text:
|Publication number
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|Publication date
|Oct 19, 2004
|Filing date
|Sep 8, 1999
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|Sep 8, 1998
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|Joseph R. Lakowicz, Ignacy Gryczynski
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|University Of Maryland, Baltimore
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|Patent Citations (12), Non-Patent Citations (20), Referenced by (20), Classifications (14), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a provisional of Ser. No. 60/099,499 filed on Sep. 8, 1998.
This application was made with Government support under Grant No. RR-08119 from the National Institutes of Health National Center for Research Resources. The federal government may have certain rights in this invention.
Fluorescence is widely used in analytical and clinical chemistry (Schulman, 1993; Spichiger-Keller, 1998; Wolfbeis, 1991a; Wolfbeis, 1991b; Kunz. 1996; Lakowicz, 1994a; Thompson, 1997). In the past several years there has been increasing interest in the use of time-resolved fluorescence as an analytical tool, e.g., for non-invasive sensing (Szmacinski and Lakowicz, 1994a; Lippitsch et al., 1997; Lippitsch et al., 1988; Bambot et al., 1995; Spichiger-Keller, 1998; Fraser, 1997). The basic idea is to identify fluorophores or sensing schemes in which the decay time of the sample changes in response to the analyte, and to use the decay time to determine the analyte concentration. Such lifetime-based sensing is most often performed using the phase-modulation method. The use of phase angles or decay times rather than intensities is advantageous because decay times are mostly independent of the signal level and can be measured in turbid media and even through skin (Bambot et al., 1995; Szmacinski and Lakowicz, 1996).
Most fluorophores used for sensing display decay times on the nanosecond timescale. It now appears possible to design low cost instruments for sensors with ns decay times. For instance, it is known that blue and UV light emitting diodes (LEDs) can be modulated to over 100 MHz and used as the excitation source in phase-modulation fluorometry (Sipior et al., 1997; Sipior et al., 1996; Lakowicz et al., 1994a; Randers-Eichorn et al., 1997). However, it may be useful to avoid the use of frequencies near 100 MHz which are needed for ns decay time measurements, and thus use the simpler electronics for lower frequencies. Also, a significant fraction of sensing fluorophores display changes in intensity without changes in lifetime.
What are the advantages of low frequency modulation sensing? It is now accepted that lifetime-based sensing can be preferable to intensity-based sensing because the lifetimes are mostly independent of changes in probe concentration and/or signal level. Modulation sensing shows many of these advantages. The modulation will be independent of the total signal level. Hence, modulation sensing can be accurate even if the overall signal level changes due to flexing in fiber optics or changes in the positioning of the sample. However, it is necessary that the relative proportions of the short and long lifetime fluorophores remain the same. If the relative intensities change, in a manner independent of analyte concentration, then the modulation calibration curve will also change. Hence, the calibration curves for a modulation sensor will change if the sensing and reference fluorophore photobleach at different rates.
An advantage of low frequency modulation sensing is the simple instrumentation One can imagine simple hand-held instruments for modulation intensity measurements (FIG. 1). It is now well known that light emitting diodes can be easily modulated at frequencies up to 100 MHz (Sipior et al., 1997; Sipior et al., 1996; Lakowicz et al., 1994a; Randers-Eichorn et al., 1997). Also, LEDs are available within a range of output wavelengths, even down to the near UV at 390 nm (Sipior et al., 1997). LEDs consume little power and can easily be driven by batteries. Hence, the modulation sensor could be a small device held near the skin. The long-lifetime complex can be part of the device, so none of the long-lifetime probe enters the sample or tissue. The high chemical and photochemical stability of the metal-ligand complexes suggests the signal from the long lifetime reference will be constant for long periods of time. Hence, such devices may prove valuable for quantitation of intrinsic and extrinsic fluorophores in tissues.
Additionally, there has been considerable progress in the design and synthesis of long lifetime metal-ligand complexes. Ruthenium, osmium and rhenium complexes have been reported (Lakowicz et al., 1995; Terpetschnig et al., 1997; Castellano et al., 1998). The rhenium complexes are particularly useful in that they display high quantum yields and lifetimes up to 3 μs in oxygenated solution (Guo et al., 1998; Guo et al., 1997).
And finally, the most important advantage of modulation sensing may be the expanded range of analytes. Any sensing fluorophore which changes intensity can be used in this model. A change in probe lifetime is not needed. Hence, modulation sensing can be used with probes such as a sodium-binding benzofuran isophthalate (SBFI) and a potassium-binding benzofuran isophthalate (PBFI), which are poor wavelength-ratiometric probes for sodium and potassium.
The possibility of non-invasive sensing is based on the low absorbance of tissue at red and near-infrared (NIR) wavelengths, and the increasing availability of long wavelength fluorophores (Lakowicz, 1994b; Matsuoka, 1990; Leznoff and Lever, 1989). However, it is difficult to perform intensity measurements in highly scattering media (Oelkrug, 1994), which has led to interest in the use of time-resolved fluorescence and lifetime-based sensing for non-invasive fluorometry (Szmacinski and Lakowicz, 1994a; Hutchinson et al., 1995; Szmacinski and Lakowicz, 1994b). It is now known that fluorescence lifetimes can be measured in the presence of extensive light scattering, and can even be measured through skin (Bambot et al., 1995).
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
Described here is a new method which allows quantitative measurements of fluorescence intensity with simple instrumentation. The method is self-calibrating related to a reference fluorophore. The method can also be used in highly scattering media. The measurement principle is based on observing the emission from both the fluorophore of interest with a ns decay time and of a reference fluorophore which displays a much longer microsecond lifetime. The reference fluorophore is placed on rather than in the sample to mimic a sensing device with the long lifetime reference held against the skin. The amplitude modulation of the emission is observed using the standard method of frequency-domain fluorometry. At an intermediate modulation frequency, the modulation is equivalent to the fractional intensity of the ns fluorophore. The method was tested in 0.5% intralipid, which is more highly scattering than skin. Quantitative intensity measurements were obtained for various concentrations of fluorescein in intralipid, and of the pH sensor 6carboxy fluorescein. Low frequency modulation measurements provide a general method for quantitative measurements in the presence of factors which preclude direct intensity measurements, or applications which require simple internally referenced measurements.
FIG. 1 is a schematic of a portable modulation intensity sensor.
FIG. 2 shows emission spectra of fluorescein in 0.5% intralipid. Excitation at 488 nm. The dashed line shows the transmission curve of the emission filter. Emission for [Ru(bpy)3]2+ in the PVA film is responsible for the weak emission near 600 nm.
FIGS. 3A and 3B are schematics of sensors with a ns fluorophore and [Ru(bpy)3]2+.
FIG. 4 shows emission spectra of the model sensor containing fluorescein in the internal aqueous phase and [Ru(bpy)3]2+ in an external PVA film.
FIG. 5 shows the frequency-domain intensity decay of the fluorescein-[Ru(bpy)3]2+ model sensor.
FIG. 6 shows emission spectra of a pH sensor based on a mixture of 6-carboxyfluorescein (6-CF) and [Ru(bpy)3]2+.
FIG. 7A shows the frequency response of the pH sensor and
FIG. 7B shows the low frequency modulation of the pH sensor.
FIG. 8 shows a calculation curve of the pH sensor.
FIG. 9 shows emission spectra of a modulated calcium sensor based on Fluo-3 and [Ru(bpy)3]2+.
FIG. 10 shows a modulation frequency response of the calcium sensor.
FIG. 11 shows phase angle frequency response of the calcium sensor.
FIGS. 12A and 12B show phase and modulation calibration curves of the calcium sensor.
FIGS. 13A and 13B show frequency-domain intensity decay of the intralipid sample containing fluorescein. The cuvette did not have the external [Ru(bpy)3]2+-PVA film.
FIG. 14 shows a frequency response of the fluorescein-[Ru(bpy)3]2+ -intralipid sample shown in FIG. 1.
FIG. 15 shows low frequency modulation of the fluorescein-[Ru(bpy)3]2+ intralipid sample shown in FIG. 2.
FIG. 16 shows modulation versus fluorescein concentration.
FIG. 17 shows emission spectra of 6carboxy fluorescein in 0.5% intralipid. The experimental arrangement is similar to that shown in FIG. 2, including the long-lifetime standard.
FIGS. 18A and 18B show the frequency response of the 6-carboxy fluorescein-[Ru(bpy)3]2+-intralipid sample shown in FIG. 6. The bottom panel shows the low frequency modulation on an expanded scale.
FIG. 19 shows modulation versus pH calibration curve for 6-CF in 0.5% intralipid.
FIGS. 20A-L show representative fluorophores which are not sensitive to analytes.
Described here is a general method to perform sensing at low light modulation frequencies near 1 MHz using sensing fluorophores with ns lifetimes, without relying on a change in lifetime of the ns fluorophore. The basic idea is to use a mixture of the ns fluorophore with a fluorophore which displays a long lifetime near 1 μs. For such a mixture the modulation of the emission at intermediate frequencies becomes equivalent to the fraction of the total emission due to the short lifetime ns fluorophore. This occurs because the emission from the μs fluorophore is demodulated, and that of the ns fluorophore is near unity. This methods allows sensing based on modulation from about 1 to 10 MHz. Additionally, the ns sensing fluorophore does not need to display a change in lifetime. A simple change in intensity in response to the analyte is adequate for a low frequency modulation sensor.
The feasibility of this sensing scheme was demonstrated to be practical in several systems. First, the method was validated using a simple mixture of ns and μs fluorophores. We then devised assays for pH, and calcium. We note that this method is generic, and can be applied to any analyte for which an intensity-based sensor is available.
Abbreviations used in this disclosure are: PVA—polyvinyl alcohol; bpy—2,2′-bipyridyl; 6-CF—6-carboxyfluorescein; FD—frequency domain; BCECF is 2′,7′-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein; [Ru(bpy)2(dcbpy)]2+ is Bis(2,2′-bipyridine)(2,2′-bipyridine-4,4′-dicarboxylic acid)ruthenium(II); C. is carboxy; SNAFL is seminaphtofluorescein; and SNARF is seminaphtorhodafluor.
The methods described herein allow measurement of many analytes. These include, but are not limited to, H+, pH, Na+, K+, Li+, Mg2+, Ca2+, Cl−, HCO3 −; CO2, O2, glucose, lactate, antigens and drugs. A large number of different fluorophores can be used as the sensing fluorophores and as the long lifetime fluorophores. Analyte sensing fluorophores include, but are not limited to, Quin-2, Fura-2, Indo-1, Calcium Green, Calcium Orange, Calcium Crimson and Benzoxazine-crown which are useful as Ca2+ probes; Mag-Quin-2, Magnesium Green, and Benzoxazine-crown as Mg2− probes; PBFI as a K+ probe, Sodium Green as a Na+ probe, and SNAFL-1, C. SNAFL-1, C. SNAFL-2, C. SNARF-1, C. SNARF-2, C. SNARF-6, C. SNARF-X, BCECF, and Resorufin Acetate as pH probes. Probes for Cl− include 6-methoxy-N-ethylquinolinium chloride; N6-methoxyquinolyl)acetoethyl ester; 6-methoxy-N-ethylquinolinium chloride; 6-methoxy-N-(3-trimethylammoniumpropyl)quinolinium dibromide; 6methoxy-N-(3-trimethylammoniumpropyl)phenanthrindium dibromide; and 6methoxy-N-(4-aminoalkyl)quinolinium bromide hydrochloride. The above fluorophores are described in Szmacinski and Lakowicz 1994a. Other chloride probes include 6-methoxy-N-(3-sulfoproxyl)quinolinium; N-sulfopropylacridinium; N,N-dimethyl-9,9′-bisacridinium nitrate; N-methylacridinium-9-carboxamides; and N-methylacridinium-9-methylcarboxylate. Additional probes useful for measuring pH include 8-hydroxypyrene-1,3,6-trisulfonate; [Ru(4,4′-diethylaminomethyl-2,2′-bipyridine)(2,2′-bipyridine)2]2+; Oregon Green, DM-NERF and Cl-NERF. Additional fluorophores sensitive to Mg2+ include Mag-Quin-1; Mag-Fura-2; Mag-Fura-5, Mag-Indo-1; Mag-Fura-Red; and Mg Orange. Additional fluorophores sensitive to Na+ include sodium-binding benzofuran isophthalate and sodium-binding benzofuran oxazole. Additional fluorophores sensitive to K+ include CD222. Additional fluorophores sensitive to Ca2+ include Fura Red; BTC (coumarin benzothiazole-based indicator); Fluo-3; Rhod-2; Ca Green-2; Ca Green-5N; Ca Orange-SN; Oregon Green - BAPTA-1, BAPTA-2 and BAPTA-5N. These additional analyte sensitive fluorophores are discussed in Lakowicz, 1999.
Fluorophores which are not sensitive to the analyte being tested but are used in conjunction with the analyte sensitive fluorophores include, but are not limited to, the compounds shown in FIGS. 20A-L.
In measuring analytes such as glucose, lactate, drugs or antigens, it is desirable to use a binding agent labeled with a fluorophore such that the binding agent binds specifically to the analyte. For example, a glucose-binding protein, a glucose-galactose binding protein, or concanavalin A can be used in a method to measure glucose. A lactate binding protein can be used for measurement of lactate. Antibodies or antibody fragments can be used to measure drugs or antigens. Other binding agents known to those of skill in the art can also be used.
While the use of lifetime measurements solves many problems associated with non-invasive sensing, it is still desirable to develop methods which would allow quantitative intensity measurements through skin and in the presence of extensive scattering. Described here is a simple method to measure fluorescence intensities in scattering media. Fluorescence is defined as meaning fluorescence, phosphorescence, luminescence or mixed-state emissions. This method is based on measurement of the modulation of the emission when the sample is excited with amplitude modulated light. In this method, the emission of the scattering sample is observed along with the emission of a long lifetime reference fluorophore. The reference fluorophore need not be within the sample, but can be on the surface outside the sample. This geometry is useful for sensing devices in which the long lifetime reference is held against the skin. The light modulation and measurement frequency is chosen so that the emission from the long-lifetime reference is completely demodulated. Under these conditions, the intensity of the fluorophore of interest with a ns decay time is given by the modulation of the total signal. This method can be applied to any sample which displays changes in intensity, or any sensing fluorophore which shows intensity changes due to its response to an analyte of interest. One can imagine this method being used with a hand-held instrument for point-of-care transdermal measurements. The method can be performed on a variety of systems or samples, including in vivo, blood plasma, whole blood, saliva, any body fluid, tissue culture, a sample from an aquarium, etc. Furthermore, the method may be used to monitor a bioprocessing reaction, industrially, in process control, as part of an analytical chemistry process, etc. The incident light can be produced by, but is not limited to, a laser, a light emitting diode (LED) or an electroluminescent light source (ELL).
Intensity decays were measured using the frequency-domain method. This method and procedure for data analysis have been described in detail (Lakowicz and Gryczynski, 1991; Lakowicz, in press; Lakowicz et al., 1984; Gratton et al., 1984; Lakowicz and Gryczynski, 1991). In this method one measures the phase (φω) and modulation (mω) of the emission for various values of the light modulation frequency (ω, in radians/sec). A number of different models were used to analyze the FD data. Initially, the intensity decays were analyzed in terms of the multi-exponential model,
where αi are the pre-exponential factors and τi the decay times. The fractional steady state intensity disassociated with each decay time is given by
Generally, Σαi and the Σfi are constrained to be equal to unity.
For the present disclosure, it is instructive to consider the theory for a sample which displays two widely different decay times, the short (τS) and long (τL) decay times. For a mixture of fluorophores the phase and modulation can be calculated using
where fi is the fractional intensity, φi is the phase angle and mi is the modulation of each fluorophore. The terms Nω and Dω are typically referred to as the sin and cosine transforms of the intensity decays (Lakowicz et al., 1984; Gratton et al., 1984). If each fluorophore displays a single exponential decay time τ then
For a multi-exponential decay the phase and modulation at any given frequency w are given by
In the present study the samples display two widely different decay times, which we will refer to as the short (τS) and long (τL) decay times. In this case the values of N and D are given by
where we have dropped the subscript ω referring to the light modulation frequency. The total fractional intensity is normalized fS+fL=1.0. If the two lifetimes are very different one can identify intermediate frequencies where the modulated emission of the short component is high is and that of the long lifetime component is low. Suppose the sample is examined at an intermediate modulation frequency such that the modulation of the short component is unity (mS=1.0) and the modulation of the long component is near zero (mL=0.0). In this case
Using equation 8, and recalling the sin2θ+cos2θ=1.0, one obtains
This is a useful result which indicates that the modulation of the emission is the fractional intensity of the short lifetime component. Hence, one can use the modulation of the emission to measure the fractional fluorescence intensity of the short lifetime fluorophore. In the present disclosure the short lifetime fluorophores were selected to display changes in intensity in response to the analyte. The long lifetime fluorophore is [Ru(bpy)3]2+, and is not sensitive to changes in pH or calcium over the investigated concentration ranges.
Measurements of nanosecond and subnanosecond lifetimes in scattering media can be affected by the time-dependent migration of photons due to the multiple scattering events. This topic has been described in detail (Hutchinson et al., 1995; Szmacinski and Kakowicz, 1994b). When a pulse of light enters the scattering media, the diffusely scattered light is delayed in time and the pulse is broadened. These effects can be described phenomenologically in terms of a time delay (tL), a decay time for the light in the tissues (tD) and a term describing the pulse broadening (Δt) (Szmacinski and Lakowicz, 1994b). For a fluorophore which displays a lifetime τ the phase and modulation measurement in scattering media is give by (Szmacinski and Lakowicz, 1994b),
Examination of Eqs. 14 and 15 indicate that the effects of the lifetime (τ) and the photon decay time (tD) are similar. Fortunately, the values of the decay time tD are modest for all but the shortest lifetimes. For instance, the value of tD for a position 4 mm deep in the 1% intralipid is about 140 ps (Szmacinski and Lakowicz, 1994b). Hence, for lifetimes 1 ns and longer, use of the values of φS ω and mS ω provides a good approximation (τapp) to the true lifetime (τ)
Some of the data were analyzed in terms of this model. Hence, we can expect an apparent lifetime for a fluorophore in scattering media to be 0.1 to 0.2 ns longer than in the absence of light scatter. We note the difference between the true and apparent lifetime depends on the method used to measure the phase and modulation of the excitation light. Importantly, the effects due to time-dependent photon migration occur mostly on the subnanosecond timescale and at frequencies above 100 MHz. Hence, it is not necessary to consider these effects for the low-frequency modulation measurements.
The parameters describing the intensity decays were obtained by non-linear least squares and minimization of χR 2,
In this expression, the subscript c indicates calculated values for assumed parameter values. The terms δφ and δm are the uncertainties in the phase and modulation data, respectively, and ν is the number of degrees of freedom. For the present measurements, we used of δφ=0.3� and δm=0.007.
The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.
The following reagent grade chemicals were obtained from commercial sources and used as received: Ru(bpy)3Cl26H2O (Aldrich), 6-carboxyfluorescein (6-CF) (Eastman/Kodak), Fluo-3 pentapotassium salt (Molecular Probes), calcium calibration buffer kit #2 (C-3009, Molecular Probes), and TRIS (Sigma). Disodium fluorescein was from Exciton, Inc. Intralipid (20%) was obtained from Kabi Vitrum, Inc. and diluted 40-fold to 0.5%. Water was deionized with a Milli-Q purification system. Solutions ranging from pH 5.0 to 9.1 were prepared in 50 mM TRIS buffer for the 6-carboxyfluorescein studies. Experiments using the Ca2+ indicator Fluo-3 were performed solely in plastic media to prevent uptake of Ca2+ from glass surfaces. All solutions were prepared in plastic vials from the calcium calibration buffer. Luminescence measurements were performed in polystyrene plastic cuvettes. In one experiment Ru(bpy)3Cl2 was in a polyvinyl alcohol film on the outside of the cuvette. In all other studies a small aliquot of aqueous Ru(bpy)3Cl26H2O was added to solutions containing the nanosecond fluorophore (6-carboxyfluorescein or Fluo-3) with analyte. We used these two different configurations to demonstrate the versatility of this approach to sensing. Typically, the steady-state emission intensity of Ru(bpy)3Cl26H2O contributed about 20-30% of the total sample intensity at the highest pH (6-carboxyfluorescein) or at the highest Ca2+ concentration (Fluo-3). All samples were optically dilute (<0.1 OD) at 488 nm.
Experiments were performed using the geometry shown as an insert in FIG. 2. The intralipid sample was contained in a 2 mm�10 mm cuvette, with the 488 nm excitation incident on the wider 10 nm surface. This surface of the cuvette was covered with a film of polyvinyl alcohol (PVA) which contained the long-lifetime fluorophore Ru(bpy)3Cl2. The emission was observed from the 2 mm side through a filter which transmitted part of the fluorescein emission and most of the [Ru(bpy)3]2+ emission. The transmission curve of this filter is shown as a dashed line in FIG. 2.
UV-Vis absorption spectra were measured on a Hewlett Packard 8453 diode array spectrophotometer with �1 nm resolution. Uncorrected steady-state emission spectra were obtained on a SLM AB-2 fluorimeter under magic angle polarization conditions. Time-resolved luminescence decays were measured in the frequency domain (ISS, Koala) using an air cooled cw-Ar+ laser (Omnichrome, 543-AP) operating at 488.0 nm (80 mW) as the excitation source. The 488 nm output was amplitude modulated using an electro-optic modulator, which provided modulated excitation from 300 kHz to 150 MHz. This frequency-domain instrument was comparable to those described previously (Gratton and Limkeman, 1983; Lakowicz and Maliwal, 1985).
The laser was passed through a Pockels cell which provided modulated light from 300 kHz to 150 MHz Two different PTS frequency synthesizers (PTS-500) were used to modulate the Pockels cell and detection system. The output of the PTS synthesizer driving the Pockels cell was amplified by an ENI 25 W linear RF amplifier (325 LA, 250 kHz-150 MHz) prior to Pockels cell input. The other PTS synthesizer output was directed into an ENI 3 W linear RF amplifier (403 LA, 150 kHz-300 MHz) for proper modulation of the detection system. The emission was observed as usual at 90� to the excitation through an appropriate combination of long-pass filters which eliminated scattered light at the excitation wavelength. We used this experimental configuration because it was available in this laboratory. In the actual use of modulation sensing we expect the light source will be an intensity modulated LED or some other solid state light source.
The intensity decays were analyzed in terms of the multi-exponential model,
where αi are the pre-exponential factors and τi the decay times. The fractional steady state intensity dissociated with each decay time is given by
Generally, Σαi and the Σfi are constrained to be equal to unity. Rhodamine B in water with a lifetime of 1.68 ns was used as a lifetime reference in the frequency-domain experiments (Gryczynski et al., 1997). Luminescence decays were analyzed by non-linear least squares procedures described previously (Lakowicz et al., 1984, Gratton et al., 1984). Global analysis of frequency-domain emission decay data was performed with programs developed at the Center for Fluorescence Spectroscopy, In the global analysis, the lifetimes were the global parameters and the amplitudes were fitted as non-global parameters. This means that the lifetimes were fitted parameters, but were constrained to be the same values at all analyte concentrations or all pH values for 6-CF. The amplitudes were also fitted parameters, but were allowed to vary at each analyte concentration or pH value.
To test the principal of modulation sensing we performed frequency domain measurements for a sample which displayed emission from both [Ru(bpy)3]2+ and fluorescein. In this case the two fluorophores were physically separated, which is a useful configuration if the sensing fluorophore can interact with the reference fluorophore. The long lifetime fluorophore [Ru(bpy)3]2+ was dissolved in melted polyvinyl alcohol (PVA) which was painted on the outer surface of the cuvette (FIG. 3A). Aqueous solutions of fluorescein were placed within the cuvette. This model assay was excited at 488 nm with an argon ion laser. Emission spectra of this sample are shown in FIG. 4. The fluorescein dominates the emission spectra with a maximum near 510 nm. The emission from [Ru(bpy)3]2+ occurs at longer wavelengths with an emission maxima near 590 nm.
For FD measurements the emission was observed through a Corning 3-72 filter, which transmitted the emission from both 6-CF and [Ru(bpy)3]2+ (FIG. 4). The relative proportion of the two emissions were varied by dilution of the fluorescein within the cuvette (FIG. 4). The concentration and emission intensity of [Ru(bpy)3]2+ remained the same as the fluorescein was diluted.
FIG. 5 shows the frequency-domain intensity decays of this model assay. The emission is already demodulated below 1.0 for the lowest measurement frequency near 1.8 MHz. This effect is due to the long decay time of [Ru(bpy)3]2+ in PVA. In separate measurements the decay time of [Ru(bpy)3)2+ in PVA was found to be 1180 ns. The modulation is nearly constant from 2 to 8 MHz. This occurs because the modulation of [Ru(bpy)3]2+ is near zero, and that of fluorescein with a lifetime of 2.85 ns is near 1.0. In this low frequency range the modulation is expected to represent the fractional steady state intensity of the short lifetime emission (eq. 13). Comparison of FIGS. 4 and 5 shows that the modulation values at intermediate frequencies are approximately equal to the intensity of fluorescein relative to that of [Ru(bpy)3]2+.
We use the concept described above to create a pH assay based on the modulation at the intermediate frequencies. As a pH-sensitive fluorophore we selected 6-carboxyfluorescein (6-CF). Fluorescein and its derivatives have been widely used for pH sensing (Thomas et al., 1979; Babcock, 1983: Klonis et al., 1998). Fluorescein and its derivatives display a pH-dependent dissociation of the carboxyl group. The ionized form which exists at pH values above 7.5 is highly fluorescent, and the protonated low pH form is essentially non-fluorescent. For this reason fluorescein is not known to display a change in lifetime when this dissociation reaction occurs.
For the modulation pH assay the 6-CF and [Ru(bpy)3]2+ were both dissolved in the aqueous phase (FIG. 3B). We did not use the external PVA film. Emission spectra of this mixture shows that the 6-CF emission is strongly affected by pH, while the intensity of the long lifetime [Ru(bpy)3]2+ is essentially the same at all pH values (FIG. 6). The emission filter was selected to transmit all the [Ru(bpy)3]2+ emission and part of the 6-CF emission. In this way one can modify the fractional intensity from each fluorophore without changing the actual concentrations.
Frequency-domain intensity decay data for the mixture of 6-CF and [Ru(bpy)3]2+ are shown in FIG. 7A. As expected based on the lifetimes of 6-CF (4.07 ns) and [Ru(bpy)3]2+ (370 ns) there is a region of constant modulation from 2 to 10 MHz. In this region the modulation of the emission increases due to the higher intensity of 6-CF at high pH. The frequency data were analyzed using the multi-exponential model (eq. 1). At all pH values the frequency responses could be fit to two decay times of 4.0 and 370 ns, as can be seen from the global analysis in Table I. As the pH value increases the fractional steady state intensity of the short component (f1) also increases. For mixtures with such different lifetimes it is preferable to use the f1 values, rather than the pre-exponential factors. The value of α1 is over 0.98 at all pH values.
Global Double-Exponential Analysis of the Intensity
Decays of pH Sensor
τ1 (ns)
τ2 (ns)
χR 2
For clinical applications the pH values are typically accurate to �0.02 or better (Mahutte et al., 1994a; Mahutte et al., 1994b; Shapiro et al., 1993; Mahutte, 1994). The present sensor was found to be sensitive to changes in pH of �0.1, as can be seen in the modulation data on an expanded scale (FIG. 7B). For instance, pH values of 6.4 and 6.5 are easily distinguishable. Such data can be used to prepare a calibration curve for pH based on the modulation at 3.7 MHz (FIG. 5). Modulation measurements are readily accurate to �0.007, which results in a pH accuracy of �0.1 (FIG. 8). These results were obtained without optimizing the assay based on the relative intensities of the two species, and the overall change in intensity of the pH-sensitive fluorophore. Inclusion of measurements of the phase angle and modulation at more than one frequency will improve the pH accuracy.
Calcium is known to be an intracellular messenger. Measurements of calcium concentrations have been the subject of numerous publications (Nuccitelli, 1994; Grynkiewicz et al., 1985). Calcium can be measured using intensity-ratiometric probes (Tsien et al., 1985; Kao, 1994; Tsien, 1989; Akkaya and Lakowicz, 1993) or using lifetime-based sensing (Lakowicz et al., 1992a; Hirschfield et al., 1993; Miyoshi et al., 1991; Lakowicz et al., 1992b; Lakowicz and Szmacinski. 1992). However, some calcium probes do not display spectral shifts upon binding calcium, and some probes do not display changes in lifetime. One such calcium probe is Fluo-3, which is highly fluorescent in the presence of bound calcium, but essentially non-fluorescent in the absence of calcium. Because the calcium-free probe does not fluoresce, the emission is due to only the calcium-bound form. For this reason only the calcium-bound form of Fluo-3 contributes to the lifetime, and thus Fluo-3 displays the same lifetime at all calcium concentrations.
While Fluo-3 cannot be used as wavelength-ratiometric or lifetime sensor for calcium, it can be used in our method of modulation sensing. This is shown in FIG. 9. In this assay both Fluo-3 and [Ru(bpy)3]2+ were contained within the cuvette. The emission of Fluo-3 at 510 nm increases dramatically in the presence of Ca2+. The emission intensity of [Ru(bpy)3]2+ is not sensitive to calcium and is the same at all calcium concentrations. The frequency-dependent modulation of the Fluo-3 [Ru-(bpy)3]2+ mixture are shown in FIG. 10. As the calcium concentration increases so does the modulation from 2 to 30 MHz. We also examined the frequency-dependent phase angles (FIG. 11). The phase angles show a maximum near 1 MHz. The largest values near 1 MHz are seen in the absence of calcium, where the emission is dominated by the long lifetime Ru complex. As the calcium concentration increases the phase angles decrease to smaller values as the emission becomes dominated by the shorter lived emission from Fluo-3.
The modulation and phase angle frequency responses (FIGS. 10 and 11) were analyzed in terms of the multi-exponential model (eq. 1). Two decay times were needed to account for the emission from Fluo-3, 0.72 and 1.89 ns (Table II). The data at all calcium concentrations could be globally fit to the same three decay times, with the fractional intensities dependent on the calcium concentration.
The data in FIGS. 10 and 11 were used to prepare calibration curves for calcium (FIGS. 12A and 12B). The modulation values 12.7 MHz show an apparent calcium dissociation constant (KD) for Fluo-3 near 100 nM, which is lower than but comparable to the known dissociation constant near 400 mM (Minta et al., 1989; Kao et al., 1989; Harkins et al., 1993). Precise agreement between the thermodynamic KD value, and the apparent value from time-resolved data is not expected. Depending on the nature of the time-resolved measurements the apparent KD values can be larger or smaller than the true KD value (Szmacinski and Lakowicz, 1994a).
Global Intensity Decay Analysis of the Fluo-3 [Ru(bpy)3]2+
Calcium Sensora Intensity Decay
τ1 = 0.72 ns
τ2 = 1.89 ns
τ3 = 359.8 ns
Ca2+ (μM)
χR 2
Ca2+ Saturated
aIn a calcium saturated solution, without [Ru(bpy)3]2−, Fluo-3 displayed a double exponential intensity decay with τ1 = 0.78 ns, τ2 = 1.92 ns, α1 = 0.768, α3 = 0.232, f1 = 0.575 and f2 = 0.425, χR 2 = 0.9. The χR 2 value for the single decay time fit for Fluo-3 alone was 18.2. In water
# [Ru(bpy)3]2+ displayed a single exponential decay of 370 ns.
The low frequency phase data were also used to prepare a calcium calibration curve (FIG. 12B). In this case the phase angles are sensitive to lower concentrations of calcium, with an apparent KD near 40 nM. This suggests the combined use of both the phase and modulation data to allow measurement of calcium over an extended range of concentrations, or to provide increased accuracy over a critical range of concentrations.
Quantitative intensity measurements in scattering media were tested using various concentrations of fluorescein in intralipid. Emission spectra are shown in FIG. 2. The maximum fluorescein concentration is 12 μm. The emission from this concentration of fluorescein can be easily seen even in 0.5% intralipid. As the fluorescein concentration is decreased, the intensity at 520 nm decreases. All the emission spectra shown in FIG. 2 were collected with the external PVA film containing [Ru(bpy)3]2+, which served as the reference fluorophore. The emission of [Ru(bpy)3]2+ occurs near 600 nm, and is visible on the long wavelength side of the fluorescein emission.
We examined the frequency response of fluorescein in 0.5% intralipid., without the long-lifetime reference (FIGS. 13A and 13B). The data could be fit reasonably well to a single decay time of 4.22 ns, but with a somewhat elevated value of XR 2=4.7. The fit was improved using the models which included the effects of time-dependent photon migration (Eqs. 14-18). Use of this model resulted in a decrease of XR 2 to 1.2. While the differences between the two fits in FIGS. 13A and 13B is not dramatic, our experience with the frequency domain measurements allows us to accept the model resulting in XR 2=1.2. Importantly, the effects due to the scattering media are only significant at frequencies above 100 MHz. Hence. these effects can be neglected in the low-frequency modulation measurements which can be performed near 2 MHz.
We next used the configuration shown in FIG. 2 to test the concept of using the A modulation to measure the intensity. FIG. 14 shows frequency-domain measurements of the emission due to both fluorescein in intralipid and the long-lifetime reference. These data were analyzed globally using equations 14-18 (Table III). The long-lifetime of 1575 ns is essentially equivalent to that observed for [Ru(bpy)3]2+ alone in the PVA film. The lifetime of 3.89 ns is assigned to fluorescein. The modulation data over the range of frequencies is shown on an expanded scale in FIG. 15. One notices a region from 0.3 to 8 MHz over which the modulation is nearly independent of the light modulation frequency.
The modulation data at 1.8 MHz were used to prepare a calibration curve for the fluorescein concentration (FIG. 16). This curve suggests that the intensity measurements can be rather accurate. Using the concentration range from 0 to 6 μM, an uncertainly in the modulation of +/−0.007 results in a fluorescein concentration uncertainty of +/−0.1 μM. Hence, the intensity can be accurate to about one part in sixty, which is probably adequate for most clinical applications.
Global Intensity Decay Analysis of Fluorescein
in 0.5% Intralipid - [Ru(byp)3]2= Doped PVA Film System
τ1 = 3.89 ns
τ2 = 1575 ns
χR 2
Examination of FIG. 16 reveals the modulation does not decrease linearly as the fluorescein concentration is decreased. This effect can be understood by noting that the modulation is approximately equal to the fractional intensity of the short component,
One has to recall that the total intensity in a FD measurement is always normalized to unity, fS+fL=1.0. A simple calculation reveals the origin of the non-linear dependence of the modulation on the total intensity. Suppose the initial intensity of the short component is fS=0.9, and that fL=0.1, resulting in a modulation of 0.9. Now suppose the intensity of the fluorescein emission is decreased two-fold. This can be simulated by setting fS=0.45 and fL=0.1, these values being appropriate prior to normalization. Hence, the two-fold decrease in fluorescein intensity results in a modulation near 0.81, consistent with the data in FIG. 16.
Another use of modulation sensing is for measurement of the intensity of a sensing fluorophore in tissues or through skin. This was examined using 6-carboxy fluorescein. Fluorescein and its derivatives are well known to display intensities which depend on pH and dissociation of the carboxyl group (Thomas et al., 1979; Babcock, 1983; Klonis et al., 1998). The ionized form which exists at pH values above 7.5 is highly fluorescent, and the protonated low pH form is essentially non-fluorescent.
To test pH sensing in scattering media, 6-CF was diluted in 0.5% intralipid, and the long-lifetime reference was again on the outer surface of the cuvette. The emission spectra shows that the intensity of 6-CF decreases as the pH decreases (FIG. 17). The emission from [Ru(bpy)3]2+ in PVA film is centered near 600 nm and its intensity is independent of pH. The frequency responses for this pH sensor (FIGS. 18A and 18B) show that the modulation from 0.5 to 8 MHz is dependent on the pH of the intralipid solution. These modulation data were used to create a calibration curve for pH (FIG. 19). For this particular sensor. modulation values accurate to +/−0.007 result in an accuracy of +/−0.1 pH units. For clinical applications, pH values are typically accurate to +/−0.02 (Shapiro et al., 1993; Mahutte et al., 1994a; Mahutte, 1994). Optimization of the method will improve the accuracy of the method for such clinical applications.
Global Intensity Decay Analysis of pH Sensor in 0.5% Intralipid
τ1 = 3.79 ns
τ2 = 1949 ns
χR 2
While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.
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|U.S. Classification
|436/68, 436/79, 436/95, 436/74, 600/316, 600/317, 436/172
|International Classification
|Cooperative Classification
|European Classification
|Jun 14, 2001
Owner name: UNIVERSITY OF MARYLAND, BALTIMORE, A CONSTITUTENT
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|Jan 14, 2008
|Fee payment
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|May 6, 2008
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|May 28, 2008
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF
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|Jun 4, 2012
|Maintenance fee reminder mailed
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|Lapse for failure to pay maintenance fees
|Dec 11, 2012
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|attached to What If the Next Presidential Limo Was a Tesla?
|Re:Yeah, but women want it all
|attached to All Else Being Equal: Disputing Claims of a Gender Pay Gap In Tech
|Re:Reinforcing the term
|attached to Google Glass User Fights Speeding Ticket, Saying She's Defending the Future
|Re:What about other people?
|attached to British Spies To Be Allowed To Break Speed Limit
|Re:in the context of society..
|Re:in the context of society..
|Re:in the context of society..
|Re:in the context of society..
|attached to Daily Pot Use Tied To Age of First Psychotic Episode
|Re:Never put your name to it
|attached to Australian Teen Reports SQL Injection Vulnerability, Company Calls Police
|attached to Weapons Systems That Kill According To Algorithms Are Coming. What To Do?
Real computer scientists don't comment their code. The identifiers are so long they can't afford the disk space.

subset: commoncrawl


text:
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Invest in ME has as its objectives to change how myalgic encephalomyelitis (ME) is perceived and treated in the press, by health departments and by healthcare professionals. We aim to do this by raising funding for biomedical research and improving education about the disease.
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- Acupressure
- Acupuncture
- Caffeine Withdrawal
- Cancer
- Chelation Therapy
- Depression
- Dietary Suppliments
- Dysphonia
- Echinacea
- Fibromyasia/Chronic Pain
- Ginseng
- Headaches
- Herbal Suppliments
- Homeopathy
- Hormone Replacement
- Hot Flashes
- Hyperhidrosis (Sweaty Palms)
- Insomnia/Trouble Sleeping
- Massage
- Meditation
- Menopause
- Migraines
- Nicotine Addiction
- Reki
- SAD (Seasonal Affective Disorder)
- Sleep Apnea
- Stress Reduction
- Veisalgia (hangover)
- Yoga
Alternative Medicine Doctors:
- San Diego
- Bridgeport
- Fairfield
- New Haven
- Norwalk
- Carson City
- Henderson
- Las Vegas
- Reno
- Bergenfield
- Clifton
- Dumont
- Little Falls
- Tenafly
- New York City
- Portland
New Jersey
New York
Alternative Medicine Articles & Stories:
Number of Children Getting Chiropractic Care Increases
The number of American children receiving chiropractic care is steadily increasing, recent surveys have showed. Since 1991, according to survey data released by the American Chiropractic Association, the number of chiropractic patients 16 years of age and younger ros... Read More
Marriage Lowers Alzheimer's Risk
If you're married at midlife, you'll have a 50 percent lower chance of developing Alzheimer's disease or other forms of dementia in your older years, according to a recent study. And if you were once married but find yourself divorced at midlife, you'll have a 200 pe... Read More
Multivitamins Found Useless Against Top Killers
In a large, well-regarded study on women, daily multivitamin use was found to provide no protection at all against breast cancer, colon cancer, heart attack, stroke, blood clots or premature death. Previous research has shown that eating a diet rich in fruits and vegetable... Read More
Vitamin D for Diabetes
Vitamin D, previously shown to benefit cancer, osteoporosis and heart disease, appears to prevent or delay the inception of diabetes, and to alleviate its symptoms among those already afflicted with it, according to a recent review of various studies. "Vitamin D has ... Read More
Stress Deforms Brain and Behavior
Stress is not just an uncomfortable feeling of nervousness and tension. It's a treacherous force that actually remolds the brain at the same time as it warps behavior, according to a bevy of neuroscientists at a recent conference in Washington, D.C. "Stress cau... Read More
Mental Disorders Associated With Migraines
Migraine sufferers seem to have an increased tendency to experience mood and anxiety disorders, according to a recent study - though it's not clear whether one causes the other or if both are caused by some unknown third factor. "An expanding body of literature has shown that migraine head... Read More
Brain Scan Reveals Bullies' Pleasure Pattern
In two unexpected findings of a recent brain-scan study, bullies appear to gain pleasure from fighting, lying and destroying property - and they seem to lack the ability to control their violent and thrill-seeking emotions. In the study, which appeared in the journal Biological Psychology, t... Read More
Elimination of Long Dying Process Could Alter Society
A group of still-hypothetical drugs that may serve to rejuvenate the body's cellular energy system and thus eliminate the diseases of aging could have the effect of removing a huge burden from the U.S. health care system and state and federal budgets. It could also promote closer, more intima... Read More
Yet More Natural Cures for Workaday Woes
Like Narcissus in the Roman legend, modern Western humanity, critics say, has become so infatuated with our own technological and pharmaceutical prowess that we've lost sight of the host of simple and natural things that could handle many of today's problems effectively. Lest we become like N... Read More
More Natural Cures for Workaday Woes
Modern medicine is coming to agree that the new ways are not always necessarily the best ways. It's reaching the conclusion that the latest pharmaceuticals are not always necessarily the only way to assault an ailment, that homespun remedies can often be the most helpful and least expensive a... Read More
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Empire Physical Medicine & Pain Management
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text:
Most of Portsmouth's beaten FA Cup finalists have played their last game for the bankrupt club, who are to sell as many players as possible to alleviate debts of £138m. Portsmouth managed to hold Chelsea for almost an hour on Saturday but the reward for the players will be a move to a new club, one that will pay their wages on time.
The Portsmouth manager, Avram Grant, is all set to be unveiled as the new manager of West Ham United, in a deal brokered by his close friend Pini Zahavi. The futures of the players, however, are far less certain and they left Wembley unsure where their next game would be coming from.
The veteran defender Steve Finnan said: "Some of them are off to the World Cup but a lot of players don't know where their futures lie. It's difficult for the younger lads. It doesn't matter so much for the likes of myself. I'm 34 now but a lot of players will have to sort their futures out as soon as possible. I don't think I'll be at Portsmouth next season. We all said: 'See you whenever', really. There's going to be a lot of players going."
Finnan is one of three players from Portsmouth's starting XI at Wembley who are out of contract this summer, along with the goalkeeper David James and defender Ricardo Rocha. Of the others, three will return to their parent clubs after their loan deals expired – Jamie O'Hara to Tottenham Hotspur, Aruna Dindae to Lens and Frédéric Piquionne to Lyons.
James said: "For us at Portsmouth, it's a funny feeling we have now. Win or lose, we were always going to have to come back to reality. The club will be in the Championship next season, and in all likelihood the squad then will bear scant resemblance to the squad we have now, which in itself is quite a sobering thought."
Of the five players left, Kevin-Prince Boateng, Hayden Mullins and Aaron Mokoena have two years remaining on their deals, while Michael Brown and Papa Bouba Diop have one. The club's administrator, Andrew Andronikou, would love to receive offers for them.
Brown said: "I am not sure how many will be left next season, maybe four or five. It's the million-dollar question. Nobody will know what happens with the administration. It's a strange feeling as we have lost and so are disappointed and we are also saying goodbye to people maybe for the last time."
O'Hara returns to Tottenham believing he deserves a place in the first team. The 23-year-old, who played at Wembley despite two stress fractures in his lower back, said: "They have some fantastic players at Spurs, but I will go back there and see what happens. It is up to the manager whether he wants to keep me. No one likes to sit on the bench. I have proven that I can play in the Premier League by what I have achieved this season."
There was also news about the future of O'Hara's Spurs team-mate Ledley King yesterday, the England centre-back signing a new two-year deal.Reuse content


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text:
Flat Out 2 DVD-RELOADED
PC Game | Window | Vivendi Games | 2.521 GB
The sequel to Bugbear Entertainment's breakthrough racing game, FlatOut 2 is an over-the-top racing game where players experience the drive of their lives as they race around fully destructible environments in one of 16 fully customizable and upgradeable cars. Tracks are filled with crash hotspots, thousands of dynamics objects, risky but rewarding alternative routes, all designed with battle racing in mind. And battle you must as you take to the races against seven fierce AI opponents each complete with personal driving style and hidden agendas. Featuring even more craze in the form of destruction derbies and ragdoll stunt events, you'll be punishing your poor driver in twelve ragdoll stunt events. Destruction fans will be delighted with full online support on all platforms.
Publisher:Vivendi Games
Genre: Demolition Derby
Release Date: Aug 1, 2006
ESRB Descriptors:Strong Lyrics, Violence
* Hurl your driver in 12 wild mini-games that you can now play online
* City tracks, country tracks, and racing tracks test your driving prowess!
* Thousands and thousands of destructible items on every track!
* Gain nitro boost and a clear advantage by crashing with control!
* Take your destructive habits to the demolition arena and compete online!
* Join the racing circuit and learn how to make your way through the competition!
* Practice, practice, practice and collect rewards for your driving ability!
* Jam away to the high-octane soundtrack featuring bands like Fallout Boy, and Wolfmother!
Información (Cómo descargar - NUEVO: Ver Enlaces de Descarga actualizados al 2017):
http://hotfile.com/dl/23407750/32ea44c/Flat.Out.2.DVD-RELOADED.part01.rar.html http://hotfile.com/dl/23407892/11a581d/Flat.Out.2.DVD-RELOADED.part02.rar.html http://hotfile.com/dl/23242183/1576a4c/Flat.Out.2.DVD-RELOADED.part03.rar.html http://hotfile.com/dl/23408059/6ec4ecd/Flat.Out.2.DVD-RELOADED.part04.rar.html http://hotfile.com/dl/23242206/a602dea/Flat.Out.2.DVD-RELOADED.part05.rar.html http://hotfile.com/dl/23408228/6bbe9cf/Flat.Out.2.DVD-RELOADED.part06.rar.html http://hotfile.com/dl/23408336/81cd430/Flat.Out.2.DVD-RELOADED.part07.rar.html http://hotfile.com/dl/23408491/f1f1c1f/Flat.Out.2.DVD-RELOADED.part08.rar.html http://hotfile.com/dl/23408620/cc66ede/Flat.Out.2.DVD-RELOADED.part09.rar.html http://hotfile.com/dl/23408805/b4ed333/Flat.Out.2.DVD-RELOADED.part10.rar.html http://hotfile.com/dl/23242252/6bb0b78/Flat.Out.2.DVD-RELOADED.part11.rar.html http://hotfile.com/dl/23408947/f5df97a/Flat.Out.2.DVD-RELOADED.part12.rar.html http://hotfile.com/dl/23409134/6cca676/Flat.Out.2.DVD-RELOADED.part13.rar.html http://hotfile.com/dl/23409155/a4a1c88/Flat.Out.2.DVD-RELOADED.part14.rar.html


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(average rating based on 11 reviews)
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Reviewed by 11 customers
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( 1 of 1 customers found this review helpful)
So Disappointed
By BlingedOutGirl
from Calgary AB
I am sure the one ring that's still in stock is the one I returned. While it looks beautiful on the website, in person, looks are deceiving. :( The stones are gray-coloured and not the bright, sparkling, beautiful Diamonelle I've purchased in the past. The ring also does not run true to size. I bought it in my size (6) as a right hand ring but couldn't get it on my finger . Even when I put it on my left hand, I thought I was going to have to have my finger amputated to get it off. So back it went.
Bottom Line No, I would not recommend this to a friend
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( 8 of 8 customers found this review helpful)
Sent it back
By M.L.
from Montreal
When I first put this ring on I stood in the sunlight to look at it, and it sparkled beautifully. Then I went indoors and I could see was a lot of tiny grey stones. The stones are too small and too dark. I didn't have any stones fall out like the other reviewers, but that's probably because I wore it all of five minutes before deciding to send it back.I hope TSC has taken the reviews seriously and learned from what the purchasers / returners complained about.
( 17 of 17 customers found this review helpful)
Disappointment Personified
By Fabulous Blonde
from Burlington
I had been looking for this style of ring for some time, so I was quite excited to find and order this one. I did not check any ratings, as I was not ordering online at that time. It certainly did not look the same when I received it, as it did on air! The casting is terribly rough and unfinished, but that part was mostly hidden. (I told myself.) The diamonelles that were haphazardly smacked into the "setting" were accidentally chosen from the "rejects" pile and they do not shine. I thought the ring just needed cleaning, as even the rhodium plating was not shiny, but no, that was probably the fault of the casting's rough finish. The poor little reject diamonelles could not shine, and so, upon gentle cleaning with mild soap and water, many of them took the honourable way out and committed suicide down the drain...I should have said this was going to be a sad tale--sorry.
( 15 of 15 customers found this review helpful)
Disappointing purchase
By safety first
from Winnipeg
I wish I had read the reviews. I really liked the style but it has no sparkle. I have ordered Diamonelle in the past and have liked it so expected more. I did not have any missing stones but did return it as it just had no "diamonelle" life to it and was very rough as other reviews have mentioned
( 10 of 10 customers found this review helpful)
I returned it
By Barb
I ordered the ring then read the reviews. I couldn't cancel the order due to the fact i had ordered it too close to midnight so had to physically send the ring beck. It definitely wasn't up to usual Diamonelle standards. I have many others and love them. This one was definitely disappointing.
( 18 of 18 customers found this review helpful)
Terrible Buy!!!
By UNHAPPY CUSTOMER!!
from Labrador, NL
This particular ring has a rough finish - and is not very shiny at all. On top of that I have had my ring for a little over a month and the stones have already started to fall out. DO NOT BUY THIS RING!!!!
By Newby
from Brantford, Ontario
This is the first item I ordered of diamonelle, unfortunately I had to return the ring after having it for a short time as it soon had two stones missing.... Disappointing
poor quality for diamonelle
By Paulette
from Fredericton, N.B.
I have purchased several diamonelle pieces and particularly love sterling diamonelle because it's affordable. This was NOT up to sterling diamonelle standards. It was very roughly finished. I hope this is not what I can expect in future for sterling diamonelle!
( 19 of 19 customers found this review helpful)
Too Good To Be True???
By shop till I drop
from Collingwood
If you think this ring will be too good to be true, it is. The diamonelle looks grey without any glitter at all. I'm so disappointed because they can do better than this. It seems like all the small rejected diamonelles were used for this ring. You get what you pay for in this instance.
( 20 of 20 customers found this review helpful)
nice ring but...
By michie
from windsor, ontario
the first time i wore this ring a stone fell out. i was disappointed because i did like the ring but figured more stones would fall out the more i wore the ring. i returned it.


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Finally, some good news for Wii U owners.
After a couple of pretty tough weeks, focused mainly around EA basically unfriending the Wii U on Facebook, Nintendo finally had something positive announcements to placate owners of its increasingly beleaguered console.
During their latest Nintendo Direct update, the big N announced that Pikmin 3 will be coming to Europe on 26th July, with Platinum Games' highly anticipated new title, The Wonderful 101, arriving on 23rd August.
Finally, the Luigi-based downloadable content for New Super Mario Bros. U, cunningly names, New Super Luigi Bros. U will be hitting the Nintendo eShop on 20th June, with a standalone release following on 26th June.
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Evidence found in defendants' automobile following a vehicle search and impoundment after a traffic stop was correctly admitted during their prosecutions for being felons in possession of a firearm. However, one defendant was incorrectly sentenced under the Armed Career Criminal ...Read More »
Criminal Law – Leave granted to review sentencing jurisdiction issue
On order of the Court, the application for leave to appeal the May 7, 2013 order of the Court of Appeals is considered, and it is GRANTED. The parties shall address: (1) whether a trial court loses jurisdiction, for purposes ...Read More »
Criminal Law – Undisclosed impeachment evidence was not prejudicial
Although the district court erred by ruling defendant did not exercise due diligence when pursuing his claim for a Brady violation, the court correctly ruled that the government's failure to disclose plea deals with prosecution witnesses did not prejudice defendant. ...Read More »
Criminal Law – Murder-for-hire conviction reversed for evidentiary error
Defendant's murder-for-hire conviction is reversed because the federal district court incorrectly allowed an FBI agent to provide lay testimony under Federal Rule of Evidence 701 to interpret coded telephone conversations between defendant and others. Overview As part of a ...Read More »
Criminal Law – Was defendant correctly sentenced as a fourth habitual offender?
On order of the Court, the application for leave to appeal the December 27, 2012 judgment of the Court of Appeals is considered. We direct the Clerk to schedule oral argument on whether to grant the application or take other ...Read More »
Criminal Law – Ban on lesser-included offense jury instruction is unconstitutional
"The prosecution appeals by leave granted the trial court's order granting defendant's motion to instruct the jury on the lesser included offense of moving violation causing death, MCL 257.601d, contrary to the prohibition against doing so under MCL 257.626(5), in ...Read More »
Criminal Law – Charges reinstated against medical marijuana dispensary workers
Marijuana charges are reinstated against defendants, who are owners, operators and employees of a medical marijuana dispensary where undercover police agents purchased marijuana. Overview The seven defendants in these consolidated cases were charged with various marijuana offenses after undercover ...Read More »
Habeas Corpus – 'Full and fair' consideration of suppression issue in state court
The federal district court correctly denied defendant habeas relief on his claim that the state court erred by denying his motion to suppress without conducting an evidentiary hearing. Background "Stone v. Powell in the main prohibits federal habeas corpus ...Read More »
Reconsideration Granted – Court of Appeals must reconsider whether drug-dog sniff was search
On order of the Court, the motion for reconsideration of this Court's June 7, 2013 order is considered, and it is GRANTED. We VACATE our order dated June 7, 2013. On reconsideration, the application for leave to appeal the April ...Read More »
Criminal Law – Defendant's habeas corpus petition properly denied
The federal district court properly denied defendant's petition for habeas corpus relief. Murder conviction Defendant was convicted of murdering his wife. The Supreme Court of Ohio affirmed his conviction and death sentence under State v. Lilly, 717 N.E.2d 322 ...Read More »


text:
Top Definition
A person who spreads joy, happiness and a good atmosphere around him or her. A person whose opinions are considered serious and reliable as well as accurate. Someone that has a way of illuminating the surrounding crowd.
His best attribute is illuminos. He must be a really good and trustworthy person.
by Azart December 29, 2008
The Urban Dictionary Mug
One side has the word, one side has the definition. Microwave and dishwasher safe. Lotsa space for your liquids.Buy the mug
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Smooth, soft, slim fit American Apparel shirt. Custom printed. 100% fine jersey cotton, except for heather grey (90% cotton).Buy the t-shirt


text:
The Adiministrative and Mentoring Core will be responsible for all fiscal activities related to the COBRE program, including the placement of all orders and providing financial reports to the module directors and investigators. Dr. Dhanasekaran will direct the Adiministrative and Mentoring Core, which will provide all administrative functions of the COBRE program. He will be assisted by a Business Administrator and a Staff Assistant. The Business Administrator, Mr. Bobby Thomas (25%), will oversee the activities of an Administrative Assistant, Ms. Alisha Booth (100%). Mr. Thomas has been with the OUHSC for 10 years and is very convensant with the fiscal and grant management policies of OUHSC and federal funding agencies including NIH. Mr. Thomas will meet monthly with each investigator to review their accounts and mentor them in the fiscal management of sponsored research grants. Salaries for the PI, administrative staff, mentoring faculty, core directors will be paid through this core module. In addition, this core will administer the fiscal accounting for the proposed pilot projects. Coordinating meetings, travel and activities of the Internal Advisory Committee and the External Advisory Committee will be the responsibility of the Administrative Assistant, Ms. Booth, under the direction of the PI.
Cancer represents a major health burden in the US. Ultimately, alleviating the burden of cancer will come from research: the generation of new ideas and, consequently, the development of novel agents. A COBRE grant will allow the Oklahoma Cancer Center to provide PJIs a focused mentoring experience to help them mature into independently-funded investigators contributing to the overall fight against cancer.
|Boswell-Casteel, Rebba C; Johnson, Jennifer M; Duggan, Kelli D et al. (2014) FUN26 (function unknown now 26) protein from saccharomyces cerevisiae is a broad selectivity, high affinity, nucleoside and nucleobase transporter. J Biol Chem 289:24440-51
|Aravindan, Natarajan; Aravindan, Sheeja; Pandian, Vijayabaskar et al. (2014) Acquired tumor cell radiation resistance at the treatment site is mediated through radiation-orchestrated intercellular communication. Int J Radiat Oncol Biol Phys 88:677-85
|Bio, Moses; Rajaputra, Pallavi; Nkepang, Gregory et al. (2014) Far-red light activatable, multifunctional prodrug for fluorescence optical imaging and combinational treatment. J Med Chem 57:3401-9
|Janakiram, Naveena B; Mohammed, Altaf; Brewer, Misty et al. (2014) Raloxifene and antiestrogenic gonadorelin inhibits intestinal tumorigenesis by modulating immune cells and decreasing stem-like cells. Cancer Prev Res (Phila) 7:300-9
|Babu, Anish; Wang, Qi; Muralidharan, Ranganayaki et al. (2014) Chitosan coated polylactic acid nanoparticle-mediated combinatorial delivery of cisplatin and siRNA/Plasmid DNA chemosensitizes cisplatin-resistant human ovarian cancer cells. Mol Pharm 11:2720-33
|Xiong, Xunhao; Arvizo, Rochelle R; Saha, Sounik et al. (2014) Sensitization of ovarian cancer cells to cisplatin by gold nanoparticles. Oncotarget 5:6453-65
|Aravindan, S; Natarajan, M; Ramraj, S K et al. (2014) Abscopal effect of low-LET ýý-radiation mediated through Rel protein signal transduction in a mouse model of nontargeted radiation response. Cancer Gene Ther 21:54-9
|VandenHeuvel, Katherine A; Carpentieri, David F; Chen, Jie et al. (2014) Ectomesenchymoma with embryonal rhabdomyosarcoma and ganglioneuroma, arising in association with benign triton tumor of the tongue. Pediatr Dev Pathol 17:226-30
|Aravindan, Sheeja; Natarajan, Mohan; Herman, Terence S et al. (2013) Radiation-induced TNF* cross signaling-dependent nuclear import of NF*B favors metastasis in neuroblastoma. Clin Exp Metastasis 30:807-17
|Chang, Theodore S; Lin, Hsueh-Kung; Rogers, Kyle A et al. (2013) Expression of aldo-keto reductase family 1 member C3 (AKR1C3) in neuroendocrine tumors & adenocarcinomas of pancreas, gastrointestinal tract, and lung. Int J Clin Exp Pathol 6:2419-29
Showing the most recent 10 out of 11 publications


text:
Hotel Bel Conti
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- Beach
- Free High-Speed Internet
- Continental Breakfast
- Room service
- Restaurant
- Bar/lounge
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- Beach
- Restaurant
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- Free High-Speed Internet
- Wheelchair accessibility
- Free parking
- Continental Breakfast
- Room service
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TripAdvisor Reviews Hotel Bel Conti Durres
Travel Blogs from Durres
Central Albania
... is a street of souvenir and antique shops where I bought a hat.
In Kruje, Lear stayed at the palace of the Bey, a young man of 18 or 19 leading a cloistered life amid his many servants and longing for contact with the outside world. Lear entertained him by imitating the sounds of a steamboat and locomotive, which the Bey had never seen.
On our way down the mountain we stopped for a roadside picnic, then headed for Shkoder.
Albania... Nil Points!
... on deck to watch the sunrise.
Albania didn't disappoint us and we were treated to a really special show. With us all on deck though John and Laura went below leaving Simon and I to takeover the watch.
We chatted a while and settled in to our routine. The course we would be taking kept us 20nm offshore and we would be entering Durres later on through a proper channel. Simon told me that the Italian coastguard like to come over in a rib ...
... a bit hair-raising, especially when we eventually arrived in Tirana as nobody takes any notice of lanes and sometimes on a 3 lane road there were 5 cars wide and they just change lanes as they like. There is no public transport here, no buses or trains, and they have only had cars for the last 20 years, and now everyone has a car and doesn't know how to drive it – it is ...
First day of touring
... not give her any money. Interesting take on poor people.
My daughter was not interested in going to the Shijaku House so she went back to the hotel to work out. We hoped into the van and drove around the city a bit to get to this house which is the home of one of their most famous artists who is now in his 80's I think but still painting. We drove down a small alley and when we weren't ...
Episode 22
... centre. Not that it really looks like the centre of a Capital City. The main square has a fountain and most of the streets about it are dug up at present and new sewerage and water pipes being laid.
This isn't a centre like you would expect to find in most western or even eastern cities. There are no tower blocks of offices and no large shops with plate glass windows displaying merchandise. No crowds of shoppers hurrying about either. ...