher in energy than the emission peak of the fluorophore. Second, the plasmon-enhanced fluorescence from CdSe/CdS/CdZnS/ZnS core/shell quantum dots is studied near a variety of silver and gold nanoparticles. With single-particle scattering spectroscopy, the localized surface plasmon resonance spectra of single metal nanoparticles is correlated with the photoluminescence excitation (PLE) spectra of the nearby quantum dots. The PLE

Determining absolute protein numbers by quantitative fluorescence microscopy.

PubMed

Verdaasdonk, Jolien Suzanne; Lawrimore, Josh; Bloom, Kerry

2014-01-01

Biological questions are increasingly being addressed using a wide range of quantitative analytical tools to examine protein complex composition. Knowledge of the absolute number of proteins present provides insights into organization, function, and maintenance and is used in mathematical modeling of complex cellular dynamics. In this chapter, we outline and describe three microscopy-based methods for determining absolute protein numbers--fluorescence correlation spectroscopy, stepwise photobleaching, and ratiometric comparison of fluorescence intensity to known standards. In addition, we discuss the various fluorescently labeled proteins that have been used as standards for both stepwise photobleaching and ratiometric comparison analysis. A detailed procedure for determining absolute protein number by ratiometric comparison is outlined in the second half of this chapter. Counting proteins by quantitative microscopy is a relatively simple yet very powerful analytical tool that will increase our understanding of protein complex composition. © 2014 Elsevier Inc. All rights reserved.

Resonance fluorescence revival in a voltage-controlled semiconductor quantum dot

NASA Astrophysics Data System (ADS)

Reigue, Antoine; Lemaître, Aristide; Gomez Carbonell, Carmen; Ulysse, Christian; Merghem, Kamel; Guilet, Stéphane; Hostein, Richard; Voliotis, Valia

2018-02-01

We demonstrate systematic resonance fluorescence recovery with near-unity emission efficiency in single quantum dots embedded in a charge-tunable device in a wave-guiding geometry. The quantum dot charge state is controlled by a gate voltage, through carrier tunneling from a close-lying Fermi sea, stabilizing the resonantly photocreated electron-hole pair. The electric field cancels out the charging/discharging mechanisms from nearby traps toward the quantum dots, responsible for the usually observed inhibition of the resonant fluorescence. Fourier transform spectroscopy as a function of the applied voltage shows a strong increase in the coherence time though not reaching the radiative limit. These charge controlled quantum dots can act as quasi-perfect deterministic single-photon emitters, with one laser pulse converted into one emitted single photon.

Quantitative fluorescence angiography for neurosurgical interventions.

PubMed

Weichelt, Claudia; Duscha, Philipp; Steinmeier, Ralf; Meyer, Tobias; Kuß, Julia; Cimalla, Peter; Kirsch, Matthias; Sobottka, Stephan B; Koch, Edmund; Schackert, Gabriele; Morgenstern, Ute

2013-06-01

Present methods for quantitative measurement of cerebral perfusion during neurosurgical operations require additional technology for measurement, data acquisition, and processing. This study used conventional fluorescence video angiography--as an established method to visualize blood flow in brain vessels--enhanced by a quantifying perfusion software tool. For these purposes, the fluorescence dye indocyanine green is given intravenously, and after activation by a near-infrared light source the fluorescence signal is recorded. Video data are analyzed by software algorithms to allow quantification of the blood flow. Additionally, perfusion is measured intraoperatively by a reference system. Furthermore, comparing reference measurements using a flow phantom were performed to verify the quantitative blood flow results of the software and to validate the software algorithm. Analysis of intraoperative video data provides characteristic biological parameters. These parameters were implemented in the special flow phantom for experimental validation of the developed software algorithms. Furthermore, various factors that influence the determination of perfusion parameters were analyzed by means of mathematical simulation. Comparing patient measurement, phantom experiment, and computer simulation under certain conditions (variable frame rate, vessel diameter, etc.), the results of the software algorithms are within the range of parameter accuracy of the reference methods. Therefore, the software algorithm for calculating cortical perfusion parameters from video data presents a helpful intraoperative tool without complex additional measurement technology.

Modified Facile Synthesis for Quantitatively Fluorescent Carbon Dots.

PubMed

Hou, Xiaofang; Hu, Yin; Wang, Ping; Yang, Liju; Al Awak, Mohamad M; Tang, Yongan; Twara, Fridah K; Qian, Haijun; Sun, Ya-Ping

2017-10-01

A simple yet consequential modification was made to the popular carbonization processing of citric acid - polyethylenimine precursor mixtures to produce carbon dots (CDots). The modification was primarily on pushing the carbonization processing a little harder at a higher temperature, such as the hydrothermal processing condition of around 330 °C for 6 hours. The CDots thus produced are comparable in spectroscopic and other properties to those obtained in other more controlled syntheses including the deliberate chemical functionalization of preprocessed and selected small carbon nanoparticles, demonstrating the consistency in CDots and reaffirming their general definition as carbon nanoparticles with surface passivation by organic or other species. Equally significant is the finding that the modified processing of citric acid - polyethylenimine precursor mixtures could yield CDots of record-setting fluorescence performance, approaching the upper limit of being quantitatively fluorescent. Thus, the reported work serves as a demonstration on not only the need in selecting the right processing conditions and its associated opportunities in one-pot syntheses of CDots, but also the feasibility in pursuing the preparation of quantitatively fluorescent CDots, which represents an important milestone in the development and understanding of these fluorescent carbon nanomaterials.

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

PubMed

Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

2008-09-01

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

Resonance fluorescence from an atom in a squeezed vacuum

NASA Astrophysics Data System (ADS)

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

1987-06-01

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

Resonance in quantum dot fluorescence in a photonic bandgap liquid crystal host.

PubMed

Lukishova, Svetlana G; Bissell, Luke J; Winkler, Justin; Stroud, C R

2012-04-01

Microcavity resonance is demonstrated in nanocrystal quantum dot fluorescence in a one-dimensional (1D) chiral photonic bandgap cholesteric-liquid crystal host under cw excitation. The resonance demonstrates coupling between quantum dot fluorescence and the cholesteric microcavity. Observed at a band edge of a photonic stop band, this resonance has circular polarization due to microcavity chirality with 4.9 times intensity enhancement in comparison with polarization of the opposite handedness. The circular-polarization dissymmetry factor g(e) of this resonance is ~1.3. We also demonstrate photon antibunching of a single quantum dot in a similar glassy cholesteric microcavity. These results are important in cholesteric-laser research, in which so far only dyes were used, as well as for room-temperature single-photon source applications.

Measuring iron in the brain using quantitative susceptibility mapping and X-ray fluorescence imaging

PubMed Central

Zheng, Weili; Nichol, Helen; Liu, Saifeng; Cheng, Yu-Chung N.; Haacke, E. Mark

2013-01-01

Measuring iron content in the brain has important implications for a number of neurodegenerative diseases. Quantitative susceptibility mapping (QSM), derived from magnetic resonance images, has been used to measure total iron content in vivo and in post mortem brain. In this paper, we show how magnetic susceptibility from QSM correlates with total iron content measured by X-ray fluorescence (XRF) imaging and by inductively coupled plasma mass spectrometry (ICPMS). The relationship between susceptibility and ferritin iron was estimated at 1.10 ± 0.08 ppb susceptibility per μg iron/g wet tissue, similar to that of iron in fixed (frozen/thawed) cadaveric brain and previously published data from unfixed brains. We conclude that magnetic susceptibility can provide a direct and reliable quantitative measurement of iron content and that it can be used clinically at least in regions with high iron content. PMID:23591072

Quantitative Laser-Saturated Fluorescence Measurements of Nitric Oxide in a Heptane Spray Flame

NASA Technical Reports Server (NTRS)

Cooper, Clayton S.; Laurendeau, Normand M.; Lee, Chi (Technical Monitor)

1997-01-01

We report spatially resolved laser-saturated fluorescence measurements of NO concentration in a pre-heated, lean-direct injection (LDI) spray flame at atmospheric pressure. The spray is produced by a hollow-cone, pressure-atomized nozzle supplied with liquid heptane. NO is excited via the Q2(26.5) transition of the gamma(0,0) band. Detection is performed in a 2-nm region centered on the gamma(0,1) band. Because of the relatively close spectral spacing between the excitation (226 nm) and detection wavelengths (236 nm), the gamma(0,1) band of NO cannot be isolated from the spectral wings of the Mie scattering signal produced by the spray. To account for the resulting superposition of the fluorescence and scattering signals, a background subtraction method has been developed that utilizes a nearby non-resonant wavelength. Excitation scans have been performed to locate the optimum off-line wavelength. Detection scans have been performed at problematic locations in the flame to determine possible fluorescence interferences from UHCs and PAHs at both the on-line and off-line excitation wavelengths. Quantitative radial NO profiles are presented and analyzed so as to better understand the operation of lean-direct injectors for gas turbine combustors.

Resonance fluorescence spectrum in a two-band photonic bandgap crystal

NASA Astrophysics Data System (ADS)

Lee, Ray-Kuang; Lai, Yinchieh

2003-05-01

Steady state resonance fluorescence spectra from a two-level atom embedded in a photonic bandgap crystal and resonantly driven by a classical pump light are calculated. The photonic crystal is considered to be with a small bandgap which is in the order of magnitude of the Rabi frequency and is modeled by the anisotropic two-band dispersion relation. Non-Markovian noises caused by the non-uniform distribution of photon density states near the photonic bandgap are taken into account by a new approach which linearizes the optical Bloch equations by using the Liouville operator expansion. Fluorescence spectra that only exhibit sidebands of the Mollow triplet are found, indicating that there is no coherent Rayleigh scattering process.

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

Real-time quantitative fluorescence imaging using a single snapshot optical properties technique for neurosurgical guidance

NASA Astrophysics Data System (ADS)

Valdes, Pablo A.; Angelo, Joseph; Gioux, Sylvain

2015-03-01

Fluorescence imaging has shown promise as an adjunct to improve the extent of resection in neurosurgery and oncologic surgery. Nevertheless, current fluorescence imaging techniques do not account for the heterogeneous attenuation effects of tissue optical properties. In this work, we present a novel imaging system that performs real time quantitative fluorescence imaging using Single Snapshot Optical Properties (SSOP) imaging. We developed the technique and performed initial phantom studies to validate the quantitative capabilities of the system for intraoperative feasibility. Overall, this work introduces a novel real-time quantitative fluorescence imaging method capable of being used intraoperatively for neurosurgical guidance.

Quantitative and qualitative 5-aminolevulinic acid–induced protoporphyrin IX fluorescence in skull base meningiomas

PubMed Central

Bekelis, Kimon; Valdés, Pablo A.; Erkmen, Kadir; Leblond, Frederic; Kim, Anthony; Wilson, Brian C.; Harris, Brent T.; Paulsen, Keith D.; Roberts, David W.

2011-01-01

Object Complete resection of skull base meningiomas provides patients with the best chance for a cure; however, surgery is frequently difficult given the proximity of lesions to vital structures, such as cranial nerves, major vessels, and venous sinuses. Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative assessment of protoporphyrin IX (PpIX) fluorescence following the exogenous administration of 5-aminolevulinic acid (ALA) has demonstrated utility in malignant glioma resection but limited use in meningiomas. Here the authors demonstrate the use of ALA-induced PpIX fluorescence guidance in resecting a skull base meningioma and elaborate on the advantages and disadvantages provided by both quantitative and qualitative fluorescence methodologies in skull base meningioma resection. Methods A 52-year-old patient with a sphenoid wing WHO Grade I meningioma underwent tumor resection as part of an institutional review board–approved prospective study of fluorescence-guided resection. A surgical microscope modified for fluorescence imaging was used for the qualitative assessment of visible fluorescence, and an intraoperative probe for in situ fluorescence detection was utilized for quantitative measurements of PpIX. The authors assessed the detection capabilities of both the qualitative and quantitative fluorescence approaches. Results The patient harboring a sphenoid wing meningioma with intraorbital extension underwent radical resection of the tumor with both visibly and nonvisibly fluorescent regions. The patient underwent a complete resection without any complications. Some areas of the tumor demonstrated visible fluorescence. The quantitative probe detected neoplastic tissue better than the qualitative modified surgical microscope. The intraoperative probe was particularly useful in areas that did not reveal visible fluorescence, and tissue from these areas was confirmed as tumor following histopathological

Quantitative magnetic resonance micro-imaging methods for pharmaceutical research.

PubMed

Mantle, M D

2011-09-30

The use of magnetic resonance imaging (MRI) as a tool in pharmaceutical research is now well established and the current literature covers a multitude of different pharmaceutically relevant research areas. This review focuses on the use of quantitative magnetic resonance micro-imaging techniques and how they have been exploited to extract information that is of direct relevance to the pharmaceutical industry. The article is divided into two main areas. The first half outlines the theoretical aspects of magnetic resonance and deals with basic magnetic resonance theory, the effects of nuclear spin-lattice (T(1)), spin-spin (T(2)) relaxation and molecular diffusion upon image quantitation, and discusses the applications of rapid magnetic resonance imaging techniques. In addition to the theory, the review aims to provide some practical guidelines for the pharmaceutical researcher with an interest in MRI as to which MRI pulse sequences/protocols should be used and when. The second half of the article reviews the recent advances and developments that have appeared in the literature concerning the use of quantitative micro-imaging methods to pharmaceutically relevant research. Copyright © 2010 Elsevier B.V. All rights reserved.

Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

PubMed

Wu, Yong; Wu, Xundong; Lu, Rong; Zhang, Jin; Toro, Ligia; Stefani, Enrico

2015-10-01

Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

Kα resonance fluorescence in Al, Ti, Cu and potential applications for X-ray sources

NASA Astrophysics Data System (ADS)

Nahar, Sultana N.; Pradhan, Anil K.

2015-04-01

The Kα resonance fluorescence (RFL) effect via photoabsorptions of inner shell electrons as the element goes through multiple ionization states is studied. We demonstrate that the resonances observed recently in Kα (1s-2p) fluorescence in aluminum plasmas by using a high-intensity X-ray free-electron laser [1] are basically K-shell resonances in hollow atoms going through multiple ionization states at resonant energies as predicted earlier for gold and iron ions [2]. These resonances are formed below the K-shell ionization edge and shift toward higher energies with ionization states, as observed. Fluorescence emission intensities depend on transition probabilities for each ionization stage of the given element for all possible Kα (1 s → 2 p) transition arrays. The present calculations for resonant photoabsorptions of Kα photons in Al have reproduced experimentally observed features. Resonant cross sections and absorption coefficients are presented for possible observation of Kα RFL in the resonant energy ranges of 4.5-5.0 keV for Ti ions and 8.0-8.7 keV for Cu ions respectively. We suggest that theoretically the Kα RFL process may be driven to enhance the Auger cycle by a twin-beam monochromatic X-ray source, tuned to the K-edge and Kα energies, with potential applications such as the development of narrow-band biomedical X-ray devices.

Calibration of Wide-Field Deconvolution Microscopy for Quantitative Fluorescence Imaging

PubMed Central

Lee, Ji-Sook; Wee, Tse-Luen (Erika); Brown, Claire M.

2014-01-01

Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure. PMID:24688321

Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

PubMed Central

Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

2015-01-01

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving

Quantitative fluorescence tomography using a trimodality system: in vivo validation

PubMed Central

Lin, Yuting; Barber, William C.; Iwanczyk, Jan S.; Roeck, Werner W.; Nalcioglu, Orhan; Gulsen, Gultekin

2010-01-01

A fully integrated trimodality fluorescence, diffuse optical, and x-ray computed tomography (FT∕DOT∕XCT) system for small animal imaging is reported in this work. The main purpose of this system is to obtain quantitatively accurate fluorescence concentration images using a multimodality approach. XCT offers anatomical information, while DOT provides the necessary background optical property map to improve FT image accuracy. The quantitative accuracy of this trimodality system is demonstrated in vivo. In particular, we show that a 2-mm-diam fluorescence inclusion located 8 mm deep in a nude mouse can only be localized when functional a priori information from DOT is available. However, the error in the recovered fluorophore concentration is nearly 87%. On the other hand, the fluorophore concentration can be accurately recovered within 2% error when both DOT functional and XCT structural a priori information are utilized together to guide and constrain the FT reconstruction algorithm. PMID:20799770

Supramolecular assembly affording a ratiometric two-photon fluorescent nanoprobe for quantitative detection and bioimaging.

PubMed

Wang, Peng; Zhang, Cheng; Liu, Hong-Wen; Xiong, Mengyi; Yin, Sheng-Yan; Yang, Yue; Hu, Xiao-Xiao; Yin, Xia; Zhang, Xiao-Bing; Tan, Weihong

2017-12-01

Fluorescence quantitative analyses for vital biomolecules are in great demand in biomedical science owing to their unique detection advantages with rapid, sensitive, non-damaging and specific identification. However, available fluorescence strategies for quantitative detection are usually hard to design and achieve. Inspired by supramolecular chemistry, a two-photon-excited fluorescent supramolecular nanoplatform ( TPSNP ) was designed for quantitative analysis with three parts: host molecules (β-CD polymers), a guest fluorophore of sensing probes (Np-Ad) and a guest internal reference (NpRh-Ad). In this strategy, the TPSNP possesses the merits of (i) improved water-solubility and biocompatibility; (ii) increased tissue penetration depth for bioimaging by two-photon excitation; (iii) quantitative and tunable assembly of functional guest molecules to obtain optimized detection conditions; (iv) a common approach to avoid the limitation of complicated design by adjustment of sensing probes; and (v) accurate quantitative analysis by virtue of reference molecules. As a proof-of-concept, we utilized the two-photon fluorescent probe NHS-Ad-based TPSNP-1 to realize accurate quantitative analysis of hydrogen sulfide (H 2 S), with high sensitivity and good selectivity in live cells, deep tissues and ex vivo -dissected organs, suggesting that the TPSNP is an ideal quantitative indicator for clinical samples. What's more, TPSNP will pave the way for designing and preparing advanced supramolecular sensors for biosensing and biomedicine.

Quantitative magnetic resonance imaging phantoms: A review and the need for a system phantom.

PubMed

Keenan, Kathryn E; Ainslie, Maureen; Barker, Alex J; Boss, Michael A; Cecil, Kim M; Charles, Cecil; Chenevert, Thomas L; Clarke, Larry; Evelhoch, Jeffrey L; Finn, Paul; Gembris, Daniel; Gunter, Jeffrey L; Hill, Derek L G; Jack, Clifford R; Jackson, Edward F; Liu, Guoying; Russek, Stephen E; Sharma, Samir D; Steckner, Michael; Stupic, Karl F; Trzasko, Joshua D; Yuan, Chun; Zheng, Jie

2018-01-01

The MRI community is using quantitative mapping techniques to complement qualitative imaging. For quantitative imaging to reach its full potential, it is necessary to analyze measurements across systems and longitudinally. Clinical use of quantitative imaging can be facilitated through adoption and use of a standard system phantom, a calibration/standard reference object, to assess the performance of an MRI machine. The International Society of Magnetic Resonance in Medicine AdHoc Committee on Standards for Quantitative Magnetic Resonance was established in February 2007 to facilitate the expansion of MRI as a mainstream modality for multi-institutional measurements, including, among other things, multicenter trials. The goal of the Standards for Quantitative Magnetic Resonance committee was to provide a framework to ensure that quantitative measures derived from MR data are comparable over time, between subjects, between sites, and between vendors. This paper, written by members of the Standards for Quantitative Magnetic Resonance committee, reviews standardization attempts and then details the need, requirements, and implementation plan for a standard system phantom for quantitative MRI. In addition, application-specific phantoms and implementation of quantitative MRI are reviewed. Magn Reson Med 79:48-61, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Magnetic resonance tracking of fluorescent nanodiamond fabrication

NASA Astrophysics Data System (ADS)

Shames, A. I.; Osipov, V. Yu; Boudou, J. P.; Panich, A. M.; von Bardeleben, H. J.; Treussart, F.; Vul', A. Ya

2015-04-01

Magnetic resonance techniques (electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR)) are used for tracking the multi-stage process of the fabrication of fluorescent nanodiamonds (NDs) produced by high-energy electron irradiation, annealing, and subsequent nano-milling. Pristine commercial high pressure and high temperature microdiamonds (MDs) with mean size 150 μm contain ~5  ×  1018 spins/g of singlet (S = 1/2) substitutional nitrogen defects P1, as well as sp3 C-C dangling bonds in the crystalline lattice. The half-field X-band EPR clearly shows (by the appearance of the intense ‘forbidden’ g = 4.26 line) that high-energy electron irradiation and annealing of MDs induce a large amount (~5  ×  1017 spins/g) of triplet (S = 1) magnetic centers, which are identified as negatively charged nitrogen vacancy defects (NV-). This is supported by EPR observations of the ‘allowed’ transitions between Zeeman sublevels of the triplet state. After progressive milling of the fluorescent MDs down to an ultrasubmicron scale (≤100 nm), the relative abundance of EPR active NV- defects in the resulting fluorescent NDs (FND) substantially decreases and, vice versa, the content of C-inherited singlet defects correlatively increases. In the fraction of the finest FNDs (mean particle size <20 nm), which are contained in the dried supernatant of ultracentrifuged aqueous dispersion of FNDs, the NV- content is found to be reduced by one order of magnitude whereas the singlet defects content increases up to ~2  ×  1019 spins/g. In addition, another triplet-type defect, which is characterized by the g = 4.00 ‘forbidden’ line, appears. On reduction of the particle size below the 20 nm limit, the ‘allowed’ EPR lines become practically unobservable, whereas the ‘forbidden’ lines remain as a reliable fingerprint of the presence of NV- centers in small ND systems. The same size reduction causes the disappearance of the

Imaging atoms from resonance fluorescence spectrum beyond the diffraction limit

NASA Astrophysics Data System (ADS)

Liao, Zeyang; Al-Amri, Mohammad; Zubairy, M. Suhail

2014-03-01

We calculate the resonance fluorescence spectrum of a linear chain of two-level atoms driven by a gradient coherent laser field. The result shows that we can determine the positions of atoms from the spectrum even when the atoms locate within subwavelength range and the dipole-dipole interaction is significant. This far-field resonance fluorescence localization microscopy method does not require point-by-point scanning and it may be more time-efficient. We also give a possible scheme to extract the position information in an extended region without requiring more peak power of laser. We also briefly discuss how to do a 2D imaging based on our scheme. This work is supported by grants from the King Abdulaziz City for Science and Technology (KACST) and the Qatar National Research Fund (QNRF) under the NPRP project.

Multi-Channel Hyperspectral Fluorescence Detection Excited by Coupled Plasmon-Waveguide Resonance

PubMed Central

Du, Chan; Liu, Le; Zhang, Lin; Guo, Jun; Guo, Jihua; Ma, Hui; He, Yonghong

2013-01-01

We propose in this paper a biosensor scheme based on coupled plasmon-waveguide resonance (CPWR) excited fluorescence spectroscopy. A symmetrical structure that offers higher surface electric field strengths, longer surface propagation lengths and depths is developed to support guided waveguide modes for the efficient excitation of fluorescence. The optimal parameters for the sensor films are theoretically and experimentally investigated, leading to a detection limit of 0.1 nM (for a Cy5 solution). Multiplex analysis possible with the fluorescence detection is further advanced by employing the hyperspectral fluorescence technique to record the full spectra for every pixel on the sample plane. We demonstrate experimentally that highly overlapping fluorescence (Cy5 and Dylight680) can be distinguished and ratios of different emission sources can be determined accurately. This biosensor shows great potential for multiplex detections of fluorescence analytes. PMID:24129023

Shifts due to quantum-mechanical interference from distant neighboring resonances for saturated fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Marsman, Alain; Horbatsch, Marko; Hessels, Eric A.

2014-05-01

Quantum-mechanical interference with distant neighboring resonances is found to cause shifts for precision saturated fluorescence spectroscopy of the atomic helium 23 S -to- 23 P transitions. The shifts are significant (larger than the experimental uncertainties for measurements of the intervals) despite the fact that the neighboring resonances are separated from the measured resonances by 1400 and 20 000 natural widths. The shifts depend strongly on experimental parameters such as the angular position of the fluorescence detector and the intensity and size of laser beams. These shifts must be considered for the ongoing program of determining the fine-structure constant from the helium 23 P fine structure. The work represents the first study of such interference shifts for saturated fluorescence spectroscopy and follows up on our previous study of similar shifts for laser spectroscopy. This work is supported by NSERC, CRC, ORF, CFI, NIST and SHARCNET.

Ongoing advances in quantitative PpIX fluorescence guided intracranial tumor resection (Conference Presentation)

NASA Astrophysics Data System (ADS)

Olson, Jonathan D.; Kanick, Stephen C.; Bravo, Jaime J.; Roberts, David W.; Paulsen, Keith D.

2016-03-01

Aminolevulinc-acid induced protoporphyrin IX (ALA-PpIX) is being investigated as a biomarker to guide neurosurgical resection of brain tumors. ALA-PpIX fluorescence can be observed visually in the surgical field; however, raw fluorescence emissions can be distorted by factors other than the fluorophore concentration. Specifically, fluorescence emissions are mixed with autofluorescence and attenuated by background absorption and scattering properties of the tissue. Recent work at Dartmouth has developed advanced fluorescence detection approaches that return quantitative assessments of PpIX concentration, which are independent of background optical properties. The quantitative fluorescence imaging (qFI) approach has increased sensitivity to residual disease within the resection cavity at the end of surgery that was not visible to the naked eye through the operating microscope. This presentation outlines clinical observations made during an ongoing investigation of ALA-PpIX based guidance of tumor resection. PpIX fluorescence measurements made in a wide-field hyperspectral imaging approach are co-registered with point-assessment using a fiber optic probe. Data show variations in the measured PpIX accumulation among different clinical tumor grades (i.e. high grade glioma, low grade glioma), types (i.e. primary tumors. metastases) and normal structures of interest (e.g. normal cortex, hippocampus). These results highlight the contrast enhancement and underscore the potential clinical benefit offered from quantitative measurements of PpIX concentration during resection of intracranial tumors.

Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography

NASA Astrophysics Data System (ADS)

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

2016-02-01

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

Quantitative light-induced fluorescence technology for quantitative evaluation of tooth wear

NASA Astrophysics Data System (ADS)

Kim, Sang-Kyeom; Lee, Hyung-Suk; Park, Seok-Woo; Lee, Eun-Song; de Josselin de Jong, Elbert; Jung, Hoi-In; Kim, Baek-Il

2017-12-01

Various technologies used to objectively determine enamel thickness or dentin exposure have been suggested. However, most methods have clinical limitations. This study was conducted to confirm the potential of quantitative light-induced fluorescence (QLF) using autofluorescence intensity of occlusal surfaces of worn teeth according to enamel grinding depth in vitro. Sixteen permanent premolars were used. Each tooth was gradationally ground down at the occlusal surface in the apical direction. QLF-digital and swept-source optical coherence tomography images were acquired at each grinding depth (in steps of 100 μm). All QLF images were converted to 8-bit grayscale images to calculate the fluorescence intensity. The maximum brightness (MB) values of the same sound regions in grayscale images before (MB) and phased values after (MB) the grinding process were calculated. Finally, 13 samples were evaluated. MB increased over the grinding depth range with a strong correlation (r=0.994, P<0.001). In conclusion, the fluorescence intensity of the teeth and grinding depth was strongly correlated in the QLF images. Therefore, QLF technology may be a useful noninvasive tool used to monitor the progression of tooth wear and to conveniently estimate enamel thickness.

Quantitative magnetic resonance imaging in traumatic brain injury.

PubMed

Bigler, E D

2001-04-01

Quantitative neuroimaging has now become a well-established method for analyzing magnetic resonance imaging in traumatic brain injury (TBI). A general review of studies that have examined quantitative changes following TBI is presented. The consensus of quantitative neuroimaging studies is that most brain structures demonstrate changes in volume or surface area after injury. The patterns of atrophy are consistent with the generalized nature of brain injury and diffuse axonal injury. Various clinical caveats are provided including how quantitative neuroimaging findings can be used clinically and in predicting rehabilitation outcome. The future of quantitative neuroimaging also is discussed.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images.

PubMed

Zhang, Zhiqing; Kuzmin, Nikolay V; Groot, Marie Louise; de Munck, Jan C

2018-01-01

Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation and characterization of alginate based-fluorescent magnetic nanoparticles for fluorescence/magnetic resonance multimodal imaging applications

NASA Astrophysics Data System (ADS)

Kwon, Yong-Su; Choi, Kee-Bong; Lim, Hyungjun; Lee, Sunghwi; Lee, Jae-Jong

2018-06-01

Simple and versatile methodologies have been reported that customize the surface of superparamagnetic iron oxide (SPIO) nanoparticles and impart additional fluorescence capabilities to these contrast agents. Herein, we present the rational design, synthesis, characterization, and biological applications of a new magnetic-based fluorescent probe. The dual modality imaging protocol was developed by labeling fluorophore with alginate natural polymers that have excellent biocompatibility and biodegradability, and using gelification method to form nanocomposites containing SPIO. The formation of alginate-based fluorescent magnetic (AFM) nanoparticles was observed in spherical and elliptical forms with a diameter of less than 500 nm by a transmission electron microscope (TEM). The fluorescent wavelength band in the range of 560 nm was also confirmed in the UV–visible spectrophotometer. In this study, we demonstrate that the multi-tasking design of AFM nanoparticles provides an ideal platform for building balanced dual-image probes of magnetic resonance imaging and optical imaging.

Time-Gated Raman Spectroscopy for Quantitative Determination of Solid-State Forms of Fluorescent Pharmaceuticals.

PubMed

Lipiäinen, Tiina; Pessi, Jenni; Movahedi, Parisa; Koivistoinen, Juha; Kurki, Lauri; Tenhunen, Mari; Yliruusi, Jouko; Juppo, Anne M; Heikkonen, Jukka; Pahikkala, Tapio; Strachan, Clare J

2018-04-03

Raman spectroscopy is widely used for quantitative pharmaceutical analysis, but a common obstacle to its use is sample fluorescence masking the Raman signal. Time-gating provides an instrument-based method for rejecting fluorescence through temporal resolution of the spectral signal and allows Raman spectra of fluorescent materials to be obtained. An additional practical advantage is that analysis is possible in ambient lighting. This study assesses the efficacy of time-gated Raman spectroscopy for the quantitative measurement of fluorescent pharmaceuticals. Time-gated Raman spectroscopy with a 128 × (2) × 4 CMOS SPAD detector was applied for quantitative analysis of ternary mixtures of solid-state forms of the model drug, piroxicam (PRX). Partial least-squares (PLS) regression allowed quantification, with Raman-active time domain selection (based on visual inspection) improving performance. Model performance was further improved by using kernel-based regularized least-squares (RLS) regression with greedy feature selection in which the data use in both the Raman shift and time dimensions was statistically optimized. Overall, time-gated Raman spectroscopy, especially with optimized data analysis in both the spectral and time dimensions, shows potential for sensitive and relatively routine quantitative analysis of photoluminescent pharmaceuticals during drug development and manufacturing.

Quantitative Imaging in Laboratory: Fast Kinetics and Fluorescence Quenching

ERIC Educational Resources Information Center

Cumberbatch, Tanya; Hanley, Quentin S.

2007-01-01

The process of quantitative imaging, which is very commonly used in laboratory, is shown to be very useful for studying the fast kinetics and fluorescence quenching of many experiments. The imaging technique is extremely cheap and hence can be used in many absorption and luminescence experiments.

Nuclear Resonance Fluorescence of U-235 above 3 MeV

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

Warren, Glen A.; Caggiano, Joseph A.; Miller, Erin A.

Pacific Northwest National Laboratory and Passport Systems have collaborated to conduct measurements to search for a nuclear resonance fluorescence response of U-235 from 3 to 5 MeV using an 8 g sample of highly enriched uranium. These new measurements complement previously reported measurements below 3 MeV. Preliminary analysis indicates that no strong resonances exist for U-235 in this energy range. A second set of measurements focused on a signature search in the 5 to 10 MeV range is still under analysis.

Detection of sex chromosome aneuploidies using quantitative fluorescent PCR in the Hungarian population.

PubMed

Nagy, Balint; Nagy, Richard Gyula; Lazar, Levente; Schonleber, Julianna; Papp, Csaba; Rigo, Janos

2015-05-20

Aneuploidies are the most frequent chromosomal abnormalities at birth. Autosomal aneuploidies cause serious malformations like trisomy 21, trisomy 18 and trisomy 13. However sex chromosome aneuploidies are causing less severe syndromes. For the detection of these aneuploidies, the "gold standard" method is the cytogenetic analysis of fetal cells, karyograms show all numerical and structural abnormalities, but it takes 2-4 weeks to get the reports. Molecular biological methods were developed to overcome the long culture time, thus, FISH and quantitative fluorescent PCR were introduced. In this work we show our experience with a commercial kit for the detection of sex chromosome aneuploidies. We analyzed 20.173 amniotic fluid samples for the period of 2006-2013 in our department. A conventional cytogenetic analysis was performed on the samples. We checked the reliability of quantitative fluorescent PCR and DNA fragment analysis on those samples where sex chromosomal aneuploidy was diagnosed. From the 20.173 amniotic fluid samples we found 50 samples with sex chromosome aneuploidy. There were 19 samples showing 46, XO, 17 samples with 46, XXY, 9 samples with 47, XXX and 5 samples with 47, XYY karyotypes. The applied quantitative fluorescent PCR and DNA fragment analyses method are suitable to detect all abnormal sex chromosome aneuploidies. Quantitative fluorescent PCR is a fast and reliable method for detection of sex chromosome aneuploidies. Copyright © 2015. Published by Elsevier B.V.

Velocity measurements by laser resonance fluorescence. [single atom diffusional motion

NASA Technical Reports Server (NTRS)

She, C. Y.; Fairbank, W. M., Jr.

1980-01-01

The photonburst correlation method was used to detect single atoms in a buffer gas. Real time flow velocity measurements with laser induced resonance fluorescence from single or multiple atoms was demonstrated and this method was investigated as a tool for wind tunnel flow measurement. Investigations show that single atoms and their real time diffusional motion on a buffer gas can be measured by resonance fluorescence. By averaging over many atoms, flow velocities up to 88 m/s were measured in a time of 0.5 sec. It is expected that higher flow speeds can be measured and that the measurement time can be reduced by a factor of 10 or more by careful experimental design. The method is clearly not ready for incorporation in high speed wind tunnels because it is not yet known whether the stray light level will be higher or lower, and it is not known what detection efficiency can be obtained in a wind tunnel situation.

Quantitative fluorescence nanoscopy for cancer biomedicine

NASA Astrophysics Data System (ADS)

Huang, Tao; Nickerson, Andrew; Peters, Alec; Nan, Xiaolin

2015-08-01

Cancer is a major health threat worldwide. Options for targeted cancer therapy, however, are often limited, in a large part due to our incomplete understanding of how key processes including oncogenesis and drug response are mediated at the molecular level. New imaging techniques for visualizing biomolecules and their interactions at the nanometer and single molecule scales, collectively named fluorescence nanoscopy, hold the promise to transform biomedical research by providing direct mechanistic insight into cellular processes. We discuss the principles of quantitative single-molecule localization microscopy (SMLM), a subset of fluorescence nanoscopy, and their applications to cancer biomedicine. In particular, we will examine oncogenesis and drug resistance mediated by mutant Ras, which is associated with ~1/3 of all human cancers but has remained an intractable drug target. At ~20 nm spatial and single-molecule stoichiometric resolutions, SMLM clearly showed that mutant Ras must form dimers to activate its effector pathways and drive oncogenesis. SMLM further showed that the Raf kinase, one of the most important effectors of Ras, also forms dimers upon activation by Ras. Moreover, treatment of cells expressing wild type Raf with Raf inhibitors induces Raf dimer formation in a manner dependent on Ras dimerization. Together, these data suggest that Ras dimers mediate oncogenesis and drug resistance in tumors with hyperactive Ras and can potentially be targeted for cancer therapy. We also discuss recent advances in SMLM that enable simultaneous imaging of multiple biomolecules and their interactions at the nanoscale. Our work demonstrates the power of quantitative SMLM in cancer biomedicine.

Metal Nanoparticles/Porous Silicon Microcavity Enhanced Surface Plasmon Resonance Fluorescence for the Detection of DNA.

PubMed

Wang, Jiajia; Jia, Zhenhong

2018-02-23

A porous silicon microcavity (PSiMC) with resonant peak wavelength of 635 nm was fabricated by electrochemical etching. Metal nanoparticles (NPs)/PSiMC enhanced fluorescence substrates were prepared by the electrostatic adherence of Au NPs that were distributed in PSiMC. The Au NPs/PSiMC device was used to characterize the target DNA immobilization and hybridization with its complementary DNA sequences marked with Rhodamine red (RRA). Fluorescence enhancement was observed on the Au NPs/PSiMC device substrate; and the minimum detection concentration of DNA ran up to 10 pM. The surface plasmon resonance (SPR) of the MC substrate; which is so well-positioned to improve fluorescence enhancement rather the fluorescence enhancement of the high reflection band of the Bragg reflector; would welcome such a highly sensitive in biosensor.

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.

Nuclear Resonance Fluorescence and Isotopic Mapping of Containers

NASA Astrophysics Data System (ADS)

Johnson, Micah S.; McNabb, Dennis P.

2009-03-01

National security programs have expressed interest in developing systems to isotopically map shipping containers, fuel assemblies, and waste barrels for various materials including special nuclear material (SNM). Current radiographic systems offer little more than an ambiguous density silhouette of a container's contents. In this paper we will present a system being developed at LLNL to isotopically map containers using the nuclear resonance fluorescence (NRF) method. Recent experimental measurements on NRF strengths in SNM are discussed.

Fluorescence resonance energy transfer between green fluorescent protein and doxorubicin enabled by DNA nanotechnology.

PubMed

Heger, Zbynek; Kominkova, Marketa; Cernei, Natalia; Krejcova, Ludmila; Kopel, Pavel; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

2014-12-01

DNA nanotechnology is a rapidly growing research area, where DNA may be used for wide range of applications such as construction of nanodevices serving for large scale of diverse purposes. Likewise a panel of various purified fluorescent proteins is investigated for their ability to emit their typical fluorescence spectra under influence of particular excitation. Hence these proteins may form ideal donor molecules for assembly of fluorescence resonance emission transfer (FRET) constructions. To extend the application possibilities of fluorescent proteins, while using DNA nanotechnology, we developed nanoconstruction comprising green fluorescent protein (GFP) bound onto surface of surface active nanomaghemite and functionalized with gold nanoparticles. We took advantage of natural affinity between gold and thiol moieties, which were modified to bind DNA fragment. Finally we enclosed doxorubicin into fullerene cages. Doxorubicin intercalated in DNA fragment bound on the particles and thus we were able to connect these parts together. Because GFP behaved as a donor and doxorubicin as an acceptor using excitation wavelength for GFP (395 nm) in emission wavelength of doxorubicin (590 nm) FRET was observed. This nanoconstruction may serve as a double-labeled transporter of doxorubicin guided by force of external magnetic force owing to the presence of nanomaghemite. Further nanomaghemite offers the possibility of using this technology for thermotherapy. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of quantitative light-induced fluorescence to monitor tooth whitening

NASA Astrophysics Data System (ADS)

Amaechi, Bennett T.; Higham, Susan M.

2001-04-01

The changing of tooth shade by whitening agents occurs gradually. Apart from being subjective and affected by the conditions of the surroundings, visual observation cannot detect a very slight change in tooth color. An electronic method, which can communicate the color change quantitatively, would be more reliable. Quantitative Light- induced Fluorescence (QLF) was developed to detect and assess dental caries based on the phenomenon of change of autofluorescence of a tooth by demineralization. However, stains on the tooth surface exhibit the same phenomenon, and therefore QLF can be used to measure the percentage fluorescence change of stained enamel with respect to surrounding unstained enamel. The present study described a technique of assessing the effect of a tooth-whitening agent using QLF. This was demonstrated in two experiments in which either wholly or partially stained teeth were whitened by intermittent immersion in sodium hypochlorite. Following each immersion, the integrated fluorescence change due to the stain was quantified using QLF. In either situation, the value of (Delta) Q decreased linearly as the tooth regained its natural shade. It was concluded that gradual changing of the shade of discolored teeth by a whitening agent could be quantified using QLF.

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.

Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker

PubMed Central

Valdés, Pablo A.; Leblond, Frederic; Kim, Anthony; Harris, Brent T.; Wilson, Brian C.; Fan, Xiaoyao; Tosteson, Tor D.; Hartov, Alex; Ji, Songbai; Erkmen, Kadir; Simmons, Nathan E.; Paulsen, Keith D.; Roberts, David W.

2011-01-01

Object Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative fluorescence of protoporphyrin IX (PpIX), synthesized endogenously following δ-aminolevulinic acid (ALA) administration, has been used for this purpose in high-grade glioma (HGG). The authors show that diagnostically significant but visually imperceptible concentrations of PpIX can be quantitatively measured in vivo and used to discriminate normal from neoplastic brain tissue across a range of tumor histologies. Methods The authors studied 14 patients with diagnoses of low-grade glioma (LGG), HGG, meningioma, and metastasis under an institutional review board–approved protocol for fluorescence-guided resection. The primary aim of the study was to compare the diagnostic capabilities of a highly sensitive, spectrally resolved quantitative fluorescence approach to conventional fluorescence imaging for detection of neoplastic tissue in vivo. Results A significant difference in the quantitative measurements of PpIX concentration occurred in all tumor groups compared with normal brain tissue. Receiver operating characteristic (ROC) curve analysis of PpIX concentration as a diagnostic variable for detection of neoplastic tissue yielded a classification efficiency of 87% (AUC = 0.95, specificity = 92%, sensitivity = 84%) compared with 66% (AUC = 0.73, specificity = 100%, sensitivity = 47%) for conventional fluorescence imaging (p < 0.0001). More than 81% (57 of 70) of the quantitative fluorescence measurements that were below the threshold of the surgeon's visual perception were classified correctly in an analysis of all tumors. Conclusions These findings are clinically profound because they demonstrate that ALA-induced PpIX is a targeting biomarker for a variety of intracranial tumors beyond HGGs. This study is the first to measure quantitative ALA-induced PpIX concentrations in vivo, and the results have broad implications for guidance during resection of

In vivo fluorescence lifetime optical projection tomography

PubMed Central

McGinty, James; Taylor, Harriet B.; Chen, Lingling; Bugeon, Laurence; Lamb, Jonathan R.; Dallman, Margaret J.; French, Paul M. W.

2011-01-01

We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions. PMID:21559145

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.

A quantitative characterization of the yeast heterotrimeric G protein cycle

PubMed Central

Yi, Tau-Mu; Kitano, Hiroaki; Simon, Melvin I.

2003-01-01

The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Gα and Gβγ-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose–response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of α-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose–response curve and the downstream dose–response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast. PMID:12960402

Combined magnetic resonance, fluorescence, and histology imaging strategy in a human breast tumor xenograft model

PubMed Central

Jiang, Lu; Greenwood, Tiffany R.; Amstalden van Hove, Erika R.; Chughtai, Kamila; Raman, Venu; Winnard, Paul T.; Heeren, Ron; Artemov, Dmitri; Glunde, Kristine

2014-01-01

Applications of molecular imaging in cancer and other diseases frequently require combining in vivo imaging modalities, such as magnetic resonance and optical imaging, with ex vivo optical, fluorescence, histology, and immunohistochemical (IHC) imaging, to investigate and relate molecular and biological processes to imaging parameters within the same region of interest. We have developed a multimodal image reconstruction and fusion framework that accurately combines in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI), ex vivo brightfield and fluorescence microscopic imaging, and ex vivo histology imaging. Ex vivo brightfield microscopic imaging was used as an intermediate modality to facilitate the ultimate link between ex vivo histology and in vivo MRI/MRSI. Tissue sectioning necessary for optical and histology imaging required generation of a three-dimensional (3D) reconstruction module for 2D ex vivo optical and histology imaging data. We developed an external fiducial marker based 3D reconstruction method, which was able to fuse optical brightfield and fluorescence with histology imaging data. Registration of 3D tumor shape was pursued to combine in vivo MRI/MRSI and ex vivo optical brightfield and fluorescence imaging data. This registration strategy was applied to in vivo MRI/MRSI, ex vivo optical brightfield/fluorescence, as well as histology imaging data sets obtained from human breast tumor models. 3D human breast tumor data sets were successfully reconstructed and fused with this platform. PMID:22945331

Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions

NASA Astrophysics Data System (ADS)

Sharifzadeh, Mohsen; Bernstein, Paul S.; Gellermann, Werner

2006-10-01

We have developed a CCD-camera-based nonmydriatic instrument that detects fluorescence from retinal lipofuscin chromophores ("autofluorescence") as a means to indirectly quantify and spatially image the distribution of macular pigment (MP). The lipofuscin fluorescence intensity is reduced at all retinal locations containing MP, since MP has a competing absorption in the blue-green wavelength region. Projecting a large diameter, 488 nm excitation spot onto the retina, centered on the fovea, but extending into the macular periphery, and comparing lipofuscin fluorescence intensities outside and inside the foveal area, it is possible to spatially map out the distribution of MP. Spectrally selective detection of the lipofuscin fluorescence reveals an important wavelength dependence of the obtainable image contrast and deduced MP optical density levels, showing that it is important to block out interfering fluorescence contributions in the detection setup originating from ocular media such as the lens. Measuring 70 healthy human volunteer subjects with no ocular pathologies, we find widely varying spatial extent of MP, distinctly differing distribution patterns of MP, and strongly differing absolute MP levels among individuals. Our population study suggests that MP imaging based on lipofuscin fluorescence is useful as a relatively simple, objective, and quantitative noninvasive optical technique suitable to rapidly screen MP levels and distributions in healthy humans with undilated pupils.

Dual-wavelength excitation to reduce background fluorescence for fluorescence spectroscopic quantitation of erythrocyte zinc protoporphyrin-IX and protoporphyrin-IX from whole blood and oral mucosa

NASA Astrophysics Data System (ADS)

Hennig, Georg; Vogeser, Michael; Holdt, Lesca M.; Homann, Christian; Großmann, Michael; Stepp, Herbert; Gruber, Christian; Erdogan, Ilknur; Hasmüller, Stephan; Hasbargen, Uwe; Brittenham, Gary M.

2014-02-01

Erythrocyte zinc protoporphyrin-IX (ZnPP) and protoporphyrin-IX (PPIX) accumulate in a variety of disorders that restrict or disrupt the biosynthesis of heme, including iron deficiency and various porphyrias. We describe a reagent-free spectroscopic method based on dual-wavelength excitation that can measure simultaneously both ZnPP and PPIX fluorescence from unwashed whole blood while virtually eliminating background fluorescence. We further aim to quantify ZnPP and PPIX non-invasively from the intact oral mucosa using dual-wavelength excitation to reduce the strong tissue background fluorescence while retaining the faint porphyrin fluorescence signal originating from erythrocytes. Fluorescence spectroscopic measurements were made on 35 diluted EDTA blood samples using a custom front-face fluorometer. The difference spectrum between fluorescence at 425 nm and 407 nm excitation effectively eliminated background autofluorescence while retaining the characteristic porphyrin peaks. These peaks were evaluated quantitatively and the results compared to a reference HPLC-kit method. A modified instrument using a single 1000 μm fiber for light delivery and detection was used to record fluorescence spectra from oral mucosa. For blood measurements, the ZnPP and PPIX fluorescence intensities from the difference spectra correlated well with the reference method (ZnPP: Spearman's rho rs = 0.943, p < 0.0001; PPIX: rs = 0.959, p < 0.0001). In difference spectra from oral mucosa, background fluorescence was reduced significantly, while porphyrin signals remained observable. The dual-wavelength excitation method evaluates quantitatively the ZnPP/heme and PPIX/heme ratios from unwashed whole blood, simplifying clinical laboratory measurements. The difference technique reduces the background fluorescence from measurements on oral mucosa, allowing for future non-invasive quantitation of erythrocyte ZnPP and PPIX.

Nuclear Resonance Fluorescence Response of U-235

NASA Astrophysics Data System (ADS)

Warren, Glen

2008-05-01

Nuclear resonance fluorescence (NRF) is a physical process that provides an isotopic-specific signature that could be used for the identification and characterization of materials. The technique involves the detection of prompt discrete-energy photons emitted from a sample, which is exposed to photons in the MeV energy range. Potential applications of the technique range from detection of high explosives to characterization of special nuclear materials. Pacific Northwest National Laboratory and Passport Systems have collaboratively conducted a set of measurements to search for an NRF response of U-235 in the 1.5 to 9 MeV energy range. Results from these measurements will be presented.

Simultaneous fluorescence and quantitative phase microscopy with single-pixel detectors

NASA Astrophysics Data System (ADS)

Liu, Yang; Suo, Jinli; Zhang, Yuanlong; Dai, Qionghai

2018-02-01

Multimodal microscopy offers high flexibilities for biomedical observation and diagnosis. Conventional multimodal approaches either use multiple cameras or a single camera spatially multiplexing different modes. The former needs expertise demanding alignment and the latter suffers from limited spatial resolution. Here, we report an alignment-free full-resolution simultaneous fluorescence and quantitative phase imaging approach using single-pixel detectors. By combining reference-free interferometry with single-pixel detection, we encode the phase and fluorescence of the sample in two detection arms at the same time. Then we employ structured illumination and the correlated measurements between the sample and the illuminations for reconstruction. The recovered fluorescence and phase images are inherently aligned thanks to single-pixel detection. To validate the proposed method, we built a proof-of-concept setup for first imaging the phase of etched glass with the depth of a few hundred nanometers and then imaging the fluorescence and phase of the quantum dot drop. This method holds great potential for multispectral fluorescence microscopy with additional single-pixel detectors or a spectrometer. Besides, this cost-efficient multimodal system might find broad applications in biomedical science and neuroscience.

Extended Fluorescent Resonant Energy Transfer in DNA Constructs

NASA Astrophysics Data System (ADS)

Oh, Taeseok

This study investigates the use of surfactants and metal cations for the enhancement of long range fluorescent resonant energy transfer (FRET) and the antenna effect in DNA structures with multiple fluorescent dyes. Double-stranded (ds) DNA structures were formed by hybridization of 21mer DNA oligonucleotides with different arrangements of three fluorescent TAMRA donor dyes with two different complementary 21mer oligonucleotides with one fluorescent TexasRed acceptor dye. In such DNA structures, hydrophobic interactions between the fluorescent dyes in close proximity produces dimerization which along with other quenching mechanisms leads to significant reduction of fluorescent emission properties. Addition of the surfactants Triton X-100, cetyltrimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) along with sodium cations (Na+) and divalent magnesium cations (Mg 2+) were tested for their ability to reduce quenching of the fluorescent dyes and improve overall fluorescent emission, the long range FRET and the antenna effect properties. When the neutral (uncharged) surfactant Triton X-100 was added to the FRET ds-DNA hybrid structures with three TAMRA donors and one TexasRed acceptor, dye dimerization and emission quenching remained unaffected. However, for the positively charged CTAB surfactant at concentrations of 100 uM or higher, the neutralization of the negatively charged ds-DNA backbone by the cationic surfactant micelles was found to reduce TAMRA dye dimerization and emission quenching and improve TexasRed quantum yield, resulting in much higher FRET efficiencies and an enhanced antenna effect. This improvement is likely due to the CTAB molecules covering or sheathing the fluorescent donor and acceptor dyes which breaks up the dimerized dye complexes and prevents further quenching from interactions with water molecules and guanine bases in the DNA structure. While the negatively charged SDS surfactant alone was not able to reduce dimerization and

Resonance Fluorescence of an InGaAs Quantum Dot in a Planar Cavity Using Orthogonal Excitation and Detection.

PubMed

Chen, Disheng; Lander, Gary R; Flagg, Edward B

2017-10-13

The ability to perform simultaneous resonant excitation and fluorescence detection is important for quantum optical measurements of quantum dots (QDs). Resonant excitation without fluorescence detection - for example, a differential transmission measurement - can determine some properties of the emitting system, but does not allow applications or measurements based on the emitted photons. For example, the measurement of photon correlations, observation of the Mollow triplet, and realization of single photon sources all require collection of the fluorescence. Incoherent excitation with fluorescence detection - for example, above band-gap excitation - can be used to create single photon sources, but the disturbance of the environment due to the excitation reduces the indistinguishability of the photons. Single photon sources based on QDs will have to be resonantly excited to have high photon indistinguishability, and simultaneous collection of the photons will be necessary to make use of them. We demonstrate a method to resonantly excite a single QD embedded in a planar cavity by coupling the excitation beam into this cavity from the cleaved face of the sample while collecting the fluorescence along the sample's surface normal direction. By carefully matching the excitation beam to the waveguide mode of the cavity, the excitation light can couple into the cavity and interact with the QD. The scattered photons can couple to the Fabry-Perot mode of the cavity and escape in the surface normal direction. This method allows complete freedom in the detection polarization, but the excitation polarization is restricted by the propagation direction of the excitation beam. The fluorescence from the wetting layer provides a guide to align the collection path with respect to the excitation beam. The orthogonality of the excitation and detection modes enables resonant excitation of a single QD with negligible laser scattering background.

Utilizing hyaluronic acid as a versatile platform for fluorescence resonance energy transfer-based glucose sensing.

PubMed

Ge, Minghao; Bai, Pengli; Chen, Mingli; Tian, Jingjing; Hu, Jun; Zhi, Xu; Yin, Huancai; Yin, Jian

2018-03-01

Here, we utilized the ultrasonic emulsification technique to generate hyaluronic acid microspheres incorporating a fluorescence-based glucose biosensor. We synthesized a novel lanthanide ion luminophore based on Eu 3+ . Eu sulfosuccinimidyl dextran (Eu-dextran) and Alexa Fluor 647 sulfosuccinimidyl-ConA (Alexa Fluor 647-ConA) were encapsulated in hyaluronic acid hydrogel to generate microspheres. Glucose sensing was carried out using a fluorescence resonance energy transfer (FRET)-based assay principle. A proportional fluorescence intensity increase was found within a 0.5-10-mM glucose concentration range. The glucose-sensing strategy showed an excellent tolerance for potential interferents. Meanwhile, the fluorescent signal of hyaluronic acid microspheres was very stable after testing for 72 h in glucose solution. Overall, hyaluronic acid microspheres encapsulating sensing biomolecules offer a stable and biocompatible biosensor for a variety of applications including cell culture systems, tissue engineering, detection of blood glucose, etc. Graphical abstract We report an ingenious biosensor encapsulated in hyaluronic acid microspheres for monitoring of glucose. Glucose sensing is carried out using a fluorescence resonance energy transfer-based assay principle with a novel lanthanide ions luminophore. The glucose detection system has excellent biocompatibility and stability for monitoring of glucose.

Phonon-wave-induced resonance fluorescence in semiconductor nanostructures: acoustoluminescence in the terahertz range.

PubMed

Ahn, K J; Milde, F; Knorr, A

2007-01-12

Acoustic wave excitation of semiconductor quantum dots generates resonance fluorescence of electronic intersublevel excitations. Our theoretical analysis predicts acoustoluminescence, in particular, a conversion of acoustic into electromagnetic THz waves over a broad spectral range.

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

NASA Astrophysics Data System (ADS)

Cerussi, Albert Edward

1999-09-01

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

NIR-Mediated Nanohybrids of Upconversion Nanophosphors and Fluorescent Conjugated Polymers for High-Efficiency Antibacterial Performance Based on Fluorescence Resonance Energy Transfer.

PubMed

Li, Junting; Zhao, Qi; Shi, Feng; Liu, Chenghui; Tang, Yanli

2016-12-01

A novel nanohybrid comprised of upconversion nanophosphors (UCNPs) and fluorescent conjugated polymers (PFVCN) is rationally fabricated. The new UCNP/PFVCN nanohybrids combine the excellent antibacterial ability of PFVCN and the near IR (NIR) absorbing property of UCNPs, which allows for NIR-mediated antibacterial through the effective fluorescence resonance energy transfer from UCNPs to PFVCN accompanied with generation of reactive oxygen species to kill bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of fluorescence resonance energy transfer in protein studies

PubMed Central

Ma, Linlin; Yang, Fan; Zheng, Jie

2014-01-01

Since the physical process of fluorescence resonance energy transfer (FRET) was elucidated more than six decades ago, this peculiar fluorescence phenomenon has turned into a powerful tool for biomedical research due to its compatibility in scale with biological molecules as well as rapid developments in novel fluorophores and optical detection techniques. A wide variety of FRET approaches have been devised, each with its own advantages and drawbacks. Especially in the last decade or so, we are witnessing a flourish of FRET applications in biological investigations, many of which exemplify clever experimental design and rigorous analysis. Here we review the current stage of FRET methods development with the main focus on its applications in protein studies in biological systems, by summarizing the basic components of FRET techniques, most established quantification methods, as well as potential pitfalls, illustrated by example applications. PMID:25368432

Quantitative high dynamic range beam profiling for fluorescence microscopy

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

Mitchell, T. J., E-mail: t.j.mitchell@dur.ac.uk; Saunter, C. D.; O’Nions, W.

2014-10-15

Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly withinmore » the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences.« less

Quantitative Förster resonance energy transfer analysis for kinetic determinations of SUMO-specific protease.

PubMed

Liu, Yan; Song, Yang; Madahar, Vipul; Liao, Jiayu

2012-03-01

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet-SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases. Copyright © 2011 Elsevier Inc. All rights reserved.

A dark-field microscope for background-free detection of resonance fluorescence from single semiconductor quantum dots operating in a set-and-forget mode

NASA Astrophysics Data System (ADS)

Kuhlmann, Andreas V.; Houel, Julien; Brunner, Daniel; Ludwig, Arne; Reuter, Dirk; Wieck, Andreas D.; Warburton, Richard J.

2013-07-01

Optically active quantum dots, for instance self-assembled InGaAs quantum dots, are potentially excellent single photon sources. The fidelity of the single photons is much improved using resonant rather than non-resonant excitation. With resonant excitation, the challenge is to distinguish between resonance fluorescence and scattered laser light. We have met this challenge by creating a polarization-based dark-field microscope to measure the resonance fluorescence from a single quantum dot at low temperature. We achieve a suppression of the scattered laser exceeding a factor of 107 and background-free detection of resonance fluorescence. The same optical setup operates over the entire quantum dot emission range (920-980 nm) and also in high magnetic fields. The major development is the outstanding long-term stability: once the dark-field point has been established, the microscope operates for days without alignment. The mechanical and optical designs of the microscope are presented, as well as exemplary resonance fluorescence spectroscopy results on individual quantum dots to underline the microscope's excellent performance.

Quantitative fluorescence correlation spectroscopy on DNA in living cells

NASA Astrophysics Data System (ADS)

Hodges, Cameron; Kafle, Rudra P.; Meiners, Jens-Christian

2017-02-01

FCS is a fluorescence technique conventionally used to study the kinetics of fluorescent molecules in a dilute solution. Being a non-invasive technique, it is now drawing increasing interest for the study of more complex systems like the dynamics of DNA or proteins in living cells. Unlike an ordinary dye solution, the dynamics of macromolecules like proteins or entangled DNA in crowded environments is often slow and subdiffusive in nature. This in turn leads to longer residence times of the attached fluorophores in the excitation volume of the microscope and artifacts from photobleaching abound that can easily obscure the signature of the molecular dynamics of interest and make quantitative analysis challenging.We discuss methods and procedures to make FCS applicable to quantitative studies of the dynamics of DNA in live prokaryotic and eukaryotic cells. The intensity autocorrelation is computed function from weighted arrival times of the photons on the detector that maximizes the information content while simultaneously correcting for the effect of photobleaching to yield an autocorrelation function that reflects only the underlying dynamics of the sample. This autocorrelation function in turn is used to calculate the mean square displacement of the fluorophores attached to DNA. The displacement data is more amenable to further quantitative analysis than the raw correlation functions. By using a suitable integral transform of the mean square displacement, we can then determine the viscoelastic moduli of the DNA in its cellular environment. The entire analysis procedure is extensively calibrated and validated using model systems and computational simulations.

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

PubMed

Dardikman, Gili; Nygate, Yoav N; Barnea, Itay; Turko, Nir A; Singh, Gyanendra; Javidi, Barham; Shaked, Natan T

2018-03-01

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

[Investigation of quantitative detection of water quality using spectral fluorescence signature].

PubMed

He, Jun-hua; Cheng, Yong-jin; Han, Yan-ling; Zhang, Hao; Yang, Tao

2008-08-01

A method of spectral analysis, which can simultaneously detect dissolved organic matter (DOM) and chlorophyll a (Chl-a) in natural water, was developed in the present paper with the intention of monitoring water quality fast and quantitatively. Firstly, the total luminescence spectra (TLS) of water sample from East Lake in Wuhan city were measured by the use of laser (532 nm) induced fluorescence (LIF). There were obvious peaks of relative intensity at the wavelength value of 580, 651 and 687 nm in the TLS of the sample, which correspond respectively to spectra of DOM, and the Raman scattering of water and Chl-a in the water. Then the spectral fluorescence signature (SFS) technique was adopted to analyze and distinguish spectral characteristics of DOM and Chl-a in natural water. The calibration curves and function expressions, which indicate the relation between the normalized fluorescence intensities of DOM and Chl-a in water and their concentrations, were obtained respectively under the condition of low concentration(< 40 mg x L(-1))by using normalization of Raman scattering spectrum of water. The curves have a high linearity. When the concentration of the solution with humic acid is large (> 40 mg x L(-1)), the Raman scattering signal is totally absorbed by the molecules of humic acid being on the ground state, so the normalization technique can not be adopted. However the function expression between the concentration of the solution with humic acid and its relative fluorescence peak intensity can be acquired directly with the aid of experiment of fluorescence spectrum. It is concluded that although the expression is non-linearity as a whole, there is a excellent linear relation between the fluorescence intensity and concentration of DOM when the concentration is less than 200 mg x L(-1). The method of measurement based on spectral fluorescence signature technique and the calibration curves gained will have prospects of broad application. It can recognize fast

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

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

Gonda, Kohsuke, E-mail: gonda@med.tohoku.ac.jp; Miyashita, Minoru; Watanabe, Mika

2012-09-28

Highlights: Black-Right-Pointing-Pointer Organic fluorescent material-assembled nanoparticles for IHC were prepared. Black-Right-Pointing-Pointer New nanoparticle fluorescent intensity was 10.2-fold greater than Qdot655. Black-Right-Pointing-Pointer Nanoparticle staining analyzed a wide range of ER expression levels in tissue. Black-Right-Pointing-Pointer Nanoparticle staining enhanced the quantitative sensitivity for ER diagnosis. -- Abstract: The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3 Prime -diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature andmore » substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Resonance fluorescence based two- and three-dimensional atom localization

NASA Astrophysics Data System (ADS)

Wahab, Abdul; Rahmatullah; Qamar, Sajid

2016-06-01

Two- and three-dimensional atom localization in a two-level atom-field system via resonance fluorescence is suggested. For the two-dimensional localization, the atom interacts with two orthogonal standing-wave fields, whereas for the three-dimensional atom localization, the atom interacts with three orthogonal standing-wave fields. The effect of the detuning and phase shifts associated with the corresponding standing-wave fields is investigated. A precision enhancement in position measurement of the single atom can be noticed via the control of the detuning and phase shifts.

5-Aminolevulinic acid-induced protoporphyrin IX fluorescence in meningioma: qualitative and quantitative measurements in vivo.

PubMed

Valdes, Pablo A; Bekelis, Kimon; Harris, Brent T; Wilson, Brian C; Leblond, Frederic; Kim, Anthony; Simmons, Nathan E; Erkmen, Kadir; Paulsen, Keith D; Roberts, David W

2014-03-01

The use of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence has shown promise as a surgical adjunct for maximizing the extent of surgical resection in gliomas. To date, the clinical utility of 5-ALA in meningiomas is not fully understood, with most descriptive studies using qualitative approaches to 5-ALA-PpIX. To assess the diagnostic performance of 5-ALA-PpIX fluorescence during surgical resection of meningioma. ALA was administered to 15 patients with meningioma undergoing PpIX fluorescence-guided surgery at our institution. At various points during the procedure, the surgeon performed qualitative, visual assessments of fluorescence by using the surgical microscope, followed by a quantitative fluorescence measurement by using an intraoperative probe. Specimens were collected at each point for subsequent neuropathological analysis. Clustered data analysis of variance was used to ascertain a difference between groups, and receiver operating characteristic analyses were performed to assess diagnostic capabilities. Red-pink fluorescence was observed in 80% (12/15) of patients, with visible fluorescence generally demonstrating a strong, homogenous character. Quantitative fluorescence measured diagnostically significant PpIX concentrations (cPpIx) in both visibly and nonvisibly fluorescent tissues, with significantly higher cPpIx in both visibly fluorescent (P < .001) and tumor tissue (P = .002). Receiver operating characteristic analyses also showed diagnostic accuracies up to 90% for differentiating tumor from normal dura. ALA-induced PpIX fluorescence guidance is a potential and promising adjunct in accurately detecting neoplastic tissue during meningioma resective surgery. These results suggest a broader reach for PpIX as a biomarker for meningiomas than was previously noted in the literature.

5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence in Meningioma: Qualitative and Quantitative Measurements In Vivo

PubMed Central

Valdes, Pablo A.; Bekelis, Kimon; Harris, Brent T.; Wilson, Brian C.; Leblond, Frederic; Kim, Anthony; Simmons, Nathan E.; Erkmen, Kadir; Paulsen, Keith D.; Roberts, David W.

2014-01-01

BACKGROUND The use of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence has shown promise as a surgical adjunct for maximizing the extent of surgical resection in gliomas. To date, the clinical utility of 5-ALA in meningiomas is not fully understood, with most descriptive studies using qualitative approaches to 5-ALA-PpIX. OBJECTIVE To assess the diagnostic performance of 5-ALA-PpIX fluorescence during surgical resection of meningioma. METHODS ALA was administered to 15 patients with meningioma undergoing PpIX fluorescence-guided surgery at our institution. At various points during the procedure, the surgeon performed qualitative, visual assessments of fluorescence by using the surgical microscope, followed by a quantitative fluorescence measurement by using an intra-operative probe. Specimens were collected at each point for subsequent neuropathological analysis. Clustered data analysis of variance was used to ascertain a difference between groups, and receiver operating characteristic analyses were performed to assess diagnostic capabilities. RESULTS Red-pink fluorescence was observed in 80% (12/15) of patients, with visible fluorescence generally demonstrating a strong, homogenous character. Quantitative fluorescence measured diagnostically significant PpIX concentrations (CPpIx) in both visibly and nonvisibly fluorescent tissues, with significantly higher CPpIx in both visibly fluorescent (P < .001) and tumor tissue (P = .002). Receiver operating characteristic analyses also showed diagnostic accuracies up to 90% for differentiating tumor from normal dura. CONCLUSION ALA-induced PpIX fluorescence guidance is a potential and promising adjunct in accurately detecting neoplastic tissue during meningioma resective surgery. These results suggest a broader reach for PpIX as a biomarker for meningiomas than was previously noted in the literature. PMID:23887194

Simulation-based evaluation of the resolution and quantitative accuracy of temperature-modulated fluorescence tomography.

PubMed

Lin, Yuting; Nouizi, Farouk; Kwong, Tiffany C; Gulsen, Gultekin

2015-09-01

Conventional fluorescence tomography (FT) can recover the distribution of fluorescent agents within a highly scattering medium. However, poor spatial resolution remains its foremost limitation. Previously, we introduced a new fluorescence imaging technique termed "temperature-modulated fluorescence tomography" (TM-FT), which provides high-resolution images of fluorophore distribution. TM-FT is a multimodality technique that combines fluorescence imaging with focused ultrasound to locate thermo-sensitive fluorescence probes using a priori spatial information to drastically improve the resolution of conventional FT. In this paper, we present an extensive simulation study to evaluate the performance of the TM-FT technique on complex phantoms with multiple fluorescent targets of various sizes located at different depths. In addition, the performance of the TM-FT is tested in the presence of background fluorescence. The results obtained using our new method are systematically compared with those obtained with the conventional FT. Overall, TM-FT provides higher resolution and superior quantitative accuracy, making it an ideal candidate for in vivo preclinical and clinical imaging. For example, a 4 mm diameter inclusion positioned in the middle of a synthetic slab geometry phantom (D:40  mm×W:100  mm) is recovered as an elongated object in the conventional FT (x=4.5  mm; y=10.4  mm), while TM-FT recovers it successfully in both directions (x=3.8  mm; y=4.6  mm). As a result, the quantitative accuracy of the TM-FT is superior because it recovers the concentration of the agent with a 22% error, which is in contrast with the 83% error of the conventional FT.

Simulation-based evaluation of the resolution and quantitative accuracy of temperature-modulated fluorescence tomography

PubMed Central

Lin, Yuting; Nouizi, Farouk; Kwong, Tiffany C.; Gulsen, Gultekin

2016-01-01

Conventional fluorescence tomography (FT) can recover the distribution of fluorescent agents within a highly scattering medium. However, poor spatial resolution remains its foremost limitation. Previously, we introduced a new fluorescence imaging technique termed “temperature-modulated fluorescence tomography” (TM-FT), which provides high-resolution images of fluorophore distribution. TM-FT is a multimodality technique that combines fluorescence imaging with focused ultrasound to locate thermo-sensitive fluorescence probes using a priori spatial information to drastically improve the resolution of conventional FT. In this paper, we present an extensive simulation study to evaluate the performance of the TM-FT technique on complex phantoms with multiple fluorescent targets of various sizes located at different depths. In addition, the performance of the TM-FT is tested in the presence of background fluorescence. The results obtained using our new method are systematically compared with those obtained with the conventional FT. Overall, TM-FT provides higher resolution and superior quantitative accuracy, making it an ideal candidate for in vivo preclinical and clinical imaging. For example, a 4 mm diameter inclusion positioned in the middle of a synthetic slab geometry phantom (D:40 mm × W :100 mm) is recovered as an elongated object in the conventional FT (x = 4.5 mm; y = 10.4 mm), while TM-FT recovers it successfully in both directions (x = 3.8 mm; y = 4.6 mm). As a result, the quantitative accuracy of the TM-FT is superior because it recovers the concentration of the agent with a 22% error, which is in contrast with the 83% error of the conventional FT. PMID:26368884

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

PubMed Central

Ghisaidoobe, Amar B. T.; Chung, Sang J.

2014-01-01

Förster resonance energy transfer (FRET) occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (λEX ∼ 280 nm, λEM ∼ 350 nm), in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the protein’s) local environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic Förster resonance energy transfer (iFRET), a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins. PMID:25490136

Isotopic imaging via nuclear resonance fluorescence with laser-based Thomson radiation

DOEpatents

Barty, Christopher P. J. [Hayward, CA; Hartemann, Frederic V [San Ramon, CA; McNabb, Dennis P [Alameda, CA; Pruet, Jason A [Brentwood, CA

2009-07-21

The present invention utilizes novel laser-based, high-brightness, high-spatial-resolution, pencil-beam sources of spectrally pure hard x-ray and gamma-ray radiation to induce resonant scattering in specific nuclei, i.e., nuclear resonance fluorescence. By monitoring such fluorescence as a function of beam position, it is possible to image in either two dimensions or three dimensions, the position and concentration of individual isotopes in a specific material configuration. Such methods of the present invention material identification, spatial resolution of material location and ability to locate and identify materials shielded by other materials, such as, for example, behind a lead wall. The foundation of the present invention is the generation of quasimonochromatic high-energy x-ray (100's of keV) and gamma-ray (greater than about 1 MeV) radiation via the collision of intense laser pulses from relativistic electrons. Such a process as utilized herein, i.e., Thomson scattering or inverse-Compton scattering, produces beams having diameters from about 1 micron to about 100 microns of high-energy photons with a bandwidth of .DELTA.E/E of approximately 10E.sup.-3.

Synthesis, quantitative structure-property relationship study of novel fluorescence active 2-pyrazolines and application.

PubMed

Girgis, Adel S; Basta, Altaf H; El-Saied, Houssni; Mohamed, Mohamed A; Bedair, Ahmad H; Salim, Ahmad S

2018-03-01

A variety of fluorescence-active fluorinated pyrazolines 13-33 was synthesized in good yields through cyclocondensation reaction of propenones 1-9 with aryl hydrazines 10-12 . Some of the synthesized compounds provided promising fluorescence properties with quantum yield ( Φ ) higher than that of quinine sulfate (standard reference). Quantitative structure-property relationship studies were undertaken supporting the exhibited fluorescence properties and estimating the parameters governing properties. Five synthesized fluorescence-active pyrazolines ( 13 , 15 , 18 , 19 and 23 ) with variable Φ were selected for treating two types of paper sheets (Fabriano and Bible paper). These investigated fluorescence compounds, especially compounds 19 and 23 , provide improvements in strength properties of paper sheets. Based on the observed performance they can be used as markers in security documents.

Synthesis, quantitative structure-property relationship study of novel fluorescence active 2-pyrazolines and application

NASA Astrophysics Data System (ADS)

Girgis, Adel S.; Basta, Altaf H.; El-Saied, Houssni; Mohamed, Mohamed A.; Bedair, Ahmad H.; Salim, Ahmad S.

2018-03-01

A variety of fluorescence-active fluorinated pyrazolines 13-33 was synthesized in good yields through cyclocondensation reaction of propenones 1-9 with aryl hydrazines 10-12. Some of the synthesized compounds provided promising fluorescence properties with quantum yield (Φ) higher than that of quinine sulfate (standard reference). Quantitative structure-property relationship studies were undertaken supporting the exhibited fluorescence properties and estimating the parameters governing properties. Five synthesized fluorescence-active pyrazolines (13, 15, 18, 19 and 23) with variable Φ were selected for treating two types of paper sheets (Fabriano and Bible paper). These investigated fluorescence compounds, especially compounds 19 and 23, provide improvements in strength properties of paper sheets. Based on the observed performance they can be used as markers in security documents.

Surface Plasmon Resonance Sensors on Raman and Fluorescence Spectroscopy

PubMed Central

Wang, Jiangcai; Lin, Weihua; Cao, En; Xu, Xuefeng; Liang, Wenjie; Zhang, Xiaofang

2017-01-01

The performance of chemical reactions has been enhanced immensely with surface plasmon resonance (SPR)-based sensors. In this review, the principle and application of SPR sensors are introduced and summarized thoroughly. We introduce the mechanism of the SPR sensors and present a thorough summary about the optical design, including the substrate and excitation modes of the surface plasmons. Additionally, the applications based on SPR sensors are described by the Raman and fluorescence spectroscopy in plasmon-driven surface catalytic reactions and the measurement of refractive index sensing, especially. PMID:29212139

Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dong-Shan; Zhou, Guo-Hua; Luo, Ming; Ji, Xing-Hu; He, Zhi-Ke

2012-08-21

We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.

Towards Violation of Classical Inequalities using Quantum Dot Resonance Fluorescence

NASA Astrophysics Data System (ADS)

Peiris, Manoj

Self-assembled semiconductor quantum dots have attracted considerable interest recently, ranging from fundamental studies of quantum optics to advanced applications in the field of quantum information science. With their atom-like properties, quantum dot based nanophotonic devices may also substantially contribute to the development of quantum computers. This work presents experimental progress towards the understanding of light-matter interactions that occur beyond well-understood monochromatic resonant light scattering processes in semiconductor quantum dots. First, we report measurements of resonance fluorescence under bichromatic laser excitation. With the inclusion of a second laser, both first-order and second-order correlation functions are substantially altered. Under these conditions, the scattered light exhibits a rich spectrum containing many spectral features that lead to a range of nonlinear multiphoton dynamics. These observations are discussed and compared with a theoretical model. Second, we investigated the light scattered by a quantum dot in the presence of spectral filtering. By scanning the tunable filters placed in front of each detector of a Hanbury-Brown and Twiss setup and recording coincidence measurements, a \\two-photon spectrum" has been experimentally reconstructed for the first time. The two-photon spectrum contains a wealth of information about the cascaded emission involved in the scattering process, such as transitions occurring via virtual intermediate states. Our measurements also reveal that the scattered frequency-filtered light from a quantum dot violates the Cauchy-Schwarz inequality. Finally, Franson-interferometry has been performed using spectrally filtered light from quantum dot resonance fluorescence. Visibilities exceeding the classical limit were demonstrated by using a pair of folded Mach-Zehnder interferometers, paving the way for producing single time-energy entangled photon pairs that could violate Bell

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.

Estimating background-subtracted fluorescence transients in calcium imaging experiments: a quantitative approach.

PubMed

Joucla, Sébastien; Franconville, Romain; Pippow, Andreas; Kloppenburg, Peter; Pouzat, Christophe

2013-08-01

Calcium imaging has become a routine technique in neuroscience for subcellular to network level investigations. The fast progresses in the development of new indicators and imaging techniques call for dedicated reliable analysis methods. In particular, efficient and quantitative background fluorescence subtraction routines would be beneficial to most of the calcium imaging research field. A background-subtracted fluorescence transients estimation method that does not require any independent background measurement is therefore developed. This method is based on a fluorescence model fitted to single-trial data using a classical nonlinear regression approach. The model includes an appropriate probabilistic description of the acquisition system's noise leading to accurate confidence intervals on all quantities of interest (background fluorescence, normalized background-subtracted fluorescence time course) when background fluorescence is homogeneous. An automatic procedure detecting background inhomogeneities inside the region of interest is also developed and is shown to be efficient on simulated data. The implementation and performances of the proposed method on experimental recordings from the mouse hypothalamus are presented in details. This method, which applies to both single-cell and bulk-stained tissues recordings, should help improving the statistical comparison of fluorescence calcium signals between experiments and studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Synthesis, quantitative structure–property relationship study of novel fluorescence active 2-pyrazolines and application

PubMed Central

Girgis, Adel S.; El-Saied, Houssni; Mohamed, Mohamed A.; Bedair, Ahmad H.; Salim, Ahmad S.

2018-01-01

A variety of fluorescence-active fluorinated pyrazolines 13–33 was synthesized in good yields through cyclocondensation reaction of propenones 1–9 with aryl hydrazines 10–12. Some of the synthesized compounds provided promising fluorescence properties with quantum yield (Φ) higher than that of quinine sulfate (standard reference). Quantitative structure–property relationship studies were undertaken supporting the exhibited fluorescence properties and estimating the parameters governing properties. Five synthesized fluorescence-active pyrazolines (13, 15, 18, 19 and 23) with variable Φ were selected for treating two types of paper sheets (Fabriano and Bible paper). These investigated fluorescence compounds, especially compounds 19 and 23, provide improvements in strength properties of paper sheets. Based on the observed performance they can be used as markers in security documents. PMID:29657796

Evaluation of resin infiltration using quantitative light-induced fluorescence technology.

PubMed

Min, Ji-Hyun; Inaba, Daisuke; Kim, Baek-Il

2016-09-01

To determine whether quantitative light-induced fluorescence (QLF) technology can be used to classify the colour of teeth specimens before and after resin infiltration (RI) treatment, and calculate the correlation between the ΔF value and colour difference (ΔE) in fluorescence images of the specimens obtained using a QLF-digital (QLF-D) device. Sixty sound bovine permanent teeth specimens were immersed in demineralized solution. Two exposed windows were formed in each specimen, and RI treatment was applied to one of them. The ΔE values were obtained for the differences between a sound tooth surface (SS), an early dental caries surface (ECS) and an ECS treated with RI (RS) in white-light and fluorescence images obtained using QLF-D, respectively. The ΔF value was obtained from fluorescence images using dedicated software for QLF-D. The mean differences between the ΔE values obtained from the white-light and fluorescence images were analyzed by paired t-test. Pearson correlation analysis and Bland-Altman plots were applied to the differences between the ΔF value for ECS (ΔFSS-ECS) and the ΔE value between SS and ECS (ΔESS-ECS), and between the ΔF value for RS (ΔFSS-RS) and the ΔE value between SS and RS (ΔESS-RS) in fluorescence images. The ΔE values obtained from fluorescence images were three times higher than the ΔE values obtained from white-light images (p<0.001). Significant correlations were confirmed between ΔESS-ECS and ΔFSS-ECS (r=-0.492, p<0.001) and between ΔESS-RS and ΔFSS-RS (r=-0.661, p<0.001). QLF technology can be used to confirm the presence of RI in teeth. Copyright © 2016 Elsevier B.V. All rights reserved.

Nuclear Resonance Fluorescence Measurements of High Explosives

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

Caggiano, Joseph A.; Warren, Glen A.; Korbly, Steve

Pacific Northwest National Laboratory and Passport Systems have collaborated to perform Nuclear Resonance Fluorescence experiments using several high quality high-explosive simulant samples. These measurements were conducted to determine the feasibility of finding and characterizing high explosive material by NRF interrogation. Electron beams of 5.1, 5.3, 8, and 10 MeV were used to produce bremsstrahlung photon beams, which irradiated the samples. The gamma-ray spectra were collected using high-purity germanium detectors. Nitrogen-to-carbon ratios of the high-explosive simulants were extracted from the 5.1 and 5.3 MeV data and compare favorably with accepted values. Analysis of the 8 and 10 MeV data is inmore » progress; preliminary isotopic comparisons within the samples are consistent with the expected results.« less

Synthesis and characterization of novel pyrene-dendronized porphyrins exhibiting efficient fluorescence resonance energy transfer: optical and photophysical properties.

PubMed

Zaragoza-Galán, Gerardo; Fowler, Michael A; Duhamel, Jean; Rein, Regis; Solladié, Nathalie; Rivera, Ernesto

2012-07-31

A novel series of pyrene dendronized porphyrins bearing two and four pyrenyl groups (Py(2)-TMEG1 and Py(4)-TMEG2) were successfully synthesized. First and second generation Fréchet type dendrons (Py(2)-G1OH and Py(4)-G2OH) were prepared from 1-pyrenylbutanol and 3,5-dihydroxybenzyl alcohol. These compounds were further linked to a trimesitylphenylporphyrin containing a butyric acid spacer via an esterification reaction to obtain the desired products. Dendrons and dendronized porphyrins were fully characterized by FTIR and (1)H NMR spectroscopy and their molecular weights were determined by matrix-assisted laser desorption ionization time of flight mass spectrometry. Their optical and photophysical properties were studied by absorption and fluorescence spectroscopies. The formation of dynamic excimers was detected in the pyrene-labeled dendrons, with more excimer being produced in the higher generation dendron. The fluorescence spectra of the pyrene dendronized porphyrins exhibited a significant decrease in the amount of pyrene monomer and excimer emission, jointly with the appearance of a new emission band at 661 nm characteristic of porphyrin emission, an indication that fluorescence resonance energy transfer (FRET) occurred from one of the excited pyrene species to the porphyrin. The FRET efficiency was found to be almost quantitative ranging between 97% and 99% depending on the construct. Model Free analysis of the fluorescence decays acquired with the pyrene monomer, excimer, and porphyrin core established that only residual pyrene excimer formation in the dendrons could occur before FRET from the excited pyrene monomer to the ground-state porphyrin core.

Detector Array Performance Estimates for Nuclear Resonance Fluorescence Applications

NASA Astrophysics Data System (ADS)

Johnson, Micah; Hall, J. M.; McNabb, D. P.

2012-10-01

There are a myriad of explorative efforts underway at several institutions to determine the feasibility of using photonuclear reactions to detect and assay materials of varying complexity and compositions. One photonuclear process that is being explored for several applications is nuclear resonance fluorescence (NRF). NRF is interesting because the resonant lines are unique to each isotope and the widths are sufficiently narrow and the level densities are sufficiently low so as to not cause interference. Therefore, NRF provides a means to isoptically map containers and materials. The choice of detector array is determined by the application and the source. We will present results from a variety of application studies of an assortment of detector arrays that may be useful. Our results stem from simulation and modeling exercises and benchmarking measurements. We will discuss the data requirements from basic scientific research that enables these application studies. We will discuss our results and the future outlook of this technology.

[Fluorescence Resonance Energy Transfer Detection of Cobalt Ions by Silver Triangular Nanoplates and Rhodamine 6G].

PubMed

Zhang, Xiu-qing; Peng, Jun; Ling, Jian; Liu, Chao-juan; Cao, Qiu-e; Ding, Zhong-tao

2015-04-01

In the present paper, the authors studied fluorescence resonance energy transfer (FRET) phenomenon between silver triangular nanoplates and bovine serum albumin (BSA)/Rhodamine 6G fluorescence complex, and established a fluorescence method for the detection of cobalt ions. We found that when increasing the silver triangular nanoplates added to certain concentrations of fluorescent bovine serum albumin (BSA)/Rhodamine 6G complex, the fluorescence of Rhodamine 6G would be quenched up to 80% due to the FRET between the quencher and donor. However, in the presence of cobalt ions, the disassociation of the fluorescent complex from silver triangular nanoplates occurred and the fluorescence of the Rhodamine 6G recovered. The recovery of fluorescence intensity rate (I/I0) has a good relationship with the cobalt ion concentration (cCO2+) added. Thus, the authors developed a fluorescence method for the detection of cobalt ions based on the FRET of silver triangular nanoplates and Rhodamine 6G.

Maximizing the quantitative accuracy and reproducibility of Förster resonance energy transfer measurement for screening by high throughput widefield microscopy

PubMed Central

Schaufele, Fred

2013-01-01

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) provides insights into the proximities and orientations of FPs as surrogates of the biochemical interactions and structures of the factors to which the FPs are genetically fused. As powerful as FRET methods are, technical issues have impeded their broad adoption in the biologic sciences. One hurdle to accurate and reproducible FRET microscopy measurement stems from variable fluorescence backgrounds both within a field and between different fields. Those variations introduce errors into the precise quantification of fluorescence levels on which the quantitative accuracy of FRET measurement is highly dependent. This measurement error is particularly problematic for screening campaigns since minimal well-to-well variation is necessary to faithfully identify wells with altered values. High content screening depends also upon maximizing the numbers of cells imaged, which is best achieved by low magnification high throughput microscopy. But, low magnification introduces flat-field correction issues that degrade the accuracy of background correction to cause poor reproducibility in FRET measurement. For live cell imaging, fluorescence of cell culture media in the fluorescence collection channels for the FPs commonly used for FRET analysis is a high source of background error. These signal-to-noise problems are compounded by the desire to express proteins at biologically meaningful levels that may only be marginally above the strong fluorescence background. Here, techniques are presented that correct for background fluctuations. Accurate calculation of FRET is realized even from images in which a non-flat background is 10-fold higher than the signal. PMID:23927839

Quantitative fluorescence using 5-aminolevulinic acid–induced protoporphyrin IX biomarker as a surgical adjunct in low-grade glioma surgery

PubMed Central

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

2015-01-01

OBJECT Previous studies in high-grade gliomas (HGGs) have indicated that protoporphyrin IX (PpIX) accumulates in higher concentrations in tumor tissue, and, when used to guide surgery, it has enabled improved resection leading to increased progression-free survival. Despite the benefits of complete resection and the advances in fluorescence-guided surgery, few studies have investigated the use of PpIX in low-grade gliomas (LGGs). Here, the authors describe their initial experience with 5-aminolevulinic acid (ALA)–induced PpIX fluorescence in a series of patients with LGG. METHODS Twelve patients with presumed LGGs underwent resection of their tumors after receiving 20 μg/kg of ALA approximately 3 hours prior to surgery under an institutional review board–approved protocol. Intraoperative assessments of the resulting PpIX emissions using both qualitative, visible fluorescence and quantitative measurements of PpIX concentration were obtained from tissue locations that were subsequently biopsied and evaluated histopathologically. Mixed models for random effects and receiver operating characteristic curve analysis for diagnostic performance were performed on the fluorescence data relative to the gold-standard histopathology. RESULTS Five of the 12 LGGs (1 ganglioglioma, 1 oligoastrocytoma, 1 pleomorphic xanthoastrocytoma, 1 oligodendroglioma, and 1 ependymoma) demonstrated at least 1 instance of visible fluorescence during surgery. Visible fluorescence evaluated on a specimen-by-specimen basis yielded a diagnostic accuracy of 38.0% (cutoff threshold: visible fluorescence score ≥ 1, area under the curve = 0.514). Quantitative fluorescence yielded a diagnostic accuracy of 67% (for a cutoff threshold of the concentration of PpIX [CPpIX] > 0.0056 μg/ml, area under the curve = 0.66). The authors found that 45% (9/20) of nonvisibly fluorescent tumor specimens, which would have otherwise gone undetected, accumulated diagnostically significant levels of CPpIX that were

Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals.

PubMed

Okamura, Yukio; Watanabe, Yuichiro

2006-01-01

Fluorescence resonance energy transfer (FRET) occurs when two fluorophores are in close proximity, and the emission energy of a donor fluorophore is transferred to excite an acceptor fluorophore. Using such fluorescently labeled oligonucleotides as FRET probes, makes possible specific detection of RNA molecules even if similar sequences are present in the environment. A higher ratio of signal to background fluorescence is required for more sensitive probe detection. We found that double-labeled donor probes labeled with BODIPY dye resulted in a remarkable increase in fluorescence intensity compared to single-labeled donor probes used in conventional FRET. Application of this double-labeled donor system can improve a variety of FRET techniques.

Micro-RNA detection based on fluorescence resonance energy transfer of DNA-carbon quantum dots probes.

PubMed

Khakbaz, Faeze; Mahani, Mohamad

2017-04-15

Carbon quantum dots have been proposed as an effective platform for miRNA detection. Carbon dots were synthesized by citric acid. The synthesized dots were characterized by dynamic light scattering, UV-Vis spectrophotometry, spectrofluorimetry, transmission electron microscopy and FT-IR spectrophotometry. The fluorescence quantum yield of the synthesized dots was determined using quinine sulfate as the standard. The FAM-labeled single stranded DNA, as sensing element, was adsorbed on dots by π-π interaction. The quenching of the dots fluorescence due to fluorescence resonance energy transfer (FRET) was used for mir 9-1 detection. In the presence of the complementary miRNA, the FRET did not take place and the fluorescence was recovered. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2018-05-25

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

Design and fabrication of fluorescence resonance energy transfer-mediated fluorescent polymer nanoparticles for ratiometric sensing of lysosomal pH.

PubMed

Chen, Jian; Tang, Ying; Wang, Hong; Zhang, Peisheng; Li, Ya; Jiang, Jianhui

2016-12-15

The design of effective tools capable of sensing lysosome pH is highly desirable for better understanding its biological functions in cellular behaviors and various diseases. Herein, a lysosome-targetable ratiometric fluorescent polymer nanoparticle pH sensor (RFPNS) was synthesized via incorporation of miniemulsion polymerization and surface modification technique. In this system, the donor: 4-ethoxy-9-allyl-1,8-naphthalimide (EANI) and the acceptor: fluorescein isothiocyanate (FITC) were covalently linked to the polymer nanoparticle to construct pH-responsive fluorescence resonance energy transfer (FRET) system. The FITC moieties on the surface of RFPNS underwent structural and spectral transformation as the presence of pH changes, resulting in ratiometric fluorescent sensing of pH. The as-prepared RFPNS displayed favorable water dispersibility, good pH-induced spectral reversibility and so on. Following the living cell uptake, the as-prepared RFPNS with good cell-membrane permeability can mainly stain in the lysosomes; and it can facilitate visualization of the intracellular lysosomal pH changes. This nanosensor platform offers a novel method for future development of ratiometric fluorescent probes for targeting other analytes, like ions, metabolites,and other biomolecules in biosamples. Copyright © 2016 Elsevier Inc. All rights reserved.

Numerical analysis of quantitative measurement of hydroxyl radical concentration using laser-induced fluorescence in flame

NASA Astrophysics Data System (ADS)

Shuang, Chen; Tie, Su; Yao-Bang, Zheng; Li, Chen; Ting-Xu, Liu; Ren-Bing, Li; Fu-Rong, Yang

2016-06-01

The aim of the present work is to quantitatively measure the hydroxyl radical concentration by using LIF (laser-induced fluorescence) in flame. The detailed physical models of spectral absorption lineshape broadening, collisional transition and quenching at elevated pressure are built. The fine energy level structure of the OH molecule is illustrated to understand the process with laser-induced fluorescence emission and others in the case without radiation, which include collisional quenching, rotational energy transfer (RET), and vibrational energy transfer (VET). Based on these, some numerical results are achieved by simulations in order to evaluate the fluorescence yield at elevated pressure. These results are useful for understanding the real physical processes in OH-LIF technique and finding a way to calibrate the signal for quantitative measurement of OH concentration in a practical combustor. Project supported by the National Natural Science Foundation of China (Grant No. 11272338) and the Fund from the Science and Technology on Scramjet Key Laboratory, China (Grant No. STSKFKT2013004).

A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2

PubMed Central

Xi, Gaina; Wang, Xiaoping; Chen, Tongsheng

2016-01-01

A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02–0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe. PMID:27143876

A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2.

PubMed

Xi, Gaina; Wang, Xiaoping; Chen, Tongsheng

2016-01-01

A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02-0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe.

Structured illumination multimodal 3D-resolved quantitative phase and fluorescence sub-diffraction microscopy

PubMed Central

Chowdhury, Shwetadwip; Eldridge, Will J.; Wax, Adam; Izatt, Joseph A.

2017-01-01

Sub-diffraction resolution imaging has played a pivotal role in biological research by visualizing key, but previously unresolvable, sub-cellular structures. Unfortunately, applications of far-field sub-diffraction resolution are currently divided between fluorescent and coherent-diffraction regimes, and a multimodal sub-diffraction technique that bridges this gap has not yet been demonstrated. Here we report that structured illumination (SI) allows multimodal sub-diffraction imaging of both coherent quantitative-phase (QP) and fluorescence. Due to SI’s conventionally fluorescent applications, we first demonstrate the principle of SI-enabled three-dimensional (3D) QP sub-diffraction imaging with calibration microspheres. Image analysis confirmed enhanced lateral and axial resolutions over diffraction-limited QP imaging, and established striking parallels between coherent SI and conventional optical diffraction tomography. We next introduce an optical system utilizing SI to achieve 3D sub-diffraction, multimodal QP/fluorescent visualization of A549 biological cells fluorescently tagged for F-actin. Our results suggest that SI has a unique utility in studying biological phenomena with significant molecular, biophysical, and biochemical components. PMID:28663887

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

PubMed Central

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

2016-01-01

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

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

NASA Astrophysics Data System (ADS)

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

2016-09-01

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

On-chip transduction of nucleic acid hybridization using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

PubMed

Tavares, Anthony J; Noor, M Omair; Vannoy, Charles H; Algar, W Russ; Krull, Ulrich J

2012-01-03

The glass surface of a glass-polydimethylsiloxane (PDMS) microfluidic channel was modified to develop a solid-phase assay for quantitative determination of nucleic acids. Electroosmotic flow (EOF) within channels was used to deliver and immobilize semiconductor quantum dots (QDs), and electrophoresis was used to decorate the QDs with oligonucleotide probe sequences. These processes took only minutes to complete. The QDs served as energy donors in fluorescence resonance energy transfer (FRET) for transduction of nucleic acid hybridization. Electrokinetic injection of fluorescent dye (Cy3) labeled oligonucleotide target into a microfluidic channel and subsequent hybridization (within minutes) provided the proximity for FRET, with emission from Cy3 being the analytical signal. The quantification of target concentration was achieved by measurement of the spatial length of coverage by target along a channel. Detection of femtomole quantities of target was possible with a dynamic range spanning an order of magnitude. The assay provided excellent resistance to nonspecific interactions of DNA. Further selectivity of the assay was achieved using 20% formamide, which allowed discrimination between a fully complementary target and a 3 base pair mismatch target at a contrast ratio of 4:1. © 2011 American Chemical Society

Synthesis of Water-Dispersible Mn2+ Functionalized Silicon Nanoparticles under Room Temperature and Atmospheric Pressure for Fluorescence and Magnetic Resonance Dual-Modality Imaging.

PubMed

Dou, Ya-Kun; Chen, Yang; He, Xi-Wen; Li, Wen-You; Li, Yu-Hao; Zhang, Yu-Kui

2017-11-07

Silicon nanoparticles (Si NPs) have been widely used in fluorescence imaging. However, rigorous synthesis conditions and the single modality imaging limit the further development of Si NPs in the field of biomedical imaging. Here, we reported a method for synthesizing water-dispersible Mn 2+ functionalized Si NPs (Mn-Si NPs) under mild experimental conditions for fluorescence and magnetic resonance dual-modality imaging. The whole synthesis process was completed under room temperature and atmospheric pressure, and no special and expensive equipment was required. The synthetic nanoparticles, with favorable pH stability, NaCl stability, photostability, and low toxicity, emitted green fluorescence (512 nm). At the same time, the nanoparticles also demonstrated excellent magnetic resonance imaging ability. In vitro, their T 1 -weighted magnetic resonance imaging effect was obvious, and the value of longitudinal relaxation degree r 1 reached 4.25 mM -1 s -1 . On the basis of their good biocompatibility, Mn-Si NPs were successfully used for the fluorescence imaging as well as magnetic resonance imaging in vivo.

Photon-number statistics in resonance fluorescence

NASA Astrophysics Data System (ADS)

Lenstra, D.

1982-12-01

The theory of photon-number statistics in resonance fluorescence is treated, starting with the general formula for the emission probability of n photons during a given time interval T. The results fully confirm formerly obtained results by Cook that were based on the theory of atomic motion in a traveling wave. General expressions for the factorial moments are derived and explicit results for the mean and the variance are given. It is explicitly shown that the distribution function tends to a Gaussian when T becomes much larger than the natural lifetime of the excited atom. The speed of convergence towards the Gaussian is found to be typically slow, that is, the third normalized central moment (or the skewness) is proportional to T-12. However, numerical results illustrate that the overall features of the distribution function are already well represented by a Gaussian when T is larger than a few natural lifetimes only, at least if the intensity of the exciting field is not too small and its detuning is not too large.

Upconversion nanoparticle-based fluorescence resonance energy transfer assay for organophosphorus pesticides.

PubMed

Long, Qian; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo

2015-06-15

This paper reports a novel nanosensor for organophosphorus pesticides based on the fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The detection mechanism is based on the facts that AuNPs quench the fluorescence of UCNPs and organophosphorus pesticides (OPs) inhibit the activity of acetylcholinesterase (AChE) which catalyzes the hydrolysis of acetylthiocholine (ATC) into thiocholine. Under the optimized conditions, the logarithm of the pesticides concentration was proportional to the inhibition efficiency. The detection limits of parathion-methyl, monocrotophos and dimethoate reached 0.67, 23, and 67 ng/L, respectively. Meanwhile, the biosensor shows good sensitivity, stability, and could be successfully applied to detection of OPs in real food samples, suggesting the biosensor has potentially extensive application clinic diagnoses assays. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed Central

2012-01-01

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

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.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

NASA Astrophysics Data System (ADS)

Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

2017-03-01

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands.

PubMed

He, Wei; Kularatne, Sumith A; Kalli, Kimberly R; Prendergast, Franklyn G; Amato, Robert J; Klee, George G; Hartmann, Lynn C; Low, Philip S

2008-10-15

Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients. A CTC-enriched fraction was isolated from the peripheral blood of ovarian and prostate cancer patients by an optimized density gradient centrifugation protocol and labeled with the aforementioned fluorescent ligands. CTCs were then quantitated by flow cytometry. CTCs were detected in 18 of 20 ovarian cancer patients (mean 222 CTCs/ml; median 15 CTCs/ml; maximum 3,118 CTCs/ml), whereas CTC numbers in 16 gender-matched normal volunteers were negligible (mean 0.4 CTCs/ml; median 0.3 CTCs/ml; maximum 1.5 CTCs/ml; p < 0.001, chi(2)). CTCs were also detected in 10 of 13 prostate cancer patients (mean 26 CTCs/ml, median 14 CTCs/ml, maximum 94 CTCs/ml) but not in 18 gender-matched healthy donors (mean 0.8 CTCs/ml, median 1, maximum 3 CTC/ml; p < 0.0026, chi(2)). Tumor-specific fluorescent antibodies were much less efficient in quantitating CTCs because of their lower CTC labeling efficiency. Use of tumor-specific fluorescent ligands to label CTCs in peripheral blood can provide a simple, accurate and sensitive method for determining the number of cancer cells circulating in the bloodstream.

Prenatal diagnosis of i(18q) and dup(18q) cases by quantitative fluorescent PCR

PubMed Central

Castro-Volio, Isabel; Ortíz-Morales, Fernando; Valle-Bourrouet, Luisa; Malespín-Bendaña, Wendy

2013-01-01

Particular sonographic fetal malformations are common in chromosome 18 aberrations, requiring invasive prenatal tests to confirm the diagnosis. Karyotyping is the gold standard assay in these cases, although it is a high complexity, expensive and approximately 2 weeks turnaround time test. On the contrary, quantitative fluorescent PCR is considered an accurate, simple, low cost and rapid assay, particularly useful for the diagnosis of aneuploidies of chromosomes 13, 18 and 21 and for the detection of maternal cell contamination of the sample. Clinical presentation of two cases of rare chromosome 18 defects, diagnosed using both techniques. One case was an isochromosome and the other was a partial duplication. Quantitative fluorescent PCR was an invaluable tool for the cytogenetics laboratory PMID:24045756

Culture-free, highly sensitive, quantitative detection of bacteria from minimally processed samples using fluorescence imaging by smartphone.

PubMed

Shrivastava, Sajal; Lee, Won-Il; Lee, Nae-Eung

2018-06-30

A critical unmet need in the diagnosis of bacterial infections, which remain a major cause of human morbidity and mortality, is the detection of scarce bacterial pathogens in a variety of samples in a rapid and quantitative manner. Herein, we demonstrate smartphone-based detection of Staphylococcus aureus in a culture-free, rapid, quantitative manner from minimally processed liquid samples using aptamer-functionalized fluorescent magnetic nanoparticles. The tagged S. aureus cells were magnetically captured in a detection cassette, and then fluorescence was imaged using a smartphone camera with a light-emitting diode as the excitation source. Our results showed quantitative detection capability with a minimum detectable concentration as low as 10 cfu/ml by counting individual bacteria cells, efficiently capturing S. aureus cells directly from a peanut milk sample within 10 min. When the selectivity of detection was investigated using samples spiked with other pathogenic bacteria, no significant non-specific detection occurred. Furthermore, strains of S. aureus from various origins showed comparable results, ensuring that the approach can be widely adopted. Therefore, the quantitative fluorescence imaging platform on a smartphone could allow on-site detection of bacteria, providing great potential assistance during major infectious disease outbreaks in remote and resource-limited settings. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection of proximal caries using quantitative light-induced fluorescence-digital and laser fluorescence: a comparative study.

PubMed

Yoon, Hyung-In; Yoo, Min-Jeong; Park, Eun-Jin

2017-12-01

The purpose of this study was to evaluate the in vitro validity of quantitative light-induced fluorescence-digital (QLF-D) and laser fluorescence (DIAGNOdent) for assessing proximal caries in extracted premolars, using digital radiography as reference method. A total of 102 extracted premolars with similar lengths and shapes were used. A single operator conducted all the examinations using three different detection methods (bitewing radiography, QLF-D, and DIAGNOdent). The bitewing x-ray scale, QLF-D fluorescence loss (ΔF), and DIAGNOdent peak readings were compared and statistically analyzed. Each method showed an excellent reliability. The correlation coefficient between bitewing radiography and QLF-D, DIAGNOdent were -0.644 and 0.448, respectively, while the value between QLF-D and DIAGNOdent was -0.382. The kappa statistics for bitewing radiography and QLF-D had a higher diagnosis consensus than those for bitewing radiography and DIAGNOdent. The QLF-D was moderately to highly accurate (AUC = 0.753 - 0.908), while DIAGNOdent was moderately to less accurate (AUC = 0.622 - 0.784). All detection methods showed statistically significant correlation and high correlation between the bitewing radiography and QLF-D. QLF-D was found to be a valid and reliable alternative diagnostic method to digital bitewing radiography for in vitro detection of proximal caries.

Quantitative method to assess caries via fluorescence imaging from the perspective of autofluorescence spectral analysis

NASA Astrophysics Data System (ADS)

Chen, Q. G.; Zhu, H. H.; Xu, Y.; Lin, B.; Chen, H.

2015-08-01

A quantitative method to discriminate caries lesions for a fluorescence imaging system is proposed in this paper. The autofluorescence spectral investigation of 39 teeth samples classified by the International Caries Detection and Assessment System levels was performed at 405 nm excitation. The major differences in the different caries lesions focused on the relative spectral intensity range of 565-750 nm. The spectral parameter, defined as the ratio of wavebands at 565-750 nm to the whole spectral range, was calculated. The image component ratio R/(G + B) of color components was statistically computed by considering the spectral parameters (e.g. autofluorescence, optical filter, and spectral sensitivity) in our fluorescence color imaging system. Results showed that the spectral parameter and image component ratio presented a linear relation. Therefore, the image component ratio was graded as <0.66, 0.66-1.06, 1.06-1.62, and >1.62 to quantitatively classify sound, early decay, established decay, and severe decay tissues, respectively. Finally, the fluorescence images of caries were experimentally obtained, and the corresponding image component ratio distribution was compared with the classification result. A method to determine the numerical grades of caries using a fluorescence imaging system was proposed. This method can be applied to similar imaging systems.

Resonance fluorescence microscopy via three-dimensional atom localization

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Real-time quantitative fluorescence measurement of microscale cell culture analog systems

NASA Astrophysics Data System (ADS)

Oh, Taek-il; Kim, Donghyun; Tatosian, Daniel; Sung, Jong Hwan; Shuler, Michael

2007-02-01

A microscale cell culture analog (μCCA) is a cell-based lab-on-a-chip assay that, as an animal surrogate, is applied to pharmacological studies for toxicology tests. A μCCA typically comprises multiple chambers and microfluidics that connect the chambers, which represent animal organs and blood flow to mimic animal metabolism more realistically. A μCCA is expected to provide a tool for high-throughput drug discovery. Previously, a portable fluorescence detection system was investigated for a single μCCA device in real-time. In this study, we present a fluorescence-based imaging system that provides quantitative real-time data of the metabolic interactions in μCCAs with an emphasis on measuring multiple μCCA samples simultaneously for high-throughput screening. The detection system is based on discrete optics components, with a high-power LED and a charge-coupled device (CCD) camera as a light source and a detector, for monitoring cellular status on the chambers of each μCCA sample. Multiple samples are characterized mechanically on a motorized linear stage, which is fully-automated. Each μCCA sample has four chambers, where cell lines MES-SA/DX- 5, and MES-SA (tumor cells of human uterus) have been cultured. All cell-lines have been transfected to express the fusion protein H2B-GFP, which is a human histone protein fused at the amino terminus to EGFP. As a model cytotoxic drug, 10 μM doxorubicin (DOX) was used. Real-time quantitative data of the intensity loss of enhanced green fluorescent protein (EGFP) during cell death of target cells have been collected over several minutes to 40 hours. Design issues and improvements are also discussed.

Nuclear Resonance Fluorescence off 54Cr: The Onset of the Pygmy Dipole Resonance

NASA Astrophysics Data System (ADS)

Ries, P. C.; Beck, T.; Beller, J.; Krishichayan; Gayer, U.; Isaak, J.; Löher, B.; Mertes, L.; Pai, H.; Pietralla, N.; Romig, C.; Savran, D.; Schilling, M.; Tornow, W.; Werner, V.; Zweidinger, M.

2016-06-01

Low-lying electric and magnetic dipole excitations (E1 and M1) below the neutron separation threshold, particularly the Pygmy Dipole Resonance (PDR), have drawn considerable attention in the last years. So far, mostly moderately heavy nuclei in the mass regions around A = 90 and A = 140 were examined with respect to the PDR. In the present work, the systematics of the PDR have been extended by measuring excitation strengths and parity quantum numbers of J = 1 states in lighter nuclei near A = 50 in order to gather information on the onset of the PDR. The nuclei 50,52,54Cr and 48,50Ti were examined via bremsstrahlung produced at the DArmstadt Superconducting electron Linear Accelerator (S-DALINAC) with photon energies up to 9.7 MeV with the method of nuclear resonance fluorescence. Numerous excited states were observed, many of which for the first time. The parity quantum numbers of these states have been determined at the High Intensity Gamma-ray Source (HIγS) of the Triangle Universities Nuclear Laboratory in Durham, NC, USA. Informations to the methods and the experimental setups will be provided and the results on 54Cr achieved will be discussed with respect to the onset of the PDR.

Advances in Spiropyrans/Spirooxazines and Applications Based on Fluorescence Resonance Energy Transfer (FRET) with Fluorescent Materials.

PubMed

Xia, Hongyan; Xie, Kang; Zou, Gang

2017-12-18

Studies on the following were reviewed: (1) the structure of spiropyrans and spirooxazines (two kinds of spiro compounds) under external stimuli and (2) the construction and applications of composite systems based on fluorescence resonance energy transfer (FRET) with fluorescent materials. When treated with different stimuli (light, acids and bases, solvents, metal ions, temperature, redox potential, and so on), spiropyrans/spirooxazines undergo transformations between the ring-closed form (SP), the ring-opened merocyanine (MC) form, and the protonated ring-opened form (MCH). This is due to the breakage of the spiro C-O bond and the protonation of MC, along with a color change. Various novel, multifunctional materials based on photochromic spiropyrans and spirooxazines have been successfully developed because of the vastly differently physiochemical properties posssed by the SP, MC and MCH forms. Among the three different structural forms, the MC form has been studied most extensively. The MC form not only gives complexes with various inorganic particles, biological molecules, and organic chemicals but also acts as the energy acceptor (of energy from fluorescent molecules) during energy transfer processes that take place under proper conditions. Furthermore, spiropyran and spirooxazine compounds exhibit reversible physicochemical property changes under proper stimuli; this provides more advantages compared with other photochromic compounds. Additionally, the molecular structures of spiropyrans and spirooxazines can be easily modified and extended, so better compounds can be obtained to expand the scope of already known applications. Described in detail are: (1) the structural properties of spiropyrans and spirooxazines and related photochromic mechanisms; (2) composite systems based on spiropyrans and spirooxazines, and (3) fluorescent materials which have potential applications in sensing, probing, and a variety of optical elements.

In-capillary probing of quantum dots and fluorescent protein self-assembly and displacement using Förster resonance energy transfer.

PubMed

Wang, Jianhao; Fan, Jie; Li, Jinchen; Liu, Li; Wang, Jianpeng; Jiang, Pengju; Liu, Xiaoqian; Qiu, Lin

2017-02-01

Herein, a Förster resonance energy transfer system was designed, which consisted of CdSe/ZnS quantum dots donor and mCherry fluorescent protein acceptor. The quantum dots and the mCherry proteins were conjugated to permit Förster resonance energy transfer. Capillary electrophoresis with fluorescence detection was used for the analyses for the described system. The quantum dots and mCherry were sequentially injected into the capillary, while the real-time fluorescence signal of donor and acceptor was simultaneously monitored by two channels with fixed wavelength detectors. An effective separation of complexes from free donor and acceptor was achieved. Results showed quantum dots and hexahistidine tagged mCherry had high affinity and the assembly was affected by His 6 -mCherry/quantum dot molar ratio. The kinetics of the self-assembly was calculated using the Hill equation. The microscopic dissociation constant values for out of- and in-capillary assays were 10.49 and 23.39 μM, respectively. The capillary electrophoresis with fluorescence detection that monitored ligands competition assay further delineated the different binding capacities of histidine containing peptide ligands for binding sites on quantum dots. This work demonstrated a novel approach for the improvement of Förster resonance energy transfer for higher efficiency, increased sensitivity, intuitionistic observation, and low sample requirements of the in-capillary probing system. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity.

PubMed

Gilmore, Marcella A; Williams, Dudley; Okawa, Yumiko; Holguin, Bret; James, Nicholas G; Ross, Justin A; Roger Aoki, K; Jameson, David M; Steward, Lance E

2011-06-01

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V(max) and k(cat)) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications. Copyright © 2011 Elsevier Inc. All rights reserved.

Nuclear Resonance Fluorescence to Measure Plutonium Mass in Spent Nuclear Fuel

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

Ludewigt, Bernhard A; Quiter, Brian J.; Ambers, Scott D.

2011-01-14

The Next Generation Safeguard Initiative (NGSI) of the U.S Department of Energy is supporting a multi-lab/university collaboration to quantify the plutonium (Pu) mass in spent nuclear fuel (SNF) assemblies and to detect the diversion of pins with non-destructive assay (NDA) methods. The following 14 NDA techniques are being studied: Delayed Neutrons, Differential Die-Away, Differential Die-Away Self-Interrogation, Lead Slowing Down Spectrometer, Neutron Multiplicity, Passive Neutron Albedo Reactivity, Total Neutron (Gross Neutron), X-Ray Fluorescence, {sup 252}Cf Interrogation with Prompt Neutron Detection, Delayed Gamma, Nuclear Resonance Fluorescence, Passive Prompt Gamma, Self-integration Neutron Resonance Densitometry, and Neutron Resonance Transmission Analysis. Understanding and maturity ofmore » the techniques vary greatly, ranging from decades old, well-understood methods to new approaches. Nuclear Resonance Fluorescence (NRF) is a technique that had not previously been studied for SNF assay or similar applications. Since NRF generates isotope-specific signals, the promise and appeal of the technique lies in its potential to directly measure the amount of a specific isotope in an SNF assay target. The objectives of this study were to design and model suitable NRF measurement methods, to quantify capabilities and corresponding instrumentation requirements, and to evaluate prospects and the potential of NRF for SNF assay. The main challenge of the technique is to achieve the sensitivity and precision, i.e., to accumulate sufficient counting statistics, required for quantifying the mass of Pu isotopes in SNF assemblies. Systematic errors, considered a lesser problem for a direct measurement and only briefly discussed in this report, need to be evaluated for specific instrument designs in the future. Also, since the technical capability of using NRF to measure Pu in SNF has not been established, this report does not directly address issues such as cost, size

Development of a dielectrophoresis-assisted surface plasmon resonance fluorescence biosensor for detection of bacteria

NASA Astrophysics Data System (ADS)

Kuroda, Chiaki; Iizuka, Ryota; Ohki, Yoshimichi; Fujimaki, Makoto

2018-05-01

To detect biological substances such as bacteria speedily and accurately, a dielectrophoresis-assisted surface plasmon resonance (SPR) fluorescence biosensor is being developed. Using Escherichia coli as a target organism, an appropriate voltage frequency to collect E. coli cells on indium tin oxide quadrupole electrodes by dielectrophoresis is analyzed. Then, E. coli is stained with 4‧,6-diamidino-2-phenylindole (DAPI). To clearly detect fluorescence signals from DAPI-stained E. coli cells, the sensor is optimized so that we can excite SPR on Al electrodes by illuminating 405 nm photons. As a result, the number of fluorescence signals is increased on the electrodes by the application of a low-frequency voltage. This indicates that E. coli cells with a lower permittivity than the surrounding water are collected by negative dielectrophoresis onto the electrodes where the electric field strength is lowest.

Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

PubMed

Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

2015-10-07

Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (<20 nm), high water solubility and good biocompatibility. The NPs were modified by ligand exchange reactions with citric acid (CA) to obtain carboxyl-functionalized NPs (Fe3O4@CeF3:Tb@CeF3-COOH). Folic acid (FA) as an affinity ligand was then covalently conjugated onto NPs to yield Fe3O4@CeF3:Tb@CeF3-FA NPs. They were then applied as multimodal imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

In-focal-plane characterization of excitation distribution for quantitative fluorescence microscopy applications

NASA Astrophysics Data System (ADS)

Dietrich, Klaus; Brülisauer, Martina; ćaǧin, Emine; Bertsch, Dietmar; Lüthi, Stefan; Heeb, Peter; Stärker, Ulrich; Bernard, André

2017-06-01

The applications of fluorescence microscopy span medical diagnostics, bioengineering and biomaterial analytics. Full exploitation of fluorescent microscopy is hampered by imperfections in illumination, detection and filtering. Mainly, errors stem from deviations induced by real-world components inducing spatial or angular variations of propagation properties along the optical path, and they can be addressed through consistent and accurate calibration. For many applications, uniform signal to noise ratio (SNR) over the imaging area is required. Homogeneous SNR can be achieved by quantifying and compensating for the signal bias. We present a method to quantitatively characterize novel reference materials as a calibration reference for biomaterials analytics. The reference materials under investigation comprise thin layers of fluorophores embedded in polymer matrices. These layers are highly homogeneous in their fluorescence response, where cumulative variations do not exceed 1% over the field of view (1.5 x 1.1 mm). An automated and reproducible measurement methodology, enabling sufficient correction for measurement artefacts, is reported. The measurement setup is equipped with an autofocus system, ensuring that the measured film quality is not artificially increased by out-of-focus reduction of the system modulation transfer function. The quantitative characterization method is suitable for analysis of modified bio-materials, especially through patterned protein decoration. The imaging method presented here can be used to statistically analyze protein patterns, thereby increasing both precision and throughput. Further, the method can be developed to include a reference emitter and detector pair on the image surface of the reference object, in order to provide traceable measurements.

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers.

PubMed

Lichten, Catherine A; White, Rachel; Clark, Ivan B N; Swain, Peter S

2014-02-03

To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour.

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers

PubMed Central

2014-01-01

Background To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. Results Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). Conclusions Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour. PMID:24495318

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

PubMed

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

2015-12-15

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

Fluorescent proteins for quantitative microscopy: important properties and practical evaluation.

PubMed

Shaner, Nathan Christopher

2014-01-01

More than 20 years after their discovery, fluorescent proteins (FPs) continue to be the subject of massive engineering efforts yielding continued improvements. Among these efforts are many aspects that should be of great interest to quantitative imaging users. With new variants frequently introduced into the research community, "tried and true" FPs that have been relied on for many years may now be due for upgrades to more modern variants. However, the dizzying array of FPs now available can make the initial act of narrowing down the potential choices an intimidating prospect. This chapter describes the FP properties that most strongly impact their performance in quantitative imaging experiments, along with their physical origins as they are currently understood. A workflow for evaluating a given FP in the researcher's chosen experimental system (e.g., a specific cell line) is described. © 2014 Elsevier Inc. All rights reserved.

Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

PubMed Central

Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

2014-01-01

Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions. PMID:24957323

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method

NASA Astrophysics Data System (ADS)

Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

2009-11-01

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants ( Ka) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02 × 10 7 and 2.07 × 10 4 L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S 0 → S 1 transition of esculin ( λexmax≈340 nm) appears, which is similar to the λemmax of BSA and HSA. The critical distance ( R0) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

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.

Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

NASA Astrophysics Data System (ADS)

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-02-01

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

Upconverting rare-earth nanoparticles with a paramagnetic lanthanide complex shell for upconversion fluorescent and magnetic resonance dual-modality imaging

NASA Astrophysics Data System (ADS)

Wang, Yan; Ji, Lei; Zhang, Bingbo; Yin, Peihao; Qiu, Yanyan; Song, Daqian; Zhou, Juying; Li, Qi

2013-05-01

Multi-modal imaging based on multifunctional nanoparticles is a promising alternative approach to improve the sensitivity of early cancer diagnosis. In this study, highly upconverting fluorescence and strong relaxivity rare-earth nanoparticles coated with paramagnetic lanthanide complex shells and polyethylene glycol (PEGylated UCNPs@DTPA-Gd3+) are synthesized as dual-modality imaging contrast agents (CAs) for upconverting fluorescent and magnetic resonance dual-modality imaging. PEGylated UCNPs@DTPA-Gd3+ with sizes in the range of 32-86 nm are colloidally stable. They exhibit higher longitudinal relaxivity and transverse relaxivity in water (r1 and r2 values are 7.4 and 27.8 s-1 per mM Gd3+, respectively) than does commercial Gd-DTPA (r1 and r2 values of 3.7 and 4.6 s-1 per mM Gd3+, respectively). They are found to be biocompatible. In vitro cancer cell imaging shows good imaging contrast of PEGylated UCNPs@DTPA-Gd3+. In vivo upconversion fluorescent imaging and T1-weighted MRI show excellent enhancement of both fluorescent and MR signals in the livers of mice administered PEGylated UCNPs@DTPA-Gd3+. All the experimental results indicate that the synthesized PEGylated UCNPs@DTPA-Gd3+ present great potential for biomedical upconversion of fluorescent and magnetic resonance dual-modality imaging applications.

Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

PubMed

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

2015-06-01

Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. © 2015 International Society for Advancement of Cytometry.

Three-dimensional quantitative flow diagnostics

NASA Technical Reports Server (NTRS)

Miles, Richard B.; Nosenchuck, Daniel M.

1989-01-01

The principles, capabilities, and practical implementation of advanced measurement techniques for the quantitative characterization of three-dimensional flows are reviewed. Consideration is given to particle, Rayleigh, and Raman scattering; fluorescence; flow marking by H2 bubbles, photochromism, photodissociation, and vibrationally excited molecules; light-sheet volume imaging; and stereo imaging. Also discussed are stereo schlieren methods, holographic particle imaging, optical tomography, acoustic and magnetic-resonance imaging, and the display of space-filling data. Extensive diagrams, graphs, photographs, sample images, and tables of numerical data are provided.

FLASH proton density imaging for improved surface coil intensity correction in quantitative and semi-quantitative SSFP perfusion cardiovascular magnetic resonance.

PubMed

Nielles-Vallespin, Sonia; Kellman, Peter; Hsu, Li-Yueh; Arai, Andrew E

2015-02-17

A low excitation flip angle (α < 10°) steady-state free precession (SSFP) proton-density (PD) reference scan is often used to estimate the B1-field inhomogeneity for surface coil intensity correction (SCIC) of the saturation-recovery (SR) prepared high flip angle (α = 40-50°) SSFP myocardial perfusion images. The different SSFP off-resonance response for these two flip angles might lead to suboptimal SCIC when there is a spatial variation in the background B0-field. The low flip angle SSFP-PD frames are more prone to parallel imaging banding artifacts in the presence of off-resonance. The use of FLASH-PD frames would eliminate both the banding artifacts and the uneven frequency response in the presence of off-resonance in the surface coil inhomogeneity estimate and improve homogeneity of semi-quantitative and quantitative perfusion measurements. B0-field maps, SSFP and FLASH-PD frames were acquired in 10 healthy volunteers to analyze the SSFP off-resonance response. Furthermore, perfusion scans preceded by both FLASH and SSFP-PD frames from 10 patients with no myocardial infarction were analyzed semi-quantitatively and quantitatively (rest n = 10 and stress n = 1). Intra-subject myocardial blood flow (MBF) coefficient of variation (CoV) over the whole left ventricle (LV), as well as intra-subject peak contrast (CE) and upslope (SLP) standard deviation (SD) over 6 LV sectors were investigated. In the 6 out of 10 cases where artifacts were apparent in the LV ROI of the SSFP-PD images, all three variability metrics were statistically significantly lower when using the FLASH-PD frames as input for the SCIC (CoVMBF-FLASH = 0.3 ± 0.1, CoVMBF-SSFP = 0.4 ± 0.1, p = 0.03; SDCE-FLASH = 10 ± 2, SDCE-SSFP = 32 ± 7, p = 0.01; SDSLP-FLASH = 0.02 ± 0.01, SDSLP-SSFP = 0.06 ± 0.02, p = 0.03). Example rest and stress data sets from the patient pool demonstrate that the low flip angle SSFP protocol

Status of miniature integrated UV resonance fluorescence and Raman sensors for detection and identification of biochemical warfare agents

NASA Astrophysics Data System (ADS)

Hug, William F.; Bhartia, Rohit; Taspin, Alexandre; Lane, Arthur; Conrad, Pamela; Sijapati, Kripa; Reid, Ray D.

2005-11-01

Laser induced native fluorescence (LINF) is the most sensitive method of detection of biological material including microorganisms, virus', and cellular residues. LINF is also a sensitive method of detection for many non-biological materials as well. The specificity with which these materials can be classified depends on the excitation wavelength and the number and location of observation wavelengths. Higher levels of specificity can be obtained using Raman spectroscopy but a much lower levels of sensitivity. Raman spectroscopy has traditionally been employed in the IR to avoid fluorescence. Fluorescence rarely occurs at wavelength below about 270nm. Therefore, when excitation occurs at a wavelength below 250nm, no fluorescence background occurs within the Raman fingerprint region for biological materials. When excitation occurs within electronic resonance bands of the biological target materials, Raman signal enhancement over one million typically occurs. Raman sensitivity within several hundred times fluorescence are possible in the deep UV where most biological materials have strong absorption. Since the Raman and fluorescence emissions occur at different wavelength, both spectra can be observed simultaneously, thereby providing a sensor with unique sensitivity and specificity capability. We will present data on our integrated, deep ultraviolet, LINF/Raman instruments that are being developed for several applications including life detection on Mars as well as biochemical warfare agents on Earth. We will demonstrate the ability to discriminate organic materials based on LINF alone. Together with UV resonance Raman, higher levels of specificity will be demonstrated. In addition, these instruments are being developed as on-line chemical sensors for industrial and municipal waste streams and product quality applications.

A rapid fluorescence assay for danofloxacin in beef muscle: effect of muscle type on limit of quantitation.

PubMed

Schneider, Marilyn J

2008-08-01

A simple, rapid fluorescence screening assay was applied to the analysis of beef muscle for danofloxacin at the U.S. tolerance level of 200 ng/g. Muscle samples were homogenized in acetic acid-acetonitrile, the resultant mixture centrifuged, and fluorescence of the supernatants was then measured. The significant difference between the fluorescence of control muscle sample extracts and extracts of samples fortified at 200 ng/g allowed for successful discrimination between the samples. Setting a threshold level at the average 200 ng/g fortified sample extract fluorescence -3sigma allowed for identification of potentially violative samples. Successful analysis of a group of blind fortified samples over a range of concentrations was accomplished in this manner, without any false-negative results. The limits of quantitation for danofloxacin, as well as enrofloxacin, using this assay were determined in three types of beef muscle (hanging tenderloin, neck, and eye round steak), as well as in serum. Significant differences in limits of quantitation were found among the three different muscle types examined, with hanging tenderloin muscle providing the lowest value. This work not only shows the potential for use of the fluorescence screening assay as an alternative to currently used microbial or antibody-based assays for the analysis of danofloxacin in beef muscle, but also suggests that assays using beef muscle may vary in performance depending on the specific muscle selected for analysis.

Quantitative Analysis of Protein Translocations by Microfluidic Total Internal Reflection Fluorescence Flow Cytometry

PubMed Central

Wang, Jun; Fei, Bei; Geahlen, Robert L.

2010-01-01

Protein translocation, or the change in a protein’s location between different subcellular compartments, is a critical process by which intracellular proteins carry out their cellular functions. Aberrant translocation events contribute to various diseases ranging from metabolic disorders to cancer. In this study, we demonstrate the use of a newly developed single-cell tool, microfluidic total internal reflection fluorescence flow cytometry (TIRF-FC), for detecting both cytosol to plasma membrane and cytosol to nucleus translocations using the tyrosine kinase Syk and the transcription factor NF-κB as models. This technique detects fluorescent molecules at the plasma membrane and in the membrane-proximal cytosol in single cells. We were able to record quantitatively changes in the fluorescence density in the evanescent field associated with these translocation processes for large cell populations with single cell resolution. We envision that TIRF-FC will provide a new approach to explore the molecular biology and clinical relevance of protein translocations. PMID:20820633

Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

USDA-ARS?s Scientific Manuscript database

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

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

ERIC Educational Resources Information Center

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

2004-01-01

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

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method.

PubMed

Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

2009-11-01

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants (K(a)) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02x10(7) and 2.07x10(4)L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S(0)-->S(1) transition of esculin (lambda(ex)(max) approximately 340nm) appears, which is similar to the lambda(em)(max) of BSA and HSA. The critical distance (R(0)) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

2014-11-01

A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel robust quantitative Förster resonance energy transfer assay for protease SENP2 kinetics determination against its all natural substrates.

PubMed

Liu, Yan; Shen, Yali; Zheng, Shasha; Liao, Jiayu

2015-12-01

SUMOylation (the process of adding the SUMO [small ubiquitin-like modifier] to substrates) is an important post-translational modification of critical proteins in multiple processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate the SUMO from its substrate. Determining the kinetics of SENPs is important for understanding their activities. Förster resonance energy transfer (FRET) technology has been widely used in biomedical research and is a powerful tool for elucidating protein interactions. In this paper we report a novel quantitative FRET-based protease assay for SENP2 endopeptidase activity that accounts for the self-fluorescent emissions of the donor (CyPet) and the acceptor (YPet). The kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP2 toward pre-SUMO1/2/3, were obtained by this novel design. Although we use SENP2 to demonstrate our method, the general principles of this quantitative FRET-based protease kinetic determination can be readily applied to other proteases.

Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation

NASA Astrophysics Data System (ADS)

Quinn, Kyle P.; Sridharan, Gautham V.; Hayden, Rebecca S.; Kaplan, David L.; Lee, Kyongbum; Georgakoudi, Irene

2013-12-01

The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.

Measuring the remineralization potential of different agents with quantitative light-induced fluorescence digital Biluminator.

PubMed

Kucukyilmaz, Ebru; Savas, Selcuk

2017-01-26

The aim of this study was to investigate the effectiveness of different remineralization agents by quantitative light-induced fluorescence digital BiluminatorTM (QLF-D). Artificial caries lesions were created, and the teeth were divided according to the tested materials: (i) distilled water, (ii) acidulated phosphate fluoride (APF), (iii) Curodont Repair (CR), (iv) ammonium hexafluorosilicate (SiF) and (v) ammonium hexafluorosilicate plus cetylpyridinium chloride (SiF + CPC). After treatment procedures, each of the samples was placed in artificial saliva. After demineralization and 1 and 4 weeks of remineralization procedures, fluorescence loss and lesion areas were measured with QLF-D. Data were statistically analyzed (α = 0.05). The fluorescence values of the demineralized enamel specimens treated with the various agents differed significantly compared with pretreatment values for both 1 and 4 weeks (p<0.05). At 4 weeks, the highest fluorescence gain was calculated in the CR, APF and SiF groups compared with the control (p<0.05). APF, SiF and CR groups yielded greater remineralization ability than SiF + CPC and control groups.

A novel device for quantitative measurement of chloride concentration by fluorescence indicator

NASA Astrophysics Data System (ADS)

Wang, Junsheng; Wu, Xudong; Chon, Chanhee; Gonska, Tanja; Li, Dongqing

2012-02-01

Cystic fibrosis (CF) is a life-threatening genetic disease. At present, the common method for diagnosis of CF is to detect the chloride concentration in sweat using ion-selective electrodes. However, the current sweat testing methods require a relatively large quantity of sweat sample, at least 25 µL, which is very difficult to obtain, especially for newborns. This paper presents a new method and a new device for rapid detection of the chloride concentration from a small volume of solution. In this method, the chloride concentration is determined quantitatively by the fluorescence intensity of MQAE, a chloride ion fluorescent indicator. In this device, the sample is carried by a small piece of filter paper on a cover glass exposed to an UV LED light source. The resulting fluorescent signals are detected by a Si photodiode. Data acquisition and processing are accomplished by LabVIEW software in a PDA. Based on the Stern-Volmer relationship, the effects of different parameters on the fluorescence intensity were analyzed. The observed significant difference between 40 and 60 mM (the borderline of chloride concentration for CF) is discussed in this paper. The results show that detection can be completed within 10 s. The minimum detectable volume of the chloride solution is 1 μL. The novel method and the device are of great potential for CF diagnosis.

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography.

PubMed

Maia, Ana Marly Araújo; de Freitas, Anderson Zanardi; de L Campello, Sergio; Gomes, Anderson Stevens Leônidas; Karlsson, Lena

2016-06-01

An in vitro study of morphological alterations between sound dental structure and artificially induced white spot lesions in human teeth, was performed through the loss of fluorescence by Quantitative Light-Induced Fluorescence (QLF) and the alterations of the light attenuation coefficient by Optical Coherence Tomography (OCT). To analyze the OCT images using a commercially available system, a special algorithm was applied, whereas the QLF images were analyzed using the software available in the commercial system employed. When analyzing the sound region against white spot lesions region by QLF, a reduction in the fluorescence intensity was observed, whilst an increase of light attenuation by the OCT system occurred. Comparison of the percentage of alteration between optical properties of sound and artificial enamel caries regions showed that OCT processed images through the attenuation of light enhanced the tooth optical alterations more than fluorescence detected by QLF System. QLF versus OCT imaging of enamel caries: a photonics assessment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new non-resonant laser-induced fluorescence instrument for the airborne in situ measurement of formaldehyde

NASA Astrophysics Data System (ADS)

St. Clair, Jason M.; Swanson, Andrew K.; Bailey, Steven A.; Wolfe, Glenn M.; Marrero, Josette E.; Iraci, Laura T.; Hagopian, John G.; Hanisco, Thomas F.

2017-12-01

A new in situ instrument for gas-phase formaldehyde (HCHO), COmpact Formaldehyde FluorescencE Experiment (COFFEE), is presented. COFFEE utilizes non-resonant laser-induced fluorescence (NR-LIF) to measure HCHO, with 300 mW of 40 kHz 355 nm laser output exciting multiple HCHO absorption features. The resulting HCHO fluorescence is collected at 5 ns resolution, and the fluorescence time profile is fit to yield the ambient HCHO mixing ratio. Typical 1σ precision at ˜ 0 pptv HCHO is 150 pptv for 1 s data. The compact instrument was designed to operate with minimal in-flight operator interaction and infrequent maintenance (1-2 times per year). COFFEE fits in the wing pod of the Alpha Jet stationed at the NASA Ames Research Center and has successfully collected HCHO data on 27 flights through 2017 March. The frequent flights, combined with a potentially long-term data set, makes the Alpha Jet a promising platform for validation of satellite-based column HCHO.

Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer

DOEpatents

Kwok, Pui-Yan; Chen, Xiangning

1999-01-01

A method for detecting the presence of a target nucleotide or sequence of nucleotides in a nucleic acid is disclosed. The method is comprised of forming an oligonucleotide labeled with two fluorophores on the nucleic acid target site. The doubly labeled oligonucleotide is formed by addition of a singly labeled dideoxynucleoside triphosphate to a singly labeled polynucleotide or by ligation of two singly labeled polynucleotides. Detection of fluorescence resonance energy transfer upon denaturation indicates the presence of the target. Kits are also provided. The method is particularly applicable to genotyping.

Automated image analysis for quantitative fluorescence in situ hybridization with environmental samples.

PubMed

Zhou, Zhi; Pons, Marie Noëlle; Raskin, Lutgarde; Zilles, Julie L

2007-05-01

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4',6'-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.

Study of resonance light scattering for remote optical probing

NASA Technical Reports Server (NTRS)

Penney, C. M.; Morey, W. W.; St. Peters, R. L.; Silverstein, S. D.; Lapp, M.; White, D. R.

1973-01-01

Enhanced scattering and fluorescence processes in the visible and UV were investigated which will enable improved remote measurements of gas properties. The theoretical relationship between scattering and fluorescence from an isolated molecule in the approach to resonance is examined through analysis of the time dependence of re-emitted light following excitation of pulsed incident light. Quantitative estimates are developed for the relative and absolute intensities of fluorescence and resonance scattering. New results are obtained for depolarization of scattering excited by light at wavelengths within a dissociative continuum. The experimental work was performed in two separate facilities. One of these utilizes argon and krypton lasers, single moded by a tilted etalon, and a 3/4 meter double monochromator. This facility was used to determine properties of the re-emission from NO2, I2 and O3 excited by visible light. The second facility involves a narrow-line dye laser, and a 3/4 meter single monochromator. The dye laser produces pulsed light with 5 nsec pulse duration and 0.005 nm spectral width.

Direct detection of time-resolved Rabi oscillations in a single quantum dot via resonance fluorescence

NASA Astrophysics Data System (ADS)

Schaibley, J. R.; Burgers, A. P.; McCracken, G. A.; Steel, D. G.; Bracker, A. S.; Gammon, D.; Sham, L. J.

2013-03-01

Optical Rabi oscillations are coherent population oscillations of a two-level system coupled by an electric dipole transition when driven by a strong nearly resonant optical field. In quantum dot structures, these measurements have typically been performed as a function of the total pulse area ∫Ω0(t)dt where the pulse area varies as a function of Rabi frequency. Here, we report direct detection of the time-resolved coherent transient response of the resonance fluorescence to measure the time evolution of the optical Rabi oscillations in a single charged InAs quantum dot. We extract a decoherence rate consistent with the limit from the excited state lifetime.

On the Search for Nuclear Resonance Fluorescence Signatures of 235U and 238U above 3 MeV

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

Warren, Glen A.; Caggiano, Joseph A.; Bertozzi, William

Nuclear resonance fluorescence is a physical process that provides an isotope-specific signature that could be used for the identification and characterization of materials. The technique involves the detection of prompt discrete-energy photons emitted from a sample that is exposed to MeV-energy photons. Potential applications of the technique range from detection of high explosives to characterization of special nuclear materials such as 235U. Pacific Northwest National Laboratory and Passport Systems have collaborated to conduct a pair of measurements to search for a nuclear resonance fluorescence response of 235U above 3 MeV and of 238U above 5 MeV using an 8 gmore » sample of highly enriched uranium and a 90 g sample of depleted uranium. No new signatures were observed. The minimum detectable integrated cross section for 235U is presented.« less

Quantitative Analysis of Nail Polish Remover Using Nuclear Magnetic Resonance Spectroscopy Revisited

ERIC Educational Resources Information Center

Hoffmann, Markus M.; Caccamis, Joshua T.; Heitz, Mark P.; Schlecht, Kenneth D.

2008-01-01

Substantial modifications are presented for a previously described experiment using nuclear magnetic resonance (NMR) spectroscopy to quantitatively determine analytes in commercial nail polish remover. The revised experiment is intended for a second- or third-year laboratory course in analytical chemistry and can be conducted for larger laboratory…

Intramolecular fluorescence resonance energy transfer and living cell imaging of novel pyridyltriphenylamine dye

NASA Astrophysics Data System (ADS)

Cao, Duojun; Qian, Ying

2016-07-01

A novel pyridyltriphenylamine-rhodamine dye PTRh and a pyridyltriphenylamine derivative PTO were synthesized and characterized by 1H NMR and HRMS-MALDI-TOF. PTRh performed typical fluorescence resonance energy transfer (FRET) signal from pyridyltriphenylamine to rhodamine along with notable color change from green to rose when interacting with Hg2+ in EtOH/H2O. And PTRh as a ratiometric probe for Hg2+ based on FRET could achieve a very low detection limit of 32 nM and energy transfer efficiency of 83.7% in aqueous organic system. On the other hand, spectra properties of PTO in its aggregates, THF/H2O mixed solution and silica nanoparticles (Si-NPs) dispersed in water were investigated. And the results indicated PTO exhibited bright green fluorescence in solid state, and PTO was successfully encapsulated in silica matrix (30-40 nm), emitting bright blue fluorescence with 11.7% quantum yield. Additionally, living cell imaging experiments demonstrated that PTRh could effectively response to intracellular Hg2+ and PTO-doped Si-NPs were well uptaken by MCF-7 breast cancer cells. It could be concluded that the chromophores are promising materials used as biosensors.

Quantitative measurement of binary liquid distributions using multiple-tracer x-ray fluorescence and radiography

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

Halls, Benjamin R.; Meyer, Terrence R.; Kastengren, Alan L.

2015-01-01

The complex geometry and large index-of-refraction gradients that occur near the point of impingement of binary liquid jets present a challenging environment for optical interrogation. A simultaneous quadruple-tracer x-ray fluorescence and line-of-sight radiography technique is proposed as a means of distinguishing and quantifying individual liquid component distributions prior to, during, and after jet impact. Two different pairs of fluorescence tracers are seeded into each liquid stream to maximize their attenuation ratio for reabsorption correction and differentiation of the two fluids during mixing. This approach for instantaneous correction of x-ray fluorescence reabsorption is compared with a more time-intensive approach of usingmore » stereographic reconstruction of x-ray attenuation along multiple lines of sight. The proposed methodology addresses the need for a quantitative measurement technique capable of interrogating optically complex, near-field liquid distributions in many mixing systems of practical interest involving two or more liquid streams.« less

Quantitative measurement of binary liquid distributions using multiple-tracer x-ray fluorescence and radiography

DOE PAGES

Halls, Benjamin R.; Meyer, Terrence R.; Kastengren, Alan L.

2015-01-23

The complex geometry and large index-of-refraction gradients that occur near the point of impingement of binary liquid jets present a challenging environment for optical interrogation. A simultaneous quadruple-tracer x-ray fluorescence and line-of-sight radiography technique is proposed as a means of distinguishing and quantifying individual liquid component distributions prior to, during, and after jet impact. Two different pairs of fluorescence tracers are seeded into each liquid stream to maximize their attenuation ratio for reabsorption correction and differentiation of the two fluids during mixing. This approach for instantaneous correction of x-ray fluorescence reabsorption is compared with a more time-intensive approach of usingmore » stereographic reconstruction of x-ray attenuation along multiple lines of sight. The proposed methodology addresses the need for a quantitative measurement technique capable of interrogating optically complex, near-field liquid distributions in many mixing systems of practical interest involving two or more liquid streams.« less

Quantitative nuclear magnetic resonance to measure body composition in infants and children

USDA-ARS?s Scientific Manuscript database

Quantitative Nuclear Magnetic Resonance (QMR) is being used in human adults to obtain measures of total body fat (FM) with high precision. The current study assessed a device specially designed to accommodate infants and children between 3 and 50 kg (EchoMRI-AH™). Body composition of 113 infants and...

Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes

PubMed Central

Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

2015-01-01

Abstract The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes. Finally, mathematical modelling combined with quantitative FRET measurements provided novel insights into the kinetics and integration of calcineurin activation in response to myocyte Ca transients. In all, DuoCaN and UniCaN stand as valuable new tools for understanding the role of calcineurin in normal and pathological signalling. Key points Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The

Characterization of an improved donor fluorescent protein for Förster resonance energy transfer microscopy

PubMed Central

Day, Richard N.; Booker, Cynthia F.; Periasamy, Ammasi

2008-01-01

The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Typically, the Cerulean and Venus variants of the cyan and yellow FPs are used for FRET studies, but there are limitations to their use. Here, Cerulean and the newly developed monomeric Teal FP (mTFP) are compared as FRET donors for Venus using spectral and fluorescence lifetime measurements from living cells. The results demonstrate that when compared to Cerulean, mTFP has increased brightness, optimal excitation using the standard 458-nm laser line, increased photostability, and improved spectral overlap with Venus. In addition, the two-photon excitation and fluorescence lifetime characteristics are determined for mTFP. Together, these measurements indicate that mTFP is an excellent donor fluorophore for FRET studies, and that its use may improve the detection of interactions involving proteins that are difficult to express, or that need to be produced at low levels in cells. PMID:18601527

Development of two-photon fluorescence microscopy for quantitative imaging in turbid tissues

NASA Astrophysics Data System (ADS)

Coleno, Mariah Lee

Two-photon laser scanning fluorescence microscopy (TPM) is a high resolution, non-invasive biological imaging technique that can be used to image turbid tissues both in vitro and in vivo at depths of several hundred microns. Although TPM has been widely used to image tissue structures, no one has focused on using TPM to extract quantitative information from turbid tissues at depth. As a result, this thesis addresses the quantitative characterization of two-photon signals in turbid media. Initially, a two-photon microscope system is constructed, and two-photon images that validate system performance are obtained. Then TPM is established as an imaging technique that can be used to validate theoretical observations already listed in the literature. In particular, TPM is found to validate the exponential dependence of the fluorescence intensity decay with depth in turbid tissue model systems. Results from these studies next prompted experimental investigation into whether TPM could be used to determine tissue optical properties. Comparing the exponential dependence of the decay with a Monte Carlo model involving tissue optical properties, TPM is shown to be useful for determining the optical properties (total attenuation coefficient) of thick, turbid tissues on a small spatial scale. Next, a role for TPM for studying and optimizing wound healing is demonstrated. In particular, TPM is used to study the effects of perturbations (growth factors, PDT) on extracellular matrix remodeling in artificially engineered skin tissues. Results from these studies combined with tissue contraction studies are shown to demonstrate ways to modulate tissues to optimize the wound healing immune response and reduce scarring. In the end, TPM is shown to be an extremely important quantitative biological imaging technique that can be used to optimize wound repair.

[Development of Fluorescence Resonance Energy Transfer Sensor for Determination of Adenosine Monophosphate in Biological Drug].

PubMed

Dong, Ling-yu; Du, Hong-ming; Wang, Peng; Wang, Li-yun; Li, Yi-ke; Zhai, Hong; Feng, Ting; Wang, Xiang-feng; Zhu, Qiao-you; Xie, Meng-xia

2015-11-01

The biological drug of the calf-blood dialysate has various pharmacological effects. It can promote the oxygen and glucose uptake for the hypoxia cells, and has beneficial effects on the malfunction of the blood circulation and trophic disturbances in the brain, and the impairment of peripheral blood circulation. Furthermore, it is favorable to wound healing and can regulate the central nervous system. Adenosine monophosphate (AMP) is a main active ingredient of the biological drug. In this report, a fluorescence resonance energy transfer (FRET) sensor has been developed with β-CD-capped ZnS QDs as energy donor and 3-hydroxyflavone (3-HF) as energy acceptor. The results showed that AMP can lead to the fluorescence quenching of the FRET sensor at 526 nm, and the Stern-Volmer curve between the fluorescence quenching and the concentrations of AMP present a satisfactory linearity with the correlation coefficient of 0.996. The developed sensor has successfully applied for determination of the AMP in the biological drug.

Prognostic Value of Quantitative Stress Perfusion Cardiac Magnetic Resonance.

PubMed

Sammut, Eva C; Villa, Adriana D M; Di Giovine, Gabriella; Dancy, Luke; Bosio, Filippo; Gibbs, Thomas; Jeyabraba, Swarna; Schwenke, Susanne; Williams, Steven E; Marber, Michael; Alfakih, Khaled; Ismail, Tevfik F; Razavi, Reza; Chiribiri, Amedeo

2018-05-01

This study sought to evaluate the prognostic usefulness of visual and quantitative perfusion cardiac magnetic resonance (CMR) ischemic burden in an unselected group of patients and to assess the validity of consensus-based ischemic burden thresholds extrapolated from nuclear studies. There are limited data on the prognostic value of assessing myocardial ischemic burden by CMR, and there are none using quantitative perfusion analysis. Patients with suspected coronary artery disease referred for adenosine-stress perfusion CMR were included (n = 395; 70% male; age 58 ± 13 years). The primary endpoint was a composite of cardiovascular death, nonfatal myocardial infarction, aborted sudden death, and revascularization after 90 days. Perfusion scans were assessed visually and with quantitative analysis. Cross-validated Cox regression analysis and net reclassification improvement were used to assess the incremental prognostic value of visual or quantitative perfusion analysis over a baseline clinical model, initially as continuous covariates, then using accepted thresholds of ≥2 segments or ≥10% myocardium. After a median 460 days (interquartile range: 190 to 869 days) follow-up, 52 patients reached the primary endpoint. At 2 years, the addition of ischemic burden was found to increase prognostic value over a baseline model of age, sex, and late gadolinium enhancement (baseline model area under the curve [AUC]: 0.75; visual AUC: 0.84; quantitative AUC: 0.85). Dichotomized quantitative ischemic burden performed better than visual assessment (net reclassification improvement 0.043 vs. 0.003 against baseline model). This study was the first to address the prognostic benefit of quantitative analysis of perfusion CMR and to support the use of consensus-based ischemic burden thresholds by perfusion CMR for prognostic evaluation of patients with suspected coronary artery disease. Quantitative analysis provided incremental prognostic value to visual assessment and

Shifts due to quantum-mechanical interference from distant neighboring resonances for saturated fluorescence spectroscopy of the 23S to 23P intervals of helium

NASA Astrophysics Data System (ADS)

Marsman, A.; Hessels, E. A.; Horbatsch, M.

2014-04-01

Quantum-mechanical interference with distant neighboring resonances is found to cause shifts for precision saturated fluorescence spectroscopy of the atomic helium 23S-to-23P transitions. The shifts are significant (larger than the experimental uncertainties for measurements of the intervals) despite the fact that the neighboring resonances are separated from the measured resonances by 1400 and 20000 natural widths. The shifts depend strongly on experimental parameters such as the angular position of the fluorescence detector, the intensity and size of laser beams, and the properties of the atomic beam. These shifts must be considered for the ongoing program of determining the fine-structure constant from the helium 23P fine structure.

Multifunctional PHPMA-Derived Polymer for Ratiometric pH Sensing, Fluorescence Imaging, and Magnetic Resonance Imaging.

PubMed

Su, Fengyu; Agarwal, Shubhangi; Pan, Tingting; Qiao, Yuan; Zhang, Liqiang; Shi, Zhengwei; Kong, Xiangxing; Day, Kevin; Chen, Meiwan; Meldrum, Deirdre; Kodibagkar, Vikram D; Tian, Yanqing

2018-01-17

In this paper, we report synthesis and characterization of a novel multimodality (MRI/fluorescence) probe for pH sensing and imaging. A multifunctional polymer was derived from poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) and integrated with a naphthalimide-based-ratiometric fluorescence probe and a gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex (Gd-DOTA complex). The polymer was characterized using UV-vis absorption spectrophotometry, fluorescence spectrofluorophotometry, magnetic resonance imaging (MRI), and confocal microscopy for optical and MRI-based pH sensing and cellular imaging. In vitro labeling of macrophage J774 and esophageal CP-A cell lines shows the polymer's ability to be internalized in the cells. The transverse relaxation time (T 2 ) of the polymer was observed to be pH-dependent, whereas the spin-lattice relaxation time (T 1 ) was not. The pH probe in the polymer shows a strong fluorescence-based ratiometric pH response with emission window changes, exhibiting blue emission under acidic conditions and green emission under basic conditions, respectively. This study provides new materials with multimodalities for pH sensing and imaging.

On the Search for Nuclear Resonance Fluorescence Signatures of 235U and 238U above 3 MeV

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

Warren, Glen A.; Caggiano, Joseph A.; Bertozzi, William

Abstract–Nuclear resonance fluorescence is a physical process that provides an isotope-specific signature that could be used for the identification and characterization of materials. The technique involves the detection of prompt discrete-energy photons emitted from a sample that is exposed to photons in the MeV energy range. Potential applications of the technique range from detection of high explosives to characterization of special nuclear materials such as 235U. Pacific Northwest National Laboratory and Passport Systems have collaborated to conduct a a pair of measurements to search for a nuclear resonance fluorescence response of 235U above 3 MeV and of 238U above 5more » MeV using an 8 g sample of highly enriched uranium and a 90 g sample of depleted uranium. No new signatures were observed. The minimum detectable integrated cross section for 235U is presented.« less

Nuclear resonance fluorescence imaging in non-intrusive cargo inspection

NASA Astrophysics Data System (ADS)

Bertozzi, William; Ledoux, Robert J.

2005-12-01

Nuclear resonance fluorescence is able to non-intrusively interrogate a region space and measure the isotopic content of the material in that space for any element with atomic number greater than that of helium. The technique involves exposing material to a continuous energy distribution of photons and detecting the scattered photons that have a discrete energy distribution unique to an isotope. The interrogating photons, which range from 2 to 8 MeV, are the most penetrating probes and can "see" through many inches of steel. Determination of the chemical components of the material occupying a region of space greatly enhances the identification of threats such as explosives, fissile materials, toxic materials and weapons of mass destruction. Systems can be designed to involve minimal operator intervention, to minimize dose to the sample, and to provide high throughput at commercial seaports, airports and other entry points.

Effects of counter-rotating-wave terms of the driving field on the spectrum of resonance fluorescence

NASA Astrophysics Data System (ADS)

Yan, Yiying; Lü, Zhiguo; Zheng, Hang

2013-11-01

We investigate the fluorescence spectrum of a two-level system driven by a monochromatic classical field by the Born-Markovian master equation based on a unitary transformation. The main purpose is to understand the effects of counter-rotating-wave terms of the driving on spectral features of the fluorescence. We have derived an analytical expression for the fluorescence spectrum, which is different from Mollow's theory, while Mollow's result on resonance is the limiting case of ours in moderately weak driving regimes. Our results demonstrate precisely that the counter-rotating-wave terms of the driving play an important role in the fluorescence spectrum for intense driving: (i) the counter-rotating coupling suppresses the red sideband in the Mollow triplet and it enhances the blue one in explicitly contrast to the well-known equal intensity of the sideband in Mollow's theory, (ii) the higher-order Mollow triplets appear as a characteristic spectral feature arising from counter-rotating-wave terms of the driving, and (iii) a significant frequency shift of the sidebands is observed, which depends on both the detuning and driving strength.

Nondestructive application of laser-induced fluorescence spectroscopy for quantitative analyses of phenolic compounds in strawberry fruits (Fragaria x ananassa).

PubMed

Wulf, J S; Rühmann, S; Rego, I; Puhl, I; Treutter, D; Zude, M

2008-05-14

Laser-induced fluorescence spectroscopy (LIFS) was nondestructively applied on strawberries (EX = 337 nm, EM = 400-820 nm) to test the feasibility of quantitatively determining native phenolic compounds in strawberries. Eighteen phenolic compounds were identified in fruit skin by UV and MS spectroscopy and quantitatively determined by use of rp-HPLC for separation and diode-array or chemical reaction detection. Partial least-squares calibration models were built for single phenolic compounds by means of nondestructively recorded fluorescence spectra in the blue-green wavelength range using different data preprocessing methods. The direct orthogonal signal correction resulted in r (2) = 0.99 and rmsep < 8% for p-coumaroyl-glucose, and r (2) = 0.99 and rmsep < 24% for cinnamoyl-glucose. In comparison, the correction of the fluorescence spectral data with simultaneously recorded reflectance spectra did not further improve the calibration models. Results show the potential of LIFS for a rapid and nondestructive assessment of contents of p-coumaroyl-glucose and cinnamoyl-glucose in strawberry fruits.

Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

PubMed

Hagihara, Kenta; Tsukagoshi, Kazuhiko; Nakajima, Chinami; Esaki, Shinsuke; Hashimoto, Masahiko

2016-01-01

We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent.

Plasmonic Metasurfaces Based on Nanopin-Cavity Resonator for Quantitative Colorimetric Ricin Sensing.

PubMed

Fan, Jiao-Rong; Zhu, Jia; Wu, Wen-Gang; Huang, Yun

2017-01-01

In view of the toxic potential of a bioweapon threat, rapid visual recognition and sensing of ricin has been of considerable interest while remaining a challenging task up to date. In this study, a gold nanopin-based colorimetric sensor is developed realizing a multicolor variation for ricin qualitative recognition and analysis. It is revealed that such plasmonic metasurfaces based on nanopin-cavity resonator exhibit reflective color appearance, due to the excitation of standing-wave resonances of narrow bandwidth in visible region. This clear color variation is a consequence of the reflective color mixing defined by different resonant wavelengths. In addition, the colored metasurfaces appear sharp color difference in a narrow refractive index range, which makes them especially well-suited for sensing applications. Therefore, this antibody-functionalized nanopin-cavity biosensor features high sensitivity and fast response, allowing for visual quantitative ricin detection within the range of 10-120 ng mL -1 (0.15 × 10 -9 -1.8 × 10 -9 m), a limit of detection of 10 ng mL -1 , and the typical measurement time of less than 10 min. The on-chip integration of such nanopin metasurfaces to portable colorimetric microfluidic device may be envisaged for the quantitative studies of a variety of biochemical molecules. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of a capillary electrophoresis instrument with laser-induced fluorescence detection for DNA quantitation. Comparison of YO-PRO-1 and PicoGreen assays.

PubMed

Guillo, Christelle; Ferrance, Jerome P; Landers, James P

2006-04-28

Highly selective and sensitive assays are required for detection and quantitation of the small masses of DNA typically encountered in clinical and forensic settings. High detection sensitivity is achieved using fluorescent labeling dyes and detection techniques such as spectrofluorometers, microplate readers and cytometers. This work describes the use of a laser-induced fluorescence (LIF) detector in conjunction with a commercial capillary electrophoresis instrument for DNA quantitation. PicoGreen and YO-PRO-1, two fluorescent DNA labeling dyes, were used to assess the potential of the system for routine DNA analysis. Linearity, reproducibility, sensitivity, limits of detection and quantitation, and sample stability were examined for the two assays. The LIF detector response was found to be linear (R2 > 0.999) and reproducible (RSD < 9%) in both cases. The PicoGreen assay displayed lower limits of detection and quantitation (20 pg and 60 pg, respectively) than the YO-PRO-1 assay (60 pg and 260 pg, respectively). Although a small variation in fluorescence was observed for the DNA/dye complexes over time, quantitation was not significantly affected and the solutions were found to be relatively stable for 80 min. The advantages of the technique include a 4- to 40-fold reduction in the volume of sample required compared to traditional assays, a 2- to 20-fold reduction in the volume of reagents consumed, fast and automated analysis, and low cost (no specific instrumentation required).

Diiodobodipy-styrylbodipy Dyads: Preparation and Study of the Intersystem Crossing and Fluorescence Resonance Energy Transfer.

PubMed

Wang, Zhijia; Xie, Yun; Xu, Kejing; Zhao, Jianzhang; Glusac, Ksenija D

2015-07-02

2,6-Diiodobodipy-styrylbodipy dyads were prepared to study the competing intersystem crossing (ISC) and the fluorescence-resonance-energy-transfer (FRET), and its effect on the photophysical property of the dyads. In the dyads, 2,6-diiodobodipy moiety was used as singlet energy donor and the spin converter for triplet state formation, whereas the styrylbodipy was used as singlet and triplet energy acceptors, thus the competition between the ISC and FRET processes is established. The photophysical properties were studied with steady-state UV-vis absorption and fluorescence spectroscopy, electrochemical characterization, and femto/nanosecond time-resolved transient absorption spectroscopies. FRET was confirmed with steady state fluorescence quenching and fluorescence excitation spectra and ultrafast transient absorption spectroscopy (kFRET = 5.0 × 10(10) s(-1)). The singlet oxygen quantum yield (ΦΔ = 0.19) of the dyad was reduced as compared with that of the reference spin converter (2,6-diiodobodipy, ΦΔ = 0.85), thus the ISC was substantially inhibited by FRET. Photoinduced intramolecular electron transfer (ET) was studied by electrochemical data and fluorescence quenching. Intermolecular triplet energy transfer was studied with nanosecond transient absorption spectroscopy as an efficient (ΦTTET = 92%) and fast process (kTTET = 5.2 × 10(4) s(-1)). These results are useful for designing organic triplet photosensitizers and for the study of the photophysical properties.

A fluorescent nucleic acid nanodevice quantitatively images elevated cyclic adenosine monophosphate in membrane-bound compartments.

PubMed

Sharma, Suruchi; Zaveri, Anisha; Visweswariah, Sandhya S; Krishnan, Yamuna

2014-11-12

cAMPhor: In the presence of cAMP, cAMPhor folds into a structure that binds DFHBI (green), increasing its fluorescence, while Alexa 647 (red) functions as a normalizing dye. It can thus be used to spatially image cAMP quantitatively in membrane-bound compartments. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A unique charge-coupled device/xenon arc lamp based imaging system for the accurate detection and quantitation of multicolour fluorescence.

PubMed

Spibey, C A; Jackson, P; Herick, K

2001-03-01

In recent years the use of fluorescent dyes in biological applications has dramatically increased. The continual improvement in the capabilities of these fluorescent dyes demands increasingly sensitive detection systems that provide accurate quantitation over a wide linear dynamic range. In the field of proteomics, the detection, quantitation and identification of very low abundance proteins are of extreme importance in understanding cellular processes. Therefore, the instrumentation used to acquire an image of such samples, for spot picking and identification by mass spectrometry, must be sensitive enough to be able, not only, to maximise the sensitivity and dynamic range of the staining dyes but, as importantly, adapt to the ever changing portfolio of fluorescent dyes as they become available. Just as the available fluorescent probes are improving and evolving so are the users application requirements. Therefore, the instrumentation chosen must be flexible to address and adapt to those changing needs. As a result, a highly competitive market for the supply and production of such dyes and the instrumentation for their detection and quantitation have emerged. The instrumentation currently available is based on either laser/photomultiplier tube (PMT) scanning or lamp/charge-coupled device (CCD) based mechanisms. This review briefly discusses the advantages and disadvantages of both System types for fluorescence imaging, gives a technical overview of CCD technology and describes in detail a unique xenon/are lamp CCD based instrument, from PerkinElmer Life Sciences. The Wallac-1442 ARTHUR is unique in its ability to scan both large areas at high resolution and give accurate selectable excitation over the whole of the UV/visible range. It operates by filtering both the excitation and emission wavelengths, providing optimal and accurate measurement and quantitation of virtually any available dye and allows excellent spectral resolution between different fluorophores

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

Direct Detection of Time-Resolved Rabi Oscillationsin a Single Quantum Dot via Resonance Fluorescence

DTIC Science & Technology

2013-03-19

Ware, E. A. Stinaff, D. Gammon, M. F. Doty, A. S . Bracker, D. Gershoni, V. L. Korenev , S . C. Bădescu, Y. Lyanda-Geller, and T. L. Reinecke, Phys. Rev...A SINGLE QUANTUM DOT VIA RESONANCE FLUORESCENCE 5a. CONTRACT NUMBER FA8750-12-2-0333 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR( S ) J...NUMBER CH 7. PERFORMING ORGANIZATION NAME( S ) AND ADDRESS(ES) University of Michigan 450 Church Street Ann Arbor MI 48109-1040 8. PERFORMING

X-ray frequency combs from optically controlled resonance fluorescence

NASA Astrophysics Data System (ADS)

Cavaletto, Stefano M.; Harman, Zoltán; Buth, Christian; Keitel, Christoph H.

2013-12-01

An x-ray pulse-shaping scheme is put forward for imprinting an optical frequency comb onto the radiation emitted on a driven x-ray transition, thus producing an x-ray frequency comb. A four-level system is used to describe the level structure of N ions driven by narrow-bandwidth x rays, an optical auxiliary laser, and an optical frequency comb. By including many-particle enhancement of the emitted resonance fluorescence, a spectrum is predicted consisting of equally spaced narrow lines which are centered on an x-ray transition energy and separated by the same tooth spacing as the driving optical frequency comb. Given an x-ray reference frequency, our comb could be employed to determine an unknown x-ray frequency. While relying on the quality of the light fields used to drive the ensemble of ions, the model has validity at energies from the 100 eV to the keV range.

Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

PubMed

Faden, Frederik; Eschen-Lippold, Lennart; Dissmeyer, Nico

2016-01-01

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of

An artificial tongue fluorescent sensor array for identification and quantitation of various heavy metal ions.

PubMed

Xu, Wang; Ren, Changliang; Teoh, Chai Lean; Peng, Juanjuan; Gadre, Shubhankar Haribhau; Rhee, Hyun-Woo; Lee, Chi-Lik Ken; Chang, Young-Tae

2014-09-02

Herein, a small-molecule fluorescent sensor array for rapid identification of seven heavy metal ions was designed and synthesized, with its sensing mechanism mimicking that of a tongue. The photoinduced electron transfer and intramolecular charge transfer mechanism result in combinatorial interactions between sensor array and heavy metal ions, which lead to diversified fluorescence wavelength shifts and emission intensity changes. Upon principle component analysis (PCA), this result renders clear identification of each heavy metal ion on a 3D spatial dispersion graph. Further exploration provides a concentration-dependent pattern, allowing both qualitative and quantitative measurements of heavy metal ions. On the basis of this information, a "safe-zone" concept was proposed, which provides rapid exclusion of versatile hazardous species from clean water samples based on toxicity characteristic leaching procedure standards. This type of small-molecule fluorescent sensor array could open a new avenue for multiple heavy metal ion detection and simplified water quality analysis.

Efficient HOMO-LUMO separation by multiple resonance effect toward ultrapure blue thermally activated delayed fluorescence

NASA Astrophysics Data System (ADS)

Hatakeyama, Takuji; Ikuta, Toshiaki; Shiren, Kazushi; Nakajima, Kiichi; Nomura, Shintaro; Ni, Jingping

2016-09-01

Organic light-emitting diodes (OLEDs) play an important role in the new generation of flat-panel displays. Conventional OLEDs employing fluorescent materials together with triplet-triplet annihilation suffer from a relatively low internal quantum efficiency (IQE) of 62.5%. On the other hand, the IQE of OLEDs employing phosphorescent or thermally activated delayed fluorescence (TADF) materials can reach 100%. However, these materials exhibit very broad peaks with a full-width at half-maximum (FWHM) of 70-100 nm and cannot satisfy the color-purity requirements for displays. Therefore, the latest commercial OLED displays employ blue fluorescent materials with a relatively low IQE, and efficient blue emitters with a small FWHM are highly needed. In our manuscript, we present organic molecules that exhibit ultrapure blue fluorescence based on TADF. These molecules consist of three benzene rings connected by one boron and two nitrogen atoms, which establish a rigid polycyclic framework and significant localization of the highest occupied and lowest unoccupied molecular orbitals by a multiple resonance effect. An OLED device based on the new emitter exhibits ultrapure blue emission at 467 nm with an FWHM of 28 nm, Commission Internationale de l'Eclairage (CIE) coordinates of (0.12, 0.13), and an IQE of 100%, which represent record-setting performance for blue OLED devices.

Fluorescent staining for leukocyte chemotaxis. Eosinophil-specific fluorescence with aniline blue.

PubMed

McCrone, E L; Lucey, D R; Weller, P F

1988-11-10

To overcome problems associated with the quantitation of human eosinophil chemotaxis in micropore filters, we have developed a fluorescent method of specifically staining eosinophils in chemotactic filters. A neutral solution of aniline blue yielded bright green fluorescent staining of the cytoplasmic granules of eosinophils. Other leukocytes and contaminating neutrophils potentially present with eosinophils did not fluoresce with aniline blue. The fluorescent staining eosinophils within filters provided bright, non-fading images that facilitated visual microscopic counting and were of sufficiently high contrast, unlike those with conventional eosinophil stains, to allow image analyzer based enumeration of eosinophil chemotactic responses at levels through the filters. Although not cell type-specific, congo red and ethidium bromide also provided high contrast, fluorescent images of all leukocyte types within chemotactic filters. Fluorescent staining with aniline blue constitutes a rapid, stable and eosinophil-specific stain that facilitates the visual or image analyzer-based quantitation of eosinophil chemotaxis.

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.

Nuclear Resonance Fluorescence Measurements on ^237Np for Security and Safeguards Applications

NASA Astrophysics Data System (ADS)

Angell, C. T.; Joshi, T.; Yee, Ryan; Norman, E. B.; Kulp, W. D.; Warren, G. A.; Korbly, S.; Klimenko, A.; Wilson, C.; Copping, R.; Shuh, D. K.

2009-10-01

The smuggling of nuclear material and the diversion of fissile material for covert weapon programs both present grave risks to world security. Methods are needed to detect nuclear material smuggled in cargo, and for proper material accountability in civilian fuel re-processing facilities. Nuclear resonance fluorescence (NRF) is a technique that can address both needs. It is a non-destructive active interrogation method that provides isotope-specific information. It works by using a γ-ray beam to resonantly excite levels in a nucleus and observing the γ-rays emitted whose energy and intensity are characteristic of that isotope. ^237Np presents significant safeguard challenges; it is fissile yet currently has fewer safeguard restrictions. NRF measurements on ^237Np will expand the nuclear database and will permit designing interrogation and assay systems. Measurements were made using the bremsstrahlung beam at the HVRL at MIT on a 7 g target of ^237Np with two incident electron energies of 2.8 and 3.1 MeV. Results will be presented with discussion of the relevant nuclear structure necessary to predict levels in other actinides.

Combining 5-Aminolevulinic Acid Fluorescence and Intraoperative Magnetic Resonance Imaging in Glioblastoma Surgery: A Histology-Based Evaluation.

PubMed

Hauser, Sonja B; Kockro, Ralf A; Actor, Bertrand; Sarnthein, Johannes; Bernays, René-Ludwig

2016-04-01

Glioblastoma resection guided by 5-aminolevulinic acid (5-ALA) fluorescence and intraoperative magnetic resonance imaging (iMRI) may improve surgical results and prolong survival. To evaluate 5-ALA fluorescence combined with subsequent low-field iMRI for resection control in glioblastoma surgery. Fourteen patients with suspected glioblastoma suitable for complete resection of contrast-enhancing portions were enrolled. The surgery was carried out using 5-ALA-induced fluorescence and frameless navigation. Areas suspicious for tumor underwent biopsy. After complete resection of fluorescent tissue, low-field iMRI was performed. Areas suspicious for tumor remnant underwent biopsy under navigation guidance and were resected. The histological analysis was blinded. In 13 of 14 cases, the diagnosis was glioblastoma multiforme. One lymphoma and 1 case without fluorescence were excluded. In 11 of 12 operations, residual contrast enhancement on iMRI was found after complete resection of 5-ALA fluorescent tissue. In 1 case, the iMRI enhancement was in an eloquent area and did not undergo a biopsy. The 28 biopsies of areas suspicious for tumor on iMRI in the remaining 10 cases showed tumor in 39.3%, infiltration zone in 25%, reactive central nervous system tissue in 32.1%, and normal brain in 3.6%. Ninety-three fluorescent and 24 non-fluorescent tissue samples collected before iMRI contained tumor in 95.7% and 87.5%, respectively. 5-ALA fluorescence-guided resection may leave some glioblastoma tissue undetected. MRI might detect areas suspicious for tumor even after complete resection of all fluorescent tissue; however, due to the limited accuracy of iMRI in predicting tumor remnant (64.3%), resection of this tissue has to be considered with caution in eloquent regions.

Method for accurate quantitation of background tissue optical properties in the presence of emission from a strong fluorescence marker

NASA Astrophysics Data System (ADS)

Bravo, Jaime; Davis, Scott C.; Roberts, David W.; Paulsen, Keith D.; Kanick, Stephen C.

2015-03-01

Quantification of targeted fluorescence markers during neurosurgery has the potential to improve and standardize surgical distinction between normal and cancerous tissues. However, quantitative analysis of marker fluorescence is complicated by tissue background absorption and scattering properties. Correction algorithms that transform raw fluorescence intensity into quantitative units, independent of absorption and scattering, require a paired measurement of localized white light reflectance to provide estimates of the optical properties. This study focuses on the unique problem of developing a spectral analysis algorithm to extract tissue absorption and scattering properties from white light spectra that contain contributions from both elastically scattered photons and fluorescence emission from a strong fluorophore (i.e. fluorescein). A fiber-optic reflectance device was used to perform measurements in a small set of optical phantoms, constructed with Intralipid (1% lipid), whole blood (1% volume fraction) and fluorescein (0.16-10 μg/mL). Results show that the novel spectral analysis algorithm yields accurate estimates of tissue parameters independent of fluorescein concentration, with relative errors of blood volume fraction, blood oxygenation fraction (BOF), and the reduced scattering coefficient (at 521 nm) of <7%, <1%, and <22%, respectively. These data represent a first step towards quantification of fluorescein in tissue in vivo.

Quantitative magnetic resonance (QMR) measurement of changes in body composition of neonatal pigs

USDA-ARS?s Scientific Manuscript database

The survival of low birth weight pigs in particular may depend on energy stores in the body. QMR (quantitative magnetic resonance) is a new approach to measuring total body fat, lean and water. These measurements are based on quantifying protons associated with lipid and water molecules in the body...

Measuring evaporation rates of laser-trapped droplets by use of fluorescent morphology-dependent resonances.

PubMed

Pastel, R; Struthers, A

2001-05-20

Morphology-dependent resonances (MDRs) are used to measure accurately the evaporation rates of laser-trapped 1- to 2-mum droplets of ethylene glycol. Droplets containing 3 x 10(-5) M Rhodamine-590 laser dye are optically trapped in a 20-mum hollow fiber by two counterpropagating 150-mW, 800-nm laser beams. A weaker 532-nm laser excites the dye, and fluorescence emission is observed near 560 nm as the droplet evaporates. A complete series of first-order TE and TM MDRs dominates the fluorescent output. MDR mode identification sizes the droplets and provides accurate evaporation rates. We verify the automated MDR mode identification by counting fringes in a videotape of the experiment. The longitudinal spring constant of the trap, measured by analysis of the videotaped motion of droplets perturbed from the trap center, provides independent verification of the laser's intensity within the trap.

Measuring Evaporation Rates of Laser-Trapped Droplets by Use of Fluorescent Morphology-Dependent Resonances

NASA Astrophysics Data System (ADS)

Pastel, Robert; Struthers, Allan

2001-05-01

Morphology-dependent resonances (MDRs) are used to measure accurately the evaporation rates of laser-trapped 1- to 2- m droplets of ethylene glycol. Droplets containing 3 x10-5 M Rhodamine-590 laser dye are optically trapped in a 20- m hollow fiber by two counterpropagating 150-mW, 800-nm laser beams. A weaker 532-nm laser excites the dye, and fluorescence emission is observed near 560 nm as the droplet evaporates. A complete series of first-order TE and TM MDRs dominates the fluorescent output. MDR mode identification sizes the droplets and provides accurate evaporation rates. We verify the automated MDR mode identification by counting fringes in a videotape of the experiment. The longitudinal spring constant of the trap, measured by analysis of the videotaped motion of droplets perturbed from the trap center, provides independent verification of the laser s intensity within the trap.

Estimation of the number of fluorescent end-members for quantitative analysis of multispectral FLIM data.

PubMed

Gutierrez-Navarro, Omar; Campos-Delgado, Daniel U; Arce-Santana, Edgar R; Maitland, Kristen C; Cheng, Shuna; Jabbour, Joey; Malik, Bilal; Cuenca, Rodrigo; Jo, Javier A

2014-05-19

Multispectral fluorescence lifetime imaging (m-FLIM) can potentially allow identifying the endogenous fluorophores present in biological tissue. Quantitative description of such data requires estimating the number of components in the sample, their characteristic fluorescent decays, and their relative contributions or abundances. Unfortunately, this inverse problem usually requires prior knowledge about the data, which is seldom available in biomedical applications. This work presents a new methodology to estimate the number of potential endogenous fluorophores present in biological tissue samples from time-domain m-FLIM data. Furthermore, a completely blind linear unmixing algorithm is proposed. The method was validated using both synthetic and experimental m-FLIM data. The experimental m-FLIM data include in-vivo measurements from healthy and cancerous hamster cheek-pouch epithelial tissue, and ex-vivo measurements from human coronary atherosclerotic plaques. The analysis of m-FLIM data from in-vivo hamster oral mucosa identified healthy from precancerous lesions, based on the relative concentration of their characteristic fluorophores. The algorithm also provided a better description of atherosclerotic plaques in term of their endogenous fluorophores. These results demonstrate the potential of this methodology to provide quantitative description of tissue biochemical composition.

Dynamic nuclear protein interactions investigated using fluorescence lifetime and fluorescence fluctuation spectroscopy

NASA Astrophysics Data System (ADS)

Siegel, Amanda P.; Hays, Nicole M.; Day, Richard N.

2012-03-01

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic interactions in pituitary specific gene regulation.

Quantitative Magnetic Resonance Thermometry and Its Use with MR-Guided Focused Ultrasound

NASA Astrophysics Data System (ADS)

Pauly, Kim

2014-03-01

Focused ultrasound (FUS) uses a large area array, typically outside the body, that is geometrically or electronically focused to a point deep in the body. Such focusing provides amplification of the ultrasound intensity, thereby allowing heating of tissue to the point of coagulation at the focus, without damage to the intervening tissue. Guidance of FUS treatments deep in the body can be done quantitatively with magnetic resonance (MR) thermometry, termed MRgFUS. The physics behind MR thermometry lie in the changes in hydrogen bonding with temperature. As tissue temperature rises, hydrogen bonds break, allowing the return of the electron cloud to shield water protons, reducing the magnetic field seen by the protons, and the resonant frequency. The change in resonant frequency is -0.01 ppm per degree C and is the same for all aqueous tissues. The result of the shift in proton resonant frequency is seen in the phase of gradient echo images. Subtraction of the phase of images acquired before and during heating allows the removal of background phase from other sources, yielding quantitative temperature maps. Temperature standard deviations less than 1 degree C are readily achievable and thermal dose maps are easily calculated. Thermal dose is found from a conversion of the whole temperature-time curve to an equivalent number of minutes at 43 degrees C. A thermal dose of 240 minutes is often taken as the threshold for tissue damage. MR thermometry is complicated by the motion of the target tissue and/or motion of other organs such as occurs during respiration. More sophisticated algorithms than the simple baseline subtraction take advantage of the facts that motion can be repetitive (in the case of respiratory motion) and/or the fact that the focal region in MRgFUS is small, allowing for extraction of the heat from the phase profile without subtraction of a background phase.

Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

PubMed

Mahen, Robert; Koch, Birgit; Wachsmuth, Malte; Politi, Antonio Z; Perez-Gonzalez, Alexis; Mergenthaler, Julia; Cai, Yin; Ellenberg, Jan

2014-11-05

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells. © 2014 Mahen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.

PubMed

Vedula, Pavan; Cruz, Lissette A; Gutierrez, Natasha; Davis, Justin; Ayee, Brian; Abramczyk, Rachel; Rodriguez, Alexis J

2016-06-30

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.

Electrosprayed synthesis of red-blood-cell-like particles with dual modality for magnetic resonance and fluorescence imaging.

PubMed

Hayashi, Koichiro; Ono, Kenji; Suzuki, Hiromi; Sawada, Makoto; Moriya, Makoto; Sakamoto, Wataru; Yogo, Toshinobu

2010-11-05

Red blood cells (RBCs) are able to avoid filtration in the spleen to prolong their half-time in the body because of their flexibility and unique shape, or a concave disk with diameter of some 10 μm. In addition, they can flow through capillary blood vessels, which are smaller than the diameter of RBCs, by morphing into a parachute-like shape. In this study, flexible RBC-like polymer particles are synthesized by electrospraying based on electrospinning. Furthermore, magnetite nanoparticles and fluorescent dye are encapsulated in the particles via in situ hydrolysis of an iron-organic compound in the presence of celluloses. The superparamagnetic behavior of the particles is confirmed by low-temperature magnetic measurements. The particles exhibited not only a dark contrast in magnetic resonance imaging (MRI), but also effective fluorescence. The RBC-like particles with flexibility are demonstrated to have a dual-modality for MRI and fluorescence imaging.

Segmentation-based retrospective shading correction in fluorescence microscopy E. coli images for quantitative analysis

NASA Astrophysics Data System (ADS)

Mai, Fei; Chang, Chunqi; Liu, Wenqing; Xu, Weichao; Hung, Yeung S.

2009-10-01

Due to the inherent imperfections in the imaging process, fluorescence microscopy images often suffer from spurious intensity variations, which is usually referred to as intensity inhomogeneity, intensity non uniformity, shading or bias field. In this paper, a retrospective shading correction method for fluorescence microscopy Escherichia coli (E. Coli) images is proposed based on segmentation result. Segmentation and shading correction are coupled together, so we iteratively correct the shading effects based on segmentation result and refine the segmentation by segmenting the image after shading correction. A fluorescence microscopy E. Coli image can be segmented (based on its intensity value) into two classes: the background and the cells, where the intensity variation within each class is close to zero if there is no shading. Therefore, we make use of this characteristics to correct the shading in each iteration. Shading is mathematically modeled as a multiplicative component and an additive noise component. The additive component is removed by a denoising process, and the multiplicative component is estimated using a fast algorithm to minimize the intra-class intensity variation. We tested our method on synthetic images and real fluorescence E.coli images. It works well not only for visual inspection, but also for numerical evaluation. Our proposed method should be useful for further quantitative analysis especially for protein expression value comparison.

Quantitative Light-induced Fluorescence-Digital as an oral hygiene evaluation tool to assess plaque accumulation and enamel demineralization in orthodontics.

PubMed

Miller, Cara C; Burnside, Girvan; Higham, Susan M; Flannigan, Norah L

2016-11-01

  To assess the use of Quantitative Light-induced Fluorescence-Digital as an oral hygiene evaluation tool during orthodontic treatment.   In this prospective, randomized clinical trial, 33 patients undergoing fixed orthodontic appliance treatment were randomly allocated to receive oral hygiene reinforcement at four consecutive appointments using either white light (WL) or Quantitative Light-induced Fluorescence-Digital (QLF) images, taken with a device, as visual aids. Oral hygiene was recorded assessing the QLF images for demineralization, by fluorescence loss (ΔF), and plaque coverage (ΔR30). A debriefing questionnaire ascertained patient perspectives.   There were no significant differences in demineralization (P  =  .56) or plaque accumulation (P  =  .82) between the WL and QLF groups from T0 to T4. There was no significant reduction in demineralization, ΔF, in the WL, or the QLF group from T0-T4 (P > .05); however, there was a significant reduction in ΔR30 plaque scores (P < .05). All the participants found being shown the images helpful, with 100% of the QLF group reflecting that it would be useful to have oral hygiene reinforcement for the full duration of treatment compared with 81% of the WL group (OR 2.3; P < .05).   Quantitative Light-induced Fluorescence-Digital can be used to detect and monitor demineralization and plaque during orthodontics. Oral hygiene reinforcement at consecutive appointments using WL or QLF images as visual aids is effective in reducing plaque coverage. In terms of clinical benefits, QLF and WL images are of similar effectiveness; however, patients preferred the QLF images.

A fluorescent-photochrome method for the quantitative characterization of solid phase antibody orientation.

PubMed

Ahluwalia, Arti; De Rossi, Danilo; Giusto, Giuseppe; Chen, Oren; Papper, Vladislav; Likhtenshtein, Gertz I

2002-06-15

A fluorescent-photochrome method of quantifying the orientation and surface density of solid phase antibodies is described. The method is based on measurements of quenching and rates of cis-trans photoisomerization and photodestruction of a stilbene-labeled hapten by a quencher in solution. These experimental parameters enable a quantitative description of the order of binding sites of antibodies immobilized on a surface and can be used to characterize the microviscosity and steric hindrance in the vicinity of the binding site. Furthermore, a theoretical method for the determination of the depth of immersion of the fluorescent label in a two-phase system was developed. The model exploits the concept of dynamic interactions and is based on the empirical dependence of parameters of static exchange interactions on distances between exchangeable centers. In the present work, anti-dinitrophenyl (DNP) antibodies and stilbene-labeled DNP were used to investigate three different protein immobilization methods: physical adsorption, covalent binding, and the Langmuir-Blodgett technique. Copyright 2002 Elsevier Science (USA).

Smart Drug Delivery System-Inspired Enzyme-Linked Immunosorbent Assay Based on Fluorescence Resonance Energy Transfer and Allochroic Effect Induced Dual-Modal Colorimetric and Fluorescent Detection.

PubMed

Miao, Luyang; Zhu, Chengzhou; Jiao, Lei; Li, He; Du, Dan; Lin, Yuehe; Wei, Qin

2018-02-06

Numerous analytical techniques have been undertaken for the detection of protein biomarkers because of their extensive and significant applications in clinical diagnosis, whereas there are few strategies to develop dual-readout immunosensors to achieve more accurate results. To the best of our knowledge, inspired by smart drug delivery system (DDS), a novel pH-responsive modified enzyme-linked immunosorbent assay (ELISA) was innovatively developed for the first time, realizing dual-modal colorimetric and fluorescent detection of cardiac troponin I (cTnI). Curcumin (CUR) was elaborately selected as a reporter molecule, which played the same role of drugs in DDS based on the following considerations: (1) CUR can be used as a kind of pH indicator by the inherited allochroic effect induced by basic pH value; (2) the fluorescence of CUR can be quenched by certain nanocarriers as the acceptor because of the occurrence of fluorescence resonance energy transfer (FRET), while recovered by the stimuli of basic pH value, which can produce "signal-on" fluorescence detection. Three-dimensional MoS 2 nanoflowers (3D-MoS 2 NFs) were employed in immobilizing CUR to constitute a nanoprobe for the determination of cTnI by virtue of good biocompatibility, high absorption capacity, and fluorescence quench efficiency toward CUR. The proposed DDS-inspired ELISA offered dual-modal colorimetric and fluorescent detection of cTnI, thereby meeting the reliable and precise analysis requirements. We believe that the developed dual-readout ELISA will create a new avenue and bring innovative inspirations for biological detections.

Mode-splitting for refractive index sensing in fluorescent whispering gallery mode resonators with broken symmetry

NASA Astrophysics Data System (ADS)

Kang, Yvonne Q.; François, Alexandre; Riesen, Nicolas; Monro, Tanya M.

2018-02-01

Whispering Gallery Mode (WGM) biosensors have been widely exploited over the past decade, owing to their unprecedented detection limits and label free capability. WGM based sensing mechanisms, such as resonance frequency shift, linewidth broadening, and splitting of the two counter-propagating WGMs, have been extensively researched and applied for bio-chemical sensing. However, the mode-splitting of the originally degenerate WGMs from different equatorial planes on a fluorescent microsphere has not been fully investigated. In this work, we break the symmetry of the surrounding environment outside the microsphere by partially embedding the sphere into a high-refractive-index medium (i.e. glue), to lift the degeneracy of the modes from different WGM planes. The split-modes from multiple planes of the fluorescent microsphere are indiscriminately collected. It is found that the effective quality factor Q of the WGMs increases non-conventionally as the Refractive Index (RI) of the probing liquid increases up to the point where it is equal to that of the glue. This presents a new methodology for quantifying changes in the probing environment based on the Q spoiling of the resonances as determined by the RI difference between the environment and that of the reference glue. Furthermore, we find that this sensing platform opens the door to simple self-referenced sensing techniques based on the analysis of the spectral positions of subsets of the split modes.

Quantitative performance measurements of bent crystal Laue analyzers for X-ray fluorescence spectroscopy.

PubMed

Karanfil, C; Bunker, G; Newville, M; Segre, C U; Chapman, D

2012-05-01

Third-generation synchrotron radiation sources pose difficult challenges for energy-dispersive detectors for XAFS because of their count rate limitations. One solution to this problem is the bent crystal Laue analyzer (BCLA), which removes most of the undesired scatter and fluorescence before it reaches the detector, effectively eliminating detector saturation due to background. In this paper experimental measurements of BCLA performance in conjunction with a 13-element germanium detector, and a quantitative analysis of the signal-to-noise improvement of BCLAs are presented. The performance of BCLAs are compared with filters and slits.

Raman and fluorescence characteristics of resonant inelastic X-ray scattering from doped superconducting cuprates

DOE PAGES

Huang, H. Y.; Jia, C. J.; Chen, Z. Y.; ...

2016-01-22

Measurements of spin excitations are essential for an understanding of spin-mediated pairing for superconductivity; and resonant inelastic X-ray scattering (RIXS) provides a considerable opportunity to probe high-energy spin excitations. However, whether RIXS correctly measures the collective spin excitations of doped superconducting cuprates remains under debate. Here we demonstrate distinct Raman- and fluorescence-like RIXS excitations of Bi1.5Pb0.6Sr1.54CaCu2O8+δ. Combining photon-energy and momentum dependent RIXS measurements with theoretical calculations using exact diagonalization provides conclusive evidence that the Raman-like RIXS excitations correspond to collective spin excitations, which are magnons in the undoped Mott insulators and evolve into paramagnons in doped superconducting compounds. In contrast,more » the fluorescence-like shifts are due primarily to the continuum of particle-hole excitations in the charge channel. Our results show that under the proper experimental conditions RIXS indeed can be used to probe paramagnons in doped high-Tc cuprate superconductors.« less

Fluorescent material concentration dependency: Förster resonance energy transfer in quasi-solid state DSSCs

NASA Astrophysics Data System (ADS)

Kim, Dong Woo; Jo, Hyun-Jun; Thogiti, Suresh; Yang, Weon Ki; Cheruku, Rajesh; Kim, Jae Hong

2017-05-01

Förster resonance energy transfer (FRET) is critical for wide spectral absorption, an increased dye loading, and photocurrent generation of dye-sensitized solar cells (DSSCs). This process consists of organic fluorescent materials (as an energy donor), and an organic dye (as an energy acceptor on TiO2 surfaces) with quasi-solid electrolyte. The judicious choice of the energy donor and acceptor facilitates a strong spectral overlap between the emission and absorption regions of the fluorescent materials and dye. This FRET process enhances the light-harvesting characteristics of quasi-solid state DSSCs. In this study, DSSCs containing different concentrations (0, 1, and 1.5 wt%) of a fluorescent material (FM) as the energy donor are investigated using FRET. The power conversion efficiency of DSSCs containing FMs in a quasi-solid electrolyte increased by 33% over a pristine cell. The optimized cell fabricated with the quasi-solid state DSSC containing 1.0 wt% FM shows a maximum efficiency of 3.38%, with a short-circuit current density ( J SC ) of 4.32 mA/cm-2, and an open-circuit voltage ( V OC ) of 0.68 V under illumination of simulated solar light (AM 1.5G, 100 mW/cm-2). [Figure not available: see fulltext.

A FRET-Based Ratiometric Chemosensor for in Vitro Cellular Fluorescence Analyses of pH

PubMed Central

Zhou, Xianfeng; Su, Fengyu; Lu, Hongguang; Senechal-Willis, Patti; Tian, Yanqing; Johnson, Roger H.; Meldrum, Deirdre R.

2011-01-01

Ratiometric fluorescence sensing is an important technique for precise and quantitative analysis of biological events occurring under complex conditions by simultaneously recording fluorescence intensities at two wavelengths and calculating their ratios. Herein, we design a ratiometric chemosensor for pH that is based on photo-induced electron transfer (PET) and binding-induced modulation of fluorescence resonance energy transfer (FRET) mechanisms. This ratiometric chemosensor was constructed by introduction of a pH-insensitive coumarin fluorophore as a FRET donor into a pH-sensitive amino-naphthalimide derivative as the FRET acceptor. The sensor exhibited clear dual-mission signal changes in blue and green spectral windows upon pH changes. The pH sensor was applied for not only measuring cellular pH, but also for visualizing stimulus-responsive changes of intracellular pH values. PMID:21982292

Measurement of a heavy-hole hyperfine interaction in InGaAs quantum dots using resonance fluorescence.

PubMed

Fallahi, P; Yilmaz, S T; Imamoğlu, A

2010-12-17

We measure the strength and the sign of hyperfine interaction of a heavy hole with nuclear spins in single self-assembled quantum dots. Our experiments utilize the locking of a quantum dot resonance to an incident laser frequency to generate nuclear spin polarization. By monitoring the resulting Overhauser shift of optical transitions that are split either by electron or exciton Zeeman energy with respect to the locked transition using resonance fluorescence, we find that the ratio of the heavy-hole and electron hyperfine interactions is -0.09 ± 0.02 in three quantum dots. Since hyperfine interactions constitute the principal decoherence source for spin qubits, we expect our results to be important for efforts aimed at using heavy-hole spins in quantum information processing.

Molecular and Cellular Quantitative Microscopy: theoretical investigations, technological developments and applications to neurobiology

NASA Astrophysics Data System (ADS)

Esposito, Alessandro

2006-05-01

This PhD project aims at the development and evaluation of microscopy techniques for the quantitative detection of molecular interactions and cellular features. The primarily investigated techniques are Fαrster Resonance Energy Transfer imaging and Fluorescence Lifetime Imaging Microscopy. These techniques have the capability to quantitatively probe the biochemical environment of fluorophores. An automated microscope capable of unsupervised operation has been developed that enables the investigation of molecular and cellular properties at high throughput levels and the analysis of cellular heterogeneity. State-of-the-art Förster Resonance Energy Transfer imaging, Fluorescence Lifetime Imaging Microscopy, Confocal Laser Scanning Microscopy and the newly developed tools have been combined with cellular and molecular biology techniques for the investigation of protein-protein interactions, oligomerization and post-translational modifications of α-Synuclein and Tau, two proteins involved in Parkinson’s and Alzheimer’s disease, respectively. The high inter-disciplinarity of this project required the merging of the expertise of both the Molecular Biophysics Group at the Debye Institute - Utrecht University and the Cell Biophysics Group at the European Neuroscience Institute - Gαttingen University. This project was conducted also with the support and the collaboration of the Center for the Molecular Physiology of the Brain (Göttingen), particularly with the groups associated with the Molecular Quantitative Microscopy and Parkinson’s Disease and Aggregopathies areas. This work demonstrates that molecular and cellular quantitative microscopy can be used in combination with high-throughput screening as a powerful tool for the investigation of the molecular mechanisms of complex biological phenomena like those occurring in neurodegenerative diseases.

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

Quantitative nondestructive evaluation of ceramic matrix composite by the resonance method

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

Watanabe, T.; Aizawa, T.; Kihara, J.

The resonance method was developed to make quantitative nondestructive evaluation on the mechanical properties without any troublesome procedure. Since the present method is indifferent to the geometry of specimen, both monolithic and ceramic matrix composite materials in process can be evaluated in the nondestructive manner. Al{sub 2}O{sub 3}, Si{sub 3}N{sub 4}, SiC/Si{sub 3}N{sub 4}, and various C/C composite materials are employed to demonstrate the validity and effectiveness of the present method.

Quantitative monitoring of activity-dependent bulk endocytosis of synaptic vesicle membrane by fluorescent dextran imaging

PubMed Central

Clayton, Emma Louise; Cousin, Michael Alan

2012-01-01

Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) retrieval mode in central nerve terminals during periods of intense neuronal activity. Despite this fact there are very few real time assays that report the activity of this critical SV retrieval mode. In this paper we report a simple and quantitative assay of ADBE using uptake of large flourescent dextrans as fluid phase markers. We show that almost all dextran uptake occurs in nerve terminals, using co-localisation with the fluorescent probe FM1-43. We also demonstrate that accumulated dextran cannot be unloaded by neuronal stimulation, indicating its specific loading into bulk endosomes and not SVs. Quantification of dextran uptake was achieved by using thresholding analysis to count the number of loaded nerve terminals, since monitoring the average fluorescence intensity of these nerve terminals did not accurately report the extent of ADBE. Using this analysis we showed that dextran uptake occurs very soon after stimulation and that it does not persist when stimulation terminates. Thus we have devised a simple and quantitative method to monitor ADBE in living neurones, which will be ideal for real time screening of small molecule inhibitors of this key SV retrieval mode. PMID:19766140

Basic quantitative polymerase chain reaction using real-time fluorescence measurements.

PubMed

Ares, Manuel

2014-10-01

This protocol uses quantitative polymerase chain reaction (qPCR) to measure the number of DNA molecules containing a specific contiguous sequence in a sample of interest (e.g., genomic DNA or cDNA generated by reverse transcription). The sample is subjected to fluorescence-based PCR amplification and, theoretically, during each cycle, two new duplex DNA molecules are produced for each duplex DNA molecule present in the sample. The progress of the reaction during PCR is evaluated by measuring the fluorescence of dsDNA-dye complexes in real time. In the early cycles, DNA duplication is not detected because inadequate amounts of DNA are made. At a certain threshold cycle, DNA-dye complexes double each cycle for 8-10 cycles, until the DNA concentration becomes so high and the primer concentration so low that the reassociation of the product strands blocks efficient synthesis of new DNA and the reaction plateaus. There are two types of measurements: (1) the relative change of the target sequence compared to a reference sequence and (2) the determination of molecule number in the starting sample. The first requires a reference sequence, and the second requires a sample of the target sequence with known numbers of the molecules of sequence to generate a standard curve. By identifying the threshold cycle at which a sample first begins to accumulate DNA-dye complexes exponentially, an estimation of the numbers of starting molecules in the sample can be extrapolated. © 2014 Cold Spring Harbor Laboratory Press.

Highly-sensitive aptasensor based on fluorescence resonance energy transfer between l-cysteine capped ZnS quantum dots and graphene oxide sheets for the determination of edifenphos fungicide.

PubMed

Arvand, Majid; Mirroshandel, Aazam A

2017-10-15

With the advantages of excellent optical properties and biocompatibility, single-strand DNA-functionalized quantum dots have been widely applied in biosensing and bioimaging. A new aptasensor with easy operation, high sensitivity, and high selectivity was developed by immobilizing the aptamer on water soluble l-cysteine capped ZnS quantum dots (QDs). Graphene oxide (GO) sheets are mixed with the aptamer-QDs. Consequently, the aptamer-conjugated QDs bind to the GO sheets to form a GO/aptamer-QDs ensemble. This aptasensor enables the energy transfer based on a fluorescence resonance energy transfer (FRET) from the QDs to the GO sheets, quenching the fluorescence of QDs. The GO/aptamer-QDs ensemble assay acts as a "turn-on'' fluorescent sensor for edifenphos (EDI) detection. When GO was replaced by EDI, the fluorescence of QDs was restored and its intensity was proportional to the EDI concentration. This GO-based aptasensor under the optimum conditions exhibited excellent analytical performance for EDI determination, ranging from 5×10 -4 to 6×10 -3 mg L -1 with the detection limit of 1.3×10 -4 mgL -1 . Furthermore, the designed aptasensor exhibited excellent selectivity toward EDI compared to other pesticides and herbicides with similar structures such as diazinon, heptachlor, endrin, dieldrin, butachlor and chlordane. Good reproducibility and precision (RSD =3.9%, n =10) of the assay indicates the high potential of the aptasensor for quantitative trace analysis of EDI. Moreover, the results demonstrate the applicability of the aptasensor for monitoring EDI fungicide in spiked real samples. Copyright © 2017 Elsevier B.V. All rights reserved.

A fluorescence resonance energy transfer-derived structure of a quantum dot-protein bioconjugate nanoassembly

PubMed Central

Medintz, I. L.; Konnert, J. H.; Clapp, A. R.; Stanish, I.; Twigg, M. E.; Mattoussi, H.; Mauro, J. M.; Deschamps, J. R.

2004-01-01

The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed. It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials. As a model system for study, we use Rhodamine red-labeled engineered variants of Escherichia coli maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core–shell QDs. Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD. In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface. Results indicate that MBP has a preferred orientation on the QD surface. The refined model is in agreement with other evidence, which indicates coordination of the protein to the QD occurs by means of its C-terminal pentahistidine tail, and the size of the QD estimated from the model is in good agreement with physical measurements of QD size. The approach detailed here may be useful in determining the orientation of proteins in other hybrid protein–nanoparticle materials. To our knowledge, this is the first structural model of a hybrid luminescent QD-protein receptor assembly elucidated by using spectroscopic measurements in conjunction with crystallographic and other data. PMID:15210939

The fluorescence resonance energy transfer (FRET) gate: a time-resolved study.

PubMed

Xu, Qing-Hua; Wang, Shu; Korystov, Dmitry; Mikhailovsky, Alexander; Bazan, Guillermo C; Moses, Daniel; Heeger, Alan J

2005-01-18

The two-step energy-transfer process in a self-assembled complex comprising a cationic conjugated polymer (CCP) and a dsDNA is investigated by using pump-dump-emission spectroscopy and time-correlated single-photon counting; energy is transferred from the CCP to an ethidium bromide (EB) molecule intercalated into the dsDNA through a fluorescein molecule linked to one terminus of the DNA. Time-dependent anisotropy measurements indicate that the inefficient direct energy transfer from the CCP to the intercalated EB results from the near orthogonality of their transition moments. These measurements also show that the transition moment of the fluorescein spans a range of angular distributions and lies between that of the CCP and EB. Consequently, the fluorescein acts as a fluorescence resonance energy-transfer gate to relay the excitation energy from the CCP to the EB.

The fluorescence resonance energy transfer (FRET) gate: A time-resolved study

PubMed Central

Xu, Qing-Hua; Wang, Shu; Korystov, Dmitry; Mikhailovsky, Alexander; Bazan, Guillermo C.; Moses, Daniel; Heeger, Alan J.

2005-01-01

The two-step energy-transfer process in a self-assembled complex comprising a cationic conjugated polymer (CCP) and a dsDNA is investigated by using pump-dump-emission spectroscopy and time-correlated single-photon counting; energy is transferred from the CCP to an ethidium bromide (EB) molecule intercalated into the dsDNA through a fluorescein molecule linked to one terminus of the DNA. Time-dependent anisotropy measurements indicate that the inefficient direct energy transfer from the CCP to the intercalated EB results from the near orthogonality of their transition moments. These measurements also show that the transition moment of the fluorescein spans a range of angular distributions and lies between that of the CCP and EB. Consequently, the fluorescein acts as a fluorescence resonance energy-transfer gate to relay the excitation energy from the CCP to the EB. PMID:15642946

Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements

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

Keller, Aaron M.; DeVore, Matthew S.; Stich, Dominik G.

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of “burst” confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering itmore » within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope’s multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope’s capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.« less

Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements

DOE PAGES

Keller, Aaron M.; DeVore, Matthew S.; Stich, Dominik G.; ...

2018-04-19

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of “burst” confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering itmore » within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope’s multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope’s capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.« less

Fluorescence acquisition during hybridization phase in quantitative real-time PCR improves specificity and signal-to-noise ratio.

PubMed

Mehndiratta, Mohit; Palanichamy, Jayanth Kumar; Ramalingam, Pradeep; Pal, Arnab; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2008-12-01

Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.

Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy

PubMed Central

Voltz, Karine; Léonard, Jérémie; Touceda, Patricia Tourón; Conyard, Jamie; Chaker, Ziyad; Dejaegere, Annick; Godet, Julien; Mély, Yves; Haacke, Stefan; Stote, Roland H.

2016-01-01

Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies. PMID:26896800

Surface plasmon resonance microscopy: achieving a quantitative optical response

PubMed Central

Peterson, Alexander W.; Halter, Michael; Plant, Anne L.; Elliott, John T.

2016-01-01

Surface plasmon resonance (SPR) imaging allows real-time label-free imaging based on index of refraction, and changes in index of refraction at an interface. Optical parameter analysis is achieved by application of the Fresnel model to SPR data typically taken by an instrument in a prism based configuration. We carry out SPR imaging on a microscope by launching light into a sample, and collecting reflected light through a high numerical aperture microscope objective. The SPR microscope enables spatial resolution that approaches the diffraction limit, and has a dynamic range that allows detection of subnanometer to submicrometer changes in thickness of biological material at a surface. However, unambiguous quantitative interpretation of SPR changes using the microscope system could not be achieved using the Fresnel model because of polarization dependent attenuation and optical aberration that occurs in the high numerical aperture objective. To overcome this problem, we demonstrate a model to correct for polarization diattenuation and optical aberrations in the SPR data, and develop a procedure to calibrate reflectivity to index of refraction values. The calibration and correction strategy for quantitative analysis was validated by comparing the known indices of refraction of bulk materials with corrected SPR data interpreted with the Fresnel model. Subsequently, we applied our SPR microscopy method to evaluate the index of refraction for a series of polymer microspheres in aqueous media and validated the quality of the measurement with quantitative phase microscopy. PMID:27782542

Quantitative intact specimen magnetic resonance microscopy at 3.0 T.

PubMed

Bath, Kevin G; Voss, Henning U; Jing, Deqiang; Anderson, Stewart; Hempstead, Barbara; Lee, Francis S; Dyke, Jonathan P; Ballon, Douglas J

2009-06-01

In this report, we discuss the application of a methodology for high-contrast, high-resolution magnetic resonance microscopy (MRM) of murine tissue using a 3.0-T imaging system. We employed a threefold strategy that included customized specimen preparation to maximize image contrast, three-dimensional data acquisition to minimize scan time and custom radiofrequency resonator design to maximize signal sensitivity. Images had a resolution of 100 x 78 x 78 microm(3) with a signal-to-noise ratio per voxel greater than 25:1 and excellent contrast-to-noise ratios over a 30-min acquisition. We quantitatively validated the methods through comparisons of neuroanatomy across two lines of genetically engineered mice. Specifically, we were able to detect volumetric differences of as little as 9% between genetically engineered mouse strains in multiple brain regions that were predictive of underlying impairments in brain development. The overall methodology was straightforward to implement and provides ready access to basic MRM at field strengths that are widely available in both the laboratory and the clinic.

Nanostructured biosensor for detecting glucose in tear by applying fluorescence resonance energy transfer quenching mechanism.

PubMed

Chen, Longyi; Tse, Wai Hei; Chen, Yi; McDonald, Matthew W; Melling, James; Zhang, Jin

2017-05-15

In this paper, a nanostructured biosensor is developed to detect glucose in tear by using fluorescence resonance energy transfer (FRET) quenching mechanism. The designed FRET pair, including the donor, CdSe/ZnS quantum dots (QDs), and the acceptor, dextran-binding malachite green (MG-dextran), was conjugated to concanavalin A (Con A), an enzyme with specific affinity to glucose. In the presence of glucose, the quenched emission of QDs through the FRET mechanism is restored by displacing the dextran from Con A. To have a dual-modulation sensor for convenient and accurate detection, the nanostructured FRET sensors were assembled onto a patterned ZnO nanorod array deposited on the synthetic silicone hydrogel. Consequently, the concentration of glucose detected by the patterned sensor can be converted to fluorescence spectra with high signal-to-noise ratio and calibrated image pixel value. The photoluminescence intensity of the patterned FRET sensor increases linearly with increasing concentration of glucose from 0.03mmol/L to 3mmol/L, which covers the range of tear glucose levels for both diabetics and healthy subjects. Meanwhile, the calibrated values of pixel intensities of the fluorescence images captured by a handhold fluorescence microscope increases with increasing glucose. Four male Sprague-Dawley rats with different blood glucose concentrations were utilized to demonstrate the quick response of the patterned FRET sensor to 2µL of tear samples. Copyright © 2016 Elsevier B.V. All rights reserved.

A laser scanning confocal imaging-surface plasmon resonance system application in real time detection of antibody-antigen interaction

NASA Astrophysics Data System (ADS)

Zhang, H. Y.; Yang, L. Q.; Liu, W. M.

2011-12-01

The laser scanning confocal microscope (LSCM) offers several advantages over conventional optical microscopy, but most LSCM work is qualitative analysis and it is very hard to achieve quantitative detection directly with the changing of the fluorescent intensity. A new real time sensor system for the antibody-antigen interaction detection was built integrating with a LSCM and a wavelength-dependent surface plasmon resonance (SPR) sensor. The system was applied to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibody in real time. The fluorescence images changing is well with that of SPR wavelengths in real time, and the trend of the resonance wavelength shift with the concentrations of antibody is similar to that of the fluorescent intensity changing. The results show that SPR makes up the short of quantificational analysis with LSCM with the high spatial resolution. The sensor system shows the merits of the of the LSCM and SPR synergetic application, which are of great importance for practical application in biosensor and life science for interesting local interaction.

Assessment of Renal Hemodynamics and Oxygenation by Simultaneous Magnetic Resonance Imaging (MRI) and Quantitative Invasive Physiological Measurements.

PubMed

Cantow, Kathleen; Arakelyan, Karen; Seeliger, Erdmann; Niendorf, Thoralf; Pohlmann, Andreas

2016-01-01

In vivo assessment of renal perfusion and oxygenation under (patho)physiological conditions by means of noninvasive diagnostic imaging is conceptually appealing. Blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) and quantitative parametric mapping of the magnetic resonance (MR) relaxation times T 2* and T 2 are thought to provide surrogates of renal tissue oxygenation. The validity and efficacy of this technique for quantitative characterization of local tissue oxygenation and its changes under different functional conditions have not been systematically examined yet and remain to be established. For this purpose, the development of an integrative multimodality approaches is essential. Here we describe an integrated hybrid approach (MR-PHYSIOL) that combines established quantitative physiological measurements with T 2* (T 2) mapping and MR-based kidney size measurements. Standardized reversible (patho)physiologically relevant interventions, such as brief periods of aortic occlusion, hypoxia, and hyperoxia, are used for detailing the relation between the MR-PHYSIOL parameters, in particular between renal T 2* and tissue oxygenation.

Quantitative Whole Body Biodistribution of Fluorescent-Labeled Agents by Non-Invasive Tomographic Imaging

PubMed Central

Vasquez, Kristine O.; Casavant, Chelsea; Peterson, Jeffrey D.

2011-01-01

When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT) offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics (therapeutic antibodies, or other large proteins) or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR) or near infrared (NIR) imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D) fluorescence reflectance imaging of excised intact organs (r2 = 0.996 and 0.969, respectively). Dynamic FMT imaging (multiple times from 0 to 24 h) performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20–50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g) in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor extravasation

Quantitative fluorescence measurements performed on typical matrix molecules in matrix-assisted laser desorption/ionisation

NASA Astrophysics Data System (ADS)

Allwood, D. A.; Dyer, P. E.

2000-11-01

Fundamental photophysical parameters have been determined for several molecules that are commonly used as matrices, e.g. ferulic acid, within matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Fluorescence quantum efficiencies ( φqe), singlet decay rates ( kl), vibrationless ground-singlet transition energies and average fluorescence wavelengths have been obtained from solid and solution samples by quantitative optical measurements. This new data will assist in modelling calculations of MALDI processes and in highlighting desirable characteristics of MALDI matrices. φqe may be as high as 0.59 whilst the radiative decay rate ( kf) appears to be within the (0.8-4)×10 8 s -1 range. Interestingly, α-cyano-4-hydroxycinnamic acid (α-CHC) has a very low φqe and fast non-radiative decay rate which would imply a rapid and efficient thermalisation of electronic excitation. This is in keeping with observations that α-CHC exhibits low threshold fluences for ion detection and the low fluences at which α-CHC tends to fragment.

Helicase Stepping Investigated with One-Nucleotide Resolution Fluorescence Resonance Energy Transfer

NASA Astrophysics Data System (ADS)

Lin, Wenxia; Ma, Jianbing; Nong, Daguan; Xu, Chunhua; Zhang, Bo; Li, Jinghua; Jia, Qi; Dou, Shuoxing; Ye, Fangfu; Xi, Xuguang; Lu, Ying; Li, Ming

2017-09-01

Single-molecule Förster resonance energy transfer is widely applied to study helicases by detecting distance changes between a pair of dyes anchored to overhangs of a forked DNA. However, it has been lacking single-base pair (1-bp) resolution required for revealing stepping kinetics of helicases. We designed a nanotensioner in which a short DNA is bent to exert force on the overhangs, just as in optical or magnetic tweezers. The strategy improved the resolution of Förster resonance energy transfer to 0.5 bp, high enough to uncover differences in DNA unwinding by yeast Pif1 and E. coli RecQ whose unwinding behaviors cannot be differentiated by currently practiced methods. We found that Pif1 exhibits 1-bp-stepping kinetics, while RecQ breaks 1 bp at a time but sequesters the nascent nucleotides and releases them randomly. The high-resolution data allowed us to propose a three-parameter model to quantitatively interpret the apparently different unwinding behaviors of the two helicases which belong to two superfamilies.

In vivo magnetic resonance and fluorescence dual imaging of tumor sites by using dye-doped silica-coated iron oxide nanoparticles

NASA Astrophysics Data System (ADS)

Jang, Haeyun; Lee, Chaedong; Nam, Gi-Eun; Quan, Bo; Choi, Hyuck Jae; Yoo, Jung Sun; Piao, Yuanzhe

2016-02-01

The difficulty in delineating tumor is a major obstacle for better outcomes in cancer treatment of patients. The use of single-imaging modality is often limited by inadequate sensitivity and resolution. Here, we present the synthesis and the use of monodisperse iron oxide nanoparticles coated with fluorescent silica nano-shells for fluorescence and magnetic resonance dual imaging of tumor. The as-synthesized core-shell nanoparticles were designed to improve the accuracy of diagnosis via simultaneous tumor imaging with dual imaging modalities by a single injection of contrast agent. The iron oxide nanocrystals ( 11 nm) were coated with Rhodamine B isothiocyanate-doped silica shells via reverse microemulsion method. Then, the core-shell nanoparticles ( 54 nm) were analyzed to confirm their size distribution by transmission electron microscopy and dynamic laser scattering. Photoluminescence spectroscopy was used to characterize the fluorescent property of the dye-doped silica shell-coated nanoparticles. The cellular compatibility of the as-prepared nanoparticles was confirmed by a trypan blue dye exclusion assay and the potential as a dual-imaging contrast agent was verified by in vivo fluorescence and magnetic resonance imaging. The experimental results show that the uniform-sized core-shell nanoparticles are highly water dispersible and the cellular toxicity of the nanoparticles is negligible. In vivo fluorescence imaging demonstrates the capability of the developed nanoparticles to selectively target tumors by the enhanced permeability and retention effects and ex vivo tissue analysis was corroborated this. Through in vitro phantom test, the core/shell nanoparticles showed a T2 relaxation time comparable to Feridex® with smaller size, indicating that the as-made nanoparticles are suitable for imaging tumor. This new dual-modality-nanoparticle approach has promised for enabling more accurate tumor imaging.

An automated real-time microscopy system for analysis of fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

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

2010-05-01

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

A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer.

PubMed

Murakoshi, Hideji; Shibata, Akihiro C E; Nakahata, Yoshihisa; Nabekura, Junichi

2015-10-15

Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.

Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology

NASA Astrophysics Data System (ADS)

Wei, Linpeng; Chen, Ye; Yin, Chengbo; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2017-04-01

Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

PubMed Central

Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

2001-01-01

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

Dynamic Measurement of Tumor Vascular Permeability and Perfusion using a Hybrid System for Simultaneous Magnetic Resonance and Fluorescence Imaging.

PubMed

Ren, Wuwei; Elmer, Andreas; Buehlmann, David; Augath, Mark-Aurel; Vats, Divya; Ripoll, Jorge; Rudin, Markus

2016-04-01

Assessing tumor vascular features including permeability and perfusion is essential for diagnostic and therapeutic purposes. The aim of this study was to compare fluorescence and magnetic resonance imaging (MRI)-based vascular readouts in subcutaneously implanted tumors in mice by simultaneous dynamic measurement of tracer uptake using a hybrid fluorescence molecular tomography (FMT)/MRI system. Vascular permeability was measured using a mixture of extravascular imaging agents, GdDOTA and the dye Cy5.5, and perfusion using a mixture of intravascular agents, Endorem and a fluorescent probe (Angiosense). Dynamic fluorescence reflectance imaging (dFRI) was integrated into the hybrid system for high temporal resolution. Excellent correspondence between uptake curves of Cy5.5/GdDOTA and Endorem/Angiosense has been found with correlation coefficients R > 0.98. The two modalities revealed good agreement regarding permeability coefficients and centers-of-gravity of the imaging agent distribution. The FMT/dFRI protocol presented is able to accurately map physiological processes and poses an attractive alternative to MRI for characterizing tumor neoangiogenesis.

Plasma-Based Mixing Actuation in Airflow, Quantitated by Probe Breakdown Fluorescence

NASA Astrophysics Data System (ADS)

Leonov, Sergey; Firsov, Alexander; Shurupov, Michail; Yarantsev, Dmitry; Ohio State University Team; JIHT RAS Team

2013-09-01

Effective mixing of fuel and oxidizer in air-breathing engine at compressible conditions is an essential problem of high-speed combustion due to short residence time of gas mixture in the combustor of limited length. The effect of the mixing actuation by plasma is observed because of the gasdynamic instability arisen after the long filamentary discharge of submicrosecond duration generated along the contact zone of two co-flown gases. The work is focused on detail consideration of the mechanism of gas instability, promoted by plasma, on effect of the discharge specific localization, and on diagnostics development for qualitative and quantitative estimation of the mixing efficiency. The dynamics of relative concentration of gas components is examined quantitatively by means of Probe Discharge Breakdown Fluorescence (PBF). In this method an optical emission spectra of weak filamentary high-voltage nanosecond probe discharge are collected from local zone of interest in airflow. The first measurements of the mixing efficiency in vicinity of wall-injected secondary gas are presented. It is shown that the method of PBF could deliver experimental data on state of the two-component medium with <1 mcs and <5 mm of time and spatial resolution, correspondingly. Funded by AFOSR under Dr Chiping Li supervision

Quantitative blood group typing using surface plasmon resonance.

PubMed

Then, Whui Lyn; Aguilar, Marie-Isabel; Garnier, Gil

2015-11-15

The accurate and reliable typing of blood groups is essential prior to blood transfusion. While current blood typing methods are well established, results are subjective and heavily reliant on analysis by trained personnel. Techniques for quantifying blood group antibody-antigen interactions are also very limited. Many biosensing systems rely on surface plasmon resonance (SPR) detection to quantify biomolecular interactions. While SPR has been widely used for characterizing antibody-antigen interactions, measuring antibody interactions with whole cells is significantly less common. Previous studies utilized SPR for blood group antigen detection, however, showed poor regeneration causing loss of functionality after a single use. In this study, a fully regenerable, multi-functional platform for quantitative blood group typing via SPR detection is achieved by immobilizing anti-human IgG antibody to the sensor surface, which binds to the Fc region of human IgG antibodies. The surface becomes an interchangeable platform capable of quantifying the blood group interactions between red blood cells (RBCs) and IgG antibodies. As with indirect antiglobulin tests (IAT), which use IgG antibodies for detection, IgG antibodies are initially incubated with RBCs. This facilitates binding to the immobilized monolayer and allows for quantitative blood group detection. Using the D-antigen as an example, a clear distinction between positive (>500 RU) and negative (<100 RU) RBCs is achieved using anti-D IgG. Complete regeneration of the anti-human IgG surface is also successful, showing negligible degradation of the surface after more than 100 regenerations. This novel approach is validated with human-sourced whole blood samples to demonstrate an interesting alternative for quantitative blood grouping using SPR analysis. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Optical gating with organic building blocks. A quantitative model for the fluorescence modulation of photochromic perylene bisimide dithienylcyclopentene triads

PubMed Central

Pärs, Martti; Gradmann, Michael; Gräf, Katja; Bauer, Peter; Thelakkat, Mukundan; Köhler, Jürgen

2014-01-01

We investigated the capability of molecular triads, consisting of two strong fluorophores that were covalently linked to a photochromic molecule, for optical gating. Therefore we monitored the fluorescence intensity of the fluorophores as a function of the isomeric state of the photoswitch. From the analysis of our data we develop a kinetic model that allows us to predict quantitatively the degree of the fluorescence modulation as a function of the mutual intensities of the lasers that are used to induce the fluorescence and the switching of the photochromic unit. We find that the achievable contrast for the modulation of the fluorescence depends mainly on the intensity ratio of the two light beams and appears to be very robust against absolute changes of these intensities. The latter result provides valuable information for the development of all-optical circuits which would require to handle different signal strengths for the input and output levels. PMID:24614963

Quantitative determination of dimethylaminoethanol in cosmetic formulations by nuclear magnetic resonance spectroscopy.

PubMed

Batista, Ivani Aparecida Soares de Andrade; Gonçalves, Maria Inês de Almeida; Singh, Anil Kumar; Hackmann, Erika Rosa Maria Kedor; Santoro, Maria Inês Rocha Miritello

2008-01-01

A nuclear magnetic resonance (NMR) spectroscopic method was validated for the quantitative determination of dimethylaminoethanol (DMAE) in cosmetic formulations. The linearity in the range from 0.5000 to 1.5000 g (DMAE salt/mass maleic acid) presents a correlation coefficient > 0.99 for all DMAE salts. The repeatability (intraday), expressed as relative standard deviation, ranged from 1.08 to 1.44% for samples and 1.31 to 1.88% for raw materials. The detection limit and quantitation limit were 0.0017 and 0.0051 g for DMAE, 0.0018 and 0.0054 g for DMAE bitartrate, and 0.0023 and 0.0071 g for DMAE acetamidobenzoate, respectively. The proposed method is simple, precise, and accurate and can be used in the quality control of raw materials and cosmetic gels containing these compounds as active substances.

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

Quantitative nuclear magnetic resonance imaging: characterisation of experimental cerebral oedema.

PubMed Central

Barnes, D; McDonald, W I; Johnson, G; Tofts, P S; Landon, D N

1987-01-01

Magnetic resonance imaging (MRI) has been used quantitatively to define the characteristics of two different models of experimental cerebral oedema in cats: vasogenic oedema produced by cortical freezing and cytotoxic oedema induced by triethyl tin. The MRI results have been correlated with the ultrastructural changes. The images accurately delineated the anatomical extent of the oedema in the two lesions, but did not otherwise discriminate between them. The patterns of measured increase in T1' and T2' were, however, characteristic for each type of oedema, and reflected the protein content. The magnetisation decay characteristics of both normal and oedematous white matter were monoexponential for T1 but biexponential for T2 decay. The relative sizes of the two component exponentials of the latter corresponded with the physical sizes of the major tissue water compartments. Quantitative MRI data can provide reliable information about the physico-chemical environment of tissue water in normal and oedematous cerebral tissue, and are useful for distinguishing between acute and chronic lesions in multiple sclerosis. Images PMID:3572428

A graphene oxide based biosensor for microcystins detection by fluorescence resonance energy transfer.

PubMed

Shi, Yan; Wu, Jiazhen; Sun, Yujing; Zhang, Yue; Wen, Zhiwei; Dai, Haichao; Wang, Hongda; Li, Zhuang

2012-01-01

Water safety is one of the most pervasive problems afflicting people throughout the world. Microcystin, a hepatotoxin produced by cyanobacteria, poses a growing and serious threat of water safety. According to World Health Organization (WHO), the limit of content of microcystin-LR (MC-LR) in drinking water is as low as 1 μg/L; it is thus necessary to explore a sensitive method for the trace detection of microcystins (MCs). Based on the observation of gold nanoparticles (Au NPs) induced graphene oxide (GO) fluorescence quenching, a reliable biosensor was developed here for microcystins detection. MCs could be attached on Au NPs through the interaction with single strand-DNA (ss-DNA) modified on Au NPs, which formed Au-DNA-MCs complexes. These MCs in the complexes could be immunologically recognized by the antibodies adsorbed on GO sheets, as a result, Au NPs were close enough to quench the photoluminescence of GO by the fluorescence resonance energy transfer (FRET). The fluorescence intensity decreased with the increase of MCs as more Au NPs linked onto GO surface. The limit of detection was 0.5 and 0.3 μg/L for microcystin-LR and microcystin-RR (MC-RR), respectively, which satisfies the strictest standard of WHO. Well defined results were also obtained in natural lake water and the specificity experiment. The antibody used here could recognize Adda group, the conservative part of MCs, which allowed the biosensor to detect both single toxin and the total content of MCs existing in the water sample. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

A new assay format for NF-kappaB based on a DNA triple helix and a fluorescence resonance energy transfer.

PubMed

Altevogt, Dominik; Hrenn, Andrea; Kern, Claudia; Clima, Lilia; Bannwarth, Willi; Merfort, Irmgard

2009-10-07

Herein we report a feasibility study for a new concept to detect DNA binding protein NF-kappaB based on a DNA triple helix formation in combination with a fluorescence resonance energy transfer (FRET). The new principle avoids expensive antibodies and radioactivity and might have implications for assays of other DNA binding proteins.

Development of cell-based quantitative evaluation method for cell cycle-arrest type cancer drugs for apoptosis by high precision surface plasmon resonance sensor

NASA Astrophysics Data System (ADS)

Ona, Toshihiro; Nishijima, Hiroshi; Kosaihira, Atsushi; Shibata, Junko

2008-04-01

In vitro rapid and quantitative cell-based assay is demanded to verify the efficacy prediction of cancer drugs since a cancer patient may have unconventional aspects of tumor development. Here, we show the rapid and non-label quantitative verifying method and instrumentation of apoptosis for cell cycle-arrest type cancer drugs (Roscovitine and D-allose) by reaction analysis of living liver cancer cells cultured on a sensor chip with a newly developed high precision (50 ndeg s -1 average fluctuation) surface plasmon resonance (SPR) sensor. The time-course cell reaction as the SPR angle change rate for 10 min from 30 min cell culture with a drug was significantly related to cell viability. By the simultaneous detection of differential SPR angle change and fluorescence by specific probes using the new instrument, the SPR angle was related to the nano-order potential decrease in inner mitochondrial membrane potential. The results obtained are universally valid for the cell cycle-arrest type cancer drugs, which mediate apoptosis through different cell-signaling pathways, by a liver cancer cell line of Hep G2 (P<0.001). This system towards the application to evaluate personal therapeutic potentials of drugs using cancer cells from patients in clinical use.

Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.

PubMed

Voltz, Karine; Léonard, Jérémie; Touceda, Patricia Tourón; Conyard, Jamie; Chaker, Ziyad; Dejaegere, Annick; Godet, Julien; Mély, Yves; Haacke, Stefan; Stote, Roland H

2016-04-20

Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

A novel quantitative analysis method of three-dimensional fluorescence spectra for vegetable oils contents in edible blend oil

NASA Astrophysics Data System (ADS)

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

2015-04-01

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

Quantitative comparison between full-spectrum and filter-based imaging in hyperspectral fluorescence microscopy

PubMed Central

GAO, L.; HAGEN, N.; TKACZYK, T.S.

2012-01-01

Summary We implement a filterless illumination scheme on a hyperspectral fluorescence microscope to achieve full-range spectral imaging. The microscope employs polarisation filtering, spatial filtering and spectral unmixing filtering to replace the role of traditional filters. Quantitative comparisons between full-spectrum and filter-based microscopy are provided in the context of signal dynamic range and accuracy of measured fluorophores’ emission spectra. To show potential applications, a five-colour cell immunofluorescence imaging experiment is theoretically simulated. Simulation results indicate that the use of proposed full-spectrum imaging technique may result in three times improvement in signal dynamic range compared to that can be achieved in the filter-based imaging. PMID:22356127

Quantitative analysis of essential oils of Thymus daenensis using laser-induced fluorescence and Raman spectroscopy.

PubMed

Khoshroo, H; Khadem, H; Bahreini, M; Tavassoli, S H; Hadian, J

2015-11-10

Laser-induced fluorescence and Raman spectroscopy are used for the investigation of different genotypes of Thymus daenensis native to the Ilam province of Iran. Different genotypes of T. daenensis essential oils, labeled T1 through T7, possess slight differences with regard to the composition of the thymol. The gas chromatography-mass spectrometry (GC-MS) method is performed to determine the concentration of each constituent as a reference method. The Raman spectra of different concentrations of pure thymol dissolved in hexane as standard samples are obtained via a laboratory prototype Raman spectroscopy setup for the calculation of the calibration curve. The regression coefficient and limit of detection are calculated. The possibility of the differentiation of different genotypes of T. daenensis is also examined by laser-induced fluorescence spectroscopy, although we do not know the exact amounts of their components. All the fluorescence spectral information is used jointly by cluster analysis to differentiate between 7 genotypes. Our results demonstrate the acceptable precision of Raman spectroscopy with GC-MS and corroborate the capacity of Raman spectroscopy in applications in the quantitative analysis field. Furthermore, the cluster analysis results show that laser-induced fluorescence spectroscopy is an acceptable technique for the rapid classification of different genotypes of T. daenensis without having any previous information of their exact amount of constituents. So, the ability to rapidly and nondestructively differentiate between genotypes makes it possible to efficiently select high-quality herbs from many samples.

A combined post-mortem magnetic resonance imaging and quantitative histological study of multiple sclerosis pathology

PubMed Central

Kolasinski, James; Chance, Steven A.; DeLuca, Gabriele C.; Esiri, Margaret M.; Chang, Eun-Hyuk; Palace, Jacqueline A.; McNab, Jennifer A.; Jenkinson, Mark; Miller, Karla L.; Johansen-Berg, Heidi

2012-01-01

Multiple sclerosis is a chronic inflammatory neurological condition characterized by focal and diffuse neurodegeneration and demyelination throughout the central nervous system. Factors influencing the progression of pathology are poorly understood. One hypothesis is that anatomical connectivity influences the spread of neurodegeneration. This predicts that measures of neurodegeneration will correlate most strongly between interconnected structures. However, such patterns have been difficult to quantify through post-mortem neuropathology or in vivo scanning alone. In this study, we used the complementary approaches of whole brain post-mortem magnetic resonance imaging and quantitative histology to assess patterns of multiple sclerosis pathology. Two thalamo-cortical projection systems were considered based on their distinct neuroanatomy and their documented involvement in multiple sclerosis: lateral geniculate nucleus to primary visual cortex and mediodorsal nucleus of the thalamus to prefrontal cortex. Within the anatomically distinct thalamo-cortical projection systems, magnetic resonance imaging derived cortical thickness was correlated significantly with both a measure of myelination in the connected tract and a measure of connected thalamic nucleus cell density. Such correlations did not exist between these markers of neurodegeneration across different thalamo-cortical systems. Magnetic resonance imaging lesion analysis depicted clearly demarcated subcortical lesions impinging on the white matter tracts of interest; however, quantitation of the extent of lesion-tract overlap failed to demonstrate any appreciable association with the severity of markers of diffuse pathology within each thalamo-cortical projection system. Diffusion-weighted magnetic resonance imaging metrics in both white matter tracts were correlated significantly with a histologically derived measure of tract myelination. These data demonstrate for the first time the relevance of functional

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin.

PubMed Central

Waud, J P; Bermúdez Fajardo, A; Sudhaharan, T; Trimby, A R; Jeffery, J; Jones, A; Campbell, A K

2001-01-01

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (alpha-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20--60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as 'Rainbow' proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2 microM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor. PMID:11463339

Quantitative Connection between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome.

PubMed

Thompson, Colin D Kinz; Sharma, Ajeet K; Frank, Joachim; Gonzalez, Ruben L; Chowdhury, Debashish

2015-08-27

At equilibrium, thermodynamic and kinetic information can be extracted from biomolecular energy landscapes by many techniques. However, while static, ensemble techniques yield thermodynamic data, often only dynamic, single-molecule techniques can yield the kinetic data that describe transition-state energy barriers. Here we present a generalized framework based upon dwell-time distributions that can be used to connect such static, ensemble techniques with dynamic, single-molecule techniques, and thus characterize energy landscapes to greater resolutions. We demonstrate the utility of this framework by applying it to cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) studies of the bacterial ribosomal pre-translocation complex. Among other benefits, application of this framework to these data explains why two transient, intermediate conformations of the pre-translocation complex, which are observed in a cryo-EM study, may not be observed in several smFRET studies.

Quantitative analysis of brain magnetic resonance imaging for hepatic encephalopathy

NASA Astrophysics Data System (ADS)

Syh, Hon-Wei; Chu, Wei-Kom; Ong, Chin-Sing

1992-06-01

High intensity lesions around ventricles have recently been observed in T1-weighted brain magnetic resonance images for patients suffering hepatic encephalopathy. The exact etiology that causes magnetic resonance imaging (MRI) gray scale changes has not been totally understood. The objective of our study was to investigate, through quantitative means, (1) the amount of changes to brain white matter due to the disease process, and (2) the extent and distribution of these high intensity lesions, since it is believed that the abnormality may not be entirely limited to the white matter only. Eleven patients with proven haptic encephalopathy and three normal persons without any evidence of liver abnormality constituted our current data base. Trans-axial, sagittal, and coronal brain MRI were obtained on a 1.5 Tesla scanner. All processing was carried out on a microcomputer-based image analysis system in an off-line manner. Histograms were decomposed into regular brain tissues and lesions. Gray scale ranges coded as lesion were then brought back to original images to identify distribution of abnormality. Our results indicated the disease process involved pallidus, mesencephalon, and subthalamic regions.

Spectrophotometry near the atmospheric cutoff of the strongest Bowen resonance fluorescence lines of O III in two planetary nebulae

NASA Technical Reports Server (NTRS)

O'Dell, C. R.; Opal, Chet B.

1989-01-01

Spectrophotometric results are presented for the stronger, well-resolved Bowen O III resonance fluorescence emission lines in the planetary nebulae 7027 and NGC 7662 down to and including the intrinsically strong line at 3133 A. These data are combined with results from the IUE atlas of spectra and similar results for the longer wavelength lines by Likkel and Aller (1986) to give the first full coverage of the Bowen lines. Good agreement is found with fluorescence theory for the primary cascade lines, except for the Likkel and Aller results. The efficiency of conversion of the exciting He II Ly-alpha into O III lines is determined, and values comparable to other planetary nebulae are found.

Combined use of X-ray fluorescence microscopy, phase contrast imaging for high resolution quantitative iron mapping in inflamed cells

NASA Astrophysics Data System (ADS)

Gramaccioni, C.; Procopio, A.; Farruggia, G.; Malucelli, E.; Iotti, S.; Notargiacomo, A.; Fratini, M.; Yang, Y.; Pacureanu, A.; Cloetens, P.; Bohic, S.; Massimi, L.; Cutone, A.; Valenti, P.; Rosa, L.; Berlutti, F.; Lagomarsino, S.

2017-06-01

X-ray fluorescence microscopy (XRFM) is a powerful technique to detect and localize elements in cells. To derive information useful for biology and medicine, it is essential not only to localize, but also to map quantitatively the element concentration. Here we applied quantitative XRFM to iron in phagocytic cells. Iron, a primary component of living cells, can become toxic when present in excess. In human fluids, free iron is maintained at 10-18 M concentration thanks to iron binding proteins as lactoferrin (Lf). The iron homeostasis, involving the physiological ratio of iron between tissues/secretions and blood, is strictly regulated by ferroportin, the sole protein able to export iron from cells to blood. Inflammatory processes induced by lipopolysaccharide (LPS) or bacterial pathoge inhibit ferroportin synthesis in epithelial and phagocytic cells thus hindering iron export, increasing intracellular iron and bacterial multiplication. In this respect, Lf is emerging as an important regulator of both iron and inflammatory homeostasis. Here we studied phagocytic cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf. Quantitative mapping of iron concentration and mass fraction at high spatial resolution is obtained combining X-ray fluorescence microscopy, atomic force microscopy and synchrotron phase contrast imaging.

Resonant two-photon ionization and laser induced fluorescence spectroscopy of jet-cooled adenine

NASA Astrophysics Data System (ADS)

Kim, Nam Joon; Jeong, Gawoon; Kim, Yung Sam; Sung, Jiha; Keun Kim, Seong; Park, Young Dong

2000-12-01

Electronic spectra of the jet-cooled DNA base adenine were obtained by the resonant two-photon ionization (R2PI) and the laser induced fluorescence (LIF) techniques. The 0-0 band to the lowest electronically excited state was found to be located at 35 503 cm-1. Well-resolved vibronic structures were observed up to 1100 cm-1 above the 0-0 level, followed by a slow rise of broad structureless absorption. The lowest electronic state was proposed to be of nπ* character, which lies ˜600 cm-1 below the onset of the ππ* state. The broad absorption was attributed to the extensive vibronic mixing between the nπ* state and the high-lying ππ* state.

A Fluorescence Lecture Demonstration.

ERIC Educational Resources Information Center

Bozzelli, Joseph W.; Kemp, Marwin

1982-01-01

Describes fluorescence demonstrations related to several aspects of molecular theory and quantitized energy levels. Demonstrations use fluorescent chemical solutions having luminescence properties spanning the visible spectrum. Also describes a demonstration of spontaneous combustion of familiar substances in chlorine. (JN)

Ensemble and Single-Molecule Studies on Fluorescence Quenching in Transition Metal Bipyridine-Complexes

PubMed Central

Brox, Dominik; Kiel, Alexander; Wörner, Svenja Johanna; Pernpointner, Markus; Comba, Peter; Martin, Bodo; Herten, Dirk-Peter

2013-01-01

Beyond their use in analytical chemistry fluorescent probes continuously gain importance because of recent applications of single-molecule fluorescence spectroscopy to monitor elementary reaction steps. In this context, we characterized quenching of a fluorescent probe by different metal ions with fluorescence spectroscopy in the bulk and at the single-molecule level. We apply a quantitative model to explain deviations from existing standard models for fluorescence quenching. The model is based on a reversible transition from a bright to a dim state upon binding of the metal ion. We use the model to estimate the stability constants of complexes with different metal ions and the change of the relative quantum yield of different reporter dye labels. We found ensemble data to agree widely with results from single-molecule experiments. Our data indicates a mechanism involving close molecular contact of dye and quenching moiety which we also found in molecular dynamics simulations. We close the manuscript with a discussion of possible mechanisms based on Förster distances and electrochemical potentials which renders photo-induced electron transfer to be more likely than Förster resonance energy transfer. PMID:23483966

Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns.

PubMed

Hennig, Simon; van de Linde, Sebastian; Bergmann, Stephan; Huser, Thomas; Sauer, Markus

2015-08-25

We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ∼100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3-168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.

DNA origami-based standards for quantitative fluorescence microscopy.

PubMed

Schmied, Jürgen J; Raab, Mario; Forthmann, Carsten; Pibiri, Enrico; Wünsch, Bettina; Dammeyer, Thorben; Tinnefeld, Philip

2014-01-01

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

Resonant Mode-hopping Micromixing

PubMed Central

Jang, Ling-Sheng; Chao, Shih-Hui; Holl, Mark R.; Meldrum, Deirdre R.

2009-01-01

A common micromixer design strategy is to generate interleaved flow topologies to enhance diffusion. However, problems with these designs include complicated structures and dead volumes within the flow fields. We present an active micromixer using a resonating piezoceramic/silicon composite diaphragm to generate acoustic streaming flow topologies. Circulation patterns are observed experimentally and correlate to the resonant mode shapes of the diaphragm. The dead volumes in the flow field are eliminated by rapidly switching from one discrete resonant mode to another (i.e., resonant mode-hop). Mixer performance is characterized by mixing buffer with a fluorescence tracer containing fluorescein. Movies of the mixing process are analyzed by converting fluorescent images to two-dimensional fluorescein concentration distributions. The results demonstrate that mode-hopping operation rapidly homogenized chamber contents, circumventing diffusion-isolated zones. PMID:19551159

Direct quantitative 13 C-filtered 1 H magnetic resonance imaging of PEGylated biomacromolecules in vivo.

PubMed

Alvares, Rohan D A; Lau, Justin Y C; Macdonald, Peter M; Cunningham, Charles H; Prosser, R Scott

2017-04-01

1 H MRI is an established diagnostic method that generally relies on detection of water. Imaging specific macromolecules is normally accomplished only indirectly through the use of paramagnetic tags, which alter the water signal in their vicinity. We demonstrate a new approach in which macromolecular constituents, such as proteins and drug delivery systems, are observed directly and quantitatively in vivo using 1 H MRI of 13 C-labeled poly(ethylene glycol) ( 13 C-PEG) tags. Molecular imaging of 13 C-PEG-labeled species was accomplished by incorporating a modified heteronuclear multiple quantum coherence filter into a gradient echo imaging sequence. We demonstrate the approach by monitoring the real-time distribution of 13 C-PEG and 13 C-PEGylated albumin injected into the hind leg of a mouse. Filtering the 1 H PEG signal through the directly coupled 13 C nuclei largely eliminates background water and fat signals, thus enabling the imaging of molecules using 1 H MRI. PEGylation is widely employed to enhance the performance of a multitude of macromolecular therapeutics and drug delivery systems, and 13 C-filtered 1 H MRI of 13 C-PEG thus offers the possibility of imaging and quantitating their distribution in living systems in real time. Magn Reson Med 77:1553-1561, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

PubMed

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Fluorescence Studies of Lysozyme Nucleation

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Smith, Lori

1998-01-01

Fluorescence is one of the most powerful tools available for the study of macromolecules. For example, fluorescence can be used to study self association through methods such as anisotropy (the rotational rate of the molecule in solution), quenching (the accessibility of a bound probe to the bulk solution), and resonance energy transfer (measurement of the distance between two species). Fluorescence can also be used to study the local environment of the probe molecules, and the changes in that environment which accompany crystal nucleation and growth. However fluorescent techniques have been very much underutilized in macromolecular growth studies. One major advantage is that the fluorescent species generally must be at low concentration, typically ca 10-5 to 10-6 M. Thus one can study a very wide range of solution conditions, ranging from very high to very low protein concentration, he latter of which are not readily accessible to scattering techniques. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme (CEWL). Fluorescent probes have been attached to two different sites, ASP 101 and the N-terrninal amine, with a sought for use in different lines of study. Preliminary resonance energy transfer studies have been -carried out using pyrene acetic acid (Ex 340 mn, Em 376 nm) lysozyme as a donor and cascade blue (Ex 377 run, Em 423 nm) labeled lysozyme as an acceptor. The emission of both the pyrene and cascade blue probes was followed as a function of the salt protein concentrations. The data show an increase in cascade blue and a concomitant decrease in the pyrene fluorescence as either the salt or protein concentrations are increased, suggesting that the two species are approaching each other close enough for resonance energy transfer to occur. This data can be analyzed to measure the distance between the probe molecules and, knowing their locations on the protein molecule their distances from and orientations with respect to each

Imaging Erg and Jun transcription factor interaction in living cells using fluorescence resonance energy transfer analyses

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

Camuzeaux, Barbara; Spriet, Corentin; Heliot, Laurent

2005-07-15

Physical interactions between transcription factors play important roles in modulating gene expression. Previous in vitro studies have shown a transcriptional synergy between Erg protein, an Ets family member, and Jun/Fos heterodimer, members of the bZip family, which requires direct Erg-Jun protein interactions. Visualization of protein interactions in living cells is a new challenge in biology. For this purpose, we generated fusion proteins of Erg, Fos, and Jun with yellow and cyan fluorescent proteins, YFP and CFP, respectively. After transient expression in HeLa cells, interactions of the resulting fusion proteins were explored by fluorescence resonance energy transfer microscopy (FRET) in fixedmore » and living cells. FRET between YFP-Erg and CFP-Jun was monitored by using photobleaching FRET and fluorescence lifetime imaging microscopy. Both techniques revealed the occurrence of intermolecular FRET between YFP-Erg and CFP-Jun. This is stressed by loss of FRET with an YFP-Erg version carrying a point mutation in its ETS domain. These results provide evidence for the interaction of Erg and Jun proteins in living cells as a critical prerequisite of their transcriptional synergy, but also for the essential role of the Y371 residue, conserved in most Ets proteins, in this interaction.« less

Quantitative fluorescence imaging of protein diffusion and interaction in living cells.

PubMed

Capoulade, Jérémie; Wachsmuth, Malte; Hufnagel, Lars; Knop, Michael

2011-08-07

Diffusion processes and local dynamic equilibria inside cells lead to nonuniform spatial distributions of molecules, which are essential for processes such as nuclear organization and signaling in cell division, differentiation and migration. To understand these mechanisms, spatially resolved quantitative measurements of protein abundance, mobilities and interactions are needed, but current methods have limited capabilities to study dynamic parameters. Here we describe a microscope based on light-sheet illumination that allows massively parallel fluorescence correlation spectroscopy (FCS) measurements and use it to visualize the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue. Imaging the mobility of heterochromatin protein HP1α (ref. 4) in cell nuclei we could provide high-resolution diffusion maps that reveal euchromatin areas with heterochromatin-like HP1α-chromatin interactions. We expect that FCS imaging will become a useful method for the precise characterization of cellular reaction-diffusion processes.

Progress on Raman laser for sodium resonance fluorescence lidar

NASA Astrophysics Data System (ADS)

Li, Steven X.; Yu, Anthony W.; Krainak, Michael A.; Bai, Yingxin; Konoplev, Oleg; Fahey, Molly E.; Numata, Kenji

2018-02-01

We are developing a Q-switched narrow linewidth intra-cavity Raman laser for a space based sodium lidar application. A novel Raman laser injection seeding scheme is proposed and is experimentally verified. A Q-switched, diode pumped, c-cut Nd:YVO4 laser has been designed to emit a fundamental wavelength at 1066.6 nm. This fundamental wavelength is used as the pump in an intra-cavity Raman conversion in a Gd0.2Y0.8VO4 composite material. By tuning the temperature of the crystal, we tuned the Raman shifting to the desired sodium absorption line. A diode end pumped, T-shaped laser cavity has been built for experimental investigation. The fundamental pump laser cavity is a twisted mode cavity to eliminate the spatial hole burning for effective injection seeding. The Raman laser cavity is a linear standing wave cavity because Raman gain medium does not suffer spatial hole burning as traditional laser gain medium. The linewidth and temporal profile of the Raman laser is experimentally investigated with narrow and broadband fundamental pump emission. We have, for the first time, demonstrated an injection seeded, high peak power, narrow linewidth intra-cavity Raman laser for potential use in a sodium resonance fluorescence lidar.

Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

PubMed

Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

2015-12-01

Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

Magnetically engineered Cd-free quantum dots as dual-modality probes for fluorescence/magnetic resonance imaging of tumors.

PubMed

Ding, Ke; Jing, Lihong; Liu, Chunyan; Hou, Yi; Gao, Mingyuan

2014-02-01

Magnetically engineered Cd-free CuInS2@ZnS:Mn quantum dots (QDs) were designed, synthesized, and evaluated as potential dual-modality probes for fluorescence and magnetic resonance imaging (MRI) of tumors in vivo. The synthesis of Mn-doped core-shell structured CuInS2@ZnS mainly comprised three steps, i.e., the preparation of fluorescent CuInS2 seeds, the particle surface coating of ZnS, and the Mn-doping of the ZnS shells. Systematic spectroscopy studies were carried out to illustrate the impacts of ZnS coating and the following Mn-doping on the optical properties of the QDs. In combination with conventional fluorescence, fluorescence excitation, and time-resolved fluorescence measurements, the structure of CuInS2@ZnS:Mn QDs prepared under optimized conditions presented a Zn gradient CuInS2 core and a ZnS outer shell, while Mn ions were mainly located in the ZnS shell, which well balanced the optical and magnetic properties of the resultant QDs. For the following in vivo imaging experiments, the hydrophobic CuInS2@ZnS:Mn QDs were transferred into water upon ligand exchange reactions by replacing the 1-dodecanethiol ligand with dihydrolipoic acid-poly(ethylene glycol) (DHLA-PEG) ligand. The MTT assays based on HeLa cells were carried out to evaluate the cytotoxicity of the current Cd-free CuInS2@ZnS:Mn QDs for comparing with that of water soluble CdTe QDs. Further in vivo fluorescence and MR imaging experiments suggested that the PEGylated CuInS2@ZnS:Mn QDs could well target both subcutaneous and intraperitoneal tumors in vivo. Copyright © 2013 Elsevier Ltd. All rights reserved.

Investigation of the influence of sampling schemes on quantitative dynamic fluorescence imaging

PubMed Central

Dai, Yunpeng; Chen, Xueli; Yin, Jipeng; Wang, Guodong; Wang, Bo; Zhan, Yonghua; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

2018-01-01

Dynamic optical data from a series of sampling intervals can be used for quantitative analysis to obtain meaningful kinetic parameters of probe in vivo. The sampling schemes may affect the quantification results of dynamic fluorescence imaging. Here, we investigate the influence of different sampling schemes on the quantification of binding potential (BP) with theoretically simulated and experimentally measured data. Three groups of sampling schemes are investigated including the sampling starting point, sampling sparsity, and sampling uniformity. In the investigation of the influence of the sampling starting point, we further summarize two cases by considering the missing timing sequence between the probe injection and sampling starting time. Results show that the mean value of BP exhibits an obvious growth trend with an increase in the delay of the sampling starting point, and has a strong correlation with the sampling sparsity. The growth trend is much more obvious if throwing the missing timing sequence. The standard deviation of BP is inversely related to the sampling sparsity, and independent of the sampling uniformity and the delay of sampling starting time. Moreover, the mean value of BP obtained by uniform sampling is significantly higher than that by using the non-uniform sampling. Our results collectively suggest that a suitable sampling scheme can help compartmental modeling of dynamic fluorescence imaging provide more accurate results and simpler operations. PMID:29675325

Smartphone-Based Dual-Modality Imaging System for Quantitative Detection of Color or Fluorescent Lateral Flow Immunochromatographic Strips

NASA Astrophysics Data System (ADS)

Hou, Yafei; Wang, Kan; Xiao, Kun; Qin, Weijian; Lu, Wenting; Tao, Wei; Cui, Daxiang

2017-04-01

Nowadays, lateral flow immunochromatographic assays are increasingly popular as a diagnostic tool for point-of-care (POC) test based on their simplicity, specificity, and sensitivity. Hence, quantitative detection and pluralistic popular application are urgently needed in medical examination. In this study, a smartphone-based dual-modality imaging system was developed for quantitative detection of color or fluorescent lateral flow test strips, which can be operated anywhere at any time. In this system, the white and ultra-violet (UV) light of optical device was designed, which was tunable with different strips, and the Sobel operator algorithm was used in the software, which could enhance the identification ability to recognize the test area from the background boundary information. Moreover, this technology based on extraction of the components from RGB format (red, green, and blue) of color strips or only red format of the fluorescent strips can obviously improve the high-signal intensity and sensitivity. Fifty samples were used to evaluate the accuracy of this system, and the ideal detection limit was calculated separately from detection of human chorionic gonadotropin (HCG) and carcinoembryonic antigen (CEA). The results indicated that smartphone-controlled dual-modality imaging system could provide various POC diagnoses, which becomes a potential technology for developing the next-generation of portable system in the near future.

qF-SSOP: real-time optical property corrected fluorescence imaging

PubMed Central

Valdes, Pablo A.; Angelo, Joseph P.; Choi, Hak Soo; Gioux, Sylvain

2017-01-01

Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively. PMID:28856038

Luminescent Quantum Dot Bioconjugates in Fluorescence Resonance Energy Transfer (FRET) Assays

NASA Astrophysics Data System (ADS)

Clapp, Aaron; Medintz, Igor; Goldman, Ellen; Anderson, George; Mauro, J. Matthew; Mattoussi, Hedi

2003-03-01

Colloidal semiconductor quantum dots (QDs) such as those made of CdSe-ZnS core-shell nanocrystals offer a promising alternative to organic dyes in a variety of biological tagging applications. They exhibit high resistance to chemical and photo-degradations, are highly luminescent, and show unique size-specific optical and spectroscopic properties. We have previously demonstrated a useful method for attaching proteins to CdSe-ZnS QDs using dihydrolipoic acid (DHLA) surface capping groups and electrostatic self-assembly in aqueous environments. We have used this conjugation strategy to build solution-based QD-conjugate sensors based on fluorescence resonance energy transfer (FRET) between QD donors and dye-labeled protein acceptors. Specific binding between the QD-ligand donor and dye-labeled receptor was achieved. In another example, the dye receptor was grafted directly onto the protein, then immobilized onto the QD surface via an electrostatic self-assembly process. The QD-complexes were optically excited in a region where absorption of the dye is negligible compared to that of the nanocrystals. We observed a continuous decrease of the QD emission accompanied by a steady and pronounced increase of the acceptor emission as the ratio of dye to QD was increased. The results of these experiments suggest efficient resonance energy transfer between the QD donor and the dye acceptor upon ligand-receptor binding. We will present these data and discuss other aspects such as donor-acceptor separation distance, degree of overlap between absorption of the acceptor and emission of the QD, and reverse FRET (upon ligand-receptor release) in a reversible assay.

Semi-quantitative estimation of cellular SiO2 nanoparticles using flow cytometry combined with X-ray fluorescence measurements.

PubMed

Choi, Seo Yeon; Yang, Nuri; Jeon, Soo Kyung; Yoon, Tae Hyun

2014-09-01

In this study, we have demonstrated feasibility of a semi-quantitative approach for the estimation of cellular SiO2 nanoparticles (NPs), which is based on the flow cytometry measurements of their normalized side scattering intensity. In order to improve our understanding on the quantitative aspects of cell-nanoparticle interactions, flow cytometry, transmission electron microscopy, and X-ray fluorescence experiments were carefully performed for the HeLa cells exposed to SiO2 NPs with different core diameters, hydrodynamic sizes, and surface charges. Based on the observed relationships among the experimental data, a semi-quantitative cellular SiO2 NPs estimation method from their normalized side scattering and core diameters was proposed, which can be applied for the determination of cellular SiO2 NPs within their size-dependent linear ranges. © 2014 International Society for Advancement of Cytometry.

Toward quantitative fluorescence microscopy with DNA origami nanorulers.

PubMed

Beater, Susanne; Raab, Mario; Tinnefeld, Philip

2014-01-01

The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution. © 2014 Elsevier Inc. All rights reserved.

Application of silver nanoparticles in the detection of SYBR Green I by surface enhanced Raman and surface-enhanced fluorescence

NASA Astrophysics Data System (ADS)

Guo, Wei; Wu, Jian; Wang, Chunyan; Zhang, Tian; Chen, Tao

2018-05-01

Silver nanomaterials have remarkable application in biomedical detection due to their unique surface plasmon resonance (SPR) characteristics. It can be used for surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF). Current research elaborates a technique for improvement of SYBR Green I detection obtained from surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) by silver nanoparticles with the average size about 70 nm. Primarily, SYBR Green I is an important fluorescent dye used in polymerase chain reaction (PCR). It is found that both Raman and fluorescence can be used for detection of this dye. Furthermore, the enhanced efficiency of the Raman and fluorescence by SERS and SEF is observed in this study, the enhancement factor for Raman signals is 3.2 × 103, and the fluorescence intensity bincreased two times by SEF. The quantitative detection of SYBR Green I by SERS and SEF can be achieved. The present work can be used to improve the detection of SYBR Green I by SERS and SEF. It would also be employed for high-sensitive detection of other materials in the future.

Self-Assembled Fluorescent Nanoprobe Based on Forster Resonance Energy Transfer for Carbon Monoxide in Living Cells and Animals via Ligand Exchange.

PubMed

Jia, Ruizhen; Song, Pengfei; Wang, Jingjing; Mai, Hengtang; Li, Sixian; Cheng, Yu; Wu, Song

2018-05-29

Carbon monoxide (CO) is recognized as a biologically essential gaseous neurotransmitter that modulates many physiological processes in living subjects. Currently reported fluorescent probes for CO imaging in cells basically utilize palladium related chemistry which requires complicated synthetic work. Herein we provide a new strategy to construct a fluorescent nanoprobe, NanoCO-1, based on the Forster resonance energy transfer (FRET) mechanism by entrapping the existing dirhodium complex as the energy acceptor and the CO recognition part, and a commonly used nitrobenzoxadiazole (NBD) dye as energy donor into a micelle formed by self-assembly. The exchange of ligands in the dirhodium complex by CO in the nanoprobe disrupts the FRET and leads to the turn-on of fluorescence. The merits of NanoCO-1 including good biocompatibility, selectivity, photostability, and low cytotoxity, render this nanoprobe ability to track CO in living cells, zebrafish embryo, and larvae. Our straightforward approach can be extended to establish the CO fluorescent probes based on adsorption of CO on a variety of metal derivatives.

A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

PubMed

Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

2017-09-15

Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

Monitoring lactic acid production during milk fermentation by in situ quantitative proton nuclear magnetic resonance spectroscopy.

PubMed

Bouteille, R; Gaudet, M; Lecanu, B; This, H

2013-04-01

When fermenting milk, lactic bacteria convert part of α- and β-lactoses into d- and l- lactic acids, causing a pH decrease responsible for casein coagulation. Lactic acid monitoring during fermentation is essential for the control of dairy gel textural and organoleptic properties, and is a way to evaluate strain efficiency. Currently, titrations are used to follow the quantity of acids formed during jellification of milk but they are not specific to lactic acid. An analytical method without the use of any reagent was investigated to quantify lactic acid during milk fermentation: in situ quantitative proton nuclear magnetic resonance spectroscopy. Two methods using in situ quantitative proton nuclear magnetic resonance spectroscopy were compared: (1) d- and l-lactic acids content determination, using the resonance of their methyl protons, showing an increase from 2.06 ± 0.02 to 8.16 ± 0.74 g/L during 240 min of fermentation; and (2) the determination of the α- and β-lactoses content, decreasing from 42.68 ± 0.02 to 30.76 ± 1.75 g/L for the same fermentation duration. The ratio between the molar concentrations of produced lactic acids and consumed lactoses enabled cross-validation, as the value (2.02 ± 0.18) is consistent with lactic acid bacteria metabolism. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Rapid and quantitative detection of zoonotic influenza A virus infection utilizing coumarin-derived dendrimer-based fluorescent immunochromatographic strip test (FICT).

PubMed

Yeo, Seon-Ju; Huong, Dinh Thi; Hong, Nguyen Ngoc; Li, Chun-Ying; Choi, Kyunghan; Yu, Kyoungsik; Choi, Du-Young; Chong, Chom-Kyu; Choi, Hak Soo; Mallik, Shyam Kumar; Kim, Hak Sung; Sung, Haan Woo; Park, Hyun

2014-01-01

Great efforts have been made to develop robust signal-generating fluorescence materials which will help in improving the rapid diagnostic test (RDT) in terms of sensitivity and quantification. In this study, we developed coumarin-derived dendrimer-based fluorescent immunochromatographic strip test (FICT) assay with enhanced sensitivity as a quantitative diagnostic tool in typical RDT environments. The accuracy of the proposed FICT was compared with that of dot blot immunoassay techniques and conventional RDTs. Through conjugation of coumarin-derived dendrimers with latex beads, fluorescent emission covering broad output spectral ranges was obtained which provided a distinct advantage of easy discrimination of the fluorescent emission of the latex beads with a simple insertion of a long-pass optical filter away from the excitation wavelength. The newly developed FICT assay was able to detect 100 ng/10 μL of influenza A nucleoprotein (NP) antigen within 5 minutes, which corresponded to 2.5-fold higher sensitivity than that of the dot blot immunoassay or conventional RDTs. Moreover, the FICT assay was confirmed to detect at least four avian influenza A subtypes (H5N3, H7N1, H7N7, and H9N2). On applying the FICT to the clinical swab samples infected with respiratory viruses, our FICT assay was confirmed to differentiate influenza H1N1 infection from other respiratory viral diseases. These data demonstrate that the proposed FICT assay is able to detect zoonotic influenza A viruses with a high sensitivity, and it enables the quantitation of the infection intensity by providing the numerical diagnostic values; thus demonstrating enhanced detectability of influenza A viruses.

Method of using a nuclear magnetic resonance spectroscopy standard. [SO/sub 2/ in gases by fluorescence

DOEpatents

Spicer, L.D.; Bennett, D.W.; Davis, J.F.

1983-05-09

(CH/sub 3/)/sub 3/SiNSO is produced by the reaction of ((CH/sub 3/)/sub 3/SI)/sub 2/NH with SO/sub 2/. Also produced in the reaction are ((CH/sub 3/)/sub 3/Si)/sub 2/O and a new solid compound (NH/sub 4/)((CH/sub 3/)/sub 3/SiOSO/sub 2/). Both (CH/sub 3/)/sub 3/SiNSO and (NH/sub 4/)((CH/sub 3/)/sub 3/SiOSO/sub 2/) have fluorescent properties. The reaction of the subject invention is used in a method of measuring the concentration of SO/sub 2/ pollutants in gases. By the method, a sample of gas is bubbled through a solution of ((CH/sub 3/)/sub 3/Si)/sub 2/NH, whereby any SO/sub 2/ present in the gas will react to produce the two fluorescent products. The measured fluorescence of these products can then be used to calculate the concentration of SO/sub 2/ in the original gas sample. The solid product (NH/sub 4/)((CH/sub 3/)/sub 3/SiOSO/sub 2/) may be used as a standard in solid state NMR spectroscopy, wherein the resonance peaks of either /sup 1/H, /sup 13/C, /sup 15/N, or /sup 29/Si may be used as a reference.

Monte Carlo treatment of resonance-radiation imprisonment in fluorescent lamps—revisited

NASA Astrophysics Data System (ADS)

Anderson, James B.

2016-12-01

We reported in 1985 a Monte Carlo treatment of the imprisonment of the 253.7 nm resonance radiation from mercury in the mercury-argon discharge of fluorescent lamps. The calculated spectra of the emitted radiation were found in good agreement with measured spectra. The addition of the isotope mercury-196 to natural mercury was found, also in agreement with experiments, to increase lamp efficiency. In this paper we report the extension of the earlier work with increased accuracy, analysis of photon exit-time distributions, recycling of energy released in quenching, analysis of dynamic similarity for different lamp sizes, variation of Mrozowski transfer rates, prediction and analysis of the hyperfine ultra-violet spectra, and optimization of tailored mercury isotope mixtures for increased lamp efficiency. The spectra were found insensitive to the extent of quenching and recycling. The optimized mixtures were found to increase efficiencies by as much as 5% for several lamp configurations. Optimization without increasing the mercury-196 fraction was found to increase efficiencies by nearly 1% for several configurations.

Photobleaching of red fluorescence in oral biofilms.

PubMed

Hope, C K; de Josselin de Jong, E; Field, M R T; Valappil, S P; Higham, S M

2011-04-01

Many species of oral bacteria can be induced to fluoresce due to the presence of endogenous porphyrins, a phenomenon that can be utilized to visualize and quantify dental plaque in the laboratory or clinical setting. However, an inevitable consequence of fluorescence is photobleaching, and the effects of this on longitudinal, quantitative analysis of dental plaque have yet to be ascertained. Filter membrane biofilms were grown from salivary inocula or single species (Prevotella nigrescens and Prevotella intermedia). The mature biofilms were then examined in a custom-made lighting rig comprising 405 nm light-emitting diodes capable of delivering 220 W/m(2) at the sample, an appropriate filter and a digital camera; a set-up analogous to quantitative light-induced fluorescence digital. Longitudinal sets of images were captured and processed to assess the degradation in red fluorescence over time. Photobleaching was observed in all instances. The highest rates of photobleaching were observed immediately after initiation of illumination, specifically during the first minute. Relative rates of photobleaching during the first minute of exposure were 19.17, 13.72 and 3.43 arbitrary units/min for P. nigrescens biofilms, microcosm biofilm and P. intermedia biofilms, respectively. Photobleaching could be problematic when making quantitative measurements of porphyrin fluorescence in situ. Reducing both light levels and exposure time, in combination with increased camera sensitivity, should be the default approach when undertaking analyses by quantitative light-induced fluorescence digital. © 2010 John Wiley & Sons A/S.

Tumor acidity-activatable TAT targeted nanomedicine for enlarged fluorescence/magnetic resonance imaging-guided photodynamic therapy.

PubMed

Gao, Meng; Fan, Feng; Li, Dongdong; Yu, Yue; Mao, Kuirong; Sun, Tianmeng; Qian, Haisheng; Tao, Wei; Yang, Xianzhu

2017-07-01

Nanoparticles simultaneously integrated the photosensitizers and diagnostic agents represent an emerging approach for imaging-guided photodynamic therapy (PDT). However, the diagnostic sensitivity and therapeutic efficacy of nanoparticles as well as the heterogeneity of tumors pose tremendous challenges for clinical imaging-guided PDT treatment. Herein, a polymeric nanoparticle with tumor acidity (pH e )-activatable TAT targeting ligand that encapsulates the photosensitizer chlorin e6 (Ce6) and chelates contrast agent Gd 3+ is successfully developed for fluorescence/magnetic resonance (MR) dual-model imaging-guided precision PDT. We show clear evidence that the resulting nanoparticle DA TAT-NP [its TAT lysine residues' amines was modified by 2,3-dimethylmaleic anhydride (DA)] efficiently avoids the rapid clearance by reticuloendothelial system (RES) by masking of the TAT peptide, resulting in the significantly prolonged circulation time in the blood. Once accumulating in the tumor tissues, DA TAT-NP is reactivated by tumor acidity to promote cellular uptake, resulting in enlarged fluorescence/MR imaging signal intensity and elevated in vivo PDT therapeutic effect. This concept provides new avenues to design tumor acidity-activatable targeted nanoparticles for imaging-guided cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Accurate quantification of fluorescent targets within turbid media based on a decoupled fluorescence Monte Carlo model.

PubMed

Deng, Yong; Luo, Zhaoyang; Jiang, Xu; Xie, Wenhao; Luo, Qingming

2015-07-01

We propose a method based on a decoupled fluorescence Monte Carlo model for constructing fluorescence Jacobians to enable accurate quantification of fluorescence targets within turbid media. The effectiveness of the proposed method is validated using two cylindrical phantoms enclosing fluorescent targets within homogeneous and heterogeneous background media. The results demonstrate that our method can recover relative concentrations of the fluorescent targets with higher accuracy than the perturbation fluorescence Monte Carlo method. This suggests that our method is suitable for quantitative fluorescence diffuse optical tomography, especially for in vivo imaging of fluorophore targets for diagnosis of different diseases and abnormalities.

Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe.

PubMed

Li, Cuixia; Zuo, Jing; Zhang, Li; Chang, Yulei; Zhang, Youlin; Tu, Langping; Liu, Xiaomin; Xue, Bin; Li, Qiqing; Zhao, Huiying; Zhang, Hong; Kong, Xianggui

2016-12-09

Accurate quantitation of intracellular pH (pH i ) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pH i sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pH i . Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pH i , in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF 4 :Yb 3+ , Tm 3+ UCNPs were used as pH i response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pH i value 3.0-7.0 with deviation less than 0.43. This approach shall facilitate the researches in pH i related areas and development of the intracellular drug delivery systems.

Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe

NASA Astrophysics Data System (ADS)

Li, Cuixia; Zuo, Jing; Zhang, Li; Chang, Yulei; Zhang, Youlin; Tu, Langping; Liu, Xiaomin; Xue, Bin; Li, Qiqing; Zhao, Huiying; Zhang, Hong; Kong, Xianggui

2016-12-01

Accurate quantitation of intracellular pH (pHi) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pHi sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pHi. Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF4:Yb3+, Tm3+ UCNPs were used as pHi response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pHi value 3.0-7.0 with deviation less than 0.43. This approach shall facilitate the researches in pHi related areas and development of the intracellular drug delivery systems.

Two-Photon Fluorescent Probe for Monitoring Autophagy via Fluorescence Lifetime Imaging.

PubMed

Hou, Liling; Ning, Peng; Feng, Yan; Ding, Yaqi; Bai, Lei; Li, Lin; Yu, Haizhu; Meng, Xiangming

2018-06-19

We reported the first lysosome targeted two-photon fluorescent probe (Lyso-NP) as a viscosity probe for monitoring autophagy. The fluorescence lifetime of Lyso-NP exhibited an excellent linear relationship with viscosity value ( R 2 = 0.99, x = 0.39). Lyso-NP also showed the specific capability for imaging lysosomal viscosity under two-photon excitation at 860 nm along with good biocompatibility. More importantly, Lyso-NP could be used to monitor the autophagy process in living cells by quantitatively detecting lysosomal viscosity changes during the membrane fusion process via two-photon fluorescence lifetime imaging.

Deep Learning for Magnetic Resonance Fingerprinting: A New Approach for Predicting Quantitative Parameter Values from Time Series.

PubMed

Hoppe, Elisabeth; Körzdörfer, Gregor; Würfl, Tobias; Wetzl, Jens; Lugauer, Felix; Pfeuffer, Josef; Maier, Andreas

2017-01-01

The purpose of this work is to evaluate methods from deep learning for application to Magnetic Resonance Fingerprinting (MRF). MRF is a recently proposed measurement technique for generating quantitative parameter maps. In MRF a non-steady state signal is generated by a pseudo-random excitation pattern. A comparison of the measured signal in each voxel with the physical model yields quantitative parameter maps. Currently, the comparison is done by matching a dictionary of simulated signals to the acquired signals. To accelerate the computation of quantitative maps we train a Convolutional Neural Network (CNN) on simulated dictionary data. As a proof of principle we show that the neural network implicitly encodes the dictionary and can replace the matching process.

Recoilless Nuclear Resonance Absorption of Gamma Radiation

NASA Astrophysics Data System (ADS)

Mössbauer, Rudolf L.

It is a high distinction to be permitted to address you on the subject of recoilless nuclear resonance absorption of gamma radiation. The methods used in this special branch of experimental physics have recently found acceptance in many areas of science. I take the liberty to confine myself essentially to the work which I was able to carry out in the years 1955-1958 at the Max Planck Institute in Heidelberg, and which finally led to establishment of the field of recoilless nuclear resonance absorption. Many investigators shared in the preparations of the basis for the research we are concerned with in this lecture. As early as the middle of the last century Stokes observed, in the case of fluorite, the phenomenon now known as fluorescence - namely, that solids, liquids, and gases under certain conditions partially absorb incident electromagnetic radiation which immediately is reradiated. A special case is the so-called resonance fluorescence, a phenomenon in which the re-emitted and the incident radiation both are of the same wavelength. The resonance fluorescence of the yellow D lines of sodium in sodium vapour is a particularly notable and exhaustively studied example. In this optical type of resonance fluorescence, light sources are used in which the atoms undergo transitions from excited states to their ground states (Fig. 1.1). The light quanta emitted in these transitions (A → B) are used to initiate the inverse process of resonance absorption in the atoms of an absorber which are identical with the radiating atoms. The atoms of the absorber undergo a transition here from the ground state (B) to the excited state (A), from which they again return to the ground state, after a certain time delay, by emission of fluorescent light.

Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate production in rat parotid intralobular ducts: quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope.

PubMed

Nakamoto, Tetsuji; Shiba, Yoshiki; Hirono, Chikara; Sugita, Makoto; Takemoto, Kazuhisa; Iwasa, Yoshiko; Akagawa, Yasumasa

2002-09-01

Fluid secretion is observed at the openings of ducts in the exocrine gland. It remains unclear whether the ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the duct lumen by carbachol (CCh) in isolated parotid intralobular duct segments to clarify the ability of the ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the duct lumen were obtained under the optical sectioning at the level of the duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated duct lumen, suggesting that CCh might induce fluid secretion in the duct, leading to the clearance of the dye and small stained clumps from the duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl-, bumetanide or DIDS, suggesting that methazolamide-sensitive production of HCO3-, but not the Cl- uptake mechanism, might contribute to the CCh-induced clearance of the dye from the duct lumen. These results represent the first measurements of fluid movement in isolated duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca2+-activated, DPC-sensitive anion channels with HCO3- secretion in the rat parotid intralobular ducts.

Quantitation of Localized 31P Magnetic Resonance Spectra Based on the Reciprocity Principle

NASA Astrophysics Data System (ADS)

Kreis, R.; Slotboom, J.; Pietz, J.; Jung, B.; Boesch, C.

2001-04-01

There is a need for absolute quantitation methods in 31P magnetic resonance spectroscopy, because none of the phosphorous-containing metabolites is necessarily constant in pathology. Here, a method for absolute quantitation of in vivo31P MR spectra that provides reproducible metabolite contents in institutional or standard units is described. It relies on the reciprocity principle, i.e., the proportionality between the B1 field map and the map of reception strength for a coil with identical relative current distributions in receive and transmit mode. Cerebral tissue contents of 31P metabolites were determined in a predominantly white matter-containing location in healthy subjects. The results are in good agreement with the literature and the interexamination coefficient of variance is better than that in most previous studies. A gender difference found for some of the 31P metabolites may be explained by different voxel composition.

Advances in Surface Plasmon Resonance Imaging allowing for quantitative measurement of laterally heterogeneous samples

NASA Astrophysics Data System (ADS)

Raegen, Adam; Reiter, Kyle; Clarke, Anthony; Lipkowski, Jacek; Dutcher, John

2012-02-01

The Surface Plasmon Resonance (SPR) phenomenon is routinely exploited to qualitatively probe changes to materials on metallic surfaces for use in probes and sensors. Unfortunately, extracting truly quantitative information is usually limited to a select few cases -- uniform absorption/desorption of small biomolecules and films, in which a continuous ``slab'' model is a good approximation. We present advancements in the SPR technique that expand the number of cases for which the technique can provide meaningful results. Use of a custom, angle-scanning SPR imaging system, together with a refined data analysis method, allow for quantitative kinetic measurements of laterally heterogeneous systems. The degradation of cellulose microfibrils and bundles of microfibrils due to the action of cellulolytic enzymes will be presented as an excellent example of the capabilities of the SPR imaging system.

Fluorescence in insects

NASA Astrophysics Data System (ADS)

Welch, Victoria L.; Van Hooijdonk, Eloise; Intrater, Nurit; Vigneron, Jean-Pol

2012-10-01

Fluorescent molecules are much in demand for biosensors, solar cells, LEDs and VCSEL diodes, therefore, considerable efforts have been expended in designing and tailoring fluorescence to specific technical applications. However, naturally occurring fluorescence of diverse types has been reported from a wide array of living organisms: most famously, the jellyfish Aequorea victoria, but also in over 100 species of coral and in the cuticle of scorpions, where it is the rule, rather than the exception. Despite the plethora of known insect species, comparatively few quantitative studies have been made of insect fluorescence. Because of the potential applications of natural fluorescence, studies in this field have relevance to both physics and biology. Therefore, in this paper, we review the literature on insect fluorescence, before documenting its occurrence in the longhorn beetles Sternotomis virescens, Sternotomis variabilis var. semi rufescens, Anoplophora elegans and Stellognatha maculata, the tiger beetles Cicindela maritima and Cicindela germanica and the weevil Pachyrrhynchus gemmatus purpureus. Optical features of insect fluorescence, including emitted wavelength, molecular ageing and naturally occurring combinations of fluorescence with bioluminescence and colour-producing structures are discussed.

Investigation of Membrane Receptors' Oligomers Using Fluorescence Resonance Energy Transfer and Multiphoton Microscopy in Living Cells

NASA Astrophysics Data System (ADS)

Mishra, Ashish K.

Investigating quaternary structure (oligomerization) of macromolecules (such as proteins and nucleic acids) in living systems (in vivo) has been a great challenge in biophysics, due to molecular diffusion, fluctuations in several biochemical parameters such as pH, quenching of fluorescence by oxygen (when fluorescence methods are used), etc. We studied oligomerization of membrane receptors in living cells by means of Fluorescence (Forster) Resonance Energy Transfer (FRET) using fluorescent markers and two photon excitation fluorescence micro-spectroscopy. Using suitable FRET models, we determined the stoichiometry and quaternary structure of various macromolecular complexes. The proteins of interest for this work are : (1) sigma-1 receptor and (2) rhodopsin, are described as below. (1) Sigma-1 receptors are molecular chaperone proteins, which also regulate ion channels. S1R seems to be involved in substance abuse, as well as several diseases such as Alzheimer's. We studied S1R in the presence and absence of its ligands haloperidol (an antagonist) and pentazocine +/- (an agonist), and found that at low concentration they reside as a mixture of monomers and dimers and that they may form higher order oligomers at higher concentrations. (2) Rhodopsin is a prototypical G protein coupled receptor (GPCR) and is directly involved in vision. GPCRs form a large family of receptors that participate in cell signaling by responding to external stimuli such as drugs, thus being a major drug target (more than 40% drugs target GPCRs). Their oligomerization has been largely controversial. Understanding this may help to understand the functional role of GPCRs oligomerization, and may lead to the discovery of more drugs targeting GPCR oligomers. It may also contribute toward finding a cure for Retinitis Pigmentosa, which is caused by a mutation (G188R) in rhodopsin, a disease which causes blindness and has no cure so far. Comparing healthy rhodopsin's oligomeric structure with that

Visualization of astaxanthin localization in HT29 human colon adenocarcinoma cells by combined confocal resonance Raman and fluorescence microspectroscopy.

PubMed

Briviba, Karlis; Bornemann, Rainer; Lemmer, Ulrich

2006-11-01

Astaxanthin, a carotenoid found in plants and seafood, exhibits antiproliferative, antioxidant and anticarcinogenic properties. We show that astaxanthin delivered with tetrahydrofuran is effectively taken up by cultured colon adenocarcinoma cells and is localized mostly in the cytoplasm as detected by confocal resonance Raman and broad-band fluorescence microspectroscopy image analysis. Cells incubated with beta-carotene at the same concentration as astaxanthin (10 microM) showed about a 50-fold lower cellular amount of beta-carotene, as detected by HPLC. No detectable Raman signal of beta-carotene was found in cells, but a weak broad-band fluorescence signal of beta-carotene was observed. beta-Carotene, like astaxanthin, was localized mostly in the cytoplasm. The heterogeneity of astaxanthin and beta-carotene cellular distribution in cells of intestinal origin suggests that the possible defense against reactive molecules by carotenoids in these cells may also be heterogeneous.

Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes

PubMed Central

Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

2016-01-01

Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application. PMID:26931282

Flash photolysis resonance fluorescence investigation of the reaction of O /P-3/ atoms with ClONO2

NASA Technical Reports Server (NTRS)

Kurylo, M. J.

1977-01-01

The rate constant for the reaction of O (P-3) atoms with ClONO2 at 10 torr total pressure is assessed over the temperature range 225-273 K by the flash photolysis resonance fluorescence technique. The data, taken together with results given by Molina et al. (1977), have been used to formulate an Arrhenius expression suitable for stratospheric modeling applications. Comparison of the rate of ClONO2 destruction via the oxygen atom reaction with the solar photolysis rate shows that chemical reaction accounts for less than 15% of the CLONO2 removal at altitudes between 20 and 30 km.

Assessing the use of Quantitative Light-induced Fluorescence-Digital as a clinical plaque assessment.

PubMed

Han, Sun-Young; Kim, Bo-Ra; Ko, Hae-Youn; Kwon, Ho-Keun; Kim, Baek-Il

2016-03-01

The aims of this study were to compare the relationship between red fluorescent plaque (RF plaque) area by Quantitative Light-induced Fluorescence-Digital (QLF-D) and disclosed plaque area by two-tone disclosure, and to assess the bacterial composition of the RF plaque by real time-PCR. Fifty healthy subjects were included and 600 facial surfaces of their anterior teeth were examined. QLF-D was taken on two separate occasions (before and after disclosing), and the RF plaque area was calculated based on Plaque Percent Index (PPI). After disclosing, the stained plaque area was analyzed to investigate the relationship with the RF plaque area. The relationship was evaluated using Pearson correlation and paired t-test. Then, the RF and non-red fluorescent (non-RF) plaque samples were obtained from the same subject for real-time PCR test. Total 10 plaque samples were compared the ratio of the 6 of bacteria using Wilcoxon signed rank test. Regarding the paired t-test, the blue-staining plaque area (9.3±9.2) showed significantly similarity with the RF plaque area (9.1±14.9, p=0.80) at ΔR20, however, the red-staining plaque area (31.6±20.9) presented difference from the RF plaque area (p<0.0001). In addition, bacterial composition of Prevotella intermedia and Streptococcus anginosus was associated with substantially more the RF plaque than the non-RF plaque (p<0.05). The plaque assessment method using QLF-D has potential to detect mature plaque, and the plaque area was associated with the blue-staining area using two-tone disclosure. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of γ -ray widths in 15N using nuclear resonance fluorescence

NASA Astrophysics Data System (ADS)

Szücs, T.; Bemmerer, D.; Caciolli, A.; Fülöp, Zs.; Massarczyk, R.; Michelagnoli, C.; Reinhardt, T. P.; Schwengner, R.; Takács, M. P.; Ur, C. A.; Wagner, A.; Wagner, L.

2015-07-01

Background: The stable nucleus 15N is the mirror of 15O, the bottleneck in the hydrogen burning CNO cycle. Most of the 15N level widths below the proton emission threshold are known from just one nuclear resonance fluorescence (NRF) measurement, with limited precision in some cases. A recent experiment with the AGATA demonstrator array determined level lifetimes using the Doppler shift attenuation method in 15O. As a reference and for testing the method, level lifetimes in 15N have also been determined in the same experiment. Purpose: The latest compilation of 15N level properties dates back to 1991. The limited precision in some cases in the compilation calls for a new measurement to enable a comparison to the AGATA demonstrator data. The widths of several 15N levels have been studied with the NRF method. Method: The solid nitrogen compounds enriched in 15N have been irradiated with bremsstrahlung. The γ rays following the deexcitation of the excited nuclear levels were detected with four high-purity germanium detectors. Results: Integrated photon-scattering cross sections of 10 levels below the proton emission threshold have been measured. Partial γ -ray widths of ground-state transitions were deduced and compared to the literature. The photon-scattering cross sections of two levels above the proton emission threshold, but still below other particle emission energies have also been measured, and proton resonance strengths and proton widths were deduced. Conclusions: Gamma and proton widths consistent with the literature values were obtained, but with greatly improved precision.

In vitro evaluation of fluorescence glucose biosensor response.

PubMed

Aloraefy, Mamdouh; Pfefer, T Joshua; Ramella-Roman, Jessica C; Sapsford, Kim E

2014-07-08

Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor.

In Vitro Evaluation of Fluorescence Glucose Biosensor Response

PubMed Central

Aloraefy, Mamdouh; Pfefer, T. Joshua; Ramella-Roman, Jessica C.; Sapsford, Kim E.

2014-01-01

Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor. PMID:25006996

A meta-classifier for detecting prostate cancer by quantitative integration of in vivo magnetic resonance spectroscopy and magnetic resonance imaging

NASA Astrophysics Data System (ADS)

Viswanath, Satish; Tiwari, Pallavi; Rosen, Mark; Madabhushi, Anant

2008-03-01

Recently, in vivo Magnetic Resonance Imaging (MRI) and Magnetic Resonance Spectroscopy (MRS) have emerged as promising new modalities to aid in prostate cancer (CaP) detection. MRI provides anatomic and structural information of the prostate while MRS provides functional data pertaining to biochemical concentrations of metabolites such as creatine, choline and citrate. We have previously presented a hierarchical clustering scheme for CaP detection on in vivo prostate MRS and have recently developed a computer-aided method for CaP detection on in vivo prostate MRI. In this paper we present a novel scheme to develop a meta-classifier to detect CaP in vivo via quantitative integration of multimodal prostate MRS and MRI by use of non-linear dimensionality reduction (NLDR) methods including spectral clustering and locally linear embedding (LLE). Quantitative integration of multimodal image data (MRI and PET) involves the concatenation of image intensities following image registration. However multimodal data integration is non-trivial when the individual modalities include spectral and image intensity data. We propose a data combination solution wherein we project the feature spaces (image intensities and spectral data) associated with each of the modalities into a lower dimensional embedding space via NLDR. NLDR methods preserve the relationships between the objects in the original high dimensional space when projecting them into the reduced low dimensional space. Since the original spectral and image intensity data are divorced from their original physical meaning in the reduced dimensional space, data at the same spatial location can be integrated by concatenating the respective embedding vectors. Unsupervised consensus clustering is then used to partition objects into different classes in the combined MRS and MRI embedding space. Quantitative results of our multimodal computer-aided diagnosis scheme on 16 sets of patient data obtained from the ACRIN trial, for which

Segmentation of fluorescence microscopy images for quantitative analysis of cell nuclear architecture.

PubMed

Russell, Richard A; Adams, Niall M; Stephens, David A; Batty, Elizabeth; Jensen, Kirsten; Freemont, Paul S

2009-04-22

Considerable advances in microscopy, biophysics, and cell biology have provided a wealth of imaging data describing the functional organization of the cell nucleus. Until recently, cell nuclear architecture has largely been assessed by subjective visual inspection of fluorescently labeled components imaged by the optical microscope. This approach is inadequate to fully quantify spatial associations, especially when the patterns are indistinct, irregular, or highly punctate. Accurate image processing techniques as well as statistical and computational tools are thus necessary to interpret this data if meaningful spatial-function relationships are to be established. Here, we have developed a thresholding algorithm, stable count thresholding (SCT), to segment nuclear compartments in confocal laser scanning microscopy image stacks to facilitate objective and quantitative analysis of the three-dimensional organization of these objects using formal statistical methods. We validate the efficacy and performance of the SCT algorithm using real images of immunofluorescently stained nuclear compartments and fluorescent beads as well as simulated images. In all three cases, the SCT algorithm delivers a segmentation that is far better than standard thresholding methods, and more importantly, is comparable to manual thresholding results. By applying the SCT algorithm and statistical analysis, we quantify the spatial configuration of promyelocytic leukemia nuclear bodies with respect to irregular-shaped SC35 domains. We show that the compartments are closer than expected under a null model for their spatial point distribution, and furthermore that their spatial association varies according to cell state. The methods reported are general and can readily be applied to quantify the spatial interactions of other nuclear compartments.

Segmentation of Fluorescence Microscopy Images for Quantitative Analysis of Cell Nuclear Architecture

PubMed Central

Russell, Richard A.; Adams, Niall M.; Stephens, David A.; Batty, Elizabeth; Jensen, Kirsten; Freemont, Paul S.

2009-01-01

Abstract Considerable advances in microscopy, biophysics, and cell biology have provided a wealth of imaging data describing the functional organization of the cell nucleus. Until recently, cell nuclear architecture has largely been assessed by subjective visual inspection of fluorescently labeled components imaged by the optical microscope. This approach is inadequate to fully quantify spatial associations, especially when the patterns are indistinct, irregular, or highly punctate. Accurate image processing techniques as well as statistical and computational tools are thus necessary to interpret this data if meaningful spatial-function relationships are to be established. Here, we have developed a thresholding algorithm, stable count thresholding (SCT), to segment nuclear compartments in confocal laser scanning microscopy image stacks to facilitate objective and quantitative analysis of the three-dimensional organization of these objects using formal statistical methods. We validate the efficacy and performance of the SCT algorithm using real images of immunofluorescently stained nuclear compartments and fluorescent beads as well as simulated images. In all three cases, the SCT algorithm delivers a segmentation that is far better than standard thresholding methods, and more importantly, is comparable to manual thresholding results. By applying the SCT algorithm and statistical analysis, we quantify the spatial configuration of promyelocytic leukemia nuclear bodies with respect to irregular-shaped SC35 domains. We show that the compartments are closer than expected under a null model for their spatial point distribution, and furthermore that their spatial association varies according to cell state. The methods reported are general and can readily be applied to quantify the spatial interactions of other nuclear compartments. PMID:19383481

FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

PubMed

Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad

2015-10-01

Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

Quantitative contrast enhanced magnetic resonance imaging for the evaluation of peripheral arterial disease: a comparative study versus standard digital angiography.

PubMed

Pavlovic, Chris; Futamatsu, Hideki; Angiolillo, Dominick J; Guzman, Luis A; Wilke, Norbert; Siragusa, Daniel; Wludyka, Peter; Percy, Robert; Northrup, Martin; Bass, Theodore A; Costa, Marco A

2007-04-01

The purpose of this study is to evaluate the accuracy of semiautomated analysis of contrast enhanced magnetic resonance angiography (MRA) in patients who have undergone standard angiographic evaluation for peripheral vascular disease (PVD). Magnetic resonance angiography is an important tool for evaluating PVD. Although this technique is both safe and noninvasive, the accuracy and reproducibility of quantitative measurements of disease severity using MRA in the clinical setting have not been fully investigated. 43 lesions in 13 patients who underwent both MRA and digital subtraction angiography (DSA) of iliac and common femoral arteries within 6 months were analyzed using quantitative magnetic resonance angiography (QMRA) and quantitative vascular analysis (QVA). Analysis was repeated by a second operator and by the same operator in approximately 1 month time. QMRA underestimated percent diameter stenosis (%DS) compared to measurements made with QVA by 2.47%. Limits of agreement between the two methods were +/- 9.14%. Interobserver variability in measurements of %DS were +/- 12.58% for QMRA and +/- 10.04% for QVA. Intraobserver variability of %DS for QMRA was +/- 4.6% and for QVA was +/- 8.46%. QMRA displays a high level of agreement to QVA when used to determine stenosis severity in iliac and common femoral arteries. Similar levels of interobserver and intraobserver variability are present with each method. Overall, QMRA represents a useful method to quantify severity of PVD.

Quantitative Serum Nuclear Magnetic Resonance Metabolomics in Large-Scale Epidemiology: A Primer on -Omic Technologies

PubMed Central

Kangas, Antti J; Soininen, Pasi; Lawlor, Debbie A; Davey Smith, George; Ala-Korpela, Mika

2017-01-01

Abstract Detailed metabolic profiling in large-scale epidemiologic studies has uncovered novel biomarkers for cardiometabolic diseases and clarified the molecular associations of established risk factors. A quantitative metabolomics platform based on nuclear magnetic resonance spectroscopy has found widespread use, already profiling over 400,000 blood samples. Over 200 metabolic measures are quantified per sample; in addition to many biomarkers routinely used in epidemiology, the method simultaneously provides fine-grained lipoprotein subclass profiling and quantification of circulating fatty acids, amino acids, gluconeogenesis-related metabolites, and many other molecules from multiple metabolic pathways. Here we focus on applications of magnetic resonance metabolomics for quantifying circulating biomarkers in large-scale epidemiology. We highlight the molecular characterization of risk factors, use of Mendelian randomization, and the key issues of study design and analyses of metabolic profiling for epidemiology. We also detail how integration of metabolic profiling data with genetics can enhance drug development. We discuss why quantitative metabolic profiling is becoming widespread in epidemiology and biobanking. Although large-scale applications of metabolic profiling are still novel, it seems likely that comprehensive biomarker data will contribute to etiologic understanding of various diseases and abilities to predict disease risks, with the potential to translate into multiple clinical settings. PMID:29106475

Sensitive molecular diagnostics using surface-enhanced resonance Raman scattering (SERRS)

NASA Astrophysics Data System (ADS)

Faulds, Karen; Graham, Duncan; McKenzie, Fiona; MacRae, Douglas; Ricketts, Alastair; Dougan, Jennifer

2009-02-01

Surface enhanced resonance Raman scattering (SERRS) is an analytical technique with several advantages over competitive techniques in terms of improved sensitivity and multiplexing. We have made great progress in the development of SERRS as a quantitative analytical method, in particular for the detection of DNA. SERRS is an extremely sensitive and selective technique which when applied to the detection of labelled DNA sequences allows detection limits to be obtained which rival, and in most cases, are better than fluorescence. Here the conditions are explored which will enable the successful detection of DNA using SERRS. The enhancing surface which is used is crucial and in this case suspensions of nanoparticles were used as they allow quantitative behaviour to be achieved and allow analogous systems to current fluorescence based systems to be made. The aggregation conditions required to obtain SERRS of DNA are crucial and herein we describe the use of spermine as an aggregating agent. The nature of the label which is used, be it fluorescent, positively or negatively charged also effects the SERRS response and these conditions are again explored here. We have clearly demonstrated the ability to identify the components of a mixture of 5 analytes in solution by using two different excitation wavelengths and also of a 6-plex using data analysis techniques. These conditions will allow the use of SERRS for the detection of target DNA in a meaningful diagnostic assay.

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.

Conformational fluctuation of Synaptotagmin-1 observed with single molecule fluorescence resonance energy transfer (smFRET)

NASA Astrophysics Data System (ADS)

Choi, Ucheor; Weninger, Keith

2008-10-01

Calcium dependent neurotransmitter release at the synapses involves a synaptic vesicle protein synaptotagmin-1, a calcium sensor, to regulate exocytosis. It has been known that Synaptotagmin-1 interacts with assembled SNARE complexes, but it is unclear how their molecular mechanisms are coupled. X-ray studies in the absence of calcium revealed a closed conformation of synaptotagmin-1 and with calcium bound to the C2 domains of synaptotagmin-3 found extensive interactions holding the domains open. Suggesting the two conformations can be the key to the two functions of synaptotagmin in regulating neurotransmission. Here we use single molecule fluorescence resonance energy transfer (smFRET) to study synaptotagmin interactions with SNARE complexes and the spontaneous conformational changes of synaptotagmin-1 when calcium is induced.

Association between quantitative measures obtained using fluorescence-based methods and activity status of occlusal caries lesions in primary molars.

PubMed

Novaes, Tatiane Fernandes; Reyes, Alessandra; Matos, Ronilza; Antunes-Pontes, Laura Regina; Marques, Renata Pereira de Samuel; Braga, Mariana Minatel; Diniz, Michele Baffi; Mendes, Fausto Medeiros

2017-05-01

Fluorescence-based methods (FBM) can add objectiveness to diagnosis strategy for caries. Few studies, however, have focused on the evaluation of caries activity. To evaluate the association between quantitative measures obtained with FBM, clinical parameters acquired from the patients, caries detection, and assessment of activity status in occlusal surfaces of primary molars. Six hundred and six teeth from 113 children (4-14 years) were evaluated. The presence of a biofilm, caries experience, and the number of active lesions were recorded. The teeth were assessed using FBM: DIAGNOdent pen (Lfpen) and Quantitative light-induced fluorescence (QLF). As reference standard, all teeth were evaluated using the ICDAS (International Caries Detection and Assessment System) associated with clinical activity assessments. Multilevel regressions compared the FBM values and evaluated the association between the FBM measures and clinical variables related to the caries activity. The measures from the FBM were higher in cavitated lesions. Only, ∆F values distinguished active and inactive lesions. The LFpen measures were higher in active lesions, at the cavitated threshold (56.95 ± 29.60). Following regression analyses, only the presence of visible biofilm on occlusal surfaces (adjusted prevalence ratio = 1.43) and ∆R values of the teeth (adjusted prevalence ratio = 1.02) were associated with caries activity. Some quantitative measures from FBM parameters are associated with caries activity evaluation, which is similar to the clinical evaluation of the presence of visible biofilm. © 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Magneto-Fluorescent Core-Shell Supernanoparticles

PubMed Central

Chen, Ou; Riedemann, Lars; Etoc, Fred; Herrmann, Hendrik; Coppey, Mathieu; Barch, Mariya; Farrar, Christian T.; Zhao, Jing; Bruns, Oliver T.; Wei, He; Guo, Peng; Cui, Jian; Jensen, Russ; Chen, Yue; Harris, Daniel K.; Cordero, Jose M.; Wang, Zhongwu; Jasanoff, Alan; Fukumura, Dai; Reimer, Rudolph; Dahan, Maxime; Jain, Rakesh K.; Bawendi, Moungi G.

2014-01-01

Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface, and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close packed magnetic nanoparticle “core” which is fully surrounded by a “shell” of fluorescent quantum dots. A thin layer of silica-coating provides high colloidal stability and biocompatiblity and a versatile surface functionality. We demonstrate that after surface pegylation, these silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe. PMID:25298155

Using a Specific RNA-Protein Interaction To Quench the Fluorescent RNA Spinach.

PubMed

Roszyk, Laura; Kollenda, Sebastian; Hennig, Sven

2017-12-15

RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labeled. "Spinach" is a genetically encodable RNA aptamer that starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyze RNA-protein interactions by quenching the spinach aptamer.

Usefulness of a Dual Macro- and Micro-Energy-Dispersive X-Ray Fluorescence Spectrometer to Develop Quantitative Methodologies for Historic Mortar and Related Materials Characterization.

PubMed

García-Florentino, Cristina; Maguregui, Maite; Romera-Fernández, Miriam; Queralt, Ignasi; Margui, Eva; Madariaga, Juan Manuel

2018-05-01

Wavelength dispersive X-ray fluorescence (WD-XRF) spectrometry has been widely used for elemental quantification of mortars and cements. In this kind of instrument, samples are usually prepared as pellets or fused beads and the whole volume of sample is measured at once. In this work, the usefulness of a dual energy dispersive X-ray fluorescence spectrometer (ED-XRF), working at two lateral resolutions (1 mm and 25 μm) for macro and microanalysis respectively, to develop quantitative methods for the elemental characterization of mortars and concretes is demonstrated. A crucial step before developing any quantitative method with this kind of spectrometers is to verify the homogeneity of the standards at these two lateral resolutions. This new ED-XRF quantitative method also demonstrated the importance of matrix effects in the accuracy of the results being necessary to use Certified Reference Materials as standards. The results obtained with the ED-XRF quantitative method were compared with the ones obtained with two WD-XRF quantitative methods employing two different sample preparation strategies (pellets and fused beads). The selected ED-XRF and both WD-XRF quantitative methods were applied to the analysis of real mortars. The accuracy of the ED-XRF results turn out to be similar to the one achieved by WD-XRF, except for the lightest elements (Na and Mg). The results described in this work proved that μ-ED-XRF spectrometers can be used not only for acquiring high resolution elemental map distributions, but also to perform accurate quantitative studies avoiding the use of more sophisticated WD-XRF systems or the acid extraction/alkaline fusion required as destructive pretreatment in Inductively coupled plasma mass spectrometry based procedures.

Orderly arranged fluorescence dyes as a highly efficient chemiluminescence resonance energy transfer probe for peroxynitrite.

PubMed

Wang, Zhihua; Teng, Xu; Lu, Chao

2015-03-17

Chemiluminescence (CL) probes for reactive oxygen species (ROS) are commonly based on a redox reaction between a CL reagent and ROS, leading to poor selectivity toward a specific ROS. The energy-matching rules in the chemiluminescence resonance energy transfer (CRET) process between a specific ROS donor and a suitable fluorescence dye acceptor is a promising method for the selective detection of ROS. Nevertheless, higher concentrations of fluorescence dyes can lead to the intractable aggregation-caused quenching effect, decreasing the CRET efficiency. In this report, we fabricated an orderly arranged structure of calcein-sodium dodecyl sulfate (SDS) molecules to improve the CRET efficiency between ONOOH* donor and calcein acceptor. Such orderly arranged calcein-SDS composites can distinguish peroxynitrite (ONOO(-)) from a variety of other ROS owing to the energy matching in the CRET process between ONOOH* donor and calcein acceptor. Under the optimal experimental conditions, ONOO(-) could be assayed in the range of 1.0-20.0 μM, and the detection limit for ONOO(-) [signal-to-noise ratio (S/N) = 3] was 0.3 μM. The proposed strategy has been successfully applied in both detecting ONOO(-) in cancer mouse plasma samples and monitoring the generation of ONOO(-) from 3-morpholinosydnonimine (SIN-1). Recoveries from cancer mouse plasma samples were in the range of 96-105%. The success of this work provides a unique opportunity to develop a CL tool to monitor ONOO(-) with high selectivity in a specific manner. Improvement of selectivity and sensitivity of CL probes holds great promise as a strategy for developing a wide range of probes for various ROS by tuning the types of fluorescence dyes.

Single-molecule-sensitive fluorescence resonance energy transfer in freely-diffusing attoliter droplets

NASA Astrophysics Data System (ADS)

Rahmanseresht, Sheema; Milas, Peker; Ramos, Kieran P.; Gamari, Ben D.; Goldner, Lori S.

2015-05-01

Fluorescence resonance energy transfer (FRET) from individual, dye-labeled RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is carefully compared to FRET from unconfined RNA in solution. The use of freely-diffusing droplets is a remarkably simple and high-throughput technique that facilitates a substantial increase in signal-to-noise for single-molecular-pair FRET measurements. We show that there can be dramatic differences between FRET in solution and in droplets, which we attribute primarily to an altered pH in the confining environment. We also demonstrate that a sufficient concentration of a non-ionic surfactant mitigates this effect and restores FRET to its neutral-pH solution value. At low surfactant levels, even accounting for pH, we observe differences between the distribution of FRET values in solution and in droplets which remain unexplained. Our results will facilitate the use of nanoemulsion droplets as attoliter volume reactors for use in biophysical and biochemical assays, and also in applications such as protein crystallization or nanoparticle synthesis, where careful attention to the pH of the confined phase is required.

In vivo quantitative visualization of hypochlorous acid in the liver using a novel selective two-photon fluorescent probe

NASA Astrophysics Data System (ADS)

Wang, Haolu; Jayachandran, Aparna; Gravot, Germain; Liang, Xiaowen; Thorling, Camilla A.; Zhang, Run; Liu, Xin; Roberts, Michael S.

2016-11-01

Hypochlorous acid (HOCl) plays a vital role in physiological events and diseases. During hepatic ischemia-reperfusion (I/R) injury, HOCl is generated by neutrophils and diffuses into hepatocytes, causing oxidant stress-mediated injury. Although many probes have been developed to detect HOCl, most were difficult to be distinguished from endogenous fluorophores in intravital imaging and only can be employed under one-photon microscopy. A novel iridium(III) complex-based ferrocene dual-signaling chemosensor (Ir-Fc) was designed and synthesized. Ir-Fc exhibited a strong positive fluorescent response only in the presence of HOCl, whereas negligible fluorescent signals were observed upon the additions of other reactive oxygen/nitrogen species and metal ions. There was a good linear relationship between probe responsive fluorescent intensity and HOCl concentration. Ir-Fc was then intravenously injected into BALB/c mice at the final concentration of 50 μM and the mouse livers were imaged using multiphoton microscopy (MPM). In the I/R liver, reduced autofluorescence was detected by MPM, indicating the hepatocyte necrosis. Remarkable enhancement of red fluorescence was observed in hepatocytes with decreased autofluorescence, indicating the reaction of Ir-Fc with endogenous HOCl molecules. The cellular concentration of HOCl was first calculated based on the intensity of MPM images. No obvious toxic effects were observed in histological examination of major organs after Ir-Fc injection. In summary, Ir-Fc has low cytotoxicity, high specificity to HOCl, and rapid "off-on" fluorescence. It is suitable for dynamic quantitatively monitoring HOCl generation using MPM at the cellular level. This technique can be readily extended to examination of liver diseases and injury.

Dynamic detection of caspase-3 activation during photosensitization by fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Chen, Qun; Chen, Tongsheng; Tang, Yonghong; Wan, Qingling

2005-04-01

Apoptosis is one of the important modes in PDT-induced cell death. Activation of caspase-3 is considered to be the final step in many apoptosis pathways. In this study, we used SCAT3, a fluorescence resonance energy transfer (FRET) probe containing caspase-3 substrate, to study the dynamics of caspase-3 activation in living ASTC-a-1 cells expressing stably SCAT3. The FRET analysis results indicated that caspase-3 activation in response to tumor necrosis factor-α or PDT resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The SCAT3 was cleaved immediately after PDT treatment, but that for TNF-a treatment was delayed two hours. Our experimental results suggested that the different apoptotic pathways induced by TNF-α or PDT caused different cleavage kinetics of SCAT3. This study shows that FRET technique based on GFPs could be used to study the mechanism of PDT-induced apoptosis in living cells.

Detection of Citrus tristeza virus by using fluorescence resonance energy transfer-based biosensor

NASA Astrophysics Data System (ADS)

Shojaei, Taha Roodbar; Salleh, Mohamad Amran Mohd; Sijam, Kamaruzaman; Rahim, Raha Abdul; Mohsenifar, Afshin; Safarnejad, Reza; Tabatabaei, Meisam

2016-12-01

Due to the low titer or uneven distribution of Citrus tristeza virus (CTV) in field samples, detection of CTV by using conventional detection techniques may be difficult. Therefore, in the present work, the cadmium-telluride quantum dots (QDs) was conjugated with a specific antibody against coat protein (CP) of CTV, and the CP were immobilized on the surface of gold nanoparticles (AuNPs) to develop a specific and sensitive fluorescence resonance energy transfer (FRET)-based nanobiosensor for detecting CTV. The maximum FRET efficiency for the developed nano-biosensor was observed at 60% in AuNPs-CP/QDs-Ab ratio of 1:8.5. The designed system showed higher sensitivity and specificity over enzyme linked immunosorbent assay (ELISA) with a limit of detection of 0.13 μg mL- 1 and 93% and 94% sensitivity and specificity, respectively. As designed sensor is rapid, sensitive, specific and efficient in detecting CTV, this could be envisioned for diagnostic applications, surveillance and plant certification program.

Quantitative Investigation of Protein-Nucleic Acid Interactions by Biosensor Surface Plasmon Resonance.

PubMed

Wang, Shuo; Poon, Gregory M K; Wilson, W David

2015-01-01

Biosensor-surface plasmon resonance (SPR) technology has emerged as a powerful label-free approach for the study of nucleic acid interactions in real time. The method provides simultaneous equilibrium and kinetic characterization for biomolecular interactions with low sample requirements and without the need for external probes. A detailed and practical guide for protein-DNA interaction analyses using biosensor-SPR methods is presented. Details of SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips and samples, experimental design, quantitative and qualitative data analyses and presentation. A specific example of the interaction of a transcription factor with DNA is provided with results evaluated by both kinetic and steady-state SPR methods.

A highly sensitive and selective aptasensor based on graphene oxide fluorescence resonance energy transfer for the rapid determination of oncoprotein PDGF-BB.

PubMed

Liang, Junfei; Wei, Ran; He, Shuai; Liu, Yikan; Guo, Lin; Li, Lidong

2013-03-21

Oncoprotein platelet derived growth factor-BB (PDGF-BB) is one of the most critical growth factors that regulates tumor growth and division. In this work, a highly sensitive and selective fluorescence resonance energy transfer (FRET) aptasensor for PDGF-BB detection based on the assembly of dye-labeled aptamer and graphene oxide (GO) is developed for the first time. Due to the non-covalent assembly between aptamer and GO, fluorescence quenching of the dye takes place because of FRET. In the presence of PDGF-BB, the binding between aptamer and PDGF-BB will disturb the interaction between aptamer and GO, and release the dye-labeled aptamer from the GO surface, resulting in restoration of the fluorophore fluorescence. Because of the high fluorescence quenching efficiency, unique structure, and electronic properties of GO, the GO aptasensor exhibits extraordinarily high sensitivity. We also demonstrate that two highly related molecular variants of PDGF (AA, AB) can be distinguished from PDGF-BB, which indicates the aptasensor has excellent selectivity. Such an aptasensor opens a rapid, selective and sensitive route for the detection of PDGF-BB and provides a promising strategy for other cancer-related proteins detections.

[Experimental studies of using real-time fluorescence quantitative PCR and RT-PCR to detect E6 and E7 genes of human papillomavirus type 16 in cervical carcinoma cell lines].

PubMed

Chen, Yue-yue; Peng, Zhi-lan; Liu, Shan-ling; He, Bing; Hu, Min

2007-06-01

To establish a method of using real-time fluorescence quantitative PCR and RT-PCR to detect the E6 and E7 genes of human papillomavirus type 16 (HPV-16). Plasmids containing HPV-16 E6 or E7 were used to generate absolute standard curves. Three cervical carcinoma cell lines CaSki, SiHa and HeLa were tested by real-time fluorescence quantitative PCR and RT-PCR analyses for the expressions of HPV-16 E6 and E7. The correlation coefficients of standard curves were larger than 0. 99, and the PCR efficiency was more than 90%. The relative levels of HPV-16 E6 and E7 DNA and RNA were CaSki>SiHa>HeLa cell. HPV-16 E6 and E7 quantum by real-time fluorescence quantitative PCR and RT-PCR analyses may serve as a reliable and sensitive tool. This study provides the possibility of further researches on the relationship between HPV-16 E6 or E7 copy number and cervical carcinoma.

Lateral Distribution of NBD-PC Fluorescent Lipid Analogs in Membranes Probed by Molecular Dynamics-Assisted Analysis of Förster Resonance Energy Transfer (FRET) and Fluorescence Quenching

PubMed Central

Loura, Luís M. S.

2012-01-01

Förster resonance energy transfer (FRET) is a powerful tool used for many problems in membrane biophysics, including characterization of the lateral distribution of lipid components and other species of interest. However, quantitative analysis of FRET data with a topological model requires adequate choices for the values of several input parameters, some of which are difficult to obtain experimentally in an independent manner. For this purpose, atomistic molecular dynamics (MD) simulations can be potentially useful as they provide direct detailed information on transverse probe localization, relative probe orientation, and membrane surface area, all of which are required for analysis of FRET data. This is illustrated here for the FRET pairs involving 1,6-diphenylhexatriene (DPH) as donor and either 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)- sn-glycero-3-phosphocholine (C6-NBD-PC) or 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine (C12-NBD-PC) as acceptors, in fluid vesicles of 1,2-dipalmitoyl-sn-3-glycerophosphocholine (DPPC, 50 °C). Incorporation of results from MD simulations improves the statistical quality of model fitting to the experimental FRET data. Furthermore, the decay of DPH in the presence of moderate amounts of C12-NBD-PC (>0.4 mol%) is consistent with non-random lateral distribution of the latter, at variance with C6-NBD-PC, for which aggregation is ruled out up to 2.5 mol% concentration. These conclusions are supported by analysis of NBD-PC fluorescence self-quenching. Implications regarding the relative utility of these probes in membrane studies are discussed. PMID:23203080

Lateral distribution of NBD-PC fluorescent lipid analogs in membranes probed by molecular dynamics-assisted analysis of Förster Resonance Energy Transfer (FRET) and fluorescence quenching.

PubMed

Loura, Luís M S

2012-11-08

Förster resonance energy transfer (FRET) is a powerful tool used for many problems in membrane biophysics, including characterization of the lateral distribution of lipid components and other species of interest. However, quantitative analysis of FRET data with a topological model requires adequate choices for the values of several input parameters, some of which are difficult to obtain experimentally in an independent manner. For this purpose, atomistic molecular dynamics (MD) simulations can be potentially useful as they provide direct detailed information on transverse probe localization, relative probe orientation, and membrane surface area, all of which are required for analysis of FRET data. This is illustrated here for the FRET pairs involving 1,6-diphenylhexatriene (DPH) as donor and either 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)- sn-glycero-3-phosphocholine (C6-NBD-PC) or 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine (C12-NBD-PC) as acceptors, in fluid vesicles of 1,2-dipalmitoyl-sn-3-glycerophosphocholine (DPPC, 50 °C). Incorporation of results from MD simulations improves the statistical quality of model fitting to the experimental FRET data. Furthermore, the decay of DPH in the presence of moderate amounts of C12-NBD-PC (>0.4 mol%) is consistent with non-random lateral distribution of the latter, at variance with C6-NBD-PC, for which aggregation is ruled out up to 2.5 mol% concentration. These conclusions are supported by analysis of NBD-PC fluorescence self-quenching. Implications regarding the relative utility of these probes in membrane studies are discussed.

Understanding the interaction of concanavalin a with mannosyl glycoliposomes: A surface plasmon resonance and fluorescence study.

PubMed

Sandoval-Altamirano, Catalina; Sanchez, Susana A; Ferreyra, Nancy F; Gunther, German

2017-10-01

The specificity of carbohydrate-protein interaction is a key factor in many biological processes and it is the foundation of technologies using glycoliposomes in drug delivery. The incorporation of glycolipids in vesicles is expected to increase their specificity toward particular targets such as lectins; however, the degree of exposure of the carbohydrate moiety at the liposome surface is a crucial parameter to be considered in the interaction. Herein we report the synthesis of mannose derivatives with one or two hydrophobic chains of different length, designed with the purpose of modifying the degree of exposure of the mannose when they were incorporated into liposomes. The interaction of glycovesicles with Con A was studied using: (i) agglutination assays; measured by dynamic laser light scattering (DLS); (ii) time resolved fluorescence methods and (iii) surface plasmon resonance (SPR) kinetic measurements. DLS data showed that an increase in hydrophobic chain length promotes a decrease of liposomes hydrodynamic radius. A longer hydrocarbon chain favors a deeper insertion into the bilayer and mannose moiety results less exposed at the surface to interact with lectin. Fluorescence experiments showed changes in the structure of glycovesicles due to the interaction with the protein. From SPR measurements the kinetic and equilibrium constants associated to the interaction of ConA with the different glycolipid synthetized were determined. The combination of SPR and fluorescence techniques allowed to study the interaction of Con A with mannosyl glycovesicles at three levels: at the surface, at the interface and deeper into the bilayer. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel multistep BRET-FRET energy transfer using nanoconjugates of firefly proteins, quantum dots, and red fluorescent proteins

NASA Astrophysics Data System (ADS)

Alam, Rabeka; Zylstra, Joshua; Fontaine, Danielle M.; Branchini, Bruce R.; Maye, Mathew M.

2013-05-01

Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated.Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated. Electronic supplementary information (ESI) available: Experimental details, Fig. S1 and Table S1-S4. See DOI: 10.1039/c3nr01842c

Noninvasive evaluation of heavy metal uptake and storage in micoralgae using a fluorescence resonance energy transfer-based heavy metal biosensor.

PubMed

Rajamani, Sathish; Torres, Moacir; Falcao, Vanessa; Ewalt Gray, Jaime; Coury, Daniel A; Colepicolo, Pio; Sayre, Richard

2014-02-01

We have developed a fluorescence resonance energy transfer (FRET)-based heavy metal biosensor for the quantification of bioavailable free heavy metals in the cytoplasm of the microalga Chlamydomonas reinhardtii. The biosensor is composed of an end-to-end fusion of cyan fluorescent protein (CFP), chicken metallothionein II (MT-II), and yellow fluorescent protein (YFP). In vitro measurements of YFP/CFP fluorescence emission ratios indicated that the addition of metals to the purified biosensor enhanced FRET between CFP and YFP, consistent with heavy metal-induced folding of MT-II. A maximum YFP/CFP FRET ratio of 2.8 was observed in the presence of saturating concentrations of heavy metals. The sensitivity of the biosensor was greatest for Hg2+ followed by Cd2+≈Pb2+>Zn2+>Cu2+. The heavy metal biosensor was unresponsive to metals that do not bind to MT-II (Na+ and Mg2+). When expressed in C. reinhardtii, we observed a differential metal-dependent response to saturating external concentrations (1.6 mm) of heavy metals (Pb2+>Cd2+) that was unlike that observed for the isolated biosensor (in vitro). Significantly, analysis of metal uptake kinetics indicated that equilibration of the cytoplasm with externally applied heavy metals occurred within seconds. Our results also indicated that algae have substantial buffering capacity for free heavy metals in their cytosol, even at high external metal concentrations.

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

PubMed Central

Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

2000-01-01

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

Detection and Analysis of Enamel Cracks by Quantitative Light-induced Fluorescence Technology.

PubMed

Jun, Mi-Kyoung; Ku, Hye-Min; Kim, Euiseong; Kim, Hee-Eun; Kwon, Ho-Keun; Kim, Baek-Il

2016-03-01

The ability to accurately detect tooth cracks and quantify their depth would allow the prediction of crack progression and treatment success. The aim of this in vitro study was to determine the capabilities of quantitative light-induced fluorescence (QLF) technology in the detection of enamel cracks. Ninety-six extracted human teeth were selected for examining naturally existing or suspected cracked teeth surfaces using a photocuring unit. QLF performed with a digital camera (QLF-D) images were used to assess the ability to detect enamel cracks based on the maximum fluorescence loss value (ΔFmax, %), which was then analyzed using the QLF-D software. A histologic evaluation was then performed in which the samples were sectioned and observed with the aid of a polarized light microscope. The relationship between ΔFmax and the histology findings was assessed based on the Spearman rank correlation. The sensitivity and specificity were calculated to evaluate the validity of using QLF-D to analyze enamel inner-half cracks and cracks extending to the dentin-enamel junction. There was a strong correlation between the results of histologic evaluations of enamel cracks and the ΔFmax value, with a correlation coefficient of 0.84. The diagnostic accuracy of QLF-D had a sensitivity of 0.87 and a specificity of 0.98 for enamel inner-half cracks and a sensitivity of 0.90 and a specificity of 1.0 for cracks extending to the dentin-enamel junction. These results indicate that QLF technology would be a useful clinical tool for diagnosing enamel cracks, especially given that this is a nondestructive method. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Comparative in vivo mucoadhesion studies of thiomer formulations using magnetic resonance imaging and fluorescence detection.

PubMed

Albrecht, K; Greindl, M; Kremser, C; Wolf, C; Debbage, P; Bernkop-Schnürch, A

2006-09-28

The aim of this study was to compare different oral delivery systems based on the thiolated polymer polycarbophil-cysteine (PCP-Cys) and to provide evidence for the validity of the hypothesis that unhydrated polymers provide better mucoadhesion in vivo. To achieve dry polymer application, a new, experimental dosage form named Eutex (made of Eudragit L100-55 and latex) capsule has been developed. Magnetic resonance imaging was used to localize the point of release of the thiolated polymer from the application forms via the positive magnetic resonance signal from a gadolinium complex (Gd-DTPA). In vivo mucoadhesion was determined by ascertaining the residence time of the fluorescence-tagged thiomer on intestinal mucosa after 3 h. Results showed that in comparison to conventional application forms the Eutex capsules led to 1.9-fold higher mucoadhesive properties of PCP-Cys when compared to application with a conventional enteric-coated capsule, and to 1.4-fold higher mucoadhesion when compared to administration with an enteric-coated tablet of the thiomer. The findings of this study should contribute to the understanding of mucoadhesion and mucoadhesion influencing parameters in vivo and should therefore be of considerable interest for the development of future mucoadhesive oral drug delivery dosage forms.

Two-Color Resonant Four-Wave Mixing Spectroscopy: New Perspectives for Direct Studies of Collisional State-to-State Transfer

NASA Astrophysics Data System (ADS)

Chen, X.; Settersten, T. B.; Radi, P. P.; Kouzov, A. P.

2008-10-01

The two-color resonant four-wave mixing (TC-RFWM) is advertised as a unique spectroscopic device enabling one to directly measure the collisional state-to-state transfer characteristics (rates and correlation times). In contrast to the laser-induced fluorescence, these characteristics are phase-sensitive and open wider opportunities to study the rotational relaxation processes. Further perspectives are offered by the recently recorded collision-induced picosecond TC-RFWM signals of OH. Their quantitative interpretation is now under development.

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection.

PubMed

Brunnenberg, M; Lindenblatt, H; Gouzoulis-Mayfrank, E; Kovar, K A

1998-11-20

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCI. The maximum plasma concentrations were 235-465 ng/ml (MDE), 67-673 ng/ml (HME) and 7-33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.

Microscope-integrated quantitative analysis of intraoperative indocyanine green fluorescence angiography for blood flow assessment: first experience in 30 patients.

PubMed

Kamp, Marcel A; Slotty, Philipp; Turowski, Bernd; Etminan, Nima; Steiger, Hans-Jakob; Hänggi, Daniel; Stummer, Walter

2012-03-01

Intraoperative measurements of cerebral blood flow are of interest during vascular neurosurgery. Near-infrared indocyanine green (ICG) fluorescence angiography was introduced for visualizing vessel patency intraoperatively. However, quantitative information has not been available. To report our experience with a microscope with an integrated dynamic ICG fluorescence analysis system supplying semiquantitative information on blood flow. We recorded ICG fluorescence curves of cortex and cerebral vessels using software integrated into the surgical microscope (Flow 800 software; Zeiss Pentero) in 30 patients undergoing surgery for different pathologies. The following hemodynamic parameters were assessed: maximum intensity, rise time, time to peak, time to half-maximal fluorescence, cerebral blood flow index, and transit times from arteries to cortex. For patients without obvious perfusion deficit, maximum fluorescence intensity was 177.7 arbitrary intensity units (AIs; 5-mg ICG bolus), mean rise time was 5.2 seconds (range, 2.9-8.2 seconds; SD, 1.3 seconds), mean time to peak was 9.4 seconds (range, 4.9-15.2 seconds; SD, 2.5 seconds), mean cerebral blood flow index was 38.6 AI/s (range, 13.5-180.6 AI/s; SD, 36.9 seconds), and mean transit time was 1.5 seconds (range, 360 milliseconds-3 seconds; SD, 0.73 seconds). For 3 patients with impaired cerebral perfusion, time to peak, rise time, and transit time between arteries and cortex were markedly prolonged (>20, >9 , and >5 seconds). In single patients, the degree of perfusion impairment could be quantified by the cerebral blood flow index ratios between normal and ischemic tissue. Transit times also reflected blood flow perturbations in arteriovenous fistulas. Quantification of ICG-based fluorescence angiography appears to be useful for intraoperative monitoring of arterial patency and regional cerebral blood flow.

Preclinical Magnetic Resonance Fingerprinting (MRF) at 7 T: Effective Quantitative Imaging for Rodent Disease Models

PubMed Central

Gao, Ying; Chen, Yong; Ma, Dan; Jiang, Yun; Herrmann, Kelsey A.; Vincent, Jason A.; Dell, Katherine M.; Drumm, Mitchell L.; Brady-Kalnay, Susann M.; Griswold, Mark A.; Flask, Chris A.; Lu, Lan

2015-01-01

High field, preclinical magnetic resonance imaging (MRI) scanners are now commonly used to quantitatively assess disease status and efficacy of novel therapies in a wide variety of rodent models. Unfortunately, conventional MRI methods are highly susceptible to respiratory and cardiac motion artifacts resulting in potentially inaccurate and misleading data. We have developed an initial preclinical, 7.0 T MRI implementation of the highly novel Magnetic Resonance Fingerprinting (MRF) methodology that has been previously described for clinical imaging applications. The MRF technology combines a priori variation in the MRI acquisition parameters with dictionary-based matching of acquired signal evolution profiles to simultaneously generate quantitative maps of T1 and T2 relaxation times and proton density. This preclinical MRF acquisition was constructed from a Fast Imaging with Steady-state Free Precession (FISP) MRI pulse sequence to acquire 600 MRF images with both evolving T1 and T2 weighting in approximately 30 minutes. This initial high field preclinical MRF investigation demonstrated reproducible and differentiated estimates of in vitro phantoms with different relaxation times. In vivo preclinical MRF results in mouse kidneys and brain tumor models demonstrated an inherent resistance to respiratory motion artifacts as well as sensitivity to known pathology. These results suggest that MRF methodology may offer the opportunity for quantification of numerous MRI parameters for a wide variety of preclinical imaging applications. PMID:25639694

Quantitative detection of bovine and porcine gelatin difference using surface plasmon resonance based biosensor

NASA Astrophysics Data System (ADS)

Wardani, Devy P.; Arifin, Muhammad; Suharyadi, Edi; Abraha, Kamsul

2015-05-01

Gelatin is a biopolymer derived from collagen that is widely used in food and pharmaceutical products. Due to some religion restrictions and health issues regarding the gelatin consumption which is extracted from certain species, it is necessary to establish a robust, reliable, sensitive and simple quantitative method to detect gelatin from different parent collagen species. To the best of our knowledge, there has not been a gelatin differentiation method based on optical sensor that could detect gelatin from different species quantitatively. Surface plasmon resonance (SPR) based biosensor is known to be a sensitive, simple and label free optical method for detecting biomaterials that is able to do quantitative detection. Therefore, we have utilized SPR-based biosensor to detect the differentiation between bovine and porcine gelatin in various concentration, from 0% to 10% (w/w). Here, we report the ability of SPR-based biosensor to detect difference between both gelatins, its sensitivity toward the gelatin concentration change, its reliability and limit of detection (LOD) and limit of quantification (LOQ) of the sensor. The sensor's LOD and LOQ towards bovine gelatin concentration are 0.38% and 1.26% (w/w), while towards porcine gelatin concentration are 0.66% and 2.20% (w/w), respectively. The results show that SPR-based biosensor is a promising tool for detecting gelatin from different raw materials quantitatively.

Detailed evaluation of the performance of microfluidic T mixers using fluorescence and ultraviolet resonance Raman spectroscopy

NASA Astrophysics Data System (ADS)

Masca, Sergiu I.; Rodriguez-Mendieta, Iñigo R.; Friel, Claire T.; Radford, Sheena E.; Smith, D. Alastair

2006-05-01

A reliable device that produces efficient mixing with a short dead time has enormous utility in the kinetic analysis of biochemical and chemical processes. We have designed two different T mixers that use moderate flow rates (0.2-0.4ml/s), can monitor reactions up to several milliseconds, and achieve mixing times as low as 20μs. The two mixers are easy to build and dismantle, reliable, and can perform hundreds of experiments without blocking. The first mixer comprises a stainless steel block, containing a microchannel, glued to a quartz cuvette, containing a 200×200μm2 observation channel defining a conventional T mixer. The reactions are monitored by imaging the length of the observation channel onto a charge-coupled device camera. In the second mixer the entire T (200×200μm2 internal cross section) is contained within a 40-mm-long quartz cuvette. We have adopted a novel approach to controlling the entrance channel bore by inserting a stainless steel wire in order to increase the linear speed of the impinging fluids. Using a dye to visualize the flow profile inside the second T mixer, it was shown that in this T geometry segregation of the reactants is observed in the junction between the inlet channels and the observation channel (T junction) and mixing occurs entirely in the observation channel. We thoroughly tested the two mixers through several kinetic reactions using both fluorescence and ultraviolet resonance Raman spectroscopy measurements. We show that both mixers provide efficient mixing with nominal dead times (using 1:10 v /v dilution), calculated using the quenching of the fluorescence of N-acetyl-L-tryptophanamide by N-bromosuccinimide, of 200±20 and 100±10μs, for each mixer, respectively. However, the ability to monitor within the inlet channels and the entire observation channel of the second mixer shows that this standard approach to estimating the dead time is artifactual, since it relies on assuming a constant flow speed throughout the

Plates-formes de microscopie et fluorescence par resonance de plasmons de surface appliquees a l'imagerie cellulaire

NASA Astrophysics Data System (ADS)

Chabot, Vincent

L'elaboration de nouveaux medicaments repose sur les etudes pharmacologiques, dont le role est d'identifier de nouveaux composes actifs ou de nouvelles cibles pharmacologiques agissant entre autres au niveau cellulaire. Recemment, la detection basee sur la resonance des plasmons de surface (SPR) a ete appliquee a l'etude de reponses cellulaires. Cette methode de detection, permettant d'observer des variations d'indice de refraction associes a de faibles changements de masse a la surface d'un metal, a l'avantage de permettre l'etude d'une population de cellules vivantes en temps reel, sans necessiter l'introduction d'agents de marquage. Pour effectuer la detection au niveau de cellules individuelles, on peut employer la microscopie SPR, qui consiste a localiser spatialement la detection par un systeme d'imagerie. Cependant, la detection basee sur la SPR est une mesure sans marquage et les signaux mesures sont attribues a une reponse moyennee des differentes sources cellulaires. Afin de mieux comprendre et identifier les composantes cellulaires generant le signal mesure en SPR, il est pertinent de combiner la microscopie SPR avec une modalite complementaire, soit l'imagerie de fluorescence. C'est dans cette problematique que s'insere ce projet de these, consistant a concevoir deux plates-formes distinctes de microscopie SPR et de fluorescence optimisees pour l'etude cellulaire, de sorte a evaluer les possibilites d'integration de ces deux modalites en un seul systeme. Des substrats adaptes pour chaque plate-forme ont ete concus et realises. Ces substrats employaient une couche d'argent passivee par l'ajout d'une mince couche d'or. La stabilite et la biocompatibilite des substrats ont ete validees pour l'etude cellulaire. Deux configurations permettant d'ameliorer la sensibilite en sondant les cellules plus profondement ont ete evaluees, soit l'emploi de plasmons de surface a longue portee et de guides d'onde a gaine metallique. La sensibilite accrue de ces

PVP-coated gold nanoparticles for the selective determination of ochratoxin A via quenching fluorescence of the free aptamer.

PubMed

Lv, Lei; Jin, Yongdong; Kang, Xiaojiao; Zhao, Yangyang; Cui, Chengbi; Guo, Zhijun

2018-05-30

This paper describes an aptamer/gold nanoparticle-based assay for ochratoxin A (OTA) detection. This assay is based on the use of an aptamer labeled with carboxyfluorescein (FAM) at its 5'-end and gold nanoparticles (AuNPs) that act as quenchers of fluorescence. When OTA is absent in the system, the fluorescently labeled aptamers are adsorbed on the surface of AuNPs. The fluorescence signal of the fluorescein-labeled OTA aptamer generated is quenched by the fluorescence resonance energy transfer effect of AuNPs. When OTA is present in the system, the fluorescently labeled aptamer binds to OTA and forms a folded structure, which can resist the adsorption of AuNPs. Thus, the fluorescent signal can be retained. The detection limit of this sensing platform is 5 nM, and the linear detection range is 10-1000 nM (R 2  = 0.994). The procedure was validated by the quantitation of OTA in spiked ginger powder samples and were found to be free of interference by the sample matrix. The recoveries and the relative standard deviation varied from 89.0% to 117.8% and from 1.9% to 6.3%, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Binding investigation on the interaction between Methylene Blue (MB)/TiO2 nanocomposites and bovine serum albumin by resonance light-scattering (RLS) technique and fluorescence spectroscopy.

PubMed

Li, Yuesheng; Zhang, Yue; Sun, Shaofa; Zhang, Aiqing; Liu, Yi

2013-11-05

The interaction between Methylene Blue (MB)/TiO2 nanocomposites and bovine serum albumin (BSA) was investigated by resonance light scattering (RLS), fluorescence, three-dimension spectra and UV-vis absorbance spectroscopy. Several factors which may influence the RLS intensity were also investigated before characterizing MB/TiO2-BSA complex. It was proved that the mechanism of MB/TiO2 nanocomposites binding to BSA was mainly a result of the formation of MB/TiO2-BSA complex. The binding constant of MB/TiO2-BSA is 0.762 × 10(-5) L mol(-1) at 298K. By calculating the binding constant at different temperature, the thermodynamic parameters ΔH, ΔG, and ΔS can be observed and deduced that the hydrophobic interactions played an important role to stabilize the complex. The distance r (3.73 nm) between donor (BSA) and acceptor (MB/TiO2) was obtained according to fluorescence resonance energy transfer (FRET). The binding site for MB/TiO2 on BSA was mainly located in sub-domain IIA. The UV-vis absorbance, circular dichroism and three dimension fluorescence have also been used to investigate the effect of MB/TiO2 on the conformation of BSA. Copyright © 2013 Elsevier B.V. All rights reserved.

Absorption Reconstruction Improves Biodistribution Assessment of Fluorescent Nanoprobes Using Hybrid Fluorescence-mediated Tomography

PubMed Central

Gremse, Felix; Theek, Benjamin; Kunjachan, Sijumon; Lederle, Wiltrud; Pardo, Alessa; Barth, Stefan; Lammers, Twan; Naumann, Uwe; Kiessling, Fabian

2014-01-01

Aim: Fluorescence-mediated tomography (FMT) holds potential for accelerating diagnostic and theranostic drug development. However, for proper quantitative fluorescence reconstruction, knowledge on optical scattering and absorption, which are highly heterogeneous in different (mouse) tissues, is required. We here describe methods to assess these parameters using co-registered micro Computed Tomography (µCT) data and nonlinear whole-animal absorption reconstruction, and evaluate their importance for assessment of the biodistribution and target site accumulation of fluorophore-labeled drug delivery systems. Methods: Besides phantoms with varying degrees of absorption, mice bearing A431 tumors were imaged 15 min and 48 h after i.v. injection of a fluorophore-labeled polymeric drug carrier (pHPMA-Dy750) using µCT-FMT. The outer shape of mice and a scattering map were derived using automated segmentation of the µCT data. Furthermore, a 3D absorption map was reconstructed from the trans-illumination data. We determined the absorption of five interactively segmented regions (heart, liver, kidney, muscle, tumor). Since blood is the main near-infrared absorber in vivo, the absorption was also estimated from the relative blood volume (rBV), determined by contrast-enhanced µCT. We compared the reconstructed absorption with the rBV-based values and analyzed the effect of using the absorption map on the fluorescence reconstruction. Results: Phantom experiments demonstrated that absorption reconstruction is possible and necessary for quantitative fluorescence reconstruction. In vivo, the reconstructed absorption showed high values in strongly blood-perfused organs such as the heart, liver and kidney. The absorption values correlated strongly with the rBV-based absorption values, confirming the accuracy of the absorption reconstruction. Usage of homogenous absorption instead of the reconstructed absorption map resulted in reduced values in the heart, liver and kidney, by

CH3 NH3 PbBr3 Perovskite Nanocrystals as Efficient Light-Harvesting Antenna for Fluorescence Resonance Energy Transfer.

PubMed

Muthu, Chinnadurai; Vijayan, Anuja; Nair, Vijayakumar C

2017-05-04

Hybrid perovskites have created enormous research interest as a low-cost material for high-performance photovoltaic devices, light-emitting diodes, photodetectors, memory devices and sensors. Perovskite materials in nanocrystal form that display intense luminescence due to the quantum confinement effect were found to be particularly suitable for most of these applications. However, the potential use of perovskite nanocrystals as a light-harvesting antenna for possible applications in artificial photosynthesis systems is not yet explored. In the present work, we study the light-harvesting antenna properties of luminescent methylammonium lead bromide (CH 3 NH 3 PbBr 3 )-based perovskite nanocrystals using fluorescent dyes (rhodamine B, rhodamine 101, and nile red) as energy acceptors. Our studies revealed that CH 3 NH 3 PbBr 3 nanocrystals are an excellent light-harvesting antenna, and efficient fluorescence resonance energy transfer occurs from the nanocrystals to fluorescent dyes. Further, the energy transfer efficiency is found to be highly dependent on the number of anchoring groups and binding ability of the dyes to the surface of the nanocrystals. These observations may have significant implications for perovskite-based light-harvesting devices and their possible use in artificial photosynthesis systems. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

PubMed

Nygate, Yoav N; Singh, Gyanendra; Barnea, Itay; Shaked, Natan T

2018-06-01

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase

ERIC Educational Resources Information Center

Wang, Sheila C.; Zamble, Deborah B.

2006-01-01

A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer…

Enhancement and Passive Acoustic Mapping of Cavitation from Fluorescently Tagged Magnetic Resonance-Visible Magnetic Microbubbles In Vivo.

PubMed

Crake, Calum; Owen, Joshua; Smart, Sean; Coviello, Christian; Coussios, Constantin-C; Carlisle, Robert; Stride, Eleanor

2016-12-01

Previous work has indicated the potential of magnetically functionalized microbubbles to localize and enhance cavitation activity under focused ultrasound exposure in vitro. The aim of this study was to investigate magnetic targeting of microbubbles for promotion of cavitation in vivo. Fluorescently labelled magnetic microbubbles were administered intravenously in a murine xenograft model. Cavitation was induced using a 0.5-MHz focused ultrasound transducer at peak negative focal pressures of 1.2-2.0 MPa and monitored in real-time using B-mode imaging and passive acoustic mapping. Magnetic targeting was found to increase the amplitude of the cavitation signal by approximately 50% compared with untargeted bubbles. Post-exposure magnetic resonance imaging indicated deposition of magnetic nanoparticles in tumours. Magnetic targeting was similarly associated with increased fluorescence intensity in the tumours after the experiments. These results suggest that magnetic targeting could potentially be used to improve delivery of cavitation-mediated therapy and that passive acoustic mapping could be used for real-time monitoring of this process. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Development of L-lactate dehydrogenase biosensor based on porous silicon resonant microcavities as fluorescence enhancers.

PubMed

Jenie, S N Aisyiyah; Prieto-Simon, Beatriz; Voelcker, Nicolas H

2015-12-15

The up-regulation of L-lactate dehydrogenase (LDH), an intracellular enzyme present in most of all body tissues, is indicative of several pathological conditions and cellular death. Herein, we demonstrate LDH detection using porous silicon (pSi) microcavities as a luminescence-enhancing optical biosensing platform. Non-fluorescent resazurin was covalently attached onto the pSi surface via thermal hydrocarbonisation, thermal hydrosylilation and acylation. Each surface modification step was confirmed by means of FTIR and the optical shifts of the resonance wavelength of the microcavity. Thermal hydrocarbonisation also afforded excellent surface stability, ensuring that the resazurin was not reduced on the pSi surface. Using a pSi microcavity biosensor, the fluorescence signal upon detection of LDH was amplified by 10 and 5-fold compared to that of a single layer and a detuned microcavity, respectively, giving a limit of detection of 0.08 U/ml. The biosensor showed a linear response between 0.16 and 6.5 U/ml, covering the concentration range of LDH in normal as well as damaged tissues. The biosensor was selective for LDH and did not produce a signal upon incubation with another NAD-dependant enzyme L-glutamic dehydrogenase. The use of the pSi microcavity as a sensing platform reduced reagent usage by 30% and analysis time threefold compared to the standard LDH assay in solution. Copyright © 2015 Elsevier B.V. All rights reserved.

Fluorescence calibration method for single-particle aerosol fluorescence instruments

NASA Astrophysics Data System (ADS)

Shipley Robinson, Ellis; Gao, Ru-Shan; Schwarz, Joshua P.; Fahey, David W.; Perring, Anne E.

2017-05-01

Real-time, single-particle fluorescence instruments used to detect atmospheric bioaerosol particles are increasingly common, yet no standard fluorescence calibration method exists for this technique. This gap limits the utility of these instruments as quantitative tools and complicates comparisons between different measurement campaigns. To address this need, we have developed a method to produce size-selected particles with a known mass of fluorophore, which we use to calibrate the fluorescence detection of a Wideband Integrated Bioaerosol Sensor (WIBS-4A). We use mixed tryptophan-ammonium sulfate particles to calibrate one detector (FL1; excitation = 280 nm, emission = 310-400 nm) and pure quinine particles to calibrate the other (FL2; excitation = 280 nm, emission = 420-650 nm). The relationship between fluorescence and mass for the mixed tryptophan-ammonium sulfate particles is linear, while that for the pure quinine particles is nonlinear, likely indicating that not all of the quinine mass contributes to the observed fluorescence. Nonetheless, both materials produce a repeatable response between observed fluorescence and particle mass. This procedure allows users to set the detector gains to achieve a known absolute response, calculate the limits of detection for a given instrument, improve the repeatability of the instrumental setup, and facilitate intercomparisons between different instruments. We recommend calibration of single-particle fluorescence instruments using these methods.

δ-aminolevulinic acid–induced protoporphyrin IX concentration correlates with histopathologic markers of malignancy in human gliomas: the need for quantitative fluorescence-guided resection to identify regions of increasing malignancy

PubMed Central

Valdés, Pablo A.; Kim, Anthony; Brantsch, Marco; Niu, Carolyn; Moses, Ziev B.; Tosteson, Tor D.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.; Harris, Brent T.

2011-01-01

Extent of resection is a major goal and prognostic factor in the treatment of gliomas. In this study we evaluate whether quantitative ex vivo tissue measurements of δ-aminolevulinic acid–induced protoporphyrin IX (PpIX) identify regions of increasing malignancy in low- and high-grade gliomas beyond the capabilities of current fluorescence imaging in patients undergoing fluorescence-guided resection (FGR). Surgical specimens were collected from 133 biopsies in 23 patients and processed for ex vivo neuropathological analysis: PpIX fluorimetry to measure PpIX concentrations (CPpIX) and Ki-67 immunohistochemistry to assess tissue proliferation. Samples displaying visible levels of fluorescence showed significantly higher levels of CPpIX and tissue proliferation. CPpIX was strongly correlated with histopathological score (nonparametric) and tissue proliferation (parametric), such that increasing levels of CPpIX were identified with regions of increasing malignancy. Furthermore, a large percentage of tumor-positive biopsy sites (∼40%) that were not visibly fluorescent under the operating microscope had levels of CPpIX greater than 0.1 µg/mL, which indicates that significant PpIX accumulation exists below the detection threshold of current fluorescence imaging. Although PpIX fluorescence is recognized as a visual biomarker for neurosurgical resection guidance, these data show that it is quantitatively related at the microscopic level to increasing malignancy in both low- and high-grade gliomas. This work suggests a need for improved PpIX fluorescence detection technologies to achieve better sensitivity and quantification of PpIX in tissue during surgery. PMID:21798847

Frequency-selective quantitation of short-echo time 1H magnetic resonance spectra

NASA Astrophysics Data System (ADS)

Poullet, Jean-Baptiste; Sima, Diana M.; Van Huffel, Sabine; Van Hecke, Paul

2007-06-01

Accurate and efficient filtering techniques are required to suppress large nuisance components present in short-echo time magnetic resonance (MR) spectra. This paper discusses two powerful filtering techniques used in long-echo time MR spectral quantitation, the maximum-phase FIR filter (MP-FIR) and the Hankel-Lanczos Singular Value Decomposition with Partial ReOrthogonalization (HLSVD-PRO), and shows that they can be applied to their more complex short-echo time spectral counterparts. Both filters are validated and compared through extensive simulations. Their properties are discussed. In particular, the capability of MP-FIR for dealing with macromolecular components is emphasized. Although this property does not make a large difference for long-echo time MR spectra, it can be important when quantifying short-echo time spectra.

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.

Quantitative Multispectral Analysis Of Discrete Subcellular Particles By Digital Imaging Fluorescence Microscopy (DIFM)

NASA Astrophysics Data System (ADS)

Dorey, C. K.; Ebenstein, David B.

1988-10-01

Subcellular localization of multiple biochemical markers is readily achieved through their characteristic autofluorescence or through use of appropriately labelled antibodies. Recent development of specific probes has permitted elegant studies in calcium and pH in living cells. However, each of these methods measured fluorescence at one wavelength; precise quantitation of multiple fluorophores at individual sites within a cell has not been possible. Using DIFM, we have achieved spectral analysis of discrete subcellular particles 1-2 gm in diameter. The fluorescence emission is broken into narrow bands by an interference monochromator and visualized through the combined use of a silicon intensified target (SIT) camera, a microcomputer based framegrabber with 8 bit resolution, and a color video monitor. Image acquisition, processing, analysis and display are under software control. The digitized image can be corrected for the spectral distortions induced by the wavelength dependent sensitivity of the camera, and the displayed image can be enhanced or presented in pseudocolor to facilitate discrimination of variation in pixel intensity of individual particles. For rapid comparison of the fluorophore composition of granules, a ratio image is produced by dividing the image captured at one wavelength by that captured at another. In the resultant ratio image, a granule which has a fluorophore composition different from the majority is selectively colored. This powerful system has been utilized to obtain spectra of endogenous autofluorescent compounds in discrete cellular organelles of human retinal pigment epithelium, and to measure immunohistochemically labelled components of the extracellular matrix associated with the human optic nerve.

Quantitative detection of astaxanthin and cantaxanthin in Atlantic salmon by resonance Raman spectroscopy

NASA Astrophysics Data System (ADS)

Ermakov, Igor V.; Ermakova, Maia R.; Gellermann, Werner

2006-02-01

Two major carotenoids species found in salmonids muscle tissues are astaxanthin and cantaxanthin. They are taken up from fish food and are responsible for the attractive red-orange color of salmon filet. Since carotenoids are powerful antioxidants and biomarkers of nutrient consumption, they are thought to indicate fish health and resistance to diseases in fish farm environments. Therefore, a rapid, accurate, quantitative optical technique for measuring carotenoid content in salmon tissues is of economic interest. We demonstrate the possibility of using fast, selective, quantitative detection of astaxanthin and cantaxanthin in salmon muscle tissues, employing resonance Raman spectroscopy. Analyzing strong Raman signals originating from the carbon-carbon double bond stretch vibrations of the carotenoid molecules under blue laser excitation, we are able to characterize quantitatively the concentrations of carotenoids in salmon muscle tissue. To validate the technique, we compared Raman data with absorption measurements of carotenoid extracts in acetone. A close correspondence was observed in absorption spectra for tissue extract in acetone and a pure astaxanthin solution. Raman results show a linear dependence between Raman and absorption data. The proposed technique holds promise as a method of rapid screening of carotenoid levels in fish muscle tissues and may be attractive for the fish farm industry to assess the dietary status of salmon, risk for infective diseases, and product quality control.

Characterization of the organic ligand shell of semiconductor quantum dots by fluorescence quenching experiments.

PubMed

Boldt, Klaus; Jander, Sebastian; Hoppe, Kathrin; Weller, Horst

2011-10-25

We present the characterization of the organic ligand shell of CdSe/Cd(x)Zn(1-x)S/ZnS nanoparticles by means of fluorescence quenching experiments. Both electron scavengers and acceptors for resonance energy transfer were employed as probes. Different quenching behavior for short and long chain thiol ligands in water was found. It could be shown that poly(ethylene oxide) (PEO)-capping of the particles comprises a densely packed inner shell and a loosely packed outer shell in which ions and small molecules diffuse unhindered. A quantitative uptake of quencher molecules into the PEO shell was observed, through which the particle volume including the ligand sphere could be determined.

Rapid and quantitative chemical exchange saturation transfer (CEST) imaging with magnetic resonance fingerprinting (MRF).

PubMed

Cohen, Ouri; Huang, Shuning; McMahon, Michael T; Rosen, Matthew S; Farrar, Christian T

2018-05-13

To develop a fast magnetic resonance fingerprinting (MRF) method for quantitative chemical exchange saturation transfer (CEST) imaging. We implemented a CEST-MRF method to quantify the chemical exchange rate and volume fraction of the N α -amine protons of L-arginine (L-Arg) phantoms and the amide and semi-solid exchangeable protons of in vivo rat brain tissue. L-Arg phantoms were made with different concentrations (25-100 mM) and pH (pH 4-6). The MRF acquisition schedule varied the saturation power randomly for 30 iterations (phantom: 0-6 μT; in vivo: 0-4 μT) with a total acquisition time of ≤2 min. The signal trajectories were pattern-matched to a large dictionary of signal trajectories simulated using the Bloch-McConnell equations for different combinations of exchange rate, exchangeable proton volume fraction, and water T 1 and T 2 relaxation times. The chemical exchange rates of the N α -amine protons of L-Arg were significantly (P < 0.0001) correlated with the rates measured with the quantitation of exchange using saturation power method. Similarly, the L-Arg concentrations determined using MRF were significantly (P < 0.0001) correlated with the known concentrations. The pH dependence of the exchange rate was well fit (R 2  = 0.9186) by a base catalyzed exchange model. The amide proton exchange rate measured in rat brain cortex (34.8 ± 11.7 Hz) was in good agreement with that measured previously with the water exchange spectroscopy method (28.6 ± 7.4 Hz). The semi-solid proton volume fraction was elevated in white (12.2 ± 1.7%) compared to gray (8.1 ± 1.1%) matter brain regions in agreement with previous magnetization transfer studies. CEST-MRF provides a method for fast, quantitative CEST imaging. © 2018 International Society for Magnetic Resonance in Medicine.

A dual-colored ratiometric-fluorescent oligonucleotide probe for the detection of human telomerase RNA in cell extracts.

PubMed

Ning, Dianhua; He, Changtian; Liu, Zhengjie; Liu, Cui; Wu, Qilong; Zhao, TingTing; Liu, Renyong

2017-05-21

Human telomerase RNA (hTR), which is one component of telomerase, was deemed to be a biomarker to monitor tumor cells due to its different expression levels in tumor cells and normal somatic cells. Thus far, plentiful fluorescent probes have been designed to investigate nucleic acids. However, most of them are limited since they are time-consuming, require professional operators and even result in false positive signals in the cellular environment. Herein, we report a dual-colored ratiometric-fluorescent oligonucleotide probe to achieve the reliable detection of human telomerase RNA in cell extracts. The probe is constructed using a dual-labeled fluorescent oligonucleotide hybridized with target-complemented Dabcyl-labeled oligonucleotide. In the presence of the target, the dual-labeled fluorescent oligonucleotide translates into a hairpin structure, which leads to the generation of the fluorescence resonance energy transfer (FRET) phenomenon under UV excitation. Compared to conventional methods, this strategy could effectively avoid false positive signals, and it not only possesses the advantages of simplicity and high specificity but also has the merits of signal stability and distinguishable color variation. Moreover, the quantitative assay of hTR would have a far-reaching impact on the telomerase mechanism and even tumor diagnosis research.

NBSGSC - a FORTRAN program for quantitative x-ray fluorescence analysis. Technical note (final)

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

Tao, G.Y.; Pella, P.A.; Rousseau, R.M.

1985-04-01

A FORTRAN program (NBSGSC) was developed for performing quantitative analysis of bulk specimens by x-ray fluorescence spectrometry. This program corrects for x-ray absorption/enhancement phenomena using the comprehensive alpha coefficient algorithm proposed by Lachance (COLA). NBSGSC is a revision of the program ALPHA and CARECAL originally developed by R.M. Rousseau of the Geological Survey of Canada. Part one of the program (CALCO) performs the calculation of theoretical alpha coefficients, and part two (CALCOMP) computes the composition of the analyte specimens. The analysis of alloys, pressed minerals, and fused specimens can currently be treated by the program. In addition to using measuredmore » x-ray tube spectral distributions, spectra from seven commonly used x-ray tube targets could also be calculated with an NBS algorithm included in the program. NBSGSC is written in FORTRAN IV for a Digital Equipment Corporation (DEC PDP-11/23) minicomputer using RLO2 firm disks and an RSX 11M operating system.« less

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells

PubMed Central

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W.; Gautier, Virginie W.

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip. PMID:26485569

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells.

PubMed

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Ultratrace analysis of transuranic actinides by laser-induced fluorescence

DOEpatents

Miller, S.M.

1983-10-31

Ultratrace quantities of transuranic actinides are detected indirectly by their effect on the fluorescent emissions of a preselected fluorescent species. Transuranic actinides in a sample are coprecipitated with a host lattice material containing at least one preselected fluorescent species. The actinide either quenches or enhances the laser-induced fluorescence of the preselected fluorescent species. The degree of enhancement or quenching is quantitatively related to the concentration of actinide in the sample.

A laser flash photolysis-resonance fluorescence kinetics study of the reaction Cl/2P/ + CH4 yields CH3 + HCl

NASA Technical Reports Server (NTRS)

Ravishankara, A. R.; Wine, P. H.

1980-01-01

The technique of laser flash photolysis-resonance fluorescence is employed to study the kinetics of the reaction Cl(2P) + CH4 yields CH3 + HCl over the temperature range 221-375 K. At temperatures less than or equal to 241 K the apparent bimolecular rate constant is found to be dependent upon the identity of the chemically inert gases in the reaction mixture. For Cl2/CH4/He reaction mixtures (total pressure = 50 torr) different bimolecular rate constants are measured at low and high methane concentrations. For Cl2/CH4/CCl/He and Cl2/CH4/Ar reaction mixtures, the bimolecular rate constant is independent of methane concentration, being approximately equal to the rate constant measured at low methane concentrations for Cl2/CH4/He mixtures. These rate constants are in good agreement with previous results obtained using the discharge flow-resonance fluorescence and competitive chlorination techniques. At 298 K the measured bimolecular rate constant is independent of the identity of the chemically inert gases in the reaction mixture and in good agreement with all previous investigations. The low-temperature results obtained in this investigation and all previous investigations can be rationalized in terms of a model which assumes that the Cl(2P 1/2) state reacts with CH4 much faster than the Cl(2P 3/2) state. Extrapolation of this model to higher temperatures, however, is not straightforward.

Optically trapped atomic resonant devices for narrow linewidth spectral imaging

NASA Astrophysics Data System (ADS)

Qian, Lipeng

This thesis focuses on the development of atomic resonant devices for spectroscopic applications. The primary emphasis is on the imaging properties of optically thick atomic resonant fluorescent filters and their applications. In addition, this thesis presents a new concept for producing very narrow linewidth light as from an atomic vapor lamp pumped by a nanosecond pulse system. This research was motivated by application for missile warning system, and presents an innovative approach to a wide angle, ultra narrow linewidth imaging filter using a potassium vapor cell. The approach is to image onto and collect the fluorescent photons emitted from the surface of an optically thick potassium vapor cell, generating a 2 GHz pass-band imaging filter. This linewidth is narrow enough to fall within a Fraunhefer dark zone in the solar spectrum, thus make the detection solar blind. Experiments are conducted to measure the absorption line shape of the potassium resonant filter, the quantum efficiency of the fluorescent behavior, and the resolution of the fluorescent image. Fluorescent images with different spatial frequency components are analyzed by using a discrete Fourier transform, and the imaging capability of the fluorescent filter is described by its Modulation Transfer Function. For the detection of radiation that is spectrally broader than the linewidth of the potassium imaging filter, the fluorescent image is seen to be blurred by diffuse fluorescence from the slightly off resonant photons. To correct this, an ultra-thin potassium imaging filter is developed and characterized. The imaging property of the ultra-thin potassium imaging cell is tested with a potassium seeded flame, yielding a resolution image of ˜ 20 lines per mm. The physics behind the atomic resonant fluorescent filter is radiation trapping. The diffusion process of the resonant photons trapped in the atomic vapor is theoretically described in this thesis. A Monte Carlo method is used to simulate the

Multicolor fluorescence enhancement from a photonics crystal surface

NASA Astrophysics Data System (ADS)

Pokhriyal, A.; Lu, M.; Huang, C. S.; Schulz, S.; Cunningham, B. T.

2010-09-01

A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, fabricated uniformly from plastic materials over a ˜3×5 in.2 surface area by nanoreplica molding, utilizes two distinct resonant modes to enhance electric field stimulation of a dye excited by a λ =632.8 nm laser (cyanine-5) and a dye excited by a λ =532 nm laser (cyanine-3). Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle. Compared to detection of a dye-labeled protein on an ordinary glass surface, the photonic crystal surface exhibited a 32× increase in fluorescent signal intensity for cyanine-5 conjugated streptavidin labeling, while a 25× increase was obtained for cyanine-3 conjugated streptavidin labeling. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in the 532 nm<λ<633 nm range by selection of an appropriate incident angle. The device is designed for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays.

Multicolor fluorescence enhancement from a photonics crystal surface

PubMed Central

Pokhriyal, A.; Lu, M.; Huang, C. S.; Schulz, S.; Cunningham, B. T.

2010-01-01

A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, fabricated uniformly from plastic materials over a ∼3×5 in.2 surface area by nanoreplica molding, utilizes two distinct resonant modes to enhance electric field stimulation of a dye excited by a λ=632.8 nm laser (cyanine-5) and a dye excited by a λ=532 nm laser (cyanine-3). Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle. Compared to detection of a dye-labeled protein on an ordinary glass surface, the photonic crystal surface exhibited a 32× increase in fluorescent signal intensity for cyanine-5 conjugated streptavidin labeling, while a 25× increase was obtained for cyanine-3 conjugated streptavidin labeling. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in the 532 nm<λ<633 nm range by selection of an appropriate incident angle. The device is designed for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays. PMID:20957067

Quantitative imaging for discovery and assembly of the metabo-regulome

PubMed Central

Okumoto, Sakiko; Takanaga, Hitomi; Frommer, Wolf B.

2009-01-01

Summary Little is known about regulatory networks that control metabolic flux in plant cells. Detailed understanding of regulation is crucial for synthetic biology. The difficulty of measuring metabolites with cellular and subcellular precision is a major roadblock. New tools have been developed for monitoring extracellular, cytosolic, organellar and vacuolar ion and metabolite concentrations with a time resolution of milliseconds to hours. Genetically encoded sensors allow quantitative measurement of steady-state concentrations of ions, signaling molecules and metabolites and their respective changes over time. Fluorescence resonance energy transfer (FRET) sensors exploit conformational changes in polypeptides as a proxy for analyte concentrations. Subtle effects of analyte binding on the conformation of the recognition element are translated into a FRET change between two fused green fluorescent protein (GFP) variants, enabling simple monitoring of analyte concentrations using fluorimetry or fluorescence microscopy. Fluorimetry provides information averaged over cell populations, while microscopy detects differences between cells or populations of cells. The genetically encoded sensors can be targeted to subcellular compartments or the cell surface. Confocal microscopy ultimately permits observation of gradients or local differences within a compartment. The FRET assays can be adapted to high-throughput analysis to screen mutant populations in order to systematically identify signaling networks that control individual steps in metabolic flux. PMID:19138219

Isotropically sensitive optical filter employing atomic resonance transitions

DOEpatents

Marling, J.B.

An ultra-high Q isotropically sensitive optical filter or optical detector is disclosed employing atomic resonance transitions. More specifically, atomic resonance transitions utilized in conjunction with two optical bandpass filters provide an optical detector having a wide field of view (approx. 2 ..pi.. steradians) and very narrow acceptance bandwidth approaching 0.01A. A light signal to be detected is transmitted through an outer bandpass filter into a resonantly absorbing atomic vapor, the excited atomic vapor than providing a fluorescence signal at a different wavelength which is transmitted through an inner bandpass filters have no common transmission band, therby resulting in complete blockage of all optical signals that are not resonantly shifted in wavelength by the intervening atomic vapor. Two embodiments are disclosed, one in which the light signal raises atoms contained in the atomic vapor from the ground state to an excited state from which fluorescence occurs, and the other in which a pump laser is used to raise the atoms in the ground state to a first excited state from which the light signal then is resonantly absorbed, thereby raising the atoms to a second excited state from which fluorescence occurs. A specific application is described in which an optical detector according to the present invention can be located in an orbiting satellite.

Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer.

PubMed

Seong, Jihye; Ouyang, Mingxing; Kim, Taejin; Sun, Jie; Wen, Po-Chao; Lu, Shaoying; Zhuo, Yue; Llewellyn, Nicholas M; Schlaepfer, David D; Guan, Jun-Lin; Chien, Shu; Wang, Yingxiao

2011-07-26

Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli. © 2011 Macmillan Publishers Limited. All rights reserved.

Investigation of BOLD fMRI Resonance Frequency Shifts and Quantitative Susceptibility Changes at 7 T

PubMed Central

Bianciardi, Marta; van Gelderen, Peter; Duyn, Jeff H.

2013-01-01

Although blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) experiments of brain activity generally rely on the magnitude of the signal, they also provide frequency information that can be derived from the phase of the signal. However, because of confounding effects of instrumental and physiological origin, BOLD related frequency information is difficult to extract and therefore rarely used. Here, we explored the use of high field (7 T) and dedicated signal processing methods to extract frequency information and use it to quantify and interpret blood oxygenation and blood volume changes. We found that optimized preprocessing improves detection of task-evoked and spontaneous changes in phase signals and resonance frequency shifts over large areas of the cortex with sensitivity comparable to that of magnitude signals. Moreover, our results suggest the feasibility of mapping BOLD quantitative susceptibility changes in at least part of the activated area and its largest draining veins. Comparison with magnitude data suggests that the observed susceptibility changes originate from neuronal activity through induced blood volume and oxygenation changes in pial and intracortical veins. Further, from frequency shifts and susceptibility values, we estimated that, relative to baseline, the fractional oxygen saturation in large vessels increased by 0.02–0.05 during stimulation, which is consistent to previously published estimates. Together, these findings demonstrate that valuable information can be derived from fMRI imaging of BOLD frequency shifts and quantitative susceptibility changes. PMID:23897623

Application of Real-Time Fluorescent PCR for Quantitative Assessment of Neospora caninum Infections in Organotypic Slice Cultures of Rat Central Nervous System Tissue

PubMed Central

Müller, Norbert; Vonlaufen, Nathalie; Gianinazzi, Christian; Leib, Stephen L.; Hemphill, Andrew

2002-01-01

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection. PMID:11773124

Pilot clinical study to assess caries lesion activity using quantitative light-induced fluorescence during dehydration

NASA Astrophysics Data System (ADS)

Ando, Masatoshi; Ferreira-Zandoná, Andrea G.; Eckert, George J.; Zero, Domenick T.; Stookey, George K.

2017-03-01

This study aimed to evaluate the ability of quantitative light-induced fluorescence (QLF) to assess caries lesion activity using visual examination (VE) as the gold standard. Twenty-four visible white spot lesions on buccal surfaces were examined from 23 children, ages 9 to 14 years. At baseline, the surface was hydrated with water, and thereafter, it was dehydrated with continuous compressed air during image acquisition. QLF images were acquired at 0 (baseline), 5, and 15 s. QLF variables [QLFV: fluorescence loss (ΔF), lesion size (S), ΔQ: ΔF×S] was recorded. Changes-in-QLFV per second (ΔQLFV) were determined: ΔQLFV=(QLFVN-QLF/N), where N indicates dehydration time. One experienced dentist conducted VE independently using a dental unit's light, compressed air, and explorer. QLFV and ΔQLFV of the active group (n=11) were compared with those of the inactive group (n=13) using two-sample t-tests. As the surface was dehydrated, S and ΔQ values of the active group increased, whereas QLFV of the inactive group showed only a small change. ΔQLFV of the active group were larger than those of the inactive group; however, the difference did not reach statistical significance (p>0.11). Within the limitations of this study, QLF data indicated increments for lesions designated as active and minimal change for lesions defined as inactive.

PH-sensitive fluorescence detection by diffuse fluorescence tomography

NASA Astrophysics Data System (ADS)

Li, Jiao; Gao, Feng; Duan, Linjing; Wang, Xin; Zhang, Limin; Zhao, Huijuan

2012-03-01

The importance of cellular pH has been shown clearly in the study of cell activity, pathological feature, drug metabolism, etc. Monitoring pH changes of living cells and imaging the regions with abnormal pH values in vivo could provide the physiologic and pathologic information for the research of the cell biology, pharmacokinetics, diagnostics and therapeutics of certain diseases such as cancer. Thus, pH-sensitive fluorescence imaging of bulk tissues has been attracting great attention in the regime of near-infrared diffuse fluorescence tomography (DFT), an efficient small-animal imaging tool. In this paper, the feasibility of quantifying pH-sensitive fluorescence targets in turbid medium is investigated using both time-domain and steady-state DFT methods. By use of the specifically designed time-domain and continuous-wave systems and the previously proposed image reconstruction scheme, we validate the method through 2-dimensional imaging experiments on a small-animal-sized phantom with multiply targets of distinct pH values. The results show that the approach can localize the targets with reasonable accuracy and achieve quantitative reconstruction of the pH-sensitive fluorescent yield.

Advancing multiscale structural mapping of the brain through fluorescence imaging and analysis across length scales

PubMed Central

Hogstrom, L. J.; Guo, S. M.; Murugadoss, K.; Bathe, M.

2016-01-01

Brain function emerges from hierarchical neuronal structure that spans orders of magnitude in length scale, from the nanometre-scale organization of synaptic proteins to the macroscopic wiring of neuronal circuits. Because the synaptic electrochemical signal transmission that drives brain function ultimately relies on the organization of neuronal circuits, understanding brain function requires an understanding of the principles that determine hierarchical neuronal structure in living or intact organisms. Recent advances in fluorescence imaging now enable quantitative characterization of neuronal structure across length scales, ranging from single-molecule localization using super-resolution imaging to whole-brain imaging using light-sheet microscopy on cleared samples. These tools, together with correlative electron microscopy and magnetic resonance imaging at the nanoscopic and macroscopic scales, respectively, now facilitate our ability to probe brain structure across its full range of length scales with cellular and molecular specificity. As these imaging datasets become increasingly accessible to researchers, novel statistical and computational frameworks will play an increasing role in efforts to relate hierarchical brain structure to its function. In this perspective, we discuss several prominent experimental advances that are ushering in a new era of quantitative fluorescence-based imaging in neuroscience along with novel computational and statistical strategies that are helping to distil our understanding of complex brain structure. PMID:26855758

Correlation between quantitative whole-body muscle magnetic resonance imaging and clinical muscle weakness in Pompe disease.

PubMed

Horvath, Jeffrey J; Austin, Stephanie L; Case, Laura E; Greene, Karla B; Jones, Harrison N; Soher, Brian J; Kishnani, Priya S; Bashir, Mustafa R

2015-05-01

Previous examination of whole-body muscle involvement in Pompe disease has been limited to physical examination and/or qualitative magnetic resonance imaging (MRI). In this study we assess the feasibility of quantitative proton-density fat-fraction (PDFF) whole-body MRI in late-onset Pompe disease (LOPD) and compare the results with manual muscle testing. Seven LOPD patients and 11 disease-free controls underwent whole-body PDFF MRI. Quantitative MR muscle group assessments were compared with physical testing of muscle groups. The 95% upper limits of confidence intervals for muscle groups were 4.9-12.6% in controls and 6.8-76.4% in LOPD patients. LOPD patients showed severe and consistent tongue and axial muscle group involvement, with less marked involvement of peripheral musculature. MRI was more sensitive than physical examination for detection of abnormality in multiple muscle groups. This integrated, quantitative approach to muscle assessment provides more detailed data than physical examination and may have clinical utility for monitoring disease progression and treatment response. © 2014 Wiley Periodicals, Inc.

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

A flash photolysis-resonance fluorescence study of the formation of O(D-1) in the photolysis of water and reaction of O(D-1) with H2, Ar and He

NASA Technical Reports Server (NTRS)

Stief, L. J.; Payne, W. A.; Klemm, R. B.

1974-01-01

The relative importance of two primary processes in the photolyis of water: (1) H2O + h (nu) yields H + OH, and (2) H2O + h (nu) yields H2 + OD-1 were determined in a direct manner by time resolved detection (via resonance fluorescence) of H and O formed in processes 1 and 2 respectively. The initially formed OD-1 was deactivated to ground state OP-3 prior to detection via resonance fluorescence. The relative quantum yields for processes 1 and 2 are 0.89 and 0.11 for the wavelength interval 105 to 145nm and = to or greater than 0.99, and = to or less than 0.01 for the wavelength interval 145 to 185nm. Rate constants at 300 K for the reactions OD-1 + H2, + Ar, and + He are presented.

Rejection of fluorescence background in resonance and spontaneous Raman microspectroscopy.

PubMed

Smith, Zachary J; Knorr, Florian; Pagba, Cynthia V; Wachsmann-Hogiu, Sebastian

2011-05-18

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to < 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

Quantitative tomographic imaging of intermolecular FRET in small animals

PubMed Central

Venugopal, Vivek; Chen, Jin; Barroso, Margarida; Intes, Xavier

2012-01-01

Forster resonance energy transfer (FRET) is a nonradiative transfer of energy between two fluorescent molecules (a donor and an acceptor) in nanometer range proximity. FRET imaging methods have been applied to proteomic studies and drug discovery applications based on intermolecular FRET efficiency measurements and stoichiometric measurements of FRET interaction as quantitative parameters of interest. Importantly, FRET provides information about biomolecular interactions at a molecular level, well beyond the diffraction limits of standard microscopy techniques. The application of FRET to small animal imaging will allow biomedical researchers to investigate physiological processes occurring at nanometer range in vivo as well as in situ. In this work a new method for the quantitative reconstruction of FRET measurements in small animals, incorporating a full-field tomographic acquisition system with a Monte Carlo based hierarchical reconstruction scheme, is described and validated in murine models. Our main objective is to estimate the relative concentration of two forms of donor species, i.e., a donor molecule involved in FRETing to an acceptor close by and a nonFRETing donor molecule. PMID:23243567

Structural dynamics of catalytic RNA highlighted by fluorescence resonance energy transfer.

PubMed

Walter, N G

2001-09-01

RNA performs a multitude of essential cellular functions involving the maintenance, transfer, and processing of genetic information. The reason probably is twofold: (a) Life started as a prebiotic RNA World, in which RNA served as the genetic information carrier and catalyzed all chemical reactions required for its proliferation and (b) some of the RNA World functions were conserved throughout evolution because neither DNA nor protein is as adept in fulfilling them. A particular advantage of RNA is its high propensity to form alternative structures as required in subsequent steps of a reaction pathway. Here I describe fluorescence resonance energy transfer (FRET) as a method to monitor a crucial conformational transition on the reaction pathway of the hairpin ribozyme, a small catalytic RNA motif from a self-replicating plant virus satellite RNA and well-studied paradigm of RNA folding. Steady-state FRET measurements in solution allow one to measure the kinetics and requirements of docking of its two independently folding domains; time-resolved FRET reveals the relative thermodynamic stability of the undocked (extended, inactive) and docked (active) ribozyme conformations; while single-molecule FRET experiments will highlight the dynamics of RNA at the individual molecule level. Similar domain docking events are expected to be at the heart of many biological functions of RNA, and the described FRET techniques promise to be adaptable to most of the involved RNA systems. Copyright 2001 Academic Press.

Fabrication et caracterisation de cristaux photoniques pour exaltation de fluorescence

NASA Astrophysics Data System (ADS)

Gascon, Annabelle

2011-12-01

In today's world, there is a pressing need for point-of-care molecular analysis that is fast, inexpensive and transportable. Lab-on-a- chips are designed to fulfill that need. They are micro-electromechanical systems (MEMS), fabricated with microelectronic techniques, that use the analytes physical properties to detect their presence in liquid samples. This detection can be performed by attaching the analyte to quantum dots. These quantum dots are semiconducting nanoparticles with narrow fluorescence band. In our project, we use a tuneable system with a two-slab photonic crystal that serves as a tuneable optical filter, detecting the presence and wavelength of these quantum dots. Photonic crystals are dielectrics with a variable refractive index, with a period near the visible light wavelength. They are called photonic crystals because they have a photonic band gap just as atomic crystals, periodic structure of atoms, have an electronic band gap. They are photonic because photons instead of electrons propagate through them. They can also enhance fluorescence from quantum dots at the photonic crystals guided resonance wavelength. My project objectives are to: (1) Fabricate two-slab photonic crystal, (2) Characterize photonic crystals, (3) Place quantum dots on photonic crystals, (4) Measure fluorescence enhancement. The device made during this project consists of a silicon wafer on which were deposited a 200 nm silicon nitride layer, then a 200 nm silicon dioxide layer and finally another 200 nm silicon nitride layer. An electron-beam lithography defines the photonic crystals and the MEMS. The photonic crystals are square lattices of holes 180 nm in diameter, at a period of 460 nm, etched through the two silicon nitride slabs. The two slabs are etched in a single step of Reactive Ion Etching (RIE). Then, the silicon under the photonic crystal is etched from the backside up to the nitride by deep-RIE. Finally, the oxide layer is removed in order to completely suspend

Quantitative solid-state 13C nuclear magnetic resonance spectrometric analyses of wood xylen: effect of increasing carbohydrate content

USGS Publications Warehouse

Bates, A.L.; Hatcher, P.G.

1992-01-01

Isolated lignin with a low carbohydrate content was spiked with increasing amounts of alpha-cellulose, and then analysed by solid-state 13C nuclear magnetic resonance (NMR) using cross-polarization with magic angle spinning (CPMAS) and dipolar dephasing methods in order to assess the quantitative reliability of CPMAS measurement of carbohydrate content and to determine how increasingly intense resonances for carbohydrate carbons affect calculations of the degree of lignin's aromatic ring substitution and methoxyl carbon content. Comparisons were made of the carbohydrate content calculated by NMR with carbohydrate concentrations obtained by phenol-sulfuric acid assay and by the calculation from the known amounts of cellulose added. The NMR methods used in this study yield overestimates for carbohydrate carbons due to resonance area overlap from the aliphatic side chain carbons of lignin. When corrections are made for these overlapping resonance areas, the NMR results agree very well with results obtained by other methods. Neither the calculated methoxyl carbon content nor the degree of aromatic ring substitution in lignin, both calculated from dipolar dephasing spectra, change with cellulose content. Likewise, lignin methoxyl content does not correlate with cellulose abundance when measured by integration of CPMAS spectra. ?? 1992.

A double-label time-resolved fluorescent strip for rapidly quantitative detection of carbofuran residues in agro-products.

PubMed

Zhang, Qi; Qu, Qiaoyu; Chen, Shanshan; Liu, Xiaowei; Li, Peiwu

2017-09-15

A rapid and quantitative time-resolved fluorescent immunochromatographic assay (TRFICA) for detecting carbofuran residues in agro-products was reported in this paper. This assay was developed based on double-label immunoprobes, one of which was a carbofuran-specific antibody coupled with europium microbeads for the test (T) line signal while the other was mouse IgG coupled with europium microbeads for the control (C) line signal. Quantitative relationships between carbofuran concentrations and T/C ratios were established to determine the analyte concentration. To increase assay accuracy, four standard curves were established for the agro-products (green bean, cabbage, apple, and pear). The limits of detection (LODs) ranged from 0.04 to 0.76mgL -1 . The spiked recoveries of carbofuran in the agro-products were in the range of 81-103%, which was in good agreement with a standard HPLC method. Therefore, we provided a new and reliable method for determination of N-methylcarbamate pesticide carbofuran residues in agro-products including vegetables and fruits. Copyright © 2017. Published by Elsevier Ltd.

Non-blinking quantum dot with a plasmonic nanoshell resonator

NASA Astrophysics Data System (ADS)

Ji, Botao; Giovanelli, Emerson; Habert, Benjamin; Spinicelli, Piernicola; Nasilowski, Michel; Xu, Xiangzhen; Lequeux, Nicolas; Hugonin, Jean-Paul; Marquier, Francois; Greffet, Jean-Jacques; Dubertret, Benoit

2015-02-01

Colloidal semiconductor quantum dots are fluorescent nanocrystals exhibiting exceptional optical properties, but their emission intensity strongly depends on their charging state and local environment. This leads to blinking at the single-particle level or even complete fluorescence quenching, and limits the applications of quantum dots as fluorescent particles. Here, we show that a single quantum dot encapsulated in a silica shell coated with a continuous gold nanoshell provides a system with a stable and Poissonian emission at room temperature that is preserved regardless of drastic changes in the local environment. This novel hybrid quantum dot/silica/gold structure behaves as a plasmonic resonator with a strong Purcell factor, in very good agreement with simulations. The gold nanoshell also acts as a shield that protects the quantum dot fluorescence and enhances its resistance to high-power photoexcitation or high-energy electron beams. This plasmonic fluorescent resonator opens the way to a new family of plasmonic nanoemitters with robust optical properties.

Quantitative Reappraisal of the Helmholtz-Guyton Resonance Theory of Frequency Tuning in the Cochlea

PubMed Central

Babbs, Charles F.

2011-01-01

To explore the fundamental biomechanics of sound frequency transduction in the cochlea, a two-dimensional analytical model of the basilar membrane was constructed from first principles. Quantitative analysis showed that axial forces along the membrane are negligible, condensing the problem to a set of ordered one-dimensional models in the radial dimension, for which all parameters can be specified from experimental data. Solutions of the radial models for asymmetrical boundary conditions produce realistic deformation patterns. The resulting second-order differential equations, based on the original concepts of Helmholtz and Guyton, and including viscoelastic restoring forces, predict a frequency map and amplitudes of deflections that are consistent with classical observations. They also predict the effects of an observation hole drilled in the surrounding bone, the effects of curvature of the cochlear spiral, as well as apparent traveling waves under a variety of experimental conditions. A quantitative rendition of the classical Helmholtz-Guyton model captures the essence of cochlear mechanics and unifies the competing resonance and traveling wave theories. PMID:22028708

Blinking fluorescence of single donor-acceptor pairs: important role of "dark'' states in resonance energy transfer via singlet levels.

PubMed

Osad'ko, I S; Shchukina, A L

2012-06-01

The influence of triplet levels on Förster resonance energy transfer via singlet levels in donor-acceptor (D-A) pairs is studied. Four types of D-A pair are considered: (i) two-level donor and two-level acceptor, (ii) three-level donor and two-level acceptor, (iii) two-level donor and three-level acceptor, and (iv) three-level donor and three-level acceptor. If singlet-triplet transitions in a three-level acceptor molecule are ineffective, the energy transfer efficiency E=I_{A}/(I_{A}+I_{D}), where I_{D} and I_{A} are the average intensities of donor and acceptor fluorescence, can be described by the simple theoretical equation E(F)=FT_{D}/(1+FT_{D}). Here F is the rate of energy transfer, and T_{D} is the donor fluorescence lifetime. In accordance with the last equation, 100% of the donor electronic energy can be transferred to an acceptor molecule at FT_{D}≫1. However, if singlet-triplet transitions in a three-level acceptor molecule are effective, the energy transfer efficiency is described by another theoretical equation, E(F)=F[over ¯](F)T_{D}/[1+F[over ¯](F)T_{D}]. Here F[over ¯](F) is a function of F depending on singlet-triplet transitions in both donor and acceptor molecules. Expressions for the functions F[over ¯](F) are derived. In this case the energy transfer efficiency will be far from 100% even at FT_{D}≫1. The character of the intensity fluctuations of donor and acceptor fluorescence indicates which of the two equations for E(F) should be used to find the value of the rate F. Therefore, random time instants of photon emission in both donor and acceptor fluorescence are calculated by the Monte Carlo method for all four types of D-A pair. Theoretical expressions for start-stop correlators (waiting time distributions) in donor and acceptor fluorescence are derived. The probabilities w_{N}^{D}(t) and w_{N}^{A}(t) of finding N photons of donor and acceptor fluorescence in the time interval t are calculated for various values of the energy

Quantitative proton magnetic resonance spectroscopy without water suppression

NASA Astrophysics Data System (ADS)

Özdemir, M. S.; DeDeene, Y.; Fieremans, E.; Lemahieu, I.

2009-06-01

The suppression of the abundant water signal has been traditionally employed to decrease the dynamic range of the NMR signal in proton MRS (1H MRS) in vivo. When using this approach, if the intent is to utilize the water signal as an internal reference for the absolute quantification of metabolites, additional measurements are required for the acquisition of the water signal. This can be prohibitively time-consuming and is not desired clinically. Additionally, traditional water suppression can lead to metabolite alterations. This can be overcome by performing quantitative 1H MRS without water suppression. However, the non-water-suppressed spectra suffer from gradient-induced frequency modulations, resulting in sidebands in the spectrum. Sidebands may overlap with the metabolites, which renders the spectral analysis and quantification problematic. In this paper, we performed absolute quantification of metabolites without water suppression. Sidebands were removed by utilizing the phase of an external reference signal of single resonance to observe the time-varying the static field fluctuations induced by gradient-vibration and deconvolving this phase contamination from the desired NMR signal. The quantification of metabolites was determined after sideband correction by calibrating the metabolite signal intensities against the recorded water signal. The method was evaluated by phantom and in vivo measurements in human brain. The maximum systematic error for the quantified metabolite concentrations was found to be 10.8%, showing the feasibility of the quantification after sideband correction.

Fluorescence Excitation-Emission Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter

USGS Publications Warehouse

Chen, W.; Westerhoff, P.; Leenheer, J.A.; Booksh, K.

2003-01-01

Excitation-emission matrix (EEM) fluorescence spectroscopy has been widely used to characterize dissolved organic matter (DOM) in water and soil. However, interpreting the >10,000 wavelength-dependent fluorescence intensity data points represented in EEMs has posed a significant challenge. Fluorescence regional integration, a quantitative technique that integrates the volume beneath an EEM, was developed to analyze EEMs. EEMs were delineated into five excitation-emission regions based on fluorescence of model compounds, DOM fractions, and marine waters or freshwaters. Volumetric integration under the EEM within each region, normalized to the projected excitation-emission area within that region and dissolved organic carbon concentration, resulted in a normalized region-specific EEM volume (??i,n). Solid-state carbon nuclear magnetic resonance (13C NMR), Fourier transform infrared (FTIR) analysis, ultraviolet-visible absorption spectra, and EEMs were obtained for standard Suwannee River fulvic acid and 15 hydrophobic or hydrophilic acid, neutral, and base DOM fractions plus nonfractionated DOM from wastewater effluents and rivers in the southwestern United States. DOM fractions fluoresced in one or more EEM regions. The highest cumulative EEM volume (??T,n = ????i,n) was observed for hydrophobic neutral DOM fractions, followed by lower ??T,n values for hydrophobic acid, base, and hydrophilic acid DOM fractions, respectively. An extracted wastewater biomass DOM sample contained aromatic protein- and humic-like material and was characteristic of bacterial-soluble microbial products. Aromatic carbon and the presence of specific aromatic compounds (as indicated by solid-state 13C NMR and FTIR data) resulted in EEMs that aided in differentiating wastewater effluent DOM from drinking water DOM.

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

PubMed Central

Macdonald, Patrick J.; Chen, Yan; Mueller, Joachim D.

2012-01-01

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction. PMID:22093611

Click strategies for single-molecule protein fluorescence.

PubMed

Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

2012-03-21

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Stroke onset time estimation from multispectral quantitative magnetic resonance imaging in a rat model of focal permanent cerebral ischemia.

PubMed

McGarry, Bryony L; Rogers, Harriet J; Knight, Michael J; Jokivarsi, Kimmo T; Sierra, Alejandra; Gröhn, Olli Hj; Kauppinen, Risto A

2016-08-01

Quantitative T2 relaxation magnetic resonance imaging allows estimation of stroke onset time. We aimed to examine the accuracy of quantitative T1 and quantitative T2 relaxation times alone and in combination to provide estimates of stroke onset time in a rat model of permanent focal cerebral ischemia and map the spatial distribution of elevated quantitative T1 and quantitative T2 to assess tissue status. Permanent middle cerebral artery occlusion was induced in Wistar rats. Animals were scanned at 9.4T for quantitative T1, quantitative T2, and Trace of Diffusion Tensor (Dav) up to 4 h post-middle cerebral artery occlusion. Time courses of differentials of quantitative T1 and quantitative T2 in ischemic and non-ischemic contralateral brain tissue (ΔT1, ΔT2) and volumes of tissue with elevated T1 and T2 relaxation times (f1, f2) were determined. TTC staining was used to highlight permanent ischemic damage. ΔT1, ΔT2, f1, f2, and the volume of tissue with both elevated quantitative T1 and quantitative T2 (V(Overlap)) increased with time post-middle cerebral artery occlusion allowing stroke onset time to be estimated. V(Overlap) provided the most accurate estimate with an uncertainty of ±25 min. At all times-points regions with elevated relaxation times were smaller than areas with Dav defined ischemia. Stroke onset time can be determined by quantitative T1 and quantitative T2 relaxation times and tissue volumes. Combining quantitative T1 and quantitative T2 provides the most accurate estimate and potentially identifies irreversibly damaged brain tissue. © 2016 World Stroke Organization.

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.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

A novel graphene-based label-free fluorescence `turn-on' nanosensor for selective and sensitive detection of phosphorylated species in biological samples and living cells

NASA Astrophysics Data System (ADS)

Ke, Yaotang; Garg, Bhaskar; Ling, Yong-Chien

2016-02-01

A novel label-free fluorescence `turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti4+-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti4+) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions between phosphate groups and Ti4+. The as-prepared rGO@PDA-Ti4+-FMNs (nanosensor), fluoresce only weakly due to the ineffective Förster resonance energy transfer between the FMNs and rGO@PDA-Ti4+. The experimental findings revealed that the microwave-assisted interaction of the nanosensor with α-, β-casein, ovalbumin, human serum, non-fat milk, egg white, and living cells (all containing Ps) releases FMNs (due to the high formation constant between phosphate groups and Ti4+), leading to an excellent fluorescence `turn-on' response. The fluorescence spectroscopy, confocal microscopy, and MALDI-TOF MS spectrometry were used to detect Ps both qualitatively and quantitatively. Under the optimized conditions, the nanosensor showed a detection limit of ca. 118.5, 28.9, and 54.8 nM for the tryptic digests of α-, β-casein and ovalbumin, respectively. Furthermore, the standard addition method was used as a bench-mark proof for phosphopeptide quantification in egg white samples. We postulate that the present quantitative assay for Ps holds tremendous potential and may pave the way to disease diagnostics in the near future.A novel label-free fluorescence `turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti4+-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti4+) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions

Groping for quantitative digital 3-D image analysis: an approach to quantitative fluorescence in situ hybridization in thick tissue sections of prostate carcinoma.

PubMed

Rodenacker, K; Aubele, M; Hutzler, P; Adiga, P S

1997-01-01

In molecular pathology numerical chromosome aberrations have been found to be decisive for the prognosis of malignancy in tumours. The existence of such aberrations can be detected by interphase fluorescence in situ hybridization (FISH). The gain or loss of certain base sequences in the desoxyribonucleic acid (DNA) can be estimated by counting the number of FISH signals per cell nucleus. The quantitative evaluation of such events is a necessary condition for a prospective use in diagnostic pathology. To avoid occlusions of signals, the cell nucleus has to be analyzed in three dimensions. Confocal laser scanning microscopy is the means to obtain series of optical thin sections from fluorescence stained or marked material to fulfill the conditions mentioned above. A graphical user interface (GUI) to a software package for display, inspection, count and (semi-)automatic analysis of 3-D images for pathologists is outlined including the underlying methods of 3-D image interaction and segmentation developed. The preparative methods are briefly described. Main emphasis is given to the methodical questions of computer-aided analysis of large 3-D image data sets for pathologists. Several automated analysis steps can be performed for segmentation and succeeding quantification. However tumour material is in contrast to isolated or cultured cells even for visual inspection, a difficult material. For the present a fully automated digital image analysis of 3-D data is not in sight. A semi-automatic segmentation method is thus presented here.

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

PubMed Central

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki

2014-01-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)–Ras–extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. PMID:24958104

Fis protein induced λF-DNA bending observed by single-pair fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Chi-Cheng, Fu; Wunshain, Fann; Yuan Hanna, S.

2006-03-01

Fis, a site-specific DNA binding protein, regulates many biological processes including recombination, transcription, and replication in E.coli. Fis induced DNA bending plays an important role in regulating these functions and bending angle range from ˜50 to 95 dependent on the DNA sequence. For instance, the average bending angle of λF-DNA (26 bp, 8.8nm long, contained λF binding site on the center) measured by gel mobility shift assays was ˜ 94 . But the traditional method cannot provide information about the dynamics and the angle distribution. In this study, λF-DNA was labeled with donor (Alexa Fluor 546) and acceptor (Alexa Fluor 647) dyes on its two 5' ends and the donor-acceptor distances were measured using single-pair fluorescence resonance energy transfer (sp-FRET) with and without the present of Fis protein. Combing with structure information of Fis-DNA complex, the sp-FRET results are used to estimate the protein induced DNA bending angle distribution and dynamics.

Quantitative fluorescence-polymerase chain reaction assay for the detection of the duplication of the Charcot Marie Tooth disease type 1A critical region.

PubMed

De Toffol, Simona; Bellone, Emilia; Dulcetti, Francesca; Ruggeri, Anna Maria; Maggio, Pietro Paolo; Pulimeno, Maria Rosaria; Mandich, Paola; Maggi, Federico; Simoni, Giuseppe; Grati, Francesca Romana

2010-04-01

Charcot Marie Tooth (CMT) syndrome is the most common hereditary peripheral neuropathy, with an incidence of about 1 in 2500. The subtype 1A (CMT1A) is caused by a tandem duplication of a 1.5-Mb region encompassing the PMP22 gene. Conventional short tandem repeat (STR) analysis can reveal this imbalance if a triallelic pattern, defining with certainty the presence of duplication, is present. In case of duplication with a biallelic pattern, it can only indicate a semiquantitative dosage of the fluorescence intensity ratio of the two fragments. In this study we developed a quantitative fluorescence-PCR using seven highly informative STRs within the CMT1A critical region that successfully disclosed or excluded the presence of the pathogenic imbalance in a cohort of 60 samples including 40 DNAs from samples with the CMT1A duplication previously characterized with two different molecular approaches, and 20 diagnostic samples from 10 members of a five-generation pedigree segregating CMT1A, 8 unrelated cases and 2 prenatal samples. The application of the quantitative fluorescence-PCR using STRs located in the critical region could be a reliable method to evaluate the presence of the PMP22 duplication for the diagnosis and classification of hereditary neuropathies in asymptomatic subjects with a family history of inherited neuropathy, in prenatal samples in cases with one affected parent, and in unrelated patients with a sporadic demyelinating neuropathy with clinical features resembling CMT (i.e., pes cavus with hammer toes) or with conduction velocities in the range of CMT1A.

A Ratiometric Threshold for Determining Presence of Cancer During Fluorescence-guided Surgery

PubMed Central

Warram, Jason M; de Boer, Esther; Moore, Lindsay S.; Schmalbach, Cecelia E; Withrow, Kirk P; Carroll, William R; Richman, Joshua S; Morlandt, Anthony B; Brandwein-Gensler, Margaret; Rosenthal, Eben L

2015-01-01

Background&Objective Fluorescence-guided imaging to assist in identification of malignant margins has the potential to dramatically improve oncologic surgery. However a standardized method for quantitative assessment of disease-specific fluorescence has not been investigated. Introduced here is a ratiometric threshold derived from mean fluorescent tissue intensity that can be used to semi-quantitatively delineate tumor from normal tissue. Methods Open-field and a closed-field imaging devices were used to quantify fluorescence in punch biopsy tissues sampled from primary tumors collected during a phase 1 trial evaluating the safety of cetuximab-IRDye800 in patients (n=11) undergoing surgical intervention for head and neck cancer. Fluorescence ratios were calculated using mean fluorescence intensity (MFI) from punch biopsy normalized by MFI of patient-matched tissues. Ratios were compared to pathological assessment and a ratiometric threshold was established to predict presence of cancer. Results During open-field imaging using an intraoperative device, the threshold for muscle normalized tumor fluorescence was found to be 2.7, which produced a sensitivity of 90.5% and specificity of 78.6% for delineating disease tissue. The skin-normalized threshold generated greater sensitivity (92.9%) and specificity (81.0%). Conclusion Successful implementation of a semi-quantitative threshold can provide a scientific methodology for delineating disease from normal tissue during fluorescence-guided resection of cancer. PMID:26074273

A ratiometric threshold for determining presence of cancer during fluorescence-guided surgery.

PubMed

Warram, Jason M; de Boer, Esther; Moore, Lindsay S; Schmalbach, Cecelia E; Withrow, Kirk P; Carroll, William R; Richman, Joshua S; Morlandt, Anthony B; Brandwein-Gensler, Margaret; Rosenthal, Eben L

2015-07-01

Fluorescence-guided imaging to assist in identification of malignant margins has the potential to dramatically improve oncologic surgery. However, a standardized method for quantitative assessment of disease-specific fluorescence has not been investigated. Introduced here is a ratiometric threshold derived from mean fluorescent tissue intensity that can be used to semi-quantitatively delineate tumor from normal tissue. Open-field and a closed-field imaging devices were used to quantify fluorescence in punch biopsy tissues sampled from primary tumors collected during a phase 1 trial evaluating the safety of cetuximab-IRDye800 in patients (n = 11) undergoing surgical intervention for head and neck cancer. Fluorescence ratios were calculated using mean fluorescence intensity (MFI) from punch biopsy normalized by MFI of patient-matched tissues. Ratios were compared to pathological assessment and a ratiometric threshold was established to predict presence of cancer. During open-field imaging using an intraoperative device, the threshold for muscle normalized tumor fluorescence was found to be 2.7, which produced a sensitivity of 90.5% and specificity of 78.6% for delineating disease tissue. The skin-normalized threshold generated greater sensitivity (92.9%) and specificity (81.0%). Successful implementation of a semi-quantitative threshold can provide a scientific methodology for delineating disease from normal tissue during fluorescence-guided resection of cancer. © 2015 Wiley Periodicals, Inc.

Fluorescence resonance energy transfer analysis of escherichia coli RNA polymerase and polymerase-DNA complexes.

PubMed

Heyduk, T; Niedziela-Majka, A

Fluorescence resonance energy transfer (FRET) is a technique allowing measurements of atomic-scale distances in diluted solutions of macromolecules under native conditions. This feature makes FRET a powerful tool to study complicated biological assemblies. In this report we review the applications of FRET to studies of transcription initiation by Escherichia coli RNA polymerase. The versatility of FRET for studies of a large macromolecular assembly such as RNA polymerase is illustrated by examples of using FRET to address several different aspects of transcription initiation by polymerase. FRET has been used to determine the architecture of polymerase, its complex with single-stranded DNA, and the conformation of promoter fragment bound to polymerase. FRET has been also used as a binding assay to determine the thermodynamics of promoter DNA fragment binding to the polymerase. Functional conformational changes in the specificity subunit of polymerase responsible for the modulation of the promoter binding activity of the enzyme and the mechanistic aspects of the transition from the initiation to the elongation complex were also investigated. Copyright 2002 Wiley Periodicals, Inc.

Analysis of nuclear resonance fluorescence excitation measured with LaBr3(Ce) detectors near 2 MeV

NASA Astrophysics Data System (ADS)

Omer, Mohamed; Negm, Hani; Ohgaki, Hideaki; Daito, Izuru; Hayakawa, Takehito; Bakr, Mahmoud; Zen, Heishun; Hori, Toshitada; Kii, Toshiteru; Masuda, Kai; Hajima, Ryoichi; Shizuma, Toshiyuki; Toyokawa, Hiroyuki; Kikuzawa, Nobuhiro

2013-11-01

The performance of LaBr3(Ce) to measure nuclear resonance fluorescence (NRF) excitations is discussed in terms of limits of detection and in comparison with high-purity germanium (HPGe) detectors near the 2 MeV region where many NRF excitation levels from special nuclear materials are located. The NRF experiment was performed at the High Intensity γ-ray Source (HIγS) facility. The incident γ-rays, of 2.12 MeV energy, hit a B4C target to excite the 11B nuclei to the first excitation level. The statistical-sensitive non-linear peak clipping (SNIP) algorithm was implemented to eliminate the background and enhance the limits of detection for the spectra measured with LaBr3(Ce). Both detection and determination limits were deduced from the experimental data.

Fluorescence Molecular Tomography: Principles and Potential for Pharmaceutical Research

PubMed Central

Stuker, Florian; Ripoll, Jorge; Rudin, Markus

2011-01-01

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

Single-pair fluorescence resonance energy transfer analysis of mRNA transcripts for highly sensitive gene expression profiling in near real time.

PubMed

Peng, Zhiyong; Young, Brandon; Baird, Alison E; Soper, Steven A

2013-08-20

Expression analysis of mRNAs transcribed from certain genes can be used as important sources of biomarkers for in vitro diagnostics. While the use of reverse transcription quantitative PCR (RT-qPCR) can provide excellent analytical sensitivity for monitoring transcript numbers, more sensitive approaches for expression analysis that can report results in near real-time are needed for many critical applications. We report a novel assay that can provide exquisite limits-of-quantitation and consists of reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) to provide digital readout through molecular counting. For this assay, no PCR was employed, which enabled short assay turnaround times. To facilitate implementation of the assay, a cyclic olefin copolymer (COC) microchip, which was fabricated using hot embossing, was employed to carry out the LDR in a continuous flow format with online single-molecule detection following the LDR. As demonstrators of the assay's utility, MMP-7 mRNA was expression profiled from several colorectal cancer cell lines. It was found that the RT-LDR/spFRET assay produced highly linear calibration plots even in the low copy number regime. Comparison to RT-qPCR indicated a better linearity over the low copy number range investigated (10-10,000 copies) with an R(2) = 0.9995 for RT-LDR/spFRET and R(2) = 0.98 for RT-qPCR. In addition, differentiating between copy numbers of 10 and 50 could be performed with higher confidence using RT-LDR/spFRET. To demonstrate the short assay turnaround times obtainable using the RT-LDR/spFRET assay, a two thermal cycle LDR was carried out on amphiphysin gene transcripts that can serve as important diagnostic markers for ischemic stroke. The ability to supply diagnostic information on possible stroke events in short turnaround times using RT-LDR/spFRET will enable clinicians to treat patients effectively with appropriate time

A real-time fluorescent sensor specific to Mg2+: crystallographic evidence, DFT calculation and its use for quantitative determination of magnesium in drinking water.

PubMed

Men, Guangwen; Chen, Chunrong; Zhang, Shitong; Liang, Chunshuang; Wang, Ying; Deng, Mengyu; Shang, Hongxing; Yang, Bing; Jiang, Shimei

2015-02-14

An "off-the-shelf" fluorescence "turn-on" Mg(2+) chemosensor 3,5-dichlorosalicylaldehyde (BCSA) was rationally designed and developed. This proposed sensor works based on Mg(2+)-induced formation of the 2 : 1 BCSA-Mg(2+) complex. The coordination of BSCA to Mg(2+) increases its structural rigidity generating a chelation-enhanced fluorescence (CHEF) effect which was confirmed by single crystal XRD studies of the BSCA-Mg(2+) complex and TD/DFT calculations. This sensor exhibits high sensitivity and selectivity for the quantitative monitoring of Mg(2+) with a wide detection range (0-40 μM), a low detection limit (2.89 × 10(-7) mol L(-1)) and a short response time (<0.5 s). It can also resist the interference from the other co-existing metal ions, especially Ca(2+). Consequently, this fluorescent sensor can be utilized to monitor Mg(2+) in real time within actual samples from drinking water.

Correlations between quantitative fat–water magnetic resonance imaging and computed tomography in human subcutaneous white adipose tissue

PubMed Central

Gifford, Aliya; Walker, Ronald C.; Towse, Theodore F.; Brian Welch, E.

2015-01-01

Abstract. Beyond estimation of depot volumes, quantitative analysis of adipose tissue properties could improve understanding of how adipose tissue correlates with metabolic risk factors. We investigated whether the fat signal fraction (FSF) derived from quantitative fat–water magnetic resonance imaging (MRI) scans at 3.0 T correlates to CT Hounsfield units (HU) of the same tissue. These measures were acquired in the subcutaneous white adipose tissue (WAT) at the umbilical level of 21 healthy adult subjects. A moderate correlation exists between MRI- and CT-derived WAT values for all subjects, R2=0.54, p<0.0001, with a slope of −2.6, (95% CI [−3.3,−1.8]), indicating that a decrease of 1 HU equals a mean increase of 0.38% FSF. We demonstrate that FSF estimates obtained using quantitative fat–water MRI techniques correlate with CT HU values in subcutaneous WAT, and therefore, MRI-based FSF could be used as an alternative to CT HU for assessing metabolic risk factors. PMID:26702407

Isotropically sensitive optical filter employing atomic resonance transitions

DOEpatents

Marling, John B.

1981-01-01

An ultra-high Q isotropically sensitive optical filter or optical detector employing atomic resonance transitions. More specifically, atomic resonance transitions utilized in conjunction with two optical bandpass filters provide an optical detector having a wide field of view (.about.2.pi. steradians) and very narrow acceptance bandwidth approaching 0.01 A. A light signal to be detected is transmitted through an outer bandpass filter into a resonantly absorbing atomic vapor, the excited atomic vapor then providing a fluorescence signal at a different wavelength which is transmitted through an inner bandpass filter. The outer and inner bandpass filters have no common transmission band, thereby resulting in complete blockage of all optical signals that are not resonantly shifted in wavelength by the intervening atomic vapor. Two embodiments are disclosed, one in which the light signal raises atoms contained in the atomic vapor from the ground state to an excited state from which fluorescence occurs, and the other in which a pump laser is used to raise the atoms in the ground state to a first excited state from which the light signal then is resonantly absorbed, thereby raising the atoms to a second excited state from which fluorescence occurs. A specific application is described in which an optical detector according to the present invention can be used as an underwater detector for light from an optical transmitter which could be located in an orbiting satellite.

Quantitative analysis of comparative genomic hybridization

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

Manoir, S. du; Bentz, M.; Joos, S.

1995-01-01

Comparative genomic hybridization (CGH) is a new molecular cytogenetic method for the detection of chromosomal imbalances. Following cohybridization of DNA prepared from a sample to be studied and control DNA to normal metaphase spreads, probes are detected via different fluorochromes. The ratio of the test and control fluorescence intensities along a chromosome reflects the relative copy number of segments of a chromosome in the test genome. Quantitative evaluation of CGH experiments is required for the determination of low copy changes, e.g., monosomy or trisomy, and for the definition of the breakpoints involved in unbalanced rearrangements. In this study, a programmore » for quantitation of CGH preparations is presented. This program is based on the extraction of the fluorescence ratio profile along each chromosome, followed by averaging of individual profiles from several metaphase spreads. Objective parameters critical for quantitative evaluations were tested, and the criteria for selection of suitable CGH preparations are described. The granularity of the chromosome painting and the regional inhomogeneity of fluorescence intensities in metaphase spreads proved to be crucial parameters. The coefficient of variation of the ratio value for chromosomes in balanced state (CVBS) provides a general quality criterion for CGH experiments. Different cutoff levels (thresholds) of average fluorescence ratio values were compared for their specificity and sensitivity with regard to the detection of chromosomal imbalances. 27 refs., 15 figs., 1 tab.« less

Deciphering the fluorescence resonance energy transfer from denatured transport protein to anthracene 1,5 disulphonate in reverse micellar environment

NASA Astrophysics Data System (ADS)

Singharoy, Dipti; Bhattacharya, Subhash Chandra

2017-12-01

Constrained environmental effect inside AOT reverse micellar media has been employed in this work to collect the information about energy transfer efficacy between sodium salt of anthracene 1,5 disulphonate (1,5-AS) with model transport proteins, bovine serum albumin (BSA), and human serum albumin (HSA). Steady state, time-resolved fluorescence and circular dichroism techniques have been used for this purpose and corresponding Fӧrster-type resonance energy transfer (FRET) from tryptophan residues to 1,5-AS indicates that 1,5-AS binds in the vicinity of the tryptophan residue (BSA and HSA) with equal strength. Indication of protein damage from fluorescence data and its confirmation has been measured from CD measurement. Molecular modeling study hereby plays a crucial role to predict the minimum energy docked conformation of the probe inside the protein environment. From the docked conformation the distance between 1,5-AS and tryptophan moiety of BSA/HSA has successfully explained the FRET possibility between them. A comparative modeling study between BSA and HSA with 1,5-AS assigning their binding site within specific amino acids plays a crucial role in support of the FRET study.

Laboratory Information Bulletin: Quantitation of Aflatoxin M1 in Bovine Milk by Liquid Chromatography with Fluorescence Detection.

PubMed

Vega, Victor A; Young, Michelle; Todd, Sarah

2016-01-01

An extraction for aflatoxin M1 from bovine milk samples is described. The samples were extracted by adding 10 mL acetonitrile to 10 g of sample. The extract was salted out with sodium chloride and magnesium sulfate to separate the water and acetonitrile. The organic layer was dried down and reconstituted in water before being subjected to an immunoaffinity column for cleanup. Once the analyte was isolated, quantitation was obtained by LC with fluorescence detection. LC/fluorescence parameters were optimized with an Agilent Poroshell 120 C18 LC column resulting in a 4 min run time. To test the procedure's robustness, three different kinds of matrixes were fortified at three different levels each. Whole milk, reduced fat milk, and skim milk samples were fortified at approximately 0.25, 0.5, and 1.0 μg/kg. Recoveries from all samples ranged from 70 to 100%. Confirmation was accomplished by injecting the samples in an ion trap mass spectrometer. The method presented here entails an extraction step followed by an immunoaffinity column clean-up that leads to fast analysis time and consistent recoveries with an uncertainty measurement of 10.5% and method detection limit of less than 0.011 μg/kg.

[A new method of processing quantitative PCR data].

PubMed

Ke, Bing-Shen; Li, Guang-Yun; Chen, Shi-Min; Huang, Xiang-Yan; Chen, Ying-Jian; Xu, Jun

2003-05-01

Today standard PCR can't satisfy the need of biotechnique development and clinical research any more. After numerous dynamic research, PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700. But the error of this technique is too great to satisfy the need of biotechnique development and clinical research. A better quantitative PCR technique is needed. The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system. This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions, and can reflect the accumulating rule of PCR product molecule accurately. Accurate quantitative PCR analysis can be made use this function relation. Accumulated PCR product quantity can be obtained from initial template number. Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used. For an example, when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1,000,000, the quantitative result accuracy will be more than 99%. The difference of result error is distinct using same condition,same instrument but different analysis method. Moreover,if the PCR quantitative analysis system is used to process data, it will get result 80 times of accuracy than using CT method.

Comparative analysis of fluorescent angiography, computed tomographic angiography and magnetic resonance angiography for planning autologous breast reconstruction

PubMed Central

Chae, Michael P.; Hunter-Smith, David J.

2015-01-01

Background The high incidence of breast cancer and growing number of breast cancer patients undergoing mastectomy has led to breast reconstruction becoming an important part of holistic treatment for these patients. In planning autologous reconstructions, preoperative assessment of donor site microvascular anatomy with advanced imaging modalities has assisted in the appropriate selection of flap donor site, individual perforators, and lead to an overall improvement in flap outcomes. In this review, we compare the accuracy of fluorescent angiography, computed tomographic angiography (CTA), and magnetic resonance angiography (MRA) and their impact on clinical outcomes. Methods A review of the published English literature dating from 1950 to 2015 using databases, such as PubMed, Medline, Web of Science, and EMBASE was undertaken. Results Fluorescent angiography is technically limited by its inability to evaluate deep-lying perforators and hence, it has a minimal role in the preoperative setting. However, it may be useful intraoperatively in evaluating microvascular anastomotic patency and the mastectomy skin perfusion. CTA is currently widely considered the standard, due to its high accuracy and reliability. Multiple studies have demonstrated its ability to improve clinical outcomes, such as operative length and flap complications. However, concerns surrounding exposure to radiation and nephrotoxic contrast agents exist. MRA has been explored, however despite recent advances, the image quality of MRA is considered inferior to CTA. Conclusions Preoperative imaging is an essential component in planning autologous breast reconstruction. Fluorescent angiography presents minimal role as a preoperative imaging modality, but may be a useful intraoperative adjunct to assess the anastomosis and the mastectomy skin perfusion. Currently, CTA is the gold standard preoperatively. MRA has a role, particularly for women of younger age, iodine allergy, and renal impairment. PMID

Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.

PubMed

Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

2013-11-01

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

Imaging of activated caspase-3 in living cell by fluorescence resonance energy transfer during photosensitization-induced apoptosis

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Chen, Qun; Tang, Yonghong

2005-01-01

Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.

Investigation of tryptophan-NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy

NASA Astrophysics Data System (ADS)

Jyothikumar, Vinod; Sun, Yuansheng; Periasamy, Ammasi

2013-06-01

A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label-free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.

Magnetic resonance imaging retinal oximetry: a quantitative physiological biomarker for early diabetic retinopathy?

PubMed

Yang, Y; Zhu, X R; Xu, Q G; Metcalfe, H; Wang, Z C; Yang, J K

2012-04-01

To assess the efficacy of using magnetic resonance imaging measurements of retinal oxygenation response to detect early diabetic retinopathy in patients with Type 2 diabetes. Magnetic resonance imaging was conducted during 100% oxygen inhalation in patients with Type 2 diabetes with either no diabetic retinopathy (n = 12) or mild to moderate background diabetic retinopathy (n = 12), as well as in healthy control subjects (n = 12). Meanwhile, changes in retinal oxygenation response were measured. In the healthy control group, levels of retinal oxygenation response increased slowly during 100% oxygen inhalation. In contrast, they increased more quickly and attained homeostasis much earlier in the groups with background diabetic retinopathy (at the 20-min time point) and with no diabetic retinopathy (at the 25-min time point) than in the healthy control group (at the 42-min time point). Furthermore, levels of retinal oxygenation response in the group with background diabetic retinopathy increased more than that of the group with no diabetic retinopathy, which in turn increased more than that of the healthy control group. There are statistically significant differences between the group with background diabetic retinopathy and the healthy control group at 6-, 8-, 10-, 15-, 20- and 25-min time points (P < 0.05). According to the normal range of the healthy control group by setting fundus photography results as 'gold standard' in our research, the sensitivity, specificity, positive predictive value, negative predictive value and receiver operating characteristic area for reporting the early indications of utility of diabetic retinopathy were 83.33%, 58.33%, 50%, 87.5% and 0.774, respectively. The results indicate that magnetic resonance imaging is a potential screening method and probably a quantitative physiological biomarker to find early diabetic retinopathy in patients with Type 2 diabetes. © 2011 The Authors. Diabetic Medicine © 2011 Diabetes UK.

Double-wavelength overlapping resonance Rayleigh scattering technique for the simultaneous quantitative analysis of three β-adrenergic blockade

NASA Astrophysics Data System (ADS)

Tan, Xuanping; Yang, Jidong; Li, Qin; Yang, Qiong; Shen, Yizhong

2016-05-01

Four simple and accurate spectrophotometric methods were proposed for the simultaneous determination of three β-adrenergic blockade, e.g. atenolol, metoprolol and propranolol. The methods were based on the reaction of the three drugs with erythrosine B (EB) in a Britton-Robinson buffer solution at pH 4.6. EB could combine with the drugs to form three ion-association complexes, which resulted in the resonance Rayleigh scattering (RRS) intensity that is enhanced significantly with new RRS peaks that appeared at 337 nm and 370 nm, respectively. In addition, the fluorescence intensity of EB was also quenched. The enhanced scattering intensities of the two peaks and the fluorescence quenched intensity of EB were proportional to the concentrations of the drugs, respectively. What is more, the RRS intensity overlapped with the double-wavelength of 337 nm and 370 nm (so short for DW-RRS) was also proportional to the drugs concentrations. So, a new method with highly sensitive for simultaneous determination of three bisoprolol drugs was established. Finally, the optimum reaction conditions, influencing factors and spectral enhanced mechanism were investigated. The new DW-RRS method has been applied to simultaneously detect the three β-blockers in fresh serum with satisfactory results.