Miniaturization capable, very sensitive polarimeter as detector of an implantable glucose probe. I. Optical amplification of the measuring value]

From a clinical point of view, an implantable telemetric probe for monitoring the blood glucose profile is highly desirable. It should be capable of monitoring the blood glucose level continuously or at regular brief intervals, if necessary requirement-controlled. Apart from blood, measurement can also be made in intercellular tissue fluid, for example, in subcutaneous connective and fatty tissue, because this fluid accurately reflects blood glucose levels after only a brief, but negligible, time lag. Since the functional lifespan of an implantable probe is of decisive importance, only physical sensors, but not bio-sensors can be considered. We are in the process of developing a very sensitive miniaturised detector based on polarimetry, capable of determining the measuring parameter--the spatial orientation of the in-plane vibration of a polarised light beam--with extreme accuracy. This is a very important point, since the physiological and pathological glucose levels modify the in-plane vibration by only a very tiny angle of rotation. The high level of accuracy is achieved by various specific optical amplification mechanisms, and amplification of the electric signal. Two purely optical amplification methods are described here. Simple linear elongation of the optical path of a laser beam within the sample, resulting in a proportional amplification of the measuring signal, is obviously strictly limited in an implantable probe. We therefore developed a technique that preserves the polarisation state of the light beam during reflection. This technique makes possible multiple passage of the light beam through the fluid being sensed, thus elongating the optical path by "folding" the light beam without the need to enlarge the measuring cuvette. In a second possibility, enlargement of the rotation angle can be achieved by reflecting the light beam from a suitable surface, when the orthogonal components of the polarised light beam are reflected to different extents.     

Fully Metal-Coated Scanning Near-Field Optical Microscopy Probes with Spiral Corrugations for Superfocusing under Arbitrarily Oriented Linearly Polarised Excitation

We study the effect of a spiral corrugation on the outer surface of a fully metal-coated scanning near-field optical microscopy (SNOM) probe using the finite element method. The introduction of a novel form of asymmetry, devoid of any preferential spatial direction and covering the whole angular range of the originally axisymmetric tip, allows attaining strong field localization for a linearly polarised mode with arbitrary orientation. Compared to previously proposed asymmetric structures which require linearly polarised excitation properly oriented with respect to the asymmetry, such a configuration enables significant simplification in mode injection. In fact, not only is the need for the delicate procedure to generate radially polarised beams overcome, but also the relative alignment between the linearly polarised beam and the tip modification is no longer critical.     

Demonstration of a highly-sensitive portable double-flash kinetic spectrophotometer for measurement of electron transfer reactions in intact plants

A highly sensitive, portable spectrophotometer for use in measuring flash-induced absorbance changes in intact leaves is demonstrated. The design of the instrument is modified for whole plant use from that suggested by Joliot and Joliot (Biochim. Biophys. Acta 765, 210–218). The spectrophotometer uses trifurcated light guides to deliver measuring and actinic beams to two comparable areas of the leaf. The measuring beam is provided by a series of short, relatively intense light pulses from a xenon flashlamp in place of the constant weak measuring beam used in conventional machines. The use of a flash measuring beam and differential detection allows for a high signal-to-noise ratio (noise levels of 10^-5A) without significant actinic effects. The time resolution of the instrument is 2 sec and the noise level is independent of the experimental time range. The instrument is battery or mains powered, computer operated, and has a liquid crystal display for computer-user interface and dialogue, and to show the kinetic traces graphically. Wavelength selection is provided by interchangeable interference filters. The instrument can communicate with a laboratory-based computer, receiving programming information and sending experimental data to be processed and plotted. The instrument is demonstrated by following the kinetics of the electrochromic shift, the change in redox states of cytochrome f and the b cytochromes in an intact cucumber leaf, and in the same leaf after infiltration with DCMU.     

Combination of microdialysis and glucosensor permits continuous (on line) SC glucose monitoring in a patient operated device. II. Evaluation in animals.

The microdialysis technique was used for following the glucose content of the extracellular subcutaneous (SC) fluid under varying blood glucose levels in rats. The glucose content in the microdialysis perfusion fluid was continuously analyzed by means of the measuring flow chamber of an ex vivo glucose monitor. In six ChBB rats blood glucose levels were varied between 40 mg/dl and 575 mg/dl by intravenous (IV) infusion of glucose and by SC injections of insulin, respectively. After a running-in period of about half an hour, the glucose content in the perfusion fluid was closely related to the blood glucose concentration (r > 0.92) up to a time period of 6 hrs. The "relative recovery" rate of glucose by the microdialysis probe in the SC tissue varied within the 6 experimental sessions. The relative recovery rate could be shown to be not dependent on the absolute blood glucose levels in the individual rat within the glucose concentration range tested.     

A diffused-optics flash kinetic spectrophotometer (DOFS) for measurements of absorbance changes in intact plants in the steady-state

Measurements of steady-state light-induced absorbance changes in intact plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we present a new instrument, the diffused-optics flash spectrophotometer (DOFS), which reduces the magnitude of light scattering interference to manageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a highly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sample and reference channels. Since the measuring beam has no defined incident angle, it is essentially 'pre-scattered', and further scattering changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and differential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a technique to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are differentially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra taken with the two configurations reveals the spectral shape of the scattering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scattering interference from kinetic traces. We suggest that DOFS is ideally suited for study of steady-state electron transfer reactions in intact plants.     

The Generation of Higher-order Laguerre-Gauss Optical Beams for High-precision Interferometry

Thermal noise in high-reflectivity mirrors is a major impediment for several types of high-precision interferometric experiments that aim to reach the standard quantum limit or to cool mechanical systems to their quantum ground state. This is for example the case of future gravitational wave observatories, whose sensitivity to gravitational wave signals is expected to be limited in the most sensitive frequency band, by atomic vibration of their mirror masses. One promising approach being pursued to overcome this limitation is to employ higher-order Laguerre-Gauss (LG) optical beams in place of the conventionally used fundamental mode. Owing to their more homogeneous light intensity distribution these beams average more effectively over the thermally driven fluctuations of the mirror surface, which in turn reduces the uncertainty in the mirror position sensed by the laser light.We demonstrate a promising method to generate higher-order LG beams by shaping a fundamental Gaussian beam with the help of diffractive optical elements. We show that with conventional sensing and control techniques that are known for stabilizing fundamental laser beams, higher-order LG modes can be purified and stabilized just as well at a comparably high level. A set of diagnostic tools allows us to control and tailor the properties of generated LG beams. This enabled us to produce an LG beam with the highest purity reported to date. The demonstrated compatibility of higher-order LG modes with standard interferometry techniques and with the use of standard spherical optics makes them an ideal candidate for application in a future generation of high-precision interferometry.     

Characterization and applications of CataCleave probe in real-time detection assays

Cycling probe technology (CPT), which utilizes a chimeric DNA-RNA-DNA probe and RNase H, is a rapid, isothermal probe amplification system for the detection of target DNA. Upon hybridization of the probe to its target DNA, RNase H cleaves the RNA portion of the DNA/RNA hybrid. Utilizing CPT, we designed a catalytically cleavable fluorescence probe (CataCleave probe) containing two internal fluorophores. Fluorescence intensity of the probe itself was weak due to F?rster resonance energy transfer. Cleavage of the probe by RNase H in the presence of its target DNA caused enhancement of donor fluorescence, but this was not observed with nonspecific target DNA. Further, RNase H reactions with CataCleave probe exhibit a catalytic dose-dependent response to target DNA. This confirms the capability for the direct detection of specific target DNA through a signal amplification process. Moreover, CataCleave probe is also ideal for detecting DNA amplification processes, such as polymerase chain reaction (PCR) and isothermal rolling circle amplification (RCA). In fact, we observed signal enhancement proportional to the amount of RCA product formed. We were also able to monitor real-time PCR by measuring enhancement of donor fluorescence. Hence, CataCleave probe is useful for real-time monitoring of both isothermal and temperature-cycling nucleic acid amplification methods.     

Suprathermal electrons in a vacuum spark discharge

Results of experiments on the detection of suprathermal electron beams in the plasma of a highcurrent low-inductance vacuum spark by means of space-resolved spectral X-ray polarimetry are presented. It is shown that the observed polarization of bremsstrahlung may be caused by an ~100-keV electron beam propagating along the discharge axis from the pinching region toward the anode. The influence of the discharge initiation conditions on the parameters of the generated electron beams is analyzed.     

Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

A modification of reverse chromosome painting was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromsome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16–24 copies; the largest unit was bigger than 600 kb and was present in 16–32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13–q15. Notably, the signal on 12q13–q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13–q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13–q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.     

An optical monitor of tension for small cardiac preparations.

When a light beam is focused on a muscle preparation which is allowed to contract, large changes in the intensity of the emerging light accompany the contraction. These movement-related optical signals were studied and compared to simultaneous measurements of force in isolated cardiac Purkinje fibers. The two signals were compared in response to action potentials and to graded changes in membrane potential controlled under voltage clamp. These experiments indicate that the optical signal is a sensitive monitor of tension development under these conditions. This technique is particularly well-suited to force measurements in smaller preparations in which direct mechanical techniques are not feasible.     

Continuous monitoring of blood glucose

Continuous blood glucose monitoring aims to: better evaluate glycaemic variations; better detect hypoglycaemia; and, ultimately, automatize insulin delivery (artificial beta cell). The sensors can be fully implantable, with the challenge of constructing durable systems to avoid repeated implantations. In-dwelling needle-like electrodes and microdialysis fibres with a pump that brings the dialysate to the glucose sensor are inserted in the subcutaneous tissue through the skin. The GlucoWatch is an almost non-invasive technique that extracts the extracellular fluid by iontophoresis. In these systems, the glucose oxidase generates the electrical signal, proportional to the glucose concentration. Non-invasive techniques aim at measuring the glucose concentration without breaching the skin, using absorption of light in the infrared spectrum. These techniques have not reached the necessary reliability for use as glycaemic alarms, and even less as artificial beta cells. Currently, glucose sensors are mainly used as glycaemic holters to help in the management of insulin therapy.     

Detection of C677T mutation of MTHFR in subject with coronary heart disease by hairpin probe with enzymatic color on microarray

Molecular beacon (MB) is especially suited for detection of single nucleotide polymorphism (SNP), and the type of MB immobilized on the surface of microarray in particular, may detect multi-sample and multi-locus. However, the majority of MB needs to be labeled with fluorescence and quenching molecules on the two ends of the probe, and observed the reaction of fluorescence or complicated electrochemical signal produced hybridization of MB and target sequence by complex and expensive instruments. The "molecular beacon" and microarray designed appropriately in our study can produce visible light response signal induced by amplification effect of enzymatic color, and are avoided with the marker of fluorescence and quenching molecules and expensive instruments. The "molecular beacon" without fluorescence and quenching molecules is entitled as "hairpin DNA probe" by us for only the "hairpin" structure of traditional molecular beacon is adopted. The merits of two techniques, molecular beacon and amplification effect of enzymatic color, are successfully combined, and the technique is simple, sensitive and specific, to detect and compare the methylenetetrahydrofolate reductase (MTHFR) Gene C677T mutation of subjects between coronary heart disease (CHD) and control group. The results showed that MTHFR Gene C677T polymorphism is an independent risk factor for CHD.     

The signaller's dilemma: a cost-benefit analysis of public and private communication

Background

Understanding the diversity of animal signals requires knowledge of factors which may influence the different stages of communication, from the production of a signal by the sender up to the detection, identification and final decision-making in the receiver. Yet, many studies on signalling systems focus exclusively on the sender, and often ignore the receiver side and the ecological conditions under which signals evolve.

Methodology/Principal Findings

We study a neotropical katydid which uses airborne sound for long distance communication, but also an alternative form of private signalling through substrate vibration. We quantified the strength of predation by bats which eavesdrop on the airborne sound signal, by analysing insect remains at roosts of a bat family. Males do not arbitrarily use one or the other channel for communication, but spend more time with private signalling under full moon conditions, when the nocturnal rainforest favours predation by visually hunting predators. Measurements of metabolic CO₂-production rate indicate that the energy necessary for signalling increases 3-fold in full moon nights when private signalling is favoured. The background noise level for the airborne sound channel can amount to 70 dB SPL, whereas it is low in the vibration channel in the low frequency range of the vibration signal. The active space of the airborne sound signal varies between 22 and 35 meters, contrasting with about 4 meters with the vibration signal transmitted on the insect''s favourite roost plant. Signal perception was studied using neurophysiological methods under outdoor conditions, which is more reliable for the private mode of communication.

Conclusions/Significance

Our results demonstrate the complex effects of ecological conditions, such as predation, nocturnal ambient light levels, and masking noise levels on the performance of receivers in detecting mating signals, and that the net advantage or disadvantage of a mode of communication strongly depends on these conditions.     

Using Neutron Spin Echo Resolved Grazing Incidence Scattering to Investigate Organic Solar Cell Materials

The spin echo resolved grazing incidence scattering (SERGIS) technique has been used to probe the length-scales associated with irregularly shaped crystallites. Neutrons are passed through two well defined regions of magnetic field; one before and one after the sample. The two magnetic field regions have opposite polarity and are tuned such that neutrons travelling through both regions, without being perturbed, will undergo the same number of precessions in opposing directions. In this case the neutron precession in the second arm is said to "echo" the first, and the original polarization of the beam is preserved. If the neutron interacts with a sample and scatters elastically the path through the second arm is not the same as the first and the original polarization is not recovered. Depolarization of the neutron beam is a highly sensitive probe at very small angles (<50 μrad) but still allows a high intensity, divergent beam to be used. The decrease in polarization of the beam reflected from the sample as compared to that from the reference sample can be directly related to structure within the sample.In comparison to scattering observed in neutron reflection measurements the SERGIS signals are often weak and are unlikely to be observed if the in-plane structures within the sample under investigation are dilute, disordered, small in size and polydisperse or the neutron scattering contrast is low. Therefore, good results will most likely be obtained using the SERGIS technique if the sample being measured consist of thin films on a flat substrate and contain scattering features that contains a high density of moderately sized features (30 nm to 5 µm) which scatter neutrons strongly or the features are arranged on a lattice. An advantage of the SERGIS technique is that it can probe structures in the plane of the sample.     

A noise-free molecular hybridization procedure for measuring RNA in cell lysates

A solution hybridization technique was designed to measure RNA abundance in crude cell lysates and at the same time to maximize confidence that signals resulted from true molecular hybridization. Cell lysates were prepared in 5 M guanidine thiocyanate, then RNA molecules in the lysates were hybridized with two probes, a 32P-labeled RNA "label probe" which provided signal and an oligodeoxyribonucleotide "capture probe" containing a poly(dA) tail which provided a mechanism for selective purification. Ternary hybrids were "captured" on oligo(dT)-coated superparamagnetic beads through a readily reversible interaction with the poly(dA) of the capture probe. RNA did not bind to dT beads through poly(A) under the capture conditions used. Hybrids were purified through cycles of capture on and release from dT beads, with each cycle yielding a 100- to 1000-fold reduction in noise (unhybridized label probe) and a 50-90% recovery of signal (hybridized label probe). Noise was driven below detectable limits after three cycles of capture, thereby improving the sensitivity of measuring target RNA. As few as 15,000 target molecules, 15 fg of a 3-kb RNA, was detectable in the equivalent of 2 x 10(6) cells in concentrated cell lysates (10(8) cells/ml). Since hybridization with both probes was required in order to yield a signal, hybridization specificity could be adjusted with either or both probes. The greater specificity and lack of noise increased confidence that the signal was proportional to the amount of RNA of interest.     

Tyramide signal amplification (TSA)-FISH applied to mapping PCR-labeled probes less than 1 kb in size.

Tyramide signal amplification (TSA)-FISH was used to map one mouse and two human DNA probes of less than 1 kb in size. The two human probes were 319 and 608 bp, and the mouse probe was 855 bp. Probes, made from PCR products, were labeled by incorporating biotin-11-dUTP (human) and biotin-16-dUTP (mouse) during PCR amplification. Signals were readily observed in both interphase and metaphase cells following TSA-FISH for all three genes, whereas conventional FISH experiments produced no signals. The two human ATP-binding cassette (ABC) genes, EST883227 (GenBank Accession No. AA243820) and EST990006 (GenBank Accession No. AA348546), mapped to human chromosomes 7p21 and 17q25. The mouse gene, cmyc (exon 2) mapped to band D2 of mouse chromosome 15. These findings demonstrate the ability of this technique to map small probes (PCR products and expressed sequence tags) of less than 1 kb through highly increased signal amplification.     

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.     

A novel opto-electromagnetic actuator coupled to the tympanic membrane

A new type of electromagnetic vibration transducer designed to be placed onto the tympanic membrane was developed. The actuator consisted of two photodiodes, two permanent magnets, an aluminum ring, two opposing wound coils, a latex membrane and a Provil Novo™ membrane. An optic probe was designed to allow sound and light signals to enter the ear canal, thereby preventing the acoustic occlusion effect of traditional ear molds. Two light-emitting diodes were used for carrying the input signals. The corresponding photodiodes were used for receiving the light signals and generating currents in the actuator. The opto-electromagnetic vibration actuator was fabricated and tested using a Laser Doppler vibrometer. The actuator showed displacements of vibration between 30 and 1 nm from 300 to 6500 Hz and reduced in amplitude at higher frequencies. The average gain of the actuator with 140 μA on the umbo displacement was about 20 dB relative to 87 dBA at the distance of 6 cm from the tympanic membrane and 0 μA in actuator.     

Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.