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PubMed1997

Total internal reflection fluorescence: applications in cellular biophysics.

Molecular interactions occurring on or near cell membrane surfaces are expected to have different properties from those occurring in bulk solutions. One particularly useful technique for studying surface-associated processes at the molecular level is total internal reflection fluorescence. In this method, the evanescent field from an internally reflected excitation source selectively excites fluorescent molecules on or near a surface. Evanescent excitation has been used recently with a variety of techniques in fluorescence microscopy and spectroscopy to probe the fundamental physicochemical properties of biochemical reactions at natural or model biological surfaces. These studies are providing enhanced understanding of cellular function. Several recent developments in total internal reflection fluorescence methodology from other fields are likely to find future application in cellular biophysics.

Biophysics↗

PubMed2001

Geometrical effects on thermal neutron reflection of hydrogenous moderators using 241Am-Be source.

The variation of thermal reflection parameter with thicknesses of water and paraffin moderators was measured using an instrument consisting of 1 Ci 241Am-Be isotopic neutron source and 3He neutron detector. Based on experimental data obtained from two different source-detector-sample geometries, analytical expressions were derived for calculation of reflection parameters for various moderator thicknesses. The measured data for the two geometries exhibit saturation curves with saturation achieved at about 8 and 16 cm when sample container was placed at top and side of the source holder, respectively. By locating the source holder along the vertical axis of the sample container, measurements showed that the reflection parameter was very sensitive with the axial location and was strongly dependent on the moderator and its thickness.

Journal Article↗

PubMed2002

Simultaneous use of neutron transmission and reflection techniques for the classification of crude oil samples.

Neutron attenuation and reflection characteristics of three crude mineral oil samples from West Africa were determined using 37 GBq 241Am-Be source in a single set-up and compared with those of paraffin (oil) and benzene, an aromatic compound. Based on the trends of measured data which are similar to those exhibited by paraffin but different from those associated with benzene, the crude oil samples could be classified as paraffinic. The total hydrogen content and weight ratios (O+C)/H and C/H determined for the crude oil samples using the transmission and reflection techniques are not only independent of the technique but are also consistent with literature values of thermal reflection technique.

Journal Article↗

PubMedDate not supplied

Calibration of a reflectance pulse oximeter in fetal lambs for arterial oxygen saturations below 70%.

OBJECTIVE: To calibrate a reflectance pulse oximeter developed at the Division of Perinatal Physiology, University Hospital of Zurich, for monitoring of oxygen saturations below 70%. METHODS: Oxygen saturation measured noninvasively with the reflectance pulse oximeter in fetal sheep was compared with oxygen saturation of arterial blood samples measured by a conventional two-wavelength oximeter. Oxygen saturation was varied by stepwise reduction of the inspired oxygen concentration of the ewe. RESULTS: A total of 58 data pairs was obtained from five fetuses, with a range of 10.9-73.5% for the reference method and 6.8-69.2% for the pulse oximetry oxygen saturation values. There was good correlation between the results obtained by the two methods (r2 = .89, P < .0001), with a 95% confidence interval of +/- 12.5%. The pulse oximeter showed a precision of 6.2% oxygen saturation. CONCLUSION: The Zurich reflectance pulse oximeter offers valid measurements of oxygen saturation levels down to 10% oxygen saturation, and is therefore suitable for monitoring the fetus during delivery.

Animals↗

PubMed2002

Effect of hemolysis on oxygen and hematocrit measurements by near infrared reflectance spectroscopy.

A short review of the principles of near infrared reflectance spectroscopy (NIRS) in whole blood is followed by a discussion on the influence of hemolysis. The increase of free plasmahemoglobin (PHb) has a strong influence on the continuous measurement of hematocrit and oxygen saturation (O(2)S) by NIRS. In view of the relative stability of hematocrit values in vivo this effect may be used to detect a change of the hemolysis rate induced by blood pumps in case of malfunction. The aim of this study is, therefore, the assessment of the hemolysis rate within an in vitro mock loop comprising a rotary blood pump by means of NIRS at constant hematocrit levels compared to the photometric reference method. Reflected light is measured by an integrated optical sensor working at three wavelengths (660 nm, 730 nm, and 830 nm). The experimental results demonstrate that the increase of free hemoglobin in plasma due to mechanical pumping leads to a decrease of detected reflected light at all three wavelengths. Influencing parameters such as adhering proteins on the sensor surface and the blood flow rate are briefly discussed. Finally, the possibility of using NIRS sensors for detecting malfunctions of blood pumps in vitro and in vivo is discussed, together with the option of using these sensors for supervision of long-term implantable pumps.

Biosensing Techniques↗

PubMed2005

How complexity science can inform a reflective process for improvement in primary care practices.

BACKGROUND: Quality improvement processes have sometimes met with limited success in small, independent primary care settings. The theoretical framework for these processes uses an implied understanding of organizations as predictable with potentially controllable components. However, most organizations are not accurately described using this framework. Complexity science provides a better fit for understanding small primary care practices. METHODS: The Multimethod Assessment Process (MAP)/Reflective Adaptive Process (RAP) is informed by complexity science. This process was developed in a series of studies designed to understand and improve primary care practice. A case example illustrates the application and impact of the MAP/RAP process. RESULTS: Guiding principles for a reflective change process include the following: an understanding of practices' vision and mission is useful in guiding change, learning and reflection helps organizations adapt to and plan change, tension and discomfort are essential and normal during change, and diverse perspectives foster adaptability and new insights for positive change. DISCUSSION: A reflective change process that treats organizations as complex adaptive systems may help practices make sustainable improvements.

Organizational Case Studies↗

PubMed2005

Reflective thinking and mental imagery: a perspective on the development of posttraumatic stress disorder.

Reflective thinking occurs when information stored in long-term memory (LTM) is not sufficient to allow one to respond "automatically" to an object or event. Instead, stored information must be entered into working memory and a novel response or solution produced. In this article I argue that mental imagery plays a central role in this process, and that over the course of normal cognitive development the process of reflective thinking "programs" LTM so that an increasingly large number of tasks can be performed without reflective thinking. Normal cognitive development thus results in a decreasing reliance on imagery. However, if highly emotional images are formed, additional retrieval cues can be entered into LTM, making such images more likely to occur in the future. Such images induce arousal, similar to that induced by the actual event. This line of thinking leads to a novel perspective on the neurocognitive deficits that underlie the development of posttraumatic stress disorder, and may also help to explain some symptoms seen in hyperactivity, impulsiveness, and difficulties in self-control.

Brain↗

PubMed1994

Reflecting time-of-flight mass spectrometer with an electrospray ion source and orthogonal extraction.

An electrospray source has been coupled to a reflecting time-of-flight mass spectrometer. The ions enter as a continuous beam in a direction perpendicular to the spectrometer axis and are formed into short bursts of ions with velocities parallel to the axis by electrical pulses applied to injection electrodes. The instrument may be operated either in the linear mode, with ions detected behind the electrostatic mirror, or in the reflecting mode, with ions detected after reflection. In the latter case the time resolution is < or = 8 ns for m/z approximately 600, enabling observation of individual isotopic peaks for masses up to about 4000 u. The sensitivity is adequate to enable measurement of mass spectra for 10 fmol of cytochrome c (approximately 12,000 u). The spectrometer does not limit the range of m/z values, and ions have been observed up to m/z approximately 6000. With proper adjustment of pH and other conditions in the source, the ionization is very soft, enabling injection of weakly bound complexes; these have been accelerated and measured in the spectrometer without observable fragmentation.

Cytochrome c Group↗

PubMed2002

Visible reflectance hyperspectral imaging: characterization of a noninvasive, in vivo system for determining tissue perfusion.

We characterize a visible reflectance hyperspectral imaging system for noninvasive, in vivo, quantitative analysis of human tissue in a clinical environment. The subject area is illuminated with a quartz-tungsten-halogen light source, and the reflected light is spectrally discriminated by a liquid crystal tunable filter (LCTF) and imaged onto a silicon charge-coupled device detector. The LCTF is continuously tunable within its useful visible spectral range (525-725 nm) with an average spectral full width at half-height bandwidth of 0.38 nm and an average transmittance of 10.0%. A standard resolution target placed 5.5 ft from the system results in a field of view with a 17-cm diameter and an optimal spatial resolution of 0.45 mm. The measured reflectance spectra are quantified in terms of apparent absorbance and formatted as a hyperspectral image cube. As a clinical example, we examine a model of vascular dysfunction involving both ischemia and reactive hyperemia during tissue reperfusion. In this model, spectral images, based upon oxyhemoglobin and deoxyhemoblobin signals in the 525-645-nm region, are deconvoluted using a multivariate least-squares regression analysis to visualize the spatial distribution of the percentages of oxyhemoglobin and deoxyhemoglobin in specific skin tissue areas.

Blood Gas Monitoring, Transcutaneous↗

PubMed2002

Attenuated partial internal reflection infrared spectroscopy.

A new method for the spectroscopic study of absorbing films is proposed. In contrast to the well-established methods that take advantage of the attenuation of total internal reflection (ATR) to obtain spectra, we intentionally arrange the optics to permit partial internal reflection from the sampling prism face. Attenuated partial internal reflection (APR) spectroscopy is introduced through theoretical calculations and experimental demonstrations. The calculated APR spectra in the infrared region were generated from the Fresnel and Airy equations. Experimentally, APR spectra of water films on a NaCl prism were obtained. APR is more sensitive than ATR, and can easily distinguish water films at the monolayer level (310 pm). The determination of film thickness from interference fringes in APR spectra is also illustrated. It is shown that APR can be used for film thickness measurements that can span 6 orders of magnitude. The limitations of APR are also discussed.

Journal Article↗

PubMed2006

Analysis of Gibbs monolayer adsorbed at the toluene/water interface by UV-visible partial internal reflection spectrometry.

Gibbs monolayers of lipophilic tetraphenylporphyrinatomanganese(III) and hydrophilic diacid of meso-tetrakis(4-sulfonatopheny)porphyrin adsorbed at the liquid-liquid interface have been analyzed by UV-visible external reflection (ER) and partial internal reflection (PIR) spectra measured at different angles of incidence. The angle-dependent ER and PIR spectra over the Brewster angles (thetaERB and thetaIRB) have readily been measured at the toluene/water interface. As preliminarily expected in our previous study, the present study has first proved that the reflection-absorbance of UV-visible PIR spectra quantitatively agrees with the theoretical calculations for the Gibbs monolayer over thetaIRB. In addition, it has also been proved that the absorbance of the PIR spectra is greatly enhanced in comparison to that of the ATR spectra. The enhancement is caused by an optical effect in the monolayer sandwiched between two phases of toluene and water that have different but refractive indices close to each other. This optical enhancement requires an optically perfect contact between the phases, which is difficult to prepare for a solid-solid contact. At the liquid/liquid interface, however, an ideal optical contact is easily realized, which makes the enhancement as much as the theoretical expectation. The PIR spectrometry will be recognized to be a new high-sensitive analytical tool to study Gibbs monolayer at the liquid/liquid interface.

Acids↗

PubMed2000

Monitoring of dye adsorption phenomena at a silica glass/water interface with total internal reflection coupled with a thermal lens effect

Thermal lens spectroscopy was combined with total internal reflectance spectroscopy to develop a novel, highly sensitive analytical method that can detect nonfluorescent as well as fluorescent analytes at surfaces and interfaces. It was verified that when the total internal reflection method is coupled with thermal lens spectroscopy (TIR-TLS), the thermal lens effect is induced by only the evanescent wave. The ability of depth profiling was shown. The detection limit of TIR-TLS was at an absorbance of 3.0 x 10(-5) unit for Sudan II acetone solution, which is better than that of attenuated total reflection by a factor of hundreds. In addition, the adsorption of acridine orange on silanol groups on a glass surface could be monitored directly by TIR-TLS, and the adsorption isotherm agreed well with Langmuir's model. The dependence of surface density of anionic silanol groups on pH was determined by TIR-TLS measurements of aqueous acridine orange solutions buffered in the pH range between 2.7 and 11.5.

Journal Article↗

PubMed2005

Structural studies of the neural-cell-adhesion molecule by X-ray and neutron reflectivity.

The structures of adhesion proteins play an important role in the formation of intercellular junctions and the control of intermembrane spacing. This paper describes the combination of neutron and X-ray specular reflectivity measurements to investigate the structure of the ectodomain of the neural-cell-adhesion molecule (NCAM). The measurements with unmodified NCAM suggest the presence of a bend in the extracellular region. Measurements with the polysialic-acid-modified form of NCAM reveal that, at physiological ionic strength, the carbohydrate chains extend beyond the range of the unmodified protein. The excluded volume of the polymer is also ionic-strength-dependent, as expected for a polyelectrolyte. The structural characteristics obtained from these independent analyses of X-ray and neutron reflectivity data agree with each other, with prior reflectivity studies, and with molecular dimensions obtained from direct-force measurements. These results provide structural insights into the configuration of the NCAM ectodomain and the regulation of NCAM adhesion by post-translational modification.

Animals↗

PubMedDate not supplied

Neutron and X-ray reflectivity studies of human serum albumin adsorption onto functionalized surfaces of self-assembled monolayers.

Neutron and X-ray reflectivity (NR and XR) have been widely used for the investigation of the structure of thin organic films. Here we demonstrate how these sensitive techniques may be applied to the study of protein adsorption to well-characterized self-assembled monolayers (SAMs) with different chemical functionalities. NR can be used for in situ study, while XR provides complementary information on the initial surfaces and dried layers measured in air after protein has been adsorbed. In situ measurements of adsorption of human serum albumin onto a hydrophilic NH2-terminated monolayer clearly show the presence of a thin layer of adsorbed protein next to the SAM. Adsorption of albumin onto a hydrophobic, deuterated, CD3-terminated SAM causes even bigger changes in the NR. Upon replacing the protein solution with protein-free buffer solution, the reflectivities from both kinds of monolayers do not change, demonstrating that the albumin adsorption is irreversible after several hours of contact with the protein solution. X-ray reflectivity measurements of dried substrates performed ex situ in air provide a lower bound estimate of the amount of protein which must be at the interface in situ. This combination of techniques provides a uniquely sensitive approach for studying changes that occur upon protein adsorption at an interface.

Adsorption↗

PubMed2005

Near-infrared reflectance model for the rapid prediction of total fat in cereal foods.

AOAC method 996.01, used in cereal foods to determine total fat as defined by the U.S. Nutrition Labeling and Education Act (NLEA), is laborious and time-consuming and utilizes hazardous chemicals. Near-infrared (NIR) reflectance spectroscopy, a rapid and environmentally benign technique, was investigated as a potential method for the prediction of total fat using AOAC method 996.01 as the reference method. Near-infrared reflectance spectra (1104-2494 nm) of ground cereal products (n = 72) were obtained using a dispersive spectrometer, and total fat was determined according to AOAC method 996.01. Using multivariate analysis, a modified partial least-squares model was developed for total fat prediction. The model had a SECV of 1.12% (range = 0.5-43.2%) and a multiple coefficient of determination of 0.99. The model was tested with independent validation samples (n = 36); all samples were predicted within NLEA accuracy guidelines. The results indicate that NIR reflectance spectroscopy is an accurate means of determining the total fat content of diverse cereal products for nutrition labeling.

Analysis of Variance↗

PubMed2005

In situ, time-resolved normal incidence reflectance spectroscopy of polycrystalline platinum microelectrodes in aqueous electrolytes.

In situ normal incidence reflectance spectra of polycrystalline Pt microelectrodes have been monitored as a function of the applied potential in aqueous 0.5 M H(2)SO(4) using a He-Ne laser source (633 nm) and a beam splitter/microscope objective arrangement. Data recorded during voltammetric cycles in Ar-purged solutions revealed a linear correlation between the normalized change in reflectance, DeltaR/R = (R(s) - R(ref))/R(ref) (where R(s) and R(ref) are the light intensities measured by the detector at the sampling, s, and reference potentials, ref, respectively), and the extent of oxidation of the Pt surface over a wide coverage range. Reflectance spectra were also collected in CO-saturated 0.5 M H(2)SO(4) during chronocoulometric measurements involving judiciously selected limits for both the potential step and duty cycle parameters. Analysis of these results made it possible to extract contributions to the current derived from oxide formation during oxidation of adsorbed and bulk CO, based strictly on the optical response.

Adsorption↗

PubMed2006

Potential-modulated, attenuated total reflectance spectroscopy of poly(3,4-ethylenedioxythiophene) and poly(3,4-ethylenedioxythiophene methanol) copolymer films on indium-tin oxide.

We report the first application of a potential-modulated spectroelectrochemical ATR (PM-ATR) instrument utilizing multiple internal reflections at an optically transparent electrode to study the charge-transfer kinetics and electrochromic response of adsorbed films. A sinusoidally modulated potential waveform was applied to an indium-tin oxide (ITO) electrode while simultaneously monitoring the optical reflectivity of thin (2-6 equivalent monolayers) copolymer films of poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4-ethylenedioxythiophene methanol) (PEDTM), previously characterized in our laboratory. At high modulation frequencies the measured response of the polymer film is selective toward the fastest electrochromic processes in the film, presumably those occurring within the first adsorbed monolayer. Quantitative determination of the electrochromic switching rate, derived from the frequency response of the attenuated reflectivity, shows a linear decrease in the rate, from 11 x 10(3) s(-1) to 3 x 10(3) s(-1), with increasing proportions of PEDTM in the copolymer, suggesting that interactions between the methanol substituent on EDTM and the ITO surface slow the switching process by limiting the rate of conformational change in the polymer film.

Journal Article↗

PubMed2004

External reflection FTIR spectroscopy of the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) on an overflowing cylinder.

External reflection Fourier transform infrared spectroscopy (ER-FTIRS) has been used to study the adsorption of the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) at the air-water interface under nonequilibrium conditions. An overflowing cylinder (OFC) was used to generate a continually expanding liquid surface with a surface age of 0.1-1 s. ER-FTIR spectra were acquired by a single bounce of p- or s-polarized radiation from the flowing surface of the OFC. The C-H stretching region of CTAB spectra was analyzed both by subtraction of a reference spectrum of pure water and by a chemometric technique known as target factor analysis (TFA). The TFA method is shown to give lower scatter in the weight of the component assignable to the adsorbed CTAB monolayer and to permit analysis of spectra at lower bulk surfactant concentrations. The surface sensitivity of ER-FTIRS is demonstrated both experimentally and by theoretical modeling. Modeling shows that surfactant adsorbed at the surface and dissolved in the bulk solution can be distinguished by reflection spectroscopy but also highlights potential errors that can arise from the neglect of the bulk surfactant contribution to the ER-FTIR spectra. Polarized spectra are consistent with an isotropic distribution of transition dipole moments of the hydrocarbon chains in CTAB. Component weights of the CTAB monolayer determined by TFA are compared with an independent determination of values of the dynamic surface excess, Gammadyn, by neutron reflection and ellipsometry. The relationship between the component weights and Gammadyn shows a small but significant deviation from linearity. Possible explanations for this deviation are discussed. The feasibility of using TFA to deconvolute ER-FTIR spectra of mixtures of hydrocarbon surfactants is demonstrated.

Journal Article↗