Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis

Basic tenets related to the use of three main classes of potentiometric redistribution fluorescent dyes (carbocyanines, oxonols, and rhodamines) are discussed in detail. They include the structure/function relationship, formation of nonfluorescent (H-type) and fluorescent (J-type) dimers and higher aggregates, probe partitioning between membranes and medium and binding to membranes and intracellular components (with attendant changes in absorption and emission spectra, fluorescence quantum yield and lifetime). The crucial importance of suitable probe-to-cell concentration ratio and selection of optimum monitored fluorescence wavelength is illustrated in schematic diagrams and possible artifacts or puzzling results stemming from faulty experimental protocol are pointed out. Special attention is paid to procedures used for probe-response calibration (potential clamping by potassium in the presence of valinomycin, use of gramicidin D in combination with N-methylglucamine, activation of Ca-dependent K-channels by A23187, the null-point technique). Among other problems treated are dye toxicity, interaction with mitochondria and other organelles, and possible effects of intracellular pH and the quantity of cytosolic proteins and/or RNA on probe response. Individual techniques using redistribution dyes (fluorescence measurements in cuvettes, flow cytometry and microfluorimetry of individual cells including fluorescence confocal microscopy) are discussed in terms of reliability, limitations and drawbacks, and selection of suitable probes. Up-to-date examples of application of slow dyes illustrate the broad range of problems in which these probes can be used.     

基于金属有机骨架材料的电阻传感器在检测VOCs中的应用

化学电阻传感器由于其结构简单、分析快速等特点在众多气体传感方式中脱颖而出,因此成为了目前应用广泛的传感器类型。其中,用于检测挥发性有机化合物（VOCs）的电阻传感器中敏感材料对气体的选择性吸附和相应的检测至关重要,此外也需要一些额外措施保证检测的选择性。因此,传感材料的比表面积、孔尺寸、功能官能团以及辅助材料等决定了传感器的响应程度、选择和敏感程度。金属有机框架材料（MOF）是一类新型的有机?无机杂化材料,具有丰富多孔、高比表面积、结构多样性、化学稳定性良好等特点,除此以外一些MOF衍生物也具有比表面积大、导电性良好等特点,因此MOF及MOF衍生物已在气体传感器中得到广泛研究和应用。基于化学电阻传感器基本原理、MOF及MOF衍生物在电阻传感器检测挥发性有机化合物中起到的作用、原理、及其应用,对其发展前景和面临的挑战进行了展望。      

Accuracy of the one-point in vivo calibration of "wired" glucose oxidase electrodes implanted in jugular veins of rats in periods of rapid rise and decline of the glucose concentration

The hypothesis of the feasibility of one-point in vivo calibration of intravenously implanted glucose sensors during periods of rapid rise and decline of venous blood glucose concentration was tested. Miniature (5 x 10(-4) cm2 mass transporting area) glucose electrodes with 10-90% response times < 2 min, that did not consume oxygen, were implanted in jugular veins of systemically heparinized rats and used in 4-h experiments, during which the blood glucose concentration was amperometrically monitored. The glucose electrodes were made by electrically connecting ("wiring") reaction centers of glucose oxidase through an electron-conducting redox hydrogel to gold electrode surfaces. The redox polymer and enzyme constituting the electrode sensing layer were immobilized by cross-linking, and thus the electrodes had no diffusional and readily leached redox mediator. One hour after their implantation, the electrodes accurately tracked the blood glucose concentration when calibrated in vivo by a one-point calibration, when the glucose concentration was steady, when rising rapidly, and when declining steeply. For an assumed 2-min lag time, the sensor readings were well correlated with the true blood glucose concentrations, with linear regression analysis yielding a slope of 0.97 +/- 0.07 and an intercept (bias) of 0.3 +/- 0.3 mM. The correlation coefficient, r2, was 0.949 +/- 0.020, and the percent difference through the 2-22 mM range was 1.9 +/- 1.0%. The results suggest that, in combination with understanding and modeling of transient physiological differences between the subcutaneous and the blood glucose concentrations, it will be possible to calibrate by one-point in vivo calibration subcutaneously implanted sensors, even while the glucose concentration changes rapidly.     

Continuous analysis in extracorporeally circulating blood--a rat model applying flow-through ion-selective electrodes for the measurement of Ca2+, K+, Na+ and pH

A rat model is introduced which enables investigations in anticoagulated blood with continuous measurements by a flow-through electrode system. In the present study, a potentiometric ion-selective electrode (ISE)-system was used for measuring Ca2+, K+, Na+ and pH in rats. The setup was adjusted to an extracorporeal blood-volume of 0.750 ml. This permits indirect measurements of the analytes via a dialysis membrane, with electrical separation of the ISE's and the animal. The flow-rates of blood and dialysis-solution were adjusted in such a way that water diffusing from the aqueous dialysis solution into the blood, across the dialysis membrane, does not alter the haematocrit. Polyethyleneglycol-hirudin was used for anticoagulation, since it was superior to heparin. The assembly enables continuous measurements in the living anaesthetized rat over a time period of at least 3 hours.     

Modified spectrophotometer for multi-dimensional circular dichroism/fluorescence data acquisition in titration experiments: application to the pH and guanidine-HCI induced unfolding of apomyoglobin

In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements. This arrangement allows multiple data sets to be collected during a single experiment, resulting in a saving of time and material, and improved correlation between the different types of measurements. The usefulness of the instrument was shown by thermal melting experiments on several different protein systems. This CD/fluorometer spectrophotometer has been further modified by interfacing with a syringe pump and a pH meter. This arrangement allows ligand, pH, and chemical denaturation titration experiments to be performed while monitoring changes in the sample's CD, absorbance, fluorescence, and light scattering properties. Our data acquisition program also has an ability to check whether the signals have approached equilibrium before the data is recorded. For performing pH titrations we have developed a procedure which uses the signal from a pH meter in a feedback circuit in order to collect data at evenly spaced pH intervals. We demonstrate the use of this instrument with studies of the unfolding of sperm whale apomyoglobin, as induced by acid pH and by the addition of guanidine-HCI.     

Fiber Optic Sensors for Bridge Monitoring

Advances in the production of optical fibers made possible the recent development of innovative sensing systems for the health monitoring of civil structures. The main reasons for this development are the reduced weight and dimensions of fiber optic sensors, the strong immunity to electromagnetic interference, the improved environmental resistance and the scale flexibility for small-gauge and long-gauge measurements. These systems can provide high-resolution and measurement capabilities that are not feasible with conventional technologies. In addition, they can be manufactured at a low cost and they offer a number of key advantages, including the ability to multiplex an appreciable number of sensors along a single fiber and interrogate such systems over large distances. For these reasons, it is evident that fiber optic sensors will change the instrumentation industry in the same way fiber optics has revolutionized communications. This paper provides an overview of the intensity modulated and spectrometric fiber optic sensors and techniques to assess the condition of existing structures in order to enhance the durability of the new bridges, increasing lifetime and reliability and decreasing maintenance activities. Application of these sensors to monitoring strain, temperature, inclination, acceleration, load measurements, ice detection, vehicles speeds and weights, and corrosion and cracking of reinforced and prestressed concrete structures will be described.     

Spatial discrimination against background with different optical systems for collection of fluorescence in laser-excited atomic fluorescence spectrometry with a graphite tube electrothermal atomizer

A single 90 degrees off-axis ellipsoidal mirror fragment was used in a dispersive detection system for electrothermal atomization laser-excited atomic fluorescence spectrometry. The performance of the new optical arrangement was compared with those of optical arrangements that employed a plane mirror in combination with biconvex or plano-convex lenses. All the optical arrangements collected fluorescence in a scheme called front surface illustration. BEAM-4, an optical ray tracing program, was used for calculations of spatial ray distributions and optical collection efficiency for the various optical configurations. Experimentally, the best collection efficiency was obtained by use of the ellipsoidal mirror, in qualitative agreement with simulations done by use of the BEAM-4 software. The best detection limit for cobalt with the new optical arrangement was 20 fg, which was a factor of 5 better than that obtained with conventional optical arrangements with otherwise the same instrumentation. The signal-to-background ratio and the fluorescence collection efficiency were also studied as a function of position of the optical components for the various optical arrangements. For both cobalt and phosphorus, the signal-to-background ratio with the new optical arrangement remained stable within 10-20% during +/- 8 mm shifts in the position of the detection system from the focal plane of the optics. Overall, the new optical arrangement offered high collection efficiency, excellent sensitivity, and facile optical alignment due to efficient spatial separation between the fluorescence signal and the background radiation. The advantages of the new optical arrangement were particularly important during measurements in the presence of high levels of blackbody radiation.     

堆垛多功能起重机夹持力测量装置的检定及应用

根据压力传感器与称重显示仪表的工作原理,通过螺栓,铁板,将其结合起来,组成一个整体夹持力测量装置。这种测量装置缺少合适检定方法。本文在研究压力传感器及称重显示仪表的工作原理的基础上,提出对该装置新的检定方法及实际工作中应用。     

Photoluminescence Characterization of Sm3+ -Doped Fluoroborate Ceramics

Sm³⁺ -doped fluoroborate ceramics (BaO-ZnO-Al₂O₃-B₂O₃-NaF) were fabricated as a potential material in illumination and display. The density, surface modality, microstructure and fluorescence were measured and characterized. The ceramics were well densified without pores and they were proved to be amorphous. Under blue and UV light excitations, the opaque fluoroborate ceramics doped with Sm³⁺ absorbed the most of excitation radiation and emited intense reddish orange light. The emission spectrum of Sm³⁺ -doped fluoroborate ceramics under 404 nm excitation consisted of four intense emission bands peaking at 564, 600, 645 and 708 nm, respectively, and the 600 nm reddish-orange emission band was the most intense. Excitation and emission spectra indicated that commercial UV and blue laser diodes, blue and bluish-green LEDs and Ar⁺ optical laser were powerful pumping sources for the fluoroborate ceramics. The rare earth doped ceramics with various visible emissions were useful for developing new color light sources, fluorescent display devices and UV sensors.     

Comparison of dark-field and bright-field incident illumination for in vivo measurements of reduced pyridine nucleotides

Bright-field and dark-field illumination techniques for in vivo measurements of reduced pyridine nucleotide fluorescence were compared in 15 rats during periods of normocapnia, hypocapnia, hypercapnia, and anoxia. Parameters investigated included fluorescence, cortical reflectance, cortical blood flow, and electroencephalograms. In normal brain, with preserved autoregulation, reduced pyridine nucleotide fluorescence was constant through a wide range in Pa(CO2), cortical blood flow, and cerebral blood volume in animals studied using vertical illumination (bright-field) techniques. There was a marked increase in reduced pyridine nucleotide fluorescence at death from anoxia. Artifacts were reduced by monochromators for excitation, emission, and reflected light; low-intensity vertical excitation energy and high-sensitivity recording instrumentation; and a small avascular (123 microns) field. Potential sources of error include photodecomposition, hemoglobin interference from absorption and reflectance, and light scattering. Vertical excitation techniques using a small field appeared to give more reliable and reproducible results than circumferential techniques using a larger field of observation.     

European multicentre evaluation of the Super Aution SA-4220 urinalysis analyser

A multicentre evaluation of the urine test strip analyser Super Aution-4220 was carried out in six laboratories. The analytical performance of the instrument with regard to imprecision, linearity, detection limit, drift, carry-over and method comparison was studied. Using the Aution stick 8 test strip the pH, glucose, protein, ketones, bilirubin, blood, urobilinogen and leukocyte esterase were analysed. Specific gravity measurements were performed by refractive index method. Within-run and between-run imprecision determined at three levels of analyte were good. No carry-over was observed. Obtained results were linear through all the described analytical range. No significant drift was detected. Method comparison with some quantitative methods was performed and showed a good correlation with most of the analytes. The study of interferences showed minor interferences by common therapeutic drugs with the measurement of some analytes. During the assessment period of about 6 months no breakdown occurred in any laboratory. The Super Aution urine analyser appeared to be a highly automated analyser of urinary test strips. The operation was simple and the maintenance required only a few minutes a day.     

Optical time-of-flight chemical detection: spatially resolved analyte mapping with extended-length continuous chemically modified optical fibers

We theoretically evaluate and experimentally verify a novel strategy for spatially resolved analyte mapping over extended remote areas. The approach combines a method for the fabrication of continuous extended-length sensors with optical time-of-flight chemical detection (OTOF-CD). The use of OTOF-CD makes it possible to locate the zones in the fiber where attenuation or fluorescence takes place, to determine the magnitude of these variations, and to relate the magnitude of the variations to the local concentration or concentrations of a single analyte or several analytes. Simulation experiments suggest that OTOF-CD should provide spatial resolution close to its theoretical limit by deconvolution of the returned wave form with all time-dependent experimental variables (laser pulse width, reagent fluorescence lifetime, etc.). The signal-processing technique should be useful for a wide variety of sensors based on absorption, refractive index, or statically and dynamically quenched fluorescence. Experimental results with a model system (a 48-m-long oxygen sensor) compare favorably with those predicted by numerical simulations. Possible experimental difficulties in the realization of these novel sensors are discussed as are ways to overcome them.     

Sensors to Monitor CFRP/Concrete Bond in Beams Using Electrochemical Impedance Spectroscopy

The use of inexpensive electrochemical impedance spectroscopy based sensor technology for nondestructive evaluation (NDE) of bond degradation between external carbon fiber-reinforced polymer (CFRP) reinforcement and concrete is examined. Copper tape on the surface of the CFRP sheet, stainless steel wire embedded in the concrete, and reinforcing bars were used as the sensing elements. Laboratory experiments were designed to test the capability of the sensors to detect the debonding of the CFRP from the concrete and to study the effect of short-term (humidity and temperature fluctuations) and long-term (freeze-thaw and wet-dry exposure and rebar corrosion) environmental conditions on the measurements. The CFRP sheet was debonded from the concrete, and impedance measurements were taken between various pairs of electrodes at various interfacial crack lengths. The dependence of the impedance spectra, and of the parameters obtained from equivalent circuit analysis, on the interfacial crack length was studied. Capacitance parameters in the equivalent circuit correlated strongly with the interfacial crack length and can be used to assess the global state of the bond between CFRP sheets and concrete. Impedance measurements taken between embedded wire sensors can be used to detect the location of debonded regions.     

In-vitro and in-vivo comparative analysis of four tissue pH monitoring systems

The purpose of this investigation was to identify the type of pH-reference electrode combination that is the most suitable and reliable in clinical applications involving long-term postoperative monitoring of microvascular reconstructive transplants and diagnosis of compartment syndrome. Four types of pH-sensing devices were chosen for the study: a standard glass pH electrode, a polymer-based pH electrode, an ISFET pH sensor, and a fiberoptic pH sensor. Various combinations of electrodes were tested in vitro for typically four days. The glass and polymer electrodes maintained stable pH readings, averaging drifts of 0.14 +/- 0.07 and 0.14 +/- 0.08 pH units per 96 hours, respectively. The fiberoptic sensors displayed an average drift of 0.20 +/- 0.15 pH units per 96 hours. ISFET sensors displayed nearly linear drifts, averaging 1.36 +/- 0.54 pH units per 96 hours. When placed in healthy animal tissue, glass and polymer electrode pH readings followed the arterial blood pH values, measured by a blood gas analyzer. In compromised tissue, both glass and polymer electrodes recorded falling pH levels correctly, indicating ischemic conditions. Generally, ISFET sensors in healthy tissue produced pH readings that did not correlate well with arterial blood pH values. Fiberoptic sensors monitored healthy-tissue pH correctly; however, mechanical disturbances of the fiberoptic sensor and occasional discontinuation of computer operation would shift the pH output significantly (at times by 0.2 pH units), resulting in subsequent inaccurate pH readings. The glass electrode was the only sensor evaluated clinically. It correctly indicated tissue viability in all clinical cases, involving both healthy and ischemic tissue. The authors conclude that the glass pH electrode has the best combination of characteristics for clinical tissue pH measurements.     

Spatial dependence of the optical collection efficiency in flow cytometry

The sensitive flow cytometric detection of fluorescent species in liquid sample streams requires efficient collection of light from small [approximately 1 picoliter (pl)] sample volumes. This is often accomplished with high numerical aperture (NA) imaging collection optics used in combination with a spatial filter. A method to measure the spatial variation of the optical collection efficiency within the sample volume, using a submicrometer light source, is described. Measurements of the relative optical collection efficiency are presented for two optical collection systems used in our laboratory for single molecule detection. The measurement are in qualitative agreement with relative optical collection efficiency calculations using a simple geometrical optics model. Absolute measurements of the peak collection efficiencies for the two collection systems are also presented. These absolute collection efficiency measurements are in good quantitative agreement with ideal collection efficiencies calculated using measured transmissions and rated NAs of the collection optics. The utility of this information for the characterization and optimization of sensitive fluorescence detection apparatus is discussed.     

Initial evaluation of a 290-microm diameter subcutaneous glucose sensor: glucose monitoring with a biocompatible, flexible-wire, enzyme-based amperometric microsensor in diabetic and nondiabetic humans

Results of the initial clinical evaluation in 20 human subjects of a subcutaneously implanted microsensor-based amperometrically glycemia-monitoring system, carried out between April 1994 and June 1995, are reported. The system was based on the electrical connection ("wiring") of the reaction centers of glucose oxidase to a gold electrode and on elimination of the chemicals that interfere with glucose monitoring through their horseradish peroxidase-catalyzed oxidation by internally generated hydrogen peroxide. The sensor was finer than a 29-gauge needle and had no leachable components. Because of its high selectivity for glucose, the sensor output was virtually nil at zero glucose level. This enables prompt "one-point" in vivo calibration of the sensor with a single blood glucose sample. Microsensors were subcutaneously implanted in ten nondiabetic and ten insulin-dependent diabetes mellitus (IDDM) volunteers. All subjects underwent standard meal tests and intravenous glucose-tolerance tests (IVGTT) in addition to hourly plasma glucose measurements. The sensor signals were continuously recorded, and the glucose concentration estimates were derived by calibrating the sensor using a single blood sample (one-point calibration). Regression analysis revealed that the sensor-estimated glucose concentrations were linearly related to the plasma glucose concentrations (r2 = 0.75) over a wide glucose concentration range (2-28 mmol/L) (sensor estimate = plasma 0.96 + 0.26 mmol/L). The difference between the estimated and actual glucose concentration was -0.13+/-0.23 mmol/L [mean +/-95% confidence interval (CI), n = 546], and 95% of the estimates fell in clinically acceptable zones of the Clarke error grid. The sensing delay time was 10.4+/-2.3 min as measured by the IVGTT. The subjects reported no discomfort associated with wearing the sensors.     

Light-Based Motion Tracking of Equipment Subjected to Earthquake Motions

Traditional sensors, such as accelerometers and displacement transducers, are widely used in laboratory and field experiments in earthquake engineering to measure the motions of both structural and nonstructural components. Such sensors, however, must be physically attached to the structure and require cumbersome cabling and configurations and substantial time for setup. For reduced-scale experiments, these conventional sensors may substantially alter the dynamic properties of the system by changing the mass, stiffness, and damping properties of the specimen. Moreover, it is very difficult with traditional sensors to capture the three-dimensional motions of light or oddly shaped components such as microscopes, computers,?or other building contents. In this paper, the methodology of light-based motion tracking is applied to the measurement of the three-dimensional motions of various types of equipment and building contents commonly found in biological and chemical science laboratories. The system is comprised of six high-speed, high-resolution charge-coupled-device (CCD) cameras outfitted with a cluster of red-light emitting diodes (LEDs). Retroreflective (passive) spherical markers discretely located in a scene are tracked in time and used to describe the behavior of various types of equipment and contents subjected to a range of earthquake motions. Results from this study show that the nonintrusive, light-based approach is extremely promising in terms of its ability to capture relative displacements in three orthogonal directions and complementary rotations.     

Effect of serum proteins and osteoblasts on the surface transformation of a calcium phosphate coating: a physicochemical and ultrastructural study

We describe a practical method for the analysis of multiple analytes in a single sample. The vehicle for each separate measurement consists of a set of microspheres identifiable by characteristic fluorophores embedded in the particles. The use of robust, bench-top flow cytometers (flow microfluorimeters) for the analysis of the multiple sets of microspheres is facilitated by hardware and software, which acquire the data from the cytometer, classify the microspheres according to sets, and collate measurement information from each microsphere set in real time. This measurement system can analyze up to 64 analytes in a single sample. The advantages of multiplexed assays using flow cytometry include robust measurements, because each microsphere set is measured repeatedly. The advantage of the assay's is consistent with simultaneous measurement of many parameters as well as the speed with which the flow microfluorimeter (cytometer) makes measurements (many hundreds per second). Here, we describe the properties of the microspheres, the calibration of the cytometer, and the influence of the properties of the microspheres on the sensitivity of measurements.     

The development of a new method of cephalometric and study cast mensuration with a computer controlled, video image capture system. Part II: Study cast mensuration

A computerized system for mensuration of dental casts, developed at the Royal Berkshire Hospital in Reading was tested against a conventional manual method and a computer controlled reflex microscope. Systematic errors, calibration, and handling aspects are discussed. Results showed that the computerized system provided data which while statistically different, were clinically comparable with the same measurements carried out using conventional manual or digitizing techniques. The results indicated that future research is required with respect to object illumination for all indirect techniques.     

Determination of dapoxetine, an investigational agent with the potential for treating depression, and its mono- and di-desmethyl metabolites in human plasma using column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of dapoxetine and its mono- and di-desmethyl metabolites in human plasma. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane-ethyl acetate. The organic extract was evaporated to dryness and the residue reconstituted with acetonitrile. The analytes were separated from late-eluting endogenous substances on a Zorbax RX-C8 pre-column. The front-cut fraction containing the analytes was further separated on a second RX-C8 column. The analytes were detected by their native fluorescence, using excitation and emission wavelengths of 230 and 330 nm, respectively. The limit of quantitation was determined to be 20 ng/ml, and the response was linear from 20 to 200 ng/ml. The method has been successfully applied to human plasma samples in a Phase I study.